(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property Organization
`International Burcau
`
`(43) International Publication Date
`12 September 2003 (12.09.2003)
`
`
`
`PCT
`
`{10) International Publication Number
`WO 03/074005 A2
`
`(51) International Patent Classification’:
`
`A6IK
`
`Bossey 76, CII-1256 ‘Troinex (CH). BUSSAT, Philippe
`[R/FR]; 46, allée du Saléve, F-74160 Feigéres (PR). AR-
`BOGAST, Christophe [FR//R|; La Sardagne, F-74250
`(21) International Application Number:=PCTI/US03/06731
`Viuz-en-Sallaz (R). PILLAI, Radhakrishna [US/US|;
`12 Walnut Court, Cranbury, NJ 08512 (US). FAN, Hoag
`(22) International Filing Date:=3 March 2003 (03.03.2003)
`[US/US]; 3 Barley Court, Plainsboro, NJ 08536 (US).
`LINDER, Karen, E. [OS/US|; 14 Basin Street, Kingston,
`NJ 08528 (US). SONG, Bo [US/US];
`1 [nglish Lane,
`Princeton, NJ 08540 (0S). NANJAPPAN, Palaniappa
`[US/US]; 30 Liberty Drive, Dayton, NJ 08810 (US).
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`Lnglish
`
`Lnglish
`
`(30) Priority Data:
`60/360,85 1
`60/440,411
`
`| March 2002 (01.03.2002)
`15 January 2003 (15.01.2003)
`
`US
`Us
`
`(74) Agents: TREANNIE, Lisa, M. et al.; Hamilton, Brook,
`Smith & Reynolds, P.C., 530 Virginia Road, P.O. Box 9133,
`Concord, MA 01742-9133 (US).
`
`(71) Applicants (for aff designated States except US}; DVAX
`CORP.
`[US/US]; 300 Technology Square, Cambridge,
`MA 02139 (US) BRACCO INTERNATIONAL B.V.
`[NLANL]; Strawinskylaan 3051, NIL_L-1077 72M Amsterdam
`(NIL).
`
`(72)
`(75)
`
`Inventors; and
`SATO, Aaron,
`Inventors/Applicants (for US only}:
`K.
`[US/US];
`19 Central Street #21, Cambridge, MA
`02145 (US). SEXTON, Daniel, J. [CA/US]; 93 Yorktown
`Street, Somerville, MA 02144 (US). LADNER, Robert,
`C.
`[US/US]; 3827 Green Valley Road, Ijamsville, MD
`21754 (US). DRANSFIELD, Daniel, T.
`|US/US|;
`14
`George Street, Ianson, MA 02341 (US). SWENSON,
`Rolf, E.
`[US/US]; 35 Vicldston Road, Princeton, NJ
`08540 (US) MARINELLI, Edmund, R.
`[US/US];
`190 ildridge Avenue, Lawrenceville, NJ O8648 (US).
`RAMALINGAM, Kondareddiar [US/US]; 46 Liberty
`Drive, Dayton, NJ 08810 (US). NUNN, Adrian, D.
`[GB/US]; 33 Mill Read, Lambertville, NJ 08530 (US).
`VON WRONSKI, Mathew, A. [US/US]; 604 Oldershaw
`Avenue, Moorestown, NJ 08057 (US). SHRIVASTAVA,
`Ajay |IN/US]; 18-16 Pheasant follow Drive, Plainsboro,
`NJ 08536 (US). POCHON, Sibylle [CIM/CI]]; Route de
`
`(81) Designated States (rational): AH, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BY, CA, CIL CN, CO, CR, CU,
`C47, DE, DK, DM, D4, BC, U1, US, FL, GB, GD, GE, GH,
`GM, HR, 110, 09, IL, IN, 18, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LY, MA, MD, MG, MK, MN, MW,
`MX, M¥, NO, N¥%, OM, PE, PL, PT, RO, RU, SC, SD, SK,
`SG, SK, SL, TI, TM, TN, TR, TT, TZ, UA, UG, US, UZ,
`VC, VN, YU, 4A, 4M, ZW.
`
`(84) Designated States fregional/: ARIPO patent (GH, GM,
`Kh, LS, MW, MY, SD, SL, S¥%, TZ, UG, ZM, ZW),
`Ifurasian patent (AM, AZ, BY. KG, KZ, MD, RU, TJ, TM),
`Huropean patent (AT, Bl, BG, CIL CY, C4, DE, DK, EE,
`LS, FI, PR, GB, GR, WU, TE, IT, LU, MC, NL, PT, RO,
`SE, SL, SK, TR), OAPI patent (BE, BJ, CK CG, CL, CM,
`GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`upon receipt ofthat report
`
`hor two-letter codes and other abbreviations, refer to the "Guid-
`ance Notes on Codes and Abbreviations” appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`03/074005A2
`
`(54) Title: KDR AND VEGEKDR BINDING PEPTIDES AND TITEIR USE IN DIAGNOSIS AND THERAPY
`
`©} (57) Abstract: The present invention provides binding polypetides for KDR or VEGE/RKDR complex, which have a variety of uses
`wherever trealing, Detecting, isolating or localizing angiogenesis is advantageous. Particularly disclosed are synthetic, isolated
`polypeptides capable of binding KDR or VEGIYKDR complex with high alfinily (¢.g., having a Kn<! uM).
