EP1572724A2 - Peptides liant des recepteurs kdr et vegf/kdr et leurs utilisations en diagnostic et therapie - Google Patents

Peptides liant des recepteurs kdr et vegf/kdr et leurs utilisations en diagnostic et therapie

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Publication number
EP1572724A2
EP1572724A2 EP03711418A EP03711418A EP1572724A2 EP 1572724 A2 EP1572724 A2 EP 1572724A2 EP 03711418 A EP03711418 A EP 03711418A EP 03711418 A EP03711418 A EP 03711418A EP 1572724 A2 EP1572724 A2 EP 1572724A2
Authority
EP
European Patent Office
Prior art keywords
tyr
glu
asp
gly
ser
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03711418A
Other languages
German (de)
English (en)
Other versions
EP1572724A4 (fr
Inventor
Aaron K. Sato
Daniel J. Sexton
Robert C. Ladner
Daniel T. Dransfield
Rolf E. Swenson
Edmund R. Marinelli
Kondareddiar Ramalingam
Adrian D. Nunn
Mathew A. Von Wronski
Ajay Shrivastava
Sibylle Pochon
Philippe Bussat
Christophe Arbogast
Radhakrishna Pillai
Hong Fan
Karen E. Linder
Bo Song
Palaniappa Nanjappan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bracco International BV
Dyax Corp
Original Assignee
Bracco International BV
Dyax Corp
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Filing date
Publication date
Application filed by Bracco International BV, Dyax Corp filed Critical Bracco International BV
Priority to EP08008365A priority Critical patent/EP2014310B8/fr
Priority to EP10185498.2A priority patent/EP2301587B1/fr
Publication of EP1572724A2 publication Critical patent/EP1572724A2/fr
Publication of EP1572724A4 publication Critical patent/EP1572724A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Angiogenesis is not only involved in embryonic development and normal tissue growth and repair, it is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures, hi addition to angiogenesis that takes place in the normal individual, angiogenic events are involved in a number of 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 become an active cancer therapy (Kim, K. et al, 1993. Nature, 362:841-844).
  • Tumor-induced angiogenesis is thought to depend on the production of pro- angiogenic growth factors by the tumor cells, which overcome other 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 (NEGF) ( ⁇ eufeld, G. et al, 1999. FASEB , 13:9-22).
  • VEGF is produced naturally by a variety of cell types in response to hypoxia and some other stimuli. Many tumors also produce large amounts of VEGF, and/or induce nearby stromal cells to make VEGF (Fukumura, D. et 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. VEGF and VEGF ⁇ 65 are the main forms produced, particularly in tumors (see, eu ⁇ eld ' fG".' el al. ' 1999, “ supra) "'"" VEGF ⁇ 2 ⁇ lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does not bind to heparin or extracellular matrix, unlike VEGF ⁇ 65 .
  • VEGF family members act primarily by binding to receptor tyrosine kinases.
  • 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 component of the receptor), and a carboxy-terminal tail, which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
  • Flt-1 Flt-1
  • VEGFR-2 VEGFR-2
  • Flk-1 VEGFR-3
  • Flt4 Flt-1 and KDR have been identified as the primary high affinity VEGF receptors. While Flt-1 has higher affinity for VEGF, KDR displays more abundant endothelial cell expression (Bikfalvi, A. et al, 1991. J Cell. Physiol, 149:50-59). Moreover, KDR is thought to dominate the angiogenic response and is therefore of greater therapeutic and diagnostic interest (see, Neufeld, G. et al. 1999, supra).
  • KDR is highly upregulated in angiogenic vessels, especially in tumors that induce a strong angiogenic response
  • KDR is made up of 1336 amino acids in its mature form. Because of glycosylation, it migrates on an SDS-PAGE gel 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 al. 1999, supra).
  • NEGF itself is a homodimer capable 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 pathway that mediates the KDR-specific biological effects of NEGF.
  • 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) and fetal liver kinase- 1 (Flk-1)), and for imaging and targeting complexes formed by VEGF and KDR.
  • VEGF vascular endothelial growth factor
  • VEGFR-2 also known as kinase domain region (KDR) and fetal liver kinase- 1 (Flk-1)
  • VEGF/KDR and KDR binding polypeptides of the present invention particularly useful for imaging important sites of angiogenesis, e.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.
  • angiogenesis e.g., neoplastic tumors
  • therapeutics e.g., radiotherapeutics
  • KDR binding polypeptides or “KDR binding moieties” and homologues thereof.
  • KDR binding polypeptides will concentrate at the sites of angiogenesis, thus providing a means for detecting and imaging sites of active angiogenesis, which may include sites of neoplastic tumor growth.
  • KDR and VEGF/KDR binding polypeptides provide novel therapeutics to inhibit or promote, e.g., angiogenesis.
  • the preparation, use and screening of such polypeptides, for example as imaging agents or as fusion partners for KDR or VEGF/KDR-homing therapeutics, is described in detail herein.
  • VEGF/KDR binding polypeptides of the 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, e.g., by causing the conjugate or fusion to "home" to the site of active l, ⁇ gidg es ⁇ '' i ffi ⁇ rebypr6f ⁇ d ⁇ !ig 1, an effective means for treating pathogenic conditions associated with angiogenesis.
  • This invention pertains to KDR and VEGF/KDR binding polypeptides, and includes use of a single binding polypeptide as a monomer or in a multimeric or polymeric construct as well as use of more 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 thereof retaining the polypeptide binding site, is advantageous. A particularly advantageous use of the binding polypeptides disclosed herein is in a method of imaging 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.
  • imaging agents including magnetic resonance imaging (MRI) contrast agents, x-ray imaging agents, radiopharmaceutical imaging agents, ultrasound imaging agents, and optical imaging agents.
  • KDR and VEGF/KDR complex binding polypeptides disclosed herein are advantageous use of the KDR and VEGF/KDR complex binding polypeptides disclosed herein.
  • therapeutic agents including compounds capable of providing a therapeutic, radiotherapeutic or cytotoxic effect.
  • delivery vehicles for therapeutics including drugs, genetic material, etc.
  • 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 Experiment 5, tetrameric constructs of KDR binding polypeptides provided herein showed improved ability to bind KDR-transfected 293H cells.
  • constructs comprising two or more binding polypeptides specific for different epitopes of KDR and/or KDR/VEGF (e.g., "heteromeric” or “heteromultimeric” constructs, see U.S. application number 60/440,201, and the application, filed concurrently herewith, having attorney's docket number 50203/010004, the contents of each is incorporated herein) were made.
  • Constructs comprising two or more binding polypeptides provided herein are expected to block multiple sites on KDR or VEGF/KDR.
  • Heteromeric constructs of the binding polypeptides provided herein show improved ability to inhibit receptor tyrosine kinase function.
  • dimeric and other multimeric constructs of the present invention comprising at least two binding polypeptides specific for different epitopes of KDR and/or KDR/NEGF are expected to inhibit the function of receptor tyrosine kinases.
  • such constructs are expected to inhibit the function of VEGF-2/KDR, VEGF-l/Flt-1 and VEGF-3/Flt-4.
  • 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, induction of cell migration, or combination thereof.
  • heteromeric constructs of binding polypeptides provided herein inhibit VEGF-induced KDR receptor activation in human endothelial cells, demonstrated by the inhibition of VEGF-induced phosphorylation of the KDR receptor.
  • heteromeric constructs of binding peptides provided herein inhibit VEGF-stimulated endothelial cell migration.
  • the present invention is drawn to constructs comprising two or more binding polypeptides.
  • the multimeric constructs comprise two or more copies of a single binding polypeptide.
  • the multimeric constructs of the present invention comprise two or more binding polypeptides, such that at least two of the binding polypeptides in the construct are specific for different epitopes of KDR and/or 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 on the specific KDR and VEGF/KDR binding polypeptides shown in Tables 1-7. hi specific embodiments, KDR and VEGF/KDR binding polypeptides of the invention comprise one or more of these sequences.
  • KDR and VEGF/KDR binding polypeptides of the invention comprise one or more of these 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 sequence selected from Consensus
  • Consensus Sequence 1 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X -X 8 -X -X ⁇ o-Cys-X ⁇ 2 - Xi3-Xi4 (TN8), wherein
  • Xi is Ala, Arg, Asp, Gly, His, Leu, Lys, Pro, Ser, Thr, Trp, Tyr or Val;
  • X 2 is Asn, Asp, Glu, Gly, He, Leu, Lys, Phe, Ser, Thr, Trp, Tyr or Val;
  • X 3 is Asn, Asp, Gin, Glu, He, Leu, Met, Thr, Trp or Val;
  • X 5 is Ala, Arg, Asn, Asp, Gin, Glu, His, He, Lys, Phe, Pro, Ser, Trp or Tyr;
  • X 6 is Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, He, Lys, Met, Phe, Pro, Ser, Thr, Trp,
  • X is Ala, Asn, Asp, Glu, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
  • X 8 is Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Thr, Trp or Tyr;
  • X 9 is Arg, Gin, Glu, Gly, He, Leu, Met, Pro, Thr, Trp, Tyr or Val;
  • Xio is Ala, Arg, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Trp or Tyr;
  • X 12 is Arg, Asp, Cys, Gin, Glu, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;
  • X ⁇ 3 is Arg, Asn, Asp, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Ser, Thr, Trp or
  • Xi 4 is Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro iSef Tflr ⁇ l ⁇ 'orTyt;" and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 2 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X -X 8 -X 9 -X ⁇ o-X ⁇ -Xi2-
  • Xi 3 -X ⁇ 4 -Cys-X ⁇ 6 -X ⁇ 7 -X ⁇ 8 (TN12), wherein Xi is Ala, Asn, Asp, Gly, Leu, Pro, Ser, T ⁇ or Tyr (preferably Asn, Asp, Pro or
  • X 2 is Ala, Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, T ⁇ or Tyr (preferably Asp, Gly,
  • X is Ala, Asn, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Ser, Thr, T ⁇ , Tyr or Val (preferably T ⁇ );
  • X 5 is Arg, Asp, Gin, Glu, Gly, His, He, Lys, Met, Thr, T ⁇ , Tyr or Val (preferably
  • X 6 is Ala, Arg, Asn, Cys, Glu, He, Leu, Met, Phe, Ser, T ⁇ or Tyr (preferably Glu,
  • X 7 is Arg, Asn, Asp, Gin, Glu, His, He, Leu, Pro, Ser, Thr, T ⁇ , Tyr or Val
  • X 8 is Ala, Asn, Asp, Gin, Glu, Gly, His, Met, Phe, Pro, Ser, T ⁇ , Tyr or Val
  • X 9 is Asp, Ghi, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, T ⁇ or Tyr (preferably Asp);
  • Xio is Ala, Arg, Asn, Asp, Gin, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser, Tl r, T ⁇ , Tyr or Val (preferably Lys or Ser);
  • Xn is Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, Lys, T ⁇ , Tyr or Val (preferably Gly or Tyr);
  • X1 2 is Ala, Arg, Gin, Gly, His, He, Lys, Met, Phe, Ser, Thr, T ⁇ , Tyr or Val
  • X 13 is Arg, Gin, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, T ⁇ or Val (preferably Glu or T ⁇ );
  • Xn is Arg, Asn, Asp, Glu, His, He, Leu, Met, Phe, Pro, Thr, T ⁇ , Tyr or Val (preferably Phe);
  • Xi ⁇ is Ala, Asn, Asp, Gin, Glu, Gly, Lys, Met, Phe, Ser, Thr, T ⁇ , Tyr or Val
  • Xn is Arg, Asn, Asp, Cys, Gly, His, Phe, Pro, Ser, T ⁇ or Tyr (preferably Pro or
  • Xis is Ala, Asn, Asp, Gly, His, Leu, Phe, Pro, Ser, ' "Trp or Tyr " (p' ⁇ i eferably'A , Pf 'br ⁇ ), and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 3 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -Gly-X 9 -Cys-Xn-Xi2- X ⁇ (TN7), wherein
  • Xi is Gly or T ⁇
  • X 2 is He, Tyr or Val
  • X 3 is Gin, Glu Tlir or T ⁇ ;
  • X 5 is Asn, Asp or Glu
  • X 6 is Glu, His, Lys or Phe
  • X 7 is Asp, Gin, Leu, Lys Met or Tyr;
  • X is Arg, Gin, Leu, Lys or Val
  • Xn is Arg, Phe, Ser, T ⁇ or Val
  • X 12 is Glu, His or Ser; and X 13 is Glu, Gly, T ⁇ or Tyr, and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 4 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X -X 8 -X -X ⁇ o-Xn-Cys-
  • Xi is Arg, Asp, Gly, He, Met, Pro or Tyr (preferably Tyr);
  • X 2 is Asp, Gly, His, Pro or T ⁇ (preferably Gly or T ⁇ );
  • X 3 is Gly, Pro, Phe, Thr or T ⁇ (preferably Pro);
  • X 5 is Ala, Asp, Lys, Ser, T ⁇ or Val (preferably Lys);
  • X 6 is Asn, Glu, Gly, His or Leu;
  • X 7 is Gin, Glu, Gly, Met, Lys, Phe, Tyr or Val (preferably Met);
  • X 8 is Ala, Asn, Asp, Gly, Leu, Met, Pro, Ser or Thr;
  • X 9 is His, Pro or T ⁇ (preferably Pro);
  • Xio is Ala, Gly, His, Leu, T ⁇ or Tyr (preferably His or T ⁇ );
  • Xn is Ala, Asp, Gh , Leu, Met, Tlir or T ⁇ ;
  • X ⁇ 3 is Ala, Lys, Ser, T ⁇ or Tyr (preferably T ⁇ );
  • X 14 is Asp, Gly, Leu, His, Met, Thr, T ⁇ or Tyr (preferably His, T ⁇ , or Tyr); and
  • X 15 is Asn, Gin, Glu, Leu, Met, Pro or T ⁇ (preferably Glu, Met or T ⁇ ), and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 5 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X -X 8 -Ser-Gly-Pro-X ⁇ 2 -
  • Xi3 ⁇ Xi4-X ⁇ 5 -Cys-X ⁇ 7 -X ⁇ s-X ⁇ 9 (MTN13; SEQ D NO:l), wherein Xi is Arg, Glu, His, Ser or T ⁇ ;
  • X 2 is Asn, Asp, Leu, Phe, Thr or Val
  • X 3 is Arg, Asp, Glu, His, Lys or Thr;
  • X 5 is Asp, Glu, His or Thr;
  • X 6 is Arg, His, Lys or Phe;
  • X 7 is Gin, He, Lys, Tyr or Val
  • X 8 is Gin, He, Leu, Met or Phe
  • X 12 is Asn, Asp, Gly, His or Tyr;
  • X1 3 is Gin, Gly, Ser or Thr;
  • X 14 is Glu, Lys, Phe or Ser;
  • X ⁇ 5 is Glu, He, Ser or Val
  • Xn is Glu, Gly, Lys, Phe, Ser or Val
  • X ⁇ 8 is Arg, Asn, Ser or Tyr
  • X 19 is Asp, Gin, Glu, Gly, Met or Tyr, and wherein the polypeptide binds KDR or a VEGF/KDR complex.
  • 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 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X -Tyr-Cys-X ⁇ 2 - X 13 -X 14 , wherein
  • Xi is Ala, Arg, Asp, Leu, Lys, Pro, Ser or Val;
  • X 2 is Asn, Asp, Glu, Lys, Thr or Ser (preferably Asn, Asp, Glu or Lys);
  • X 3 is He, Leu or T ⁇
  • X 5 is Ala, Arg, Glu, Lys or Ser (preferably Glu);
  • X 6 is Ala, Asp, Gin, Glu, Thr or Val (preferably Asp or Glu);
  • X is Asp or Glu
  • X 8 is T ⁇ or Tyr
  • X 9 is Tlir or Tyr (preferably Tyr);
  • Xn is Glu, Met, Phe, T ⁇ or Tyr (preferably T ⁇ , Phe, Met, or Tyr); X 13 is He, Leu or Met; and
  • X 14 is He, Leu, Met, Phe or Tlir (preferably Thr or Leu), and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 7 T ⁇ -Tyr-T ⁇ -Cys-X 5 -X 6 -X 7 -Gly-X 9 -X ⁇ o-Cys-
  • X12-X13-X14 (SEQ ID NO:2), wherein X 5 is Asp, Gin or His;
  • X 6 is His or Tyr (preferably Tyr);
  • X is He, His or Tyr
  • X 9 is He, Met or Val; X 10 is Gly or Tyr;
  • X 12 is Asp, Lys or Pro
  • X 13 is Gin, Gly or T ⁇
  • X ⁇ is Phe, Ser or Tl r, and wherein the polypeptide binds KDR or a VEGF/KDR complex; or Consensus Sequence 8: X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -Gly-X ⁇ o-Cys-X ⁇ 2 -
  • Xi is Gly, Leu, His, Thr, T ⁇ or Tyr (preferably T ⁇ , Tyr, Leu or His);
  • X 2 is He, Leu, Thr, T ⁇ or Val (preferably Val, He or Leu);
  • X 3 is Asp, Glu, Gin, T ⁇ or Tlir, (preferably Glu, Asp or Gin);
  • X 5 is Ala, Arg, Asn, Asp, His, Phe, T ⁇ or Tyr (preferably Tyr, T ⁇ or Phe);
  • X 6 is Ala, Asp, Gin, His, Lys, Met, Ser, Thr, T ⁇ , Tyr or Val;
  • X 7 is Ala, Asn, Asp, Glu, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr or Val;
  • X 8 is Asp, Phe, Ser, Thr, T ⁇ or Tyr (preferably Thr, Ser or Asp);
  • X 10 is Ala, Arg, Gin, His, He, Leu, Lys, Met, Phe, T ⁇ or Tyr (preferably Arg or Lys);
  • X 12 is Arg, Gin, His, He, Lys, Met, Phe, Thr, T ⁇ , Tyr or Val (preferably Tyr, T ⁇ ,
  • X 13 is Arg, Asn, Asp, Glu, His, Met, Pro, Ser or Thr;
  • X 14 is Arg, Gin, Glu, Gly, Phe, Ser, T ⁇ or Tyr, and wherein the polypeptide binds KDR or a VEGF/KDR complex.