`
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`WO 03/074005
`
`PCT/US03/06731
`
`KDR AND VEGF/KDR BINDING PEPTIDES AND
`
`THEIR USE IN DIAGNOSIS AND THERAPY
`
`RELATED APPLICATIONS
`
`This application claims the benefit of U.S. Provisional Application No.
`60/360,851, filed March 1, 2002, and U.S. Provisional Application No. 60/440,411,
`
`filed January 15, 2003.
`
`BACKGROUND OF THE INVENTION
`
`In the developing embryo,the primary vascular networkis established by in
`situ differentiation of meso-dermalcells in a process called vasculogenesis. After
`
`embryonic vasculogenesis however, it is believed that all subsequent generation of
`new blood vessels, in the embryo or in adults, is governed by the sprouting or
`splitting of new capillaries fromthe pre-existing vasculature in a process called
`angiogenesis (Pepper, M.ef ai., 1996. Enzyme Protein, 49:138-162; Risau, W.,
`1997. Nature, 386:671-674). Angiogenesis is not only involved in embryonic
`development and normal tissue growth andrepair,it is also involved in the female
`reproductive cycle, establishment and maintenance of pregnancy, and in repair of
`wounds and fractures. In addition to angiogenesis that takes place in the normal
`
`individual, angiogenic events are involved in a numberof pathological processes,
`notably tumor growth and metastasis, and other conditions in which blood vessel
`proliferation is increased, such as diabetic retinopathy, psoriasis and arthropathies.
`Angiogenesis is so important in the transition of a tumor from hyperplastic to
`neoplastic growth, that inhibition of angiogenesis has becomean active cancer
`therapy (Kim,K.et al., 1993. Nature, 362:841-844).
`|
`Tumor-induced angiogenesis is thought to depend onthe production of pro-
`angiogenic growth factors by the tumor cells, which overcomeother forces that tend
`to keep existing vessels quiescent and stable (Hanahan, D. and Folkman,J., 1996.
`Cell, 86:353-364). The best characterized of these pro-angiogenic agents is vascular
`endothelial growth factor (VEGF) (Neufeld, G.et al, 1999, FASEB J., 13:9-22).
`VEGFis produced naturally by a variety of cell types in response to hypoxia
`and someotherstimuli. Many tumors also produce large amounts of VEGF,and/or
`induce nearby stromalcells to make VEGF (Fukumura, D. ef al., 1998, Cell, 94:715-
`725). VEGF,also referred to as VEGF-A,is synthesized as five different splice
`isoforms of 121, 145, 165, 189, and 206 amino acids. VEGF121 and VEGF165 are the
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`main forms produced, particularly in tumors (sée,Neiifeld,Gréfat! 1999Supra)."
`
`VEGF)2 lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does
`
`not bind to heparin or extracellular mairix, unlike VEGFigs.
`
`VEGFfamily members act primarily by binding to receptor tyrosine kinases.
`
`In general, receptor tyrosine kinases are glycoproteins having an extracellular
`
`domain capable of binding one or more specific growth factors, a transmembrane
`
`domain (usually an alpha helix), a juxtamembrane domain (where the receptor may
`
`be regulated, e.g., by phosphorylation), a tyrosine kinase domain (the catalytic
`
`componentof the receptor), and a carboxy-terminal tail, which in many receptorsis
`
`involved in recognition and binding of the substrates for the tyrosine kinase. There
`
`are three endothelial cell-specific receptor tyrosine kinases known to bind VEGF:
`
`VEGFR-1 (Fit-1), VEGFR-2 (KDR or Fik-1), and VEGFR-3 (Flt4). Flt-1 and KDR
`
`have been identified as the primary high affinity VEGF receptors. While Flt-1 has
`
`higheraffinity for VEGF, KDR displays more abundant endothelial cell expression
`
`15
`
`(Bikfalvi, A. et aZ., 1991. / Cell. Physiol., 149:50-59). Moreover, KDRis thought
`
`to dominate the angiogenic response and is therefore of greater therapeutic and
`
`diagnostic interest (see, Neufeld, G. et al. 1999, supra). Expression of KDRis
`
`highly upregulated in angiogenic vessels, especially m tumors that induce a strong
`
`angiogenic response (Veikkola, T. ef a/., 2000. Cancer Res., 60:203-212).
`
`20
`
`KDRis made up of 1336 amino acidsin its mature form. Because of
`
`glycosylation, it migrates on an SDS-PAGEgel with an apparent molecular weight
`
`of about 205 kDa. KDR contains seven immunoglobulin-like domains in its
`
`extracellular domain, of which the first three are the most important in VEGF
`
`binding (Neufeld, G. et af. 1999, supra). VEGFitself is a homodimercapable of
`
`binding to two KDR molecules simultaneously. The result is that two KDR
`
`molecules become dimerized upon binding and autophosphorylate, becoming much
`
`more active. The increased kinase activity in turn initiates a signaling pathwaythat
`
`mediates the KDR-specific biological effects of VEGF.