  • Consensus Sequence 2 revealed sub-families of preferred binding polypeptides, which are described by Consensus Sequences 9-12 as follows:
  • Consensus Sequence 9 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -T ⁇ -Gly-Gly-X ⁇ 2 - Xi 3 -Cys-Xi 5 -X 16 -X 17 (TNI 1, i.e., 11-mer binders isolated from the TN12 library;
  • Xi is Ser, Phe, T ⁇ , Tyr or Gly (preferably Ser);
  • X 2 is Arg, Gly, Ser or T ⁇ (preferably Arg);
  • X 3 is Ala, Glu, He or Val (preferably Val or He);
  • X 5 is Ala, Phe or T ⁇ (preferably T ⁇ or Phe);
  • X 6 is Glu or Lys (preferably Glu);
  • X 7 is Asp, Ser, T ⁇ or Tyr (preferably Asp, T ⁇ or Tyr);
  • X 8 is Phe, Pro or Ser (preferably Ser); X 12 is Gin or Glu (preferably Glu);
  • X 15 is Gin, He, Leu, Phe or Tyr (preferably Phe, Tyr or Leu);
  • Xi 6 is Arg, Gly or Pro (preferably Arg);
  • Xn is Gin, His, Phe, Ser, Tyr or Val (preferably Tyr, Phe, His or Val), and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 10 Tyr-Pro-X -Cys-X 5 -Glu-X 7 -Ser-X 9 -Ser-X ⁇ -
  • X 3 is Gly or T ⁇ (preferably T ⁇ );
  • X 5 is His or Tyr (preferably His, or Tyr); X 7 is His, Leu or Thr;
  • X 9 is Asp or Leu (preferably Asp);
  • Xn is Gly or Val (preferably Val);
  • X 12 is Thr or Val (preferably Thr);
  • Xi 3 is Arg or T ⁇ (preferably Arg); Xi 6 is Ala or Val (preferably Val);
  • Xn is Asp or Pro (preferably Pro);
  • Xis is Gly or T ⁇ (preferably T ⁇ ), and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 11 X ⁇ -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X ⁇ o-Gly-X ⁇ 2 - T ⁇ -X ⁇ 4 -Cys-X ⁇ 6 -X 17 -Xi8 (TNI 2; SEQ ID NO:5), wherein
  • Xi is Asp, Gly, Pro or Ser (preferably Asp);
  • X 2 is Arg, Asn, Asp, Gly or Ser (preferably Asp, Asn, or Ser);
  • X 3 is Gly, Thr, T ⁇ or Tyr (preferably T ⁇ or Tyr);
  • X 5 is Glu, Met or Thr (preferably Glu);
  • X 6 is He, Leu, Met or Phe (preferably Met, Leu, or Phe);
  • X 7 is Arg, Asp, Glu, Met, T ⁇ or Val;
  • X 8 is Asn, Gin, Gly, Ser or Val
  • X 9 is Asp or Glu
  • Xio is Lys, Ser, Thr or Val (preferably Lys);
  • X 12 is Arg, Gin, Lys or T ⁇ (preferably T ⁇ , Arg" or ' Lys);
  • X 14 is Asn, Leu, Phe or Tyr (preferably Tyr, Phe, or Asn);
  • Xi 6 is Gly, Phe, Ser or Tyr (preferably Tyr or Phe);
  • Xn is Gly, Leu, Pro or Ser (preferably Pro or Ser); and Xis is Ala, Asp, Pro, Ser, T ⁇ or Tyr, and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Consensus Sequence 12 Asn-T ⁇ -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X9-X ⁇ o--X ⁇ -
  • X 3 is Glu or Lys
  • X 5 is Glu or Gly
  • X 6 is T ⁇ or Tyr
  • X 7 is Ser or Thr
  • X 8 is Asn or Gin
  • Xn is Asp or Gin
  • X ⁇ 3 is Glu or Thr; Xi 6 is Ala or Val;
  • Xn is Arg or Tyr
  • X 18 is Leu or Pro, and wherein the polypeptide binds KDR or a VEGF/KDR complex.
  • Consensus Sequence 13 Z ⁇ -X ⁇ -X 2 -X 3 -X 4 -X 5 -Z 2 (Lin20), wherein,
  • Zi is a polypeptide of at least one amino acid or is absent
  • Xi is Ala, Asp, Gin or Glu (preferably Gin or Glu);
  • X 2 is Ala, Asp, Gin, Glu Pro (preferably Asp, Glu or Gin);
  • X 3 is Ala, Leu, Lys, Phe, Pro, T ⁇ or Tyr (preferably T ⁇ , Tyr, Phe or Leu);
  • X is Asp, Leu, Ser, T ⁇ , Tyr or Val (preferably Tyr, T ⁇ , Leu or Val);
  • X 5 is Ala, Arg, Asp, Glu, Gly, Leu, T ⁇ or Tyr (preferably T ⁇ , Tyr or Leu);
  • Z 2 is a polypeptide of at least one amino acid or is absent, and wherein the polypeptide binds KDR or a VE 7J KTOffl ⁇ -l*3 'c*r
  • Consensus Sequence 14 X ⁇ -X 2 -X 3 -Tyr-T ⁇ -Glu-X 7 -X 8 -X 9 -Leu (Lin20;
  • sequence can optionally have a N-terminal polypeptide
  • Xi is Asp, Gly or Ser (preferably Gly);
  • X 2 is He, Phe or Tyr
  • X 3 is Ala, Ser or Val
  • X 7 is Gin, Glu, He or Val
  • X 8 is Ala, He or Val (preferably He or Val);
  • X 9 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 X 3 is T ⁇ and the amino acid sequence of X -X ⁇ o is Asp-T ⁇ - Tyr-Tyr (SEQ FD NO: 8). More preferred structures include polypeptides comprising Consensus Sequence 1, wherein X 3 is T ⁇ and the amino acid sequence of X 5 -X ⁇ o is Glu-Glu-Asp-T ⁇ -Tyr-Tyr (SEQ ID NO:9).
  • Consensus Sequence 1 includes polypeptides in which: X 3 is T ⁇ and the amino acid sequence of X 5 -X ⁇ o is Glu-Glu-Asp-T ⁇ -Tyr-Tyr (SEQ ID NO: 9), and the peptide X 13 -X ⁇ 4 is fle-Thr.
  • Xi will be Pro and X ⁇ 2 will be one of Phe, T ⁇ or Tyr.
  • cyclic polypeptide families described above are disclosed in Tables 1, 2, 4, 5, and 7, infra. Additional cyclic polypeptides found to bind a KDR or VEGF/KDR target have a cyclic portion (or loop), formed by a disulfide bond between the two cysteine residues, consisting often amino acids, for example, as follows: Asn-Asn-Ser-Cys-T ⁇ -Leu-Ser-Thr-Thr-Leu-Gly-Ser-Cys-Phe-Phe-Asp (SEQ TD NO: 10), Asp-His-His-Cys-Tyr-Leu-His-Asn-Gly-Gln-T ⁇ -Ile-Cys-Ty ⁇ --Pro- Phe (SEQ TD NO: 11),
  • Additional preferred embodiments include linear polypeptides capable of binding a KDR or VEGF/KDR target comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected"frbfh the "gYb' ⁇ '"o ⁇ Mir ⁇ , 'acil" JI " sequences set forth in Table 3, infra.
  • 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 of the phage selectant from which the binding polypeptides were isolated.
  • Preferred N-terminal flanking peptides include Ala-Gly- (most preferably for TN7, TN8, TN9 sequences), Gly- Ser- (most preferably for TN10 sequences), Gly-Asp- (most preferably for TN12 sequences), Ala-Gin- (most preferably for linear sequences), and Ser-Gly- (most preferably for MTN13 sequences).
  • Preferred C-terminal flanking peptides include — Gly-Thr (most preferably for TN7, TN8, TN9 sequences), -Ala-Pro (most preferably for TN10 sequences), -Asp-Pro (most preferably for TN12 sequences), - Gly-Gly (most preferably for linear sequences), and -Gly-Ser (most preferably for MTN13 sequences).
  • N-terminal amino acids will be selected from Gly- (most preferably for TN7, TN8, TN9, MTN13 sequences), Ser- (most preferably for TN10 sequences), Asp- (most preferably for TN12 sequences), and Gin- (most preferably for linear sequences), and most preferably C-terminal amino acids will be selected from -Gly (most preferably for TN7, TN8, TN9, MTN13 and linear sequences), -Ala (most preferably for TN10 sequences), and -Asp (most preferably for TN12 sequences).
  • Loop Consensus Sequence 15 Cys-Xz-X ' ⁇ ⁇ ⁇ Xv ⁇ ys '(TN ⁇ ) 1 ;
  • X 2 is Ala, Arg, Asn, Asp, Gin, Glu, His, He, Lys, Phe, Pro, Ser, T ⁇ or Tyr (preferably Asp, Glu or Tyr);
  • X 3 is Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, He, Lys, Met, Phe, Pro, Ser, Thr, T ⁇ , Tyr or Val (preferably Glu, Met or Tyr);
  • X is Ala, Asn, Asp, Glu, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, T ⁇ , Tyr or Val (preferably Asp);
  • X 5 is Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Thr, T ⁇ or Tyr (preferably T ⁇ or Thr);
  • X 6 is Arg, Gin, Glu, Gly, He, Leu, Met, Pro, Tl r, T ⁇ , Tyr or Val (preferably Gly or Tyr);
  • X 7 is Ala, Arg, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, T ⁇ or Tyr (preferably Lys or Tyr), and wherein the polypeptide binds KDR or a VEGF/KDR complex; or
  • Loop Consensus Sequence 16 Cys-X 2 -X 3 -X 4 -X5-X 6 -X -X8-X9-X ⁇ o-X ⁇ -
  • X 2 is Arg, Asp, Gin, Glu, Gly, His, He, Lys, Met, Thr, T ⁇ , Tyr or Val (preferably
  • X 3 is Ala, Arg, Asn, Cys, Glu, He, Leu, Met, Phe, Ser, T ⁇ or Tyr (preferably Glu, Phe or Tyr);
  • X is Arg, Asn, Asp, Gin, Glu, His, He, Leu, Pro, Ser, Thr, T ⁇ , Tyr or Val
  • X 5 is Ala, Asn, Asp, Gin, Glu, Gly, His, Met, Phe, Pro, Ser, T ⁇ , Tyr or Val
  • X 6 is Asp, Gin, Glu, Gly, His, He, Leu, Met, Phe, Pro, Ser, Thr, T ⁇ or Tyr
  • X 7 is Ala, Arg, Asn, Asp, Gin, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser, Thr, T ⁇ , Tyr or Val (preferably Lys or Ser);
  • X 8 is Ala, Arg, Asn, Asp, Gin, Glu, Gly, His, Lys, T ⁇ , Tyr or Val (preferably Gly or Tyr);
  • X 9 is Ala, Arg, Gin, Gly, His, lie, Lys, Met, Phe, Ser, Thr, T ⁇ , Tyr or Val
  • Xio is Arg, Gin, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, T ⁇ or Val (preferably Glu or T ⁇ );
  • Xn is Arg, Asn, Asp, Glu, His, He, Leu, Met, Phe, Pro, Thr, T ⁇ , Tyr or Val
  • Loop Consensus Sequence 17 Cys-X 2 -X 3 -X 4 -Gly-X 6 -Cys (TN7), wherein
  • X 2 is Asn, Asp or Glu
  • X 3 is Glu, His, Lys or Phe
  • X 4 is Asp, Gin, Leu, Lys, Met or Tyr;
  • X 6 is Arg, Gin, Leu, Lys or Val, and wherein the polypeptide binds KDR or a VEGF/KDR complex;
  • Loop Consensus Sequence 18 Cys-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -Cys (TN9), wherein
  • X 2 is Ala, Asp, Lys, Ser, T ⁇ or Val (preferably Lys);
  • X 3 is Asn, Glu, Gly, His or Leu;
  • X 4 is Gin, Glu, Gly, Met, Lys, Phe, Tyr or Val (preferably Met);
  • X 5 is Ala, Asn, Asp, Gly, Leu, Met, Pro, Ser or Thr;
  • X 6 is His, Pro or T ⁇ (preferably Pro or T ⁇ );
  • X is Ala, Gly, His, Leu, T ⁇ or Tyr (preferably T ⁇ );
  • X 8 is Ala, Asp, Gin, Leu, Met, Thr or T ⁇ , and wherein the polypeptide binds KDR or a VEGF/KDR complex;
  • Loop Consensus Sequence 19 Cys-X 2 -X 3 -X 4 -X 5 -Ser-Gly-Pro-X 9 -X ⁇ o-
  • X 2 is Asp, Glu, His or Thr;
  • X 3 is Arg, His, Lys or Phe;
  • X4 is Gin, He, Lys, Tyr or Val;
  • X 5 is Gin, lie, Leu, Met or Phe;
  • X 9 is Asn, Asp, Gly, His or Tyr;
  • X10 is Gin, Gly, Ser or Thr;
  • Xn is Glu, Lys, Phe or Ser; and X12 is Glu, He, Ser or Val, and wherein the polypeptide binds KDR or a VEGF/KDR complex.
  • Consensus Sequences 20-22 as follows: Loop Consensus Sequence 20: Cys-X 2 -X 3 - - 5 -K ⁇ L T ⁇ Cy ⁇ TN8).
  • X 2 is Ala, Arg, Glu, Lys or Ser (preferably Glu);
  • X 3 is Ala, Asp, Gin, Glu, Thr or Val (preferably Asp or Glu); X is Asp or Glu;
  • X 5 is T ⁇ or Tyr
  • X 6 is Tl r or Tyr (preferably Tyr); or
  • Loop Consensus Sequence 21 Cys-X -X 3 -X 4 -Gly-X 6 -X -Cys (TN8), wherein X 2 is Asp, Gin or His;
  • X 3 is His or Tyr (preferably Tyr);
  • X is His, He or Tyr
  • X 6 is He, Met or Val
  • X is Gly or Tyr; or Loop Consensus Sequence 22: Cys-X 2 -X 3 -X 4 -X 5 -Gly-X 7 -Cys (TN8), wherein
  • X 2 is Ala, Arg, Asn, Asp, His, Phe, T ⁇ or Tyr (preferably Tyr, T ⁇ or Phe);
  • X 3 is Ala, Asp, Gin, His, Lys, Met, Ser, Thr, T ⁇ , Tyr or Val;
  • X 4 is Ala, Asn, Asp, Gin, Glu, Gly, His, He, Leu, Lys, Pro, Ser, Thr or Val;
  • X 5 is Asp, Phe, Ser, Thr, T ⁇ or Tyr (preferably Thr, Ser or Asp);
  • X is Ala, Arg, Gin, His, He, Leu, Lys, Met, Phe, T ⁇ or Tyr (preferably Arg or Lys).
  • Loop Consensus Sequence 23 Cys-X2-X3-X 4 -X 5 -T ⁇ -Gly-Gly-X 9 -X ⁇ o-
  • X 2 is Ala, Phe or T ⁇ (preferably T ⁇ or Phe);
  • X 3 is Glu or Lys (preferably Glu);
  • X4 is Asp, Ser, T ⁇ or Tyr (preferably Asp, T ⁇ or Tyr);
  • X 5 is Phe, Pro or Ser (preferably Ser);
  • X 9 is Gin or Glu (preferably Glu).
  • X10 is He, Phe or Val
  • Loop Consensus Sequence 24 Cys-X 2 -Glu-X 4 -Ser-X 6 -Ser-X 8 -X 9 -X ⁇ 0 - Phe-Cys (TN12; SEQ ID NO: 15), wherein
  • X 2 is His or Tyr; is Leu, His or Thr;
  • X 6 is Asp or Leu (preferably Asp);
  • X 8 is Gly or Val (preferably Val);
  • X 9 is Thr or Val (preferably Tlir).