`
`From the foregoing, it can be seen that not only is the VEGF binding activity
`
`30
`
`of KDR ixvivo critical to angiogenesis, but the ability to detect KDR upregulation
`
`on endothelial cells or to detect VEGF/KDR binding complexes would be extremely
`
`beneficial in detecting or monitoring angiogenesis, with particular diagnostic
`applications such as detecting malignant tumor growth. It would aiso be beneficial
`in therapeutic applications such as targeting tumorcidal agents or angiogenesis
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`inhibitors to a tumorsite or targeting KDR, VEGFACDR,of ahplogenesis"azonists"to
`
`a desired site.
`
`SUMMARY OF THE INVENTION
`The present invention relates to polypeptides and compositions useful for
`detecting and targeting primary receptors on endothelial cells for vascular
`endothelial growth factor (VEGF),i.e., vascular endothelial growth factor receptor-2
`(VEGFR-2, also known as kinase domain region (KDR)andfetal liver kinase-1
`(Flk-1)), and for imaging andtargeting complexes formed by VEGF and KDR. The
`involvement of VEGF and KDRin angiogenesis makes the VEGF/KDRand KDR
`binding polypeptides of the present invention particularly useful for imaging
`importantsites of angiogenesis, ¢.g., neoplastic tumors, for targeting substances,
`e.g., therapeutics, including radiotherapeutics,to such sites, and for treating certain
`disease states, including those associated with inappropriate angiogenesis.
`A group ofpolypeptides has been discovered that bind to KDR or
`VEGF/KDRcomplex (referred to herein as "KDR binding polypeptides" or "KDR
`binding moieties" and homologuesthereof). Such KDR and VEGF/KDRbinding
`polypeptides will concentrate at the sites of angiogenesis, thus providing a means for
`detecting and imagingsites of active angiogenesis, which may includesites of
`neoplastic tumor growth. Such KDR and VEGF/KDRbinding polypeptides provide
`novel therapeutics to inhibit or promote, ¢.g., angiogenesis. The preparation, use
`and screening of such polypeptides, for example as imaging agents or as fusion
`partners for KDR or VEGF/KDR-homung therapeutics, is described in detail herein.
`Tn answer to the need for improved materials and methods for detecting,
`localizing, measuring and possibly inhibiting affecting, e.g., angiogenesis, we have
`now surprisingly discovered seven families of non-naturally occurring polypeptides
`that bind specifically to KDR or VEGF/KDRcomplex. Appropriate labeling of such
`polypeptides provides detectable imaging agents that can bind, e.g., at high
`concentration, to KDR-expressing endothelial cells or cells exhibiting VEGF/KDR
`complexes, providing angiogenesis specific imaging agents. The KDR and
`VEGF/KDRbinding polypeptides ofthe instant invention can thus be used in the
`detection and diagnosis of such angiogenesis-related disorders. Conjugation or
`fusion of such polypeptides with effective agents such as VEGF inhibitors or
`tumorcidal agents can also be used to treat pathogenic tumors, ¢.g., by causing the
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`
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`
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`
`conjugate or fusion to "home"to the site of active'angidgériesis;"thérébyproviditig'an
`effective means for treating pathogenic conditions associated with angiogenesis.
`This invention pertains to KDR and VEGF/KDR binding polypeptides, and
`includesuse ofa single binding polypeptide as a monomeror in a multimeric or
`polymeric construct as well as use ofmore than one binding polypeptide of the
`invention in multimeric or polymeric constructs. Binding polypeptides according to
`this invention are useful in any application where binding, detecting or isolating
`KDR or VEGF/KDR complex,or fragments thereofretaining the polypeptide
`bindingsite, is advantageous. A particularly advantageous use ofthe binding
`polypeptides disclosed herein is in a method ofimaging angiogenesis in vivo. The
`method entails the use of specific binding polypeptides according to the invention
`for detecting a site of angiogenesis, where the binding polypeptides have been
`detectably labeled for use as imaging agents, including magnetic resonance imaging
`(MRI) contrast agents, x-ray imaging agents, radiopharmaceutical imaging agents,
`ultrasound imaging agents, and optical imaging agents.
`Another advantageous use ofthe KDR and VEGF/KDR complex binding
`polypeptides disclosed herein 1s to target therapeutic agents, (including compounds
`capable ofproviding a therapeutic, radiotherapeutic or cytotoxic effect.) or delivery
`vehicles for therapeutics (including drugs, genetic material, eic.) to sites of
`angiogenesis orother tissue expressing KDR.
`Constructs comprising two or more KDR or KDR/VEGF binding
`polypeptides show improved ability to bind the target molecule compared to the
`corresponding monomeric binding polypeptides. For example, as shown in.