  • X 10 is Arg or T ⁇ (preferably Arg); or
  • Loop Consensus Sequence 25 Cys-X 2 -X 3 -X -X 5 -X 6 -X 7 -Gly-X 9 -T ⁇ -
  • Xn-Cys (TN12; SEQ TD NO: 16), wherein X 2 is Glu, Met or Tlir (preferably Glu);
  • X 3 is He, Leu, Met or Phe (preferably Met, Leu or Phe);
  • X 4 is Arg, Asp, Glu, Met, T ⁇ or Val;
  • X 5 is Asn, Gin, Gly, Ser or Val
  • X 6 is Glu or Asp
  • X 7 is Lys, Ser, Thr or Val (preferably Lys)
  • X 9 is Arg, Gin, Lys or T ⁇ (preferably T ⁇ , Arg or Lys);
  • Xn is Asn, Leu, Phe or Tyr (preferably Tyr, Phe or Asn); or
  • Loop Consensus Sequence 26 Cys-X -X3-X -X 5 -X 6 -X 7 -X 8 -X 9 -X ⁇ o-Xn-
  • X 3 is T ⁇ or Tyr; is Ser or Thr;
  • X 5 is Asn or Gin
  • X 6 is Gly or Met
  • X 7 is Phe or Tyr
  • X 8 is Asp or Gin
  • X 9 is Lys or Tyr
  • X10 is Glu or Thr
  • Xn is Glu or Phe.
  • Loop Consensus Sequence 27 Cys-X 2 -X 3 -X -Gly-X 6 -Cys (TN7), wherein
  • X 2 is Asn, Asp or Glu
  • X 3 is Glu, His, Lys or Phe
  • X 4 is Asp, Gin, Leu, Lys, Met or Tyr;
  • X 6 is Arg, Gin, Leu, Lys or Val.
  • Preferred embodiments of the cyclic peptides of Loop Consensus Sequence 18 include KDR and/or VEGF/KDR complex binding polypeptides comprising sequences of Loop Consensus Sequence 28 as follows:
  • Loop Consensus Sequence 28 Cys-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -Cys (TN9), wherein
  • X 2 is Ala, Lys, Ser, T ⁇ or Val (preferably Lys);
  • X 3 is Asn, Glu, Gly, His or Leu;
  • X 4 is Glu, Gly, Lys, Met or Tyr (preferably Met);
  • X 5 is Ala, Asn, Asp, Leu, Met, Pro or Ser;
  • X 6 is His, Pro or T ⁇ (preferably Pro);
  • X is His, Leu, T ⁇ or Tyr (preferably T ⁇ or His); and X 8 is Ala, Asp, Gin, Leu, Met, Thr or T ⁇ .
  • Loop Consensus Sequence 29 Cys-X2-X 3 -X 4 -X 5 -Ser-Gly-Pro-X 9 -X ⁇ o- Xi ⁇ -Xi 2 -Cys (MTN13 ; SEQ ID NO: 17), wherein
  • X 2 is Asp, Glu, His or Thr;
  • X 3 is Arg, His, Lys or Phe
  • X 4 is Gin, He, Lys, Tyr or Val
  • X 5 is Gin, He, Leu, Met or Phe
  • X 9 is Asn, Asp, Gly, His or Tyr
  • X10 is Gin, Gly, Ser or Thr;
  • Xn is Glu, Lys, Phe or Ser
  • X 1 2 is Glu, He, Ser or Val.
  • Such detectable labeling can involve radiolabeling, enzymatic labeling, or labeling with MR paramagnetic chelates or microparticles; inco ⁇ oration into ultrasound bubbles, microparticles, microspheres, emulsions, or liposomes; or conjugation with optical dyes.
  • methods for isolating KDR or KDR-expressing cells using the present binding polypeptides are provided.
  • the KDR and VEGF/KDR complex binding polypeptides of the invention can be used as therapeutic agents, either as the sole bioactive agent in a pharmaceutically acceptable composition or conjugated to (or in combination with) other therapeutic agents to treat diseases or conditions involving angiogenesis or diseases associated with a number of pathogens, including, for example, malaria, HTV, SIV, Simian hemorrhagic fever, etc.
  • therapeutic agents it may be advantageous to enhance the serum residence time of the peptides.
  • methods of screening polypeptides identified by phage display for their ability to bind to cells expressing the target are provided. These methods permit rapid screening of the binding ability of polypeptides, including polypeptides with monomeric affinities that are too low for evaluation in standard cell-binding assays. Additionally, t ⁇ ese"met ⁇ io ⁇ s”may oe-use ⁇ to rapidly assess the stability of the peptides in the presence of serum.
  • FIG. 1 panels A and B are graphs illustrating the saturation binding curves of binding peptide/neutravidin-HRP complexes.
  • FIG. 1 A illustrates the saturation binding curve for SEQ ID NO:264 and SEQ ID NO:294.
  • FIG. IB illustrates the saturation binding curve for SEQ ID NO:277 and SEQ ID NO:356. All peptides had a C-terminal biotin and JJ spacer.
  • FIG. 1 panels A and B are graphs illustrating the saturation binding curves of binding peptide/neutravidin-HRP complexes.
  • FIG. 1 A illustrates the saturation binding curve for SEQ ID NO:264 and SEQ ID NO:294.
  • FIG. IB illustrates the saturation binding curve for SEQ ID NO:277 and SEQ ID NO:356. All peptides had a C-terminal biotin and JJ spacer.
  • FIG. 2 is a graph illustrating the binding of peptide/neutravidin-HRP complexes: control (biotinylated with spacer, and SEQ ID NOS:264, 294, 277 and 356) to KDR-transfected and Mock-transfected 293H cells at a single concentration (5.55 nM). All peptides had a C-terminal biotin and JJ spacer.
  • FIG. 3 is illustrates peptide structures, with and without both spacer (di(8- amino-3,6-dioxaoctanoic acid) "JJ") and biotin tested in Example 5((a) biotinylated SEQ ID NO:264 with a JJ spacer; (b) SEQ ID NO:264 with an N-terminal biotin; (c) biotinylated SEQ ID NO:294 with the JJ spacer (d) biotinylated SEQ ID NO:294).
  • spacer di(8- amino-3,6-dioxaoctanoic acid
  • FIG. 4 is a bar graph illustrating binding of peptide/neutravidin HRP complexes to KDR-transfected and mock-transfected 293H cells at single a concentration (2.78 nM); peptides include (a) control (with spacer); (b) control; (c) biotinylated SEQ ID NO:264 with a JJ spacer; (d) SEQ ID NO:264 with an N- terminal biotin; and (e) biotinylated SEQ ID NO:294 with the JJ spacer; and biotinylated SEQ ID NO:294.
  • FIG. 5 is a bar graph illustrating specific binding (binding to KDR transfected cells minus binding to Mock transfected cells) of peptide/neutravidin- HRP complexes with and without 40% rat serum, (a) SEQ ID NO:294; (b) SEQ ID NO:264; (c) SEQ ID NO:277; (d) SEQ ID NO:356. Concentration of peptide/avidin HRP solutions was 6.66 nM for (a) and (b), 3.33 nM (c), and 2.22 nM for (d). ). All peptides had a C-terminal biotin and JJ spacer.
  • FIG. 5 is a bar graph illustrating specific binding (binding to KDR transfected cells minus binding to Mock transfected cells) of peptide/neutravidin- HRP complexes with and without 40% rat serum, (a) SEQ ID NO:294; (b) SEQ ID NO:264; (c) SEQ ID NO:2
  • FIG. 6 is a bar graph illustrating binding of polypeptide/avidin-HRP solutions (SEQ ID NO:294 and/or SEQ ID NO:264) to mock- and KDR-transfected cells plotted as absorbance at 450 nm.
  • the proportions of control and KDR binding peptides used to form each tetrameric complex are indicated in the legend for each tested multimer.
  • FIG. 7 is a bar graph illustrating specific binding of ' a ' Bio ⁇ nylate 1 d' , S---Q H3 ' NO:264 with a JJ spacer/avidin-HRP complex to KDR transfected cells (background binding to mock-transfected cells subtracted), plotted as absorbance at 450 nm.
  • FIG. 8 illustrates structures of binding polypeptide sequences tested in Example 6: SEQ ID NOS:294 and 368-372.
  • FIG. 9 is a bar graph illustrating the binding of fluorescent beads to KDR- transfected and mock-transfected cells. Neutravidin-coated beads with the indicated biotinylated ligands attached were tested for binding to KDR-expressing and non- expressing 293H cells.
  • FIG. 10 is a bar graph illustrating percent inhibition of I-labeled VEGF binding by binding polypeptides
  • acetylated SEQ ID NO:294 without the modified C-terminus, "P6", GDSRVCWEDSWGGEVCFRYDP; SEQ ID NO:374)
  • SEQ ID NO:263 without the modified C-terminus, "P4", AGDSWCSTEYTYCEMIGT; SEQ ID NO:375
  • biotinylated SEQ ID NO:264 with a JJ spacer and
  • SEQ ID NO:277 biotinylated with the JJ spacer
  • FIG. 11 depicts chemiluminescent detection on film demonstrating that activated (phosphorylated) KDR was not detected in immunoprecipitates from unstimulated (-V) HUVECs, but was abundant in immunoprecipitates from VEGF- stimulated (+V) HUVECs (upper panel). Reprobing the blot with anti-KDR demonstrated that comparable amounts of total KDR were present in both immunoprecipitates (lower panel).
  • FIG. 12 depicts chemiluminescent detection on film demonstrating the ability of an anti-KDR antibody (1 ⁇ g/mL) to partially block VEGF-mediated phosphorylation.
  • FIG. 13 depicts chemiluminescent detection on film demonstrating the ability of a KDR-binding polypeptide SEQ ID NO:306 (10 ⁇ M) to block VEGF-mediated KDR phosphorylation.
  • FIG. 14 is a bar graph showing binding of a Tc-labeled polypeptide (SEQ ID NO:339) to KDR-transfected 293H cells.
  • FIG. 15 is a graph showing the percentage inhibition of 125 I-labeled VEGF binding by peptides P12-XB (SEQ ID NO:277) 5 l ' t>l; D3 an " ' 3-D" (AQDWYYDEILSMADQLRHAFLSGG; SEQ ID NO:376) at three different concentrations (10 ⁇ M, 0.3 ⁇ M, and 0.03 ⁇ M) to KDR-transfected 293H cells. The results are from one experiment carried out in triplicate +/- S.D.
  • FIG. 16 is a photograph showing the ability of Dl to completely block the
  • FIG. 17 is a graph showing that Dl potently blocks the migration/invasion of endothelial cells induced by VEGF. Migrating cells were quantitated by fluorescence measurement after staining the migrated cells with a fluorescent dye.
  • FIG. 18 is a graph showing the binding of 125 I-labeled D5 to mock and KDR transfected 293H cells in the absence and presence of 40% mouse serum.
  • FIG. 19 is a graph showing the specific binding (KDR-MOCK) of 125 I- labeled D5 to KDR-transfected 293H cells in the absence and presence of 40% mouse serum.
  • FIG. 20 is a graph of plasma clearance as percent injected dose per mL versus time.
  • FIG. 21 shows SE-HPLC profiles of plasma from the Superdex peptide column. Top panel, sample injected; followed by Omin, 30min, and 90min. The insert within each panel shows time point, animal number and volume injected for HPLC analysis.
  • FIG. 22 is a graph showing the results of testing of KDR peptides in HUVEC proliferation assay.
  • FIG. 23 shows the kinetic analysis of Dl (see FIG. 36), binding to murine KDR-Fc. All sensograms are fit to the bivalent analyte model.
  • FIG. 24 shows the kinetic analysis of D7, a heterodimer of SEQ ID NO:264 and SEQ ID NO:294. All sensograms are fit to the bivalent analyte model.
  • FIG. 25 shows Kinetic analysis of fluorescein labeled SEQ ID NO:277 binding to murine KDR-Fc. All sensograms are fit to the 1 : 1 Langmuir model.
  • FIG. 26 depicts examples of alpha, beta, , gamma , br ', delfa u 'dipe ⁇ tia ⁇ i at ⁇ i 1 ' ⁇ i mimics (such as ⁇ , ⁇ , ⁇ , or ⁇ turn mimics), shown in panels 1, 2 and 3
  • FIG. 27 shows an oxi e linker.
  • the amino acids containing an aminoalcohol function (4), and containing an alkoxyamino function (5), are inco ⁇ orated into the peptide chain, not necessarily at the end of the peptide chain.
  • FIG. 28 shows an Example of cyclization of cysteine with a pendant bromoacetamide function.
  • FIG. 29 is a schematic showing the formation of cyclic peptides with a thiazolidine linkage via intramolecular reaction of peptide aldehydes with cysteine moieties.
  • FIG. 30 is a schematic showing lactam surrogate for the disulfide bond via quasiorthogonal deprotection of Lys and Asp followed by on-resin cyclization and cleavage from resin.
  • FIG. 31 is a schematic showing lactam surrogate for the disulfide bond via quasiorthogonal deprotection of Lys and Asp using allyl-based protecting groups followed by on-resin cyclization and cleavage from resin.
  • FIG. 32 is a schematic depicting Grubbs Olefin Metathesis Cyclization.
  • FIG. 33 shows phospholipid structures
  • FIGS. 34A-F depict preferred structures of chelators.
  • FIG. 35 shows the structure of a chelating agent.
  • FIG. 36 shows dimer 1 (Dl; Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ
  • FIG. 41 shows dimer 8 (D8; Ac- AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ ID NO:356) ⁇ Ac- AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ TD NO:356)(J-Glut-)- NH 2 ⁇ K(Biotin-JJ)-NH 2 ).
  • FIG. 42 shows dimer 9 (D9; Ac- AQDWYYDEILSMADQLRHAFLSGGGGGK(SEQ ID NO:356) ⁇ [Ac- GDSRVCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:294)(JJ-Glut-)]-NH 2 ⁇ K- NH 2 ).
  • FIG. 43 shows dimer 10 (D 10 Ac-
  • FIG. 44 shows dimer 11 (Dll; Ac- AGPTWCEDDWYYCWLFGTGGGK(SEQ TD NO:277) ⁇ Ac-
  • FIG. 46 shows dimer 13 (D13; Ac- AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277) ⁇ Ac- VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)[JJ-Glut-K(BOA)]-NH 2 ⁇ - NH 2 ).
  • FIG. 47 shows dimer 14 (D14; Ac-
  • FIG. 48 shows dimer 15 (D15; Ac- AGPTWCEDDWYYCWLFGTGGGK(SEQ TD NO:277) ⁇ [[Ac-
  • FIG. 49 shows dimer 16 (D16; Ac- AGPTWCEDDWYYCWLFGTGGGGK(SEQ ID NO:277) ⁇ PnAO6-Glut-K [Ac- GDSRVCWEDSWGGEVCFRYDPGGGK(SECl '" it)"NO:2 ) : p i
  • FIG. 50 shows dimer 17 (D17; Ac- AQDWYYDEILJGRGGRGGRGGK(SEQ ID NO:478) ⁇ K[Ac-
  • FIG. 51 shows dimer 18 (D18; Ac- AGPTWCDYDWEYCWLGTFGGGK(SEQ ID NO:479) ⁇ PnAO6-Glut-K[Ac- GVDFRCEWSDWGEVGCRSPDYGGGK (SEQ TD NO:489)(JJ-Glut)-NH 2 ] ⁇ - NH 2 ).
  • FIG. 52 shows dimer 19 (D19; Ac- AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:294) ⁇ Biotin-K[Ac- VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH 2 ] ⁇ -NH 2 ).
  • FIG. 53 shows dimer 20 (D20; (- JJAGPTWCEDDWYYCWLFGTGGGGK(SEQ ID NO:480)-NH 2 )-Glut- VCWEDSWGGEVCFRYDPGGG(SEQ ID NO:370)-NH 2 ).
  • FIG. 54 shows dimer 21 (D21; [- JJAGPTWCEDDWYYCWLFGTGGGGK(SEQ TD NO:480)(PnAO6-Glut)-NH 2 ]- Glut-VCWEDSWGGEVCFRYDPGGG(SEQ ID NO:370)-NH 2 ).
  • FIG. 55 shows dimer 22 (D22; Ac-
  • FIG. 56 shows dimer 23 (D23; Ac- AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277) ⁇ Ac- VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337) [JJ-Glut-K(SATA)]-NH 2 ⁇ - NH 2 .
  • D23 is also D5 functionalized with the SATA (S-Acetylthioacetyl) group).
  • FIG. 57 shows dimer 24 (D24; Ac- AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO:277) ⁇ SATA-JJK[Ac- VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO:337)(JJ-Glut)-NH 2 ] ⁇ -NH 2 ).
  • FIG. 58 shows dimer 25 (D25; Ac-
  • FIG. 59 shows dimer 26 (D26; AGPTWCEDDWYYCWLFGTGGGGK(SEQ VCWEDSWGGEVCFRYDPGGG(SEQ ID NO:370)-NH2)-K ⁇ -NH 2 ).
  • FIG. 61 shows a dimeric binding peptide of the invention.
  • FIG. 62 shows a dimeric binding peptide of the invention.
  • FIG. 63 shows a dimeric binding peptide of the invention.
  • FIG. 64 shows a dimeric binding peptide of the invention.
  • FIG. 65 is a graph showing the inhibition of tumor growth by D6 as a function of D6 concentration.
  • FIG. 66 shows that D26 (squares) with its glycosylation and modified spacer is able to block the effects of VEGF in the migration assay to block VEGF- stimulated migration even more potently than D24 (diamonds), which lacks those chemical modifications.