`Experiment5, tetrameric constructs of KDR binding polypeptides provided herein
`showed improved ability to bind KDR-transfected 293H cells. Combining two or
`more binding polypeptides in a single molecular construct appears to improve the
`avidity of the construct over the monomeric binding polypeptides as shown by a
`decrease in Kp.
`In addition, as demonstrated herein, constructs comprising two or more
`binding polypeptides specific for different epitopes of KDR and/or KDR/VEGF
`(2.g., “heteromeric” or “heteromultimeric” constructs, see U.S. application number
`60/440,201, and the application,filed concurrently herewith, having attomey’s
`docket number 50203/010004, the contents of each iscorporated herein) were
`made. Constructs comprising two or more binding polypeptides provided herein are
`
`10
`
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`WO 03/074005
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`expected to block multiple sites on KDR or VEGE/KIDR. “Phe hétetontetic! oF ot.
`
`constructs show superior binding ability over both the corresponding monomers, as
`
`well as multimeric constructs comprising multiple copies of the same binding
`
`polypeptide. Furthermore, heteromeric constructs comprising two or more binding
`
`peptides specific for different epitopes, together with a control peptide were also
`
`able to efficiently bind KDR-transfected 293H cells. Thus, inclusion of two or more
`
`binding polypeptides that recognize different epitopes further improves the avidity of
`
`the constructfor the target molecule, as demonstrated by a decrease in Kp.
`
`Heteromeric constructs of the binding polypeptides provided herein show
`
`10
`
`improved ability to inhibit receptor tyrosine kinase function. Based on experiments
`
`described herein, dimeric and other multimeric constructs of the present invention
`
`comprising at least two binding polypeptides specific for different epitopes of KDR
`
`and/or KDR/VEGFare expected to inhibit the function of receptor tyrosine kinases.
`
`In particular, such constructs are expected to inhibit the function of VEGF-2/KDR,
`
`15
`
`VEGF-1/FIt-1 and VEGF-3/Flt-4,
`
`For the purposes of the present invention, receptor tyrosine kinase function
`
`can include any one of: oligomerization of the receptor, receptor phosphorylation,
`
`kinase activity of the receptor, recruitment of downstream signaling molecules,
`
`induction of genes, induction of cell proliferation,duction of cel] migration, or
`
`20
`
`combination thereof. For example, heteromeric constructs of binding polypeptides
`
`provided herein inhibit VEGF-induced KDR receptoractivation in human
`
`endothelial cells, demonstrated by the inhibition of VEGF-induced phosphorylation
`
`of the KDR receptor. In addition, heteromeric constructs of binding peptides
`
`provided herein inhibit VEGF-stimulated endothelial cell migration. As shown
`
`25
`
`herein, targeting two or more distinct epitopes on KDR with a single binding
`
`construct greatly improves the ability of the construct to inhibit receptor function.
`
`Even binding peptides with weakability to block receptor activity can be used to
`
`generate heteromeric constructs having improved ability to block VEGF-induced
`
`receptor function.
`
`30
`
`Therefore, the present invention is drawn to constructs comprising two or
`
`more binding polypeptides. In one embodiment, the multimeric constructs comprise
`
`two or more copies of a single binding polypeptide. In another embodiment, the
`multimeric constructs of the present invention comprise two or more binding
`polypeptides, such that at least two ofthe binding polypeptides in the construct are
`
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`
`specific fordifferent epitopes ofKDR and/or KDR/VEGE.”These’Eonstiuctsarealso
`referred to herein as “heteromeric constructs,” “heteromultimers,” etc. The
`constructs of the present invention can also include unrelated, or control peptide.
`The constructs can include two or more,three or more, or four or more binding
`polypeptides. Based on the teachings provided herein, one of ordinary skill in the art
`is able to assemble the binding polypeptides provided herein into multimeric
`constructs and to select multimeric constructs having improved properties, such as
`improved ability to bind the target molecule, or improved ability to inhibit receptor
`tyrosine kinase function. Such multimeric constructs having improved properties are
`included in the present invention.
`Consensus sequences 1-14) have been determined based onthe specific KDR
`and VEGF/KDRbinding polypeptides shown in Tables 1-7. In specific
`embodiments, KDR and VEGEF/KDRbinding polypeptides of the invention
`comprise one or more ofthese sequences. Such preferred KDR or VEGF/KDR.