  • FIG. 67 shows that Adjunct A enhances the potency of D6 in blocking the biological effects of VEGF in a migration assay with cultured HUVECs.
  • FIG. 68 is a schematic showing Scheme 1 (synthesis of Peptide 2).
  • FIG. 69 is a schematic showing Scheme 2 (synthesis of Peptide 4).
  • FIG. 70 is a schematic showing Scheme 3 (synthesis of D26).
  • FIG. 71 depicts % inhibition ⁇ s.d. of specific 125 I-VEGF binding to KDR- transfected cells by SEQ ID NO: 504 (squares) and Dl (diamonds).
  • FIG. 72 depicts % maximum VEGF-stimulated migration ⁇ s.d. of HUVEC cells in the presence of the indicated concentrations of SEQ ID NO: 504 (diamonds) Dl (squares).
  • FIG. 73 is a graphical representation showing total binding of complexes of control peptide and the test peptides (SEQ ID NOS:321, 320 and 323) with 125 I- streptavidin (in the presence of VEGF) to mock-transfected and KDR-transfected cells. Only the complex containing SEQ ID NO:321 showed specific binding (KDR-mock).
  • FIG. 73 is a graphical representation showing total binding of complexes of control peptide and the test peptides (SEQ ID NOS:321, 320 and 323) with 125 I- streptavidin (in the presence of VEGF) to mock-transfected and KDR-transfected cells. Only the complex containing SEQ ID NO:321 showed specific binding (KDR-
  • FIG. 74 is a graphical representation showing s 1 peci'fi , c"bMitfg' ' of ⁇ ⁇ le es" 1 '' of peptide (SEQ J-D NO:321) and 125 I-streptavidin (in the absence and presence of VEGF) to KDR-transfected cells at various cone. (0-13.33 nM) of peptide- 125 I- streptavidin complex.
  • FIG. 75 shows that homodimeric D8 (squares) does not block the effects of
  • VEGF in the migration assay as carried out in Example 28 as well the heterodimeric D17 (diamonds).
  • FIG. 76 is a schematic showing the synthesis of cyclic lactam peptides (sample procedure).
  • FIG. 77 is a graphical representation showing binding of SEQ ID NO:482 derivatives with different spacer length and biotin. Derivatives have none, one J and two J spacers respectively in between the SEQ ID NO:482 targeting sequence and biotin.
  • FIG. 78 depicts the binding of Tc-labeled D10 to KDR-transfected 293H cells as described in Example 32.
  • Panel B depicts the lack of binding of Tc-labeled D18 to KDR-transfected 293H cells as described in Example 32.
  • Mock mock- transfected.
  • Trans KDR-transfected.
  • MS mouse serum.
  • FIGS. 79A-G show derivatives of binding peptides of the invention.
  • FIG. 80 Summarizes the results of a radiotherapy study with D13 conducted in nude mice implanted with PC3 tumors. Each plotted line represents the growth over time for an individual tumor in a treated mouse, except for the heavy dashed line, which represents the average tumor growth in a set of untreated mice, as described in Example 34.
  • FIG. 83 shows uptake and retention of bubble contrast in the matrigel or tumor up to 30 minutes post injection for suspensions of microbubbles conjugated to KDR peptides of the invention.
  • the same bubbles showed only transient (no more than 10 minutes) visualization/bubble contrast in the AOI situated outside the matrigel or tumor site.
  • FIG. 84 shows uptake and retention of bubble contrast in the matrigel or tumor up to 30 minutes post injection for suspensions of microbubbles conjugated to KDR peptides of the invention, hi contrast, the same bubbles showed only transient (no more than 10 minutes) visualization bubble contrast in the AOI situated outside the matrigel or tumor site.
  • FIG. 85 shows a typical example of peptide-conjugated ultrasound contrast agents bound to KDR-or mock-transfected cells ⁇ n fjreieric ⁇ -b ⁇ W ⁇ -hM'ald'- ⁇ e- ⁇ ur . •••••*• (magnification: 1 OOx)
  • the term "recombinant” is used to describe non- naturally altered or manipulated nucleic acids, host cells transfected with exogenous nucleic acids, or polypeptides expressed non-naturally, through manipulation of isolated DNA and transformation of host cells.
  • Recombinant is a term that specifically encompasses DNA molecules which have been constructed in vitro using genetic engineering techniques, and use of the term "recombinant” as an adjective to describe a molecule, construct, vector, cell, polypeptide or polynucleotide specifically excludes naturally occurring such molecules, constructs, vectors, cells, polypeptides or polynucleotides.
  • bacteriophage is defined as a bacterial virus containing a DNA core and a protective shell built up by the aggregation of a number of different protein molecules.
  • polypeptide is used to refer to a compound of two or more amino acids joined through the main chain (as opposed to side chain) by a peptide amide bond (-C(:O)NH-).
  • peptide is used interchangeably herein with “polypeptide” but is generally used to refer to polypeptides having fewer than 40, and preferably fewer than 25 amino acids.
  • binding polypeptide refers to any polypeptide capable of forming a binding complex with another molecule.
  • An equivalent term sometimes used herein is “binding moiety”.
  • KDR binding polypeptide is a polypeptide that forms a complex in vitro or in vivo with vascular endothelial growth factor receptor-2 (or KDR, Flk-1);
  • VEGF/KDR complex binding polypeptide is a polypeptide that forms a complex in vitro or in vivo with a binding complex formed between vascular endothelial growth factor (VEGF) and KDR, in particular the complex of homodimeric VEGF and one or two KDR molecules that is believed to form at the surface of endothelial cells during angiogenesis.
  • KDR and VEGF/KDR binding polypeptides include but are not limited to the peptides presented in Tables 1-7, infra, and include hybrid and chimeric polypeptides inco ⁇ orating such peptides. Also included within the definition of KDR and VEGF/KDR complex binding polypeptides are poiypepiides wnicti' are mo Me' of" "'"" optimized as disclosed herein.
  • modifications include substitution of amino acids for those in the parent polypeptide sequence to optimize properties, obliterate an enzyme cleavage site, etc.; C- or N-terminal amino acid substitutions or elongations, e.g., for the pu ⁇ ose of linking the binding polypeptide to a detectable imaging label or other substrate, examples of which include, e.g., addition of a polyhistidine "tail" in order to assist in purification; truncations; amide bond changes; translocations; retroinverso peptides; peptoids; retroinversopeptoids; the use of N-terminal or C-terminal modifications or linkers, such as polyglycine or polylysine segments; alterations to include functional groups, notably hydrazide (-NH-NH 2 ) functionalities or the C-terminal linker -Gly-Gly-Gly- Lys (SEQ ID NO: 18), to assist in immobilization of binding
  • suitable substrates for the binding polypeptides include a tumorcidal agent or enzyme, a liposome (e.g., loaded with a therapeutic agent, an ultrasound appropriate gas, or both), or a solid support, well, plate, bead, tube, slide, filter, or dish.
  • dimers or multimers of one or more KDR or VEGF/KDR binding polypeptides may be formed. Such constructs may, for example, exhibit increased ability to bind to KDR. All such modified binding polypeptides are also considered KDR or VEGF/KDR complex binding polypeptides so long as they retain the ability to bind the KDR or VEGF/KDR targets.
  • homologous polypeptides may be produced using any of the modification or optimization techniques described herein or known to those skilled in the art. Such homologous polypeptides will be understood to fall within the scope of the present invention and the definition of KDR and VEGF/KDR complex binding polypeptides so long as the substitution, addition, or deletion of amino acids or other such modification does not eliminate its ability to bind either KDR or VEGF/KDR complex.
  • homologous refers to the degree of sequence similarity between two polymers (i.e., polypeptide molecules or nucleic acid molecules).
  • polymers are homologous at that position. For example, if the amino acid residues at 60 of 100 amino acid positions in two polypeptide sequences match or are homologous then the two sequences are 60% homologous.
  • the homology percentage figures referred to herein reflect the maximal homology possible between the two polymers, i.e., the percent homology when the two polymers are so aligned as to have the greatest number of matched (homologous) positions.
  • Polypeptide homologues within the scope of the present invention will be at least 70% and preferably greater than 80% homologous to at least one of the KDR or VEGF/KDR binding sequences disclosed herein.
  • binding refers to the determination by standard assays, including those described herein, that a binding polypeptide recognizes and binds reversibly to a given target.
  • standard assays include, but are not limited to equilibrium dialysis, gel filtration, and the monitoring of spectroscopic changes that result from binding.
  • binding specificity refers to a binding polypeptide having a higher binding affinity for one target over another.
  • KDR specificity refers to a KDR binding moiety having a higher affinity for KDR over an irrelevant target.