`complex binding polypeptides include polypeptides with the potential to form a
`cyclic or loop structure between invariant cysteine residues comprising, or
`alternatively consisting of, an amino acid sequenceselected from Consensus
`
`Sequences 1-5 below:
`
`Consensus Sequence 1: X|~X2—X3-Cys—X5~—X¢ X7—Kg—X9 —-Kyo -Cys-X12-
`
`Xi3-X14 (TN8), wherein
`%X, is Ala, Arg, Asp, Gly, His, Leu, Lys, Pro, Ser, Thr, Trp, Tyr or Val;
`X> is Asn, Asp, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr or Val;
`X3 is Asn, Asp, Gin, Glu, Ile, Leu, Met, Thr, Trp or Val;
`Xs is Ala, Arg, Asn, Asp, Gln, Glu, His,Ile, Lys, Phe, Pro, Ser, Trp or Tyr;
`X¢ is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ne, Lys, Met, Phe, Pro, Ser, Thr, Trp,
`
`Tyr or Val;
`X7 is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
`Xs is Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Thr, Trp or Tyr;
`Xo is Arg, Gln, Glu, Gly, He, Leu, Met, Pro, Thr, Trp, Tyr or Val;
`Xo is Ala, Arg, Gln, Glu, Gly, His,Tle, Leu, Lys, Met, Phe, Trp or Tyr;
`X12 is Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr
`
`or Val;
`X13 is Arg, Asn, Asp, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp or
`
`Tyr; and
`
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`Xy4 is Gin, Glu, Gly, His, We, Leu, Lys, Met, Phe, Pto; Ser,Thr‘Tip or Tyr"
`and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
`Consensus Sequence 2: KKX3-Cys—XspK7-Xg—KoKoMA
`Xi3—-Xya-Cys-Kig-Xi7-X18 (TN12), wherein
`%; is Ala, Asn, Asp, Gly, Leu, Pro, Ser, Trp or Tyr (preferably Asn, Asp, Pro or
`
`10
`
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`
`20
`
`Tyr);
`X> is Ala, Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr (preferably Asp, Gly,
`Pro, Ser or Trp);
`X; is Ala, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or
`
`Val (preferably Trp);
`X;5 is Arg, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Thr, Trp, Tyr or Val (preferably
`Glu,Ile or Tyr);
`X,¢ is Ala, Arg, Asn, Cys, Glu,Ile, Leu, Met, Phe, Ser, Trp or Tyr (preferably Glu,
`
`Phe or Tyr);
`X; is Arg, Asn, Asp, Gln, Glu, His, fle, Leu, Pro, Ser, Thr, Trp, Tyr or Val
`(preferably Glu);
`Xe is Ala, Asn, Asp, Gin, Glu, Gly, His, Met, Phe, Pro, Ser, Trp, Tyr or Val
`(preferably Gln or Ser);
`Xo is Asp, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp or Tyr
`
`(preferably Asp);
`Xyo is Ala, Arg, Asn, Asp, Gln, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr
`or Val (preferably Lys or Ser);
`Xi is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Trp, Tyr or Val (preferably Gly
`
`or Tyr);
`X17 is Ala, Arg, Gln, Gly, His, Ie, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val
`(preferably Trp or Thr);
`X13 is Arg, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, Trp or Val (preferably Glu
`
`or Trp);
`X14 is Arg, Asn, Asp, Glu, His,Ile, Leu, Met, Phe, Pro, Thr, Trp, Tyr or Val
`(preferably Phe);
`X16 is Ala, Asn, Asp, Gln, Glu, Gly, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val
`
`30
`
`(preferably Asp),
`Xi7 is Arg, Asn, Asp, Cys, Gly, His, Phe, Pro, Ser, Trp or Tyr (preferably Pro or
`Tyr); and
`
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`Xi is Ala, Asn, Asp, Gly, His, Leu, Phe, Pro, Ser,Trp or Tyi'(pieftrablyA's, Pto’br
`
`Trp),
`and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
`Consensus Sequence 3: Xy—Ko-Xy-Cys-X5s—X6-XK7-Gly—Xp—Cys-—B1Aa—
`
`X13 (TN7), wherein
`
`X, is Gly or Trp;
`
`X2 is Ile, Tyr or Val;
`
`3 is Gln, Glu Thr or Trp;
`
`Xs is Asn, Asp or Glu;
`
`10
`
`X¢ is Glu, His, Lys or Phe;
`
`X7 is Asp, Gln, Leu, Lys Met or Tyr;
`
`Xo is Arg, Gln, Leu, Lys or Val,
`
`Xj), is Arg, Phe, Ser, Trp or Val,
`
`X12 1s Giu, His or Ser; and
`
`Xy3 is Glu, Gly, Trp or Tyr,
`and wherein the polypeptide binds KDR or a VEGF/KDRcomplex;or
`
`Consensus Sequence 4: X)—X.—X3-Cys—X5—X¢ Xy—Xg—Ko—X9-KX11-“Cys—
`
`Xy3-Xg—X1s (TINY), wherein
`X, is Arg, Asp, Gly, Ile, Met, Pro or Tyr (preferably Tyr);
`X is Asp, Gly, His, Pro or Trp (preferably Gly or Trp);
`X3 is Gly, Pro, Phe, Thr or Trp (preferably Pro);
`Xs is Ala, Asp, Lys, Ser, Trp or Val (preferably Lys);
`
`X¢ is Asn, Glu, Gly, His or Leu;
`X, is Gln, Giu, Gly, Met, Lys, Phe, Tyr or Val (preferably Met);
`is Ala, Asn, Asp, Gly, Leu, Met, Pro, Ser or Thr;
`
`Xo is His, Pro or Trp (preferably Pro);
`Xo is Ala, Gly, His, Leu, Trp or Tyr (preferably His or Trp);
`X11 is Ala, Asp, Gin, Leu, Met, Thror Trp;
`Xi3 is Ala, Lys, Ser, Trp or Tyr (preferably Trp);
`X\4 is Asp, Gly, Leu, His, Met, Thr, Trp or Tyr (preferably His, Trp, or Tyr); and
`X15 is Asn, Gln, Glu, Leu, Met, Pro or Trp (preferably Glu, Met or Ttp),
`and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
`Consensus Sequence5: Xy—-X2-X3-Cys—K5-X-X7-Xg-Ser-GlyPro-Xi2-
`Xis-Xyq—Kys-Cys—X17-X1e-Xi9 (MIN13; SEQ ID NO:1), wherein
`
`20
`
`25
`
`30
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`X, is Arg, Glu, His, Ser or Trp;
`
`X2 is Asn, Asp, Leu, Phe, Thr or Val;
`
`X3 is Arg, Asp, Glu, His, Lys or Thr;
`
`Xs is Asp, Glu, His or Thr;
`
`X, 1s Arg, His, Lys or Phe;
`
`X7 is Gln, Ile, Lys, Tyr or Val;
`
`Xs is Gln, Ile, Leu, Met or Phe;
`
`Xj2 is Asn, Asp, Gly, His or Tyr;
`
`X13 is Gln, Gly, Ser or Thr;
`
`Xi4 is Glu, Lys, Phe or Ser;
`
`Xis is Glu,Ile, Ser or Val;
`
`Xi7 is Glu, Gly, Lys, Phe, Ser or Val;
`
`Xig is Arg, Asn, Ser or Tyr; and
`
`X19 is Asp, Gln, Glu, Gly, Met or Tyr,
`
`and wherein the polypeptide binds KDR or a VEGF/KDR complex,
`
`Further analysis of the polypeptides isolated from the TN8&library (see
`
`Consensus Sequence 1) revealed sub-families of preferred binding polypeptides,
`
`which are described by the Consensus Sequences 6, 7 and 8 as follows:
`
`Consensus Sequence 6: 4)—X2—X3—Cys—X5—Kg-—X7—Xg_—Xo-Tyr_Cys—X2
`
`20
`
`Xy3—-N14, wherein
`
`X; 1s Ala, Arg, Asp, Leu, Lys, Pro, Ser orVal;
`
`Xz is Asn, Asp, Glu, Lys, Thr or Ser (preferably Asn, Asp, Glu or Lys);
`
`X3 1s Ile, Leu or Trp;
`
`Xs5 1s Ala, Arg, Glu, Lys or Ser (preferably Glu);
`
`25
`
`Xe is Ala, Asp, Gln, Glu, Thr or Val (preferably Asp or Glu);
`
`X7 1s Asp or Glu;
`
`Xe is Trp or Tyr;
`
`Xg is Thr or Tyr (preferably Tyr);
`
`Xj2 1s Glu, Met, Phe, Trp or Tyr (preferably Trp, Phe, Met, or Tyr);
`
`30
`
`X13 is Ile, Leu or Met; and
`
`X14 is Ile, Leu, Met, Phe or Thr (preferably Thr or Leu),
`
`and wherein the polypeptide binds KDR or a VEGEF/KDR complex; or
`
`Consensus Sequence 7: Trp—Tyr-Trp—Cys—X'5s—Xp—X7-G]y—X9—X1 9-Cys—
`
`Xy2-K13—-X14 (SEQ ID NO:2), wherein
`
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`Xs; is Asp, Gin or Fis;
`Xs is His or Tyr (preferably Tyr);
`
`X; is Ile, His or Tyr;
`
`Xo is Ile, Met or Val;
`
`Xi is Gly or Tyr;
`
`X12 is Asp, Lys or Pro;
`
`X43 is Gln, Gly or Trp; and
`
`X14 is Phe, Ser or Thr,
`and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
`Consensus Sequence 8: Xy-K2-X3-Cys—Ks—KgKy-Xg—-Gly—Ko-Cys-Xj2-
`
`Xi3—X14, wherein.
`X, is Gly, Leu, His, Thr, Trp or Tyr (preferably Trp, Tyr, Leu or His);
`X> is Ile, Leu, Thr, Trp or Val(preferably Val, Ile or Leu);
`X; is Asp, Glu, Gln, Trp or Thr, (preferably Glu, Asp or Gln);
`Xs is Ala, Arg, Asn, Asp, His, Phe, Trp or Tyr (preferably Tyr, Trp or Phe);
`X¢ is Ala, Asp, Gln, His, Lys, Met, Ser, Thr, Trp, Tyr or Val;
`X> is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr or Val;
`X, is Asp, Phe, Ser, Thr, Trp or Tyr (preferably Thr, Ser or Asp);
`Xt is Ala, Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr (preferably Arg or
`
`Lys);
`X12 is Arg, Gln, His, Ile, Lys, Met, Phe, Thr, Trp, Tyr or Val (preferably Tyr, Trp,
`Phe, Ile or Val);
`X13 is Arg, Asn, Asp, Glu, His, Met, Pro, Ser or Thr; and.
`Xi4is Arg, Gln, Glu, Gly, Phe, Ser, Trp or Tyr,
`and wherein the polypeptide binds KDR or a VEGF/KDR complex.
`Further analysis of the polypeptides isolated from the TN12 library (see
`Consensus Sequence2) revealed sub-families of preferred binding polypeptides,
`which are described by Consensus Sequences 9-12 as follows:
`
`Consensus Sequence 9: X;-X)~X3-Cys—X5—Kg—X7—Xg Trp-Gly—Gly-X12-
`Xq3-Cys—X1s—X1e-Xi7 (TN11, i.e, 11-merbindersisolated from the TN12 library;
`SEQ ID NO:3), wherein.
`
`10
`
`15
`
`20)
`
`25
`
`30
`
`X, is Ser, Phe, Trp, Tyr or Gly (preferably Ser);
`
`X, is Arg, Gly, Ser or Trp (preferably Arg);
`
`X3 is Ala, Glu,Ile or Val (preferably Val or Ie);
`
`10
`
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`Xs is Ala, Phe or Trp (preferably Trp or Phe);
`X¢ is Glu or Lys (preferably Glu);
`X, is Asp, Ser, Trp or Tyr (preferably Asp, Trp or Tyr);
`X3 is Phe, Pro or Ser (preferably Ser);
`
`Xy2 is Gin or Glu (preferably Glu);
`
`10
`
`X13 is He, Phe or Val,
`X15 is Gln, Ile, Leu, Phe or Tyr (preferably Phe, Tyr or Leu);
`Xi¢ is Arg, Gly or Pro (preferably Arg); and
`X17 is Gln, His, Phe, Ser, Tyr or Val (preferably Tyr, Phe, His or Val),
`and wherein the polypeptide binds KDR ora VEGF/KDR complex;or
`Consensus Sequence 10: Tyr—-Pro-X3—Cys—Xs-Glu-X7-Ser—X5-Ser-Aj1
`XX13-Phe-Cys—Xis-X17-¥18 (TN12; SEQ ID NO:4), wherein
`X3 is Gly or Trp (preferably Trp);
`Xs is His or Tyr (preferably His, or Tyr);
`
`15
`
`X, is His, Leu or Thr;
`
`Xo is Asp or Leu (preferably Asp);
`X11 is Gly or Val (preferably Val);
`
`X12is Thr or Val (preferably Thr);
`
`Xi; is Arg or Trp (preferably Arg):
`
`20
`
`Xis5 is Ala or Val (preferably Val);
`X17 is Asp or Pro (preferably Pro); and
`Xs is Gly or Trp (preferably Trp),
`and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
`Consensus Sequence 11: XXX3-Cys-X5-Xgp-X7-Xg-KoMpGlyX2-
`Trp—Xy4-Cys—X16—Xi7-Xig (TN12; SEQ ID NO:5), wherein
`X,is Asp, Gly, Pro or Ser (preferably Asp);
`*> is Arg, Asn, Asp, Gly or Ser (preferably Asp, Asn,or Ser);
`X; is Gly, Thr, Trp or Tyr (preferably Trp or Tyx);
`Xs is Glu, Metor Thr (preferably Glu);
`Xz is He, Leu, Met or Phe (preferably Met, Leu, or Phe);
`X7 is Arg, Asp, Glu, Met, Trp or Val;
`
`25
`
`30
`
`Xg is Asn, Glu, Gly, Ser or Val;
`
`Xo is Asp or Glu;
`Xio is Lys, Ser, Thr or Val (preferably Lys);
`
`11
`
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`X12 is Arg, Glu, Lys or Trp (preferably Trp, Arg,ofLys);
`X14 is Asn, Leu, Phe or Tyr(preferably Tyr, Phe, or Asn);
`X16 is Gly, Phe, Ser or Tyr (preferably Tyr or Phe);
`X17 is Gly, Leu, Pro or Ser (preferably Pro or Ser); and
`Xie is Ala, Asp, Pro, Ser, Trp or Tyr,
`and wherein the polypeptide binds KDR or a VEGF/SDR complex; or
`Consensus Sequence 12: Asn~Trp—X3—Cys—X5—Xp-X7-Xg-Ko-—Kio-M-
`My-X—X—-Cys—X16—X17X18 (TN12; SEQ ID NO:6), wherein
`
`X3 is Glu or Lys;
`
`10
`
`Xs is Glu or Gly;
`
`X. is Trp or Tyr;
`
`X; is Ser or Thr;
`
`Xs is Asn or Gin;
`
`Xo is Gly or Met;
`
`15
`
`Xio 18 Phe or Tyr;
`
`X1, is Asp or Gln;
`
`Xj2 1s Lys or Tyr;
`
`Xi3 is Glu or Thr;
`
`X14 is Glu or Phe;
`
`20
`
`X16 is Ala or Val;
`
`X17 is Arg or Tyr; and
`
`25
`
`30
`
`Xig is Leu or Pro,
`and wherein the polypeptide binds KDR or a VEGF/KDR complex.
`Analysis of the binding polypeptides isolated from a linear display library
`(Lin20) defined two families ofpreferred embodiments including the amino acid
`sequences of Consensus Sequences 13 and 14 as follows:
`Consensus Sequence 13: Z)—X\—X2—-X3-Xy-K5-Zy (Lin20), wherein,
`Z, is a polypeptide of at least one aminoacid oris absent,
`X, is Ala, Asp, Gln or Glu (preferably Gln or Glu);
`X> is Ala, Asp, Gln, Glu Pro (preferably Asp, Glu or Gin);
`X; is Ala, Leu, Lys, Phe, Pro, Trp or Tyr (preferably Trp, Tyr. Phe or Leu);
`X4 is Asp, Leu, Ser, Trp, Tyr or Val(preferably Tyr, Trp, Leu or Val);
`Xs is Ala, Arg, Asp, Glu, Gly, Leu, Trp or Tyr (preferably Trp, Tyr or Leu); and
`Z, is a polypeptide of at least one amino acid oris absent,
`
`12
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`and wherein the polypeptide binds KDR or a VEGF7IKCUK cotmipiéexsor
`
`Consensus Sequence 14: X;—X3—X3—Tyr-Trp—Glu—X7—Xg—Xo—Leu (Lin20;
`
`SEQ ID NO:7), wherein, the sequence can optionally have a N-terminal polypeptide,
`
`C-terminal polypeptide, or a polypeptide at both termini ofat least one amino acid;
`
`and wherein
`
`Xj, is Asp, Gly or Ser (preferably Gly);
`
`X2 is Ile, Phe or Tyr;
`
`X3 18 Ala, Ser or Val;
`
`7 is Gln, Glu,Ile or Val;
`
`10
`
`Xg 1s Ala, Ile or Val (preferably De or Val);
`
`Xo is Ala, Glu, Val or Thr;
`
`and wherein the polypeptide binds KDR or a VEGF/KDR complex.
`
`Preferred embodiments comprising the Consensus Sequence 1 above, include
`
`polypeptides in which X3 is Trp and the amino acid sequence of X7-Xjo is Asp-Trp-
`
`Tyr-Tyr (SEQ ID NO:8). More preferred structures include polypeptides comprising
`
`Consensus Sequence 1, wherein X3 is Trp and the amino acid sequence of X5~Xjq is
`
`Glu-Glu-Asp-Trp-Tyr-Tyr (SEQ ID NO:9). Additional preferred polypeptides
`
`comprising Consensus Sequence | include polypeptides in which: X3 is Trp and the
`
`20
`
`amino acid sequence of X5-Xy9 is Glu-Glu-Asp-Trp-Tyr-Tyr (SEQ ID NO:9), and
`
`the peptide X13-Xj4 is Me-Thr. Of these preferred polypeptides,it is additionally
`
`preferred that X, will be Pro and X12 will be one of Phe, Trp or Tyr.
`Particular embodimentsofthe cyclic polypeptide families described above
`are disclosed in Tables 1, 2, 4, 5, and 7,infra.
`
`25
`
`Additional cyclic polypeptides found to bind a KDR or VEGF/KDRtarget
`
`have a cyclic portion (or loop), formed by a disulfide bond between the two cysteine
`
`residues, consisting of ten amino acids, for example, as follows:
`
`Asn—Asn-Ser—Cys—Trp—Leu-Ser—Thr-Thr—Leu—Giy—Ser-Cys—Phe—Phe—Asp (SEQ
`
`ID NO:10), Asp-His-His—Cys—Tyr-Leu—His—Asn—Gly—Gln—Trp—Ile-Cys-Tyr-Pro—
`
`30
`
`Phe (SEQ ID NO:1)),
`Asn-Ser—His—Cys—Tyr—Ile-Trp—Asp-Gly_Met—Trp-Leu—Cys~Phe-Pro—Asp (SEQ
`ID NO:12).
`
`Additional preferred embodiments include linear polypeptides capable of
`
`binding a KDR or VEGF/KDRtarget comprising, or alternatively consisting of, a
`
`13
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`polypeptide having an amino acid sequence selectedfotn the’grbtp'ofatiito"acrl*
`sequencesset forth in Table 3, infra.
`The polypeptides of the invention can optionally have additional amino acids
`attached at either or both of the N- and C-terminal ends. In prefexred embodiments,
`binding polypeptides according to the invention can be prepared having N-terminal
`and/or C-terminal flanking peptides of one or more, preferably two, amino acids
`corresponding to the flanking peptides of the display construct ofthe phage selectant
`from which the binding polypeptides were isolated. Preferred N-terminal flanking
`peptides include Ala-Gly— (mostpreferably f