  • VEGF/KDR specificity refers to a VEGF/KDR complex binding moiety having a higher affinity for a VEGF/KDR complex over an a given target. Binding specificity may be characterized by a dissociation equilibrium constant (K D ) or an association equilibrium constant (K a ) for the two tested target materials, or can be any measure of relative binding strength.
  • the binding polypeptides according to the present invention are specific for KDR or VEGF/KDR complex and preferably have a K D for KDR or VEGF/KDR complex that is lower than lO ⁇ M, more preferably less than l.O ⁇ M, most preferably less than 0.5 ⁇ M or even lower.
  • patient refers to any mammal, especially humans.
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic carrier or excipient that may be administered to a patient, together with a compound of this invention, and which does not destroy the biological or pharmacological activity thereof.
  • the present invention provides novel binding moieties that bind KDR or a complex of VEGF and KDR.
  • binding moieties make possible the efficient detection, imaging and localization of activated endothelial cells exhibiting upregulated KDR expression and binding to VEGF.
  • Such endothelial cells are characteristic of active angiogenesis, and therefore the polypeptides described herein provide a means of detecting, monitoring and localizing sites of angiogenesis.
  • the binding polypeptides of this invention when appropriately labeled, are useful for detecting, imaging and localizing rumor-induced angiogenesis.
  • the binding polypeptides can be used to form a variety of diagnostic and therapeutic agents for diagnosing and treating neoplastic tumor growth or other pathogenic instances of angiogenesis.
  • the binding polypeptides can themselves be used as therapeutic agents.
  • KDR and VEGF/KDR complex binding polypeptides according to the present invention were isolated initially by screening of phage display libraries, that is, populations of recombinant bacteriophage transfonned to express an exogenous peptide on their surface, hi order to isolate new polypeptide binding moieties for a particular target, such as KDR or VEGF/KDR, screening of large peptide libraries, for example using phage display techniques, is especially advantageous, in that very large numbers (e.g., 5 x 10 9 ) of potential binders can be tested and successful binders isolated in a short period of time.
  • phage display libraries that is, populations of recombinant bacteriophage transfonned to express an exogenous peptide on their surface, hi order to isolate new polypeptide binding moieties for a particular target, such as KDR or VEGF/KDR
  • screening of large peptide libraries, for example using phage display techniques is especially advantageous, in that very large numbers (e.g
  • a candidate binding domain is selected to serve as a structural template for the peptides to be displayed in the library.
  • the phage library is made up of a" multiplicity of analogues of the parental domain or template.
  • the binding domain template may be a naturally occurring or synthetic protein, or a region or domain of a protein.
  • the binding domain template may be selected based on knowledge of a known interaction between the binding domain template and the binding target, but this is not critical, hi fact, it is not essential that the domain selected to act as a template for the library have any affinity for the target at all: Its pvupose is to provide a structure from which a multiplicity (library) of similarly structured polypeptides (analogues) can be generated, which multiplicity of analogues will hopefully include one or more analogues that exhibit the desired binding properties (and any other properties screened for).
  • the variegated amino acid sequences of the library In selecting the parental binding domain or template on which to base the variegated amino acid sequences of the library, the most important consideration is how the variegated peptide domains will be presented to the target, i.e., in what conformation the peptide analogues will come into contact with the target, hi phage display methodologies, for example, the analogues will be generated by insertion of synthetic DNA encoding the analogues into phage, resulting in display of the analogue on the surfaces of the phage.
  • Such libraries of phage such as M13 phage, displaying a wide variety of different polypeptides, can be prepared using techniques as described, e.g. , in Kay et al. , Phage Display of Peptides and Proteins: A
  • cyclic peptide (or "loop") libraries designated TN6/VI, TN7/IV, TN8/IX, TN9/TV, TNIO/LX, TN12/I, and MTN13/I, and a linear library, designated Lin20.
  • Each library was constructed for expression of diversified polypeptides on Ml 3 phage.
  • the seven libraries having a "TN” designation were designed to display a short, variegated exogenous peptide loop of 6, 7, 8, 9, 10, 12 or 13 amino acids, respectively, on the surface of Ml 3 phage, at the amino terminus of protein HI.
  • the libraries are designated TN6/VI (having a potential 3.3 x 10 12 amino acid sequence diversity), TN7/IV (having a potential 1.2 x 10 14 amino acid sequence diversity), TN8/IX (having a potential 2.2 x 10 15 amino acid sequence diversity), TN9/IV (having a potential 4.2 x 10 16 amino acid sequence diversity, TN10/IX (having a potential 3.0 x 10 16 amino acid sequence diversity), TN12/I (having a sequence diversity of 4.6 x 10 19 ), MTN13/I (having a potential 8.0 10" amino acid sequence diversity), and Lin20 (having a potential 3.8 10 25 amino acid sequence diversity).
  • the TN6/VI library was constructed to display a single microprotein binding loop contained in a 12-amino acid template.
  • the TN6/VI library utilized a template sequence of Xaa ⁇ -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa ⁇ 5 -Xaa -Xaa 8 -Cys-Xaa ⁇ o-Xaa ⁇ i- Xaa ⁇ .
  • the amino acids at positions 2, 3, 5, 6, 7, 8, 10, and 11 of the template were varied to permit any amino acid except cysteine (Cys).
  • the amino acids at positions 1 and 12 of the template were varied to permit any amino acid except cysteine (Cys), glutamic acid (Glu), isoleucine (He), Lysine (Lys), methionine (Met), and threonine (Thr).
  • the TN7/IV library was constructed to display a single microprotein binding loop contained in a 13 -amino acid template.
  • the TN7/IV library utilized a template sequence of Xaa ⁇ -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa -Xaa 8 -Xaa 9 -Cys-Xaa ⁇ - Xaa ⁇ 2 -Xaa ⁇ 3 .
  • the amino acids at amino acid positions 1, 2, 3, 5, 6, 7, 8, 9, 11, 12, and 13 of the template were varied to permit any amino acid except cysteine (Cys).
  • the TN8/LX library was constructed to display a single microprotein binding loop contained in a 14-amino acid template.
  • the TN8/IX library utilized a template sequence of Xaa ⁇ -Xaa 2 -Xaa 3 -Cys-Xaa 5 - Xaa 6 -Xaa -Xaa 8 -Xaa 9 -Xaa ⁇ 0 -Cys- Xaa ⁇ 2 -Xaai 3 -Xaa ⁇ .
  • the amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 12, 13, and 14 in the template were varied to permit any amino acid except cysteine (Cys).
  • the TN9/IV library was constructed to display a single microprotein binding loop contained in a 15-amino acid template.
  • the TN9/TV library utilized a template sequence Xaa ⁇ -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa ⁇ o-Xaan-Cys- Xaai 3 -Xaa ⁇ 4 -Xaa ⁇ 5 .
  • the amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 in the template were varied to permit any amino acid except cysteine (Cys).
  • the TN10/IX library was constructed to display a single microprotein binding loop contained in a 16-amino acid template.
  • the TNIO/IX library utilized a template sequence Xaa ⁇ -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa ⁇ o - Xaan-Xaai 2 -Cys-Xaai 4 -Xaa ⁇ 5 -Xaa ⁇ 6 .
  • the amino acids at positions 1, 2, 15, and 16 in the template were varied to permit any amino acid selected from a group of 10 amino acids: D, F, H, L, N, P, R, S, W, or Y).
  • the amino acids at positions 3 and 14 in the template were varied to permit any amino acid selected from a group of 14 amino acids: A, D, F, G, H, L, N, P, Q, R, S, V, W, or Y).
  • the amino acids at positions 5, 6, 7, 8, 9, 10, 11, and 12 in the template were varied to permit any amino acid except cysteine (Cys).
  • the TNI 2/1 library was constructed to display a single microprotein binding loop contained in an 18-amino acid template.
  • the TN12/I library utilized a template sequence Xaa ⁇ -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa -Xaa 8 -Xaa 9 -Xaa ⁇ o-Xaan-Xaa ⁇ 2 - Xaa ⁇ 3 -Xaa ⁇ 4 -Cys-Xaa ⁇ 6 -Xaa ⁇ 7 -Xaa ⁇ 8 .
  • the amino acids at position 1, 2, 17, and 18 in the template were varied to permit any amino acid selected from a group of 12 amino acids: A, D, F, G, H, L, N, P, R, S, W, or Y).
  • the amino acids at positions 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 16 were varied to pennit any amino acid except cysteine (Cys).
  • the MTN13/I library was constructed to display a single microprotein binding loop contained in a 19-amino acid template featuring two variable regions of equal size (i.e., eight amino acids) separated by a constant region of three amino acids (Ser-Gly-Pro).
  • the MTN13/I library utilized a template sequence Xaai- Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa -Xaa 8 -Ser-Gly-Pro-Xaa ⁇ 2 -Xaa ⁇ 3 -Xaai 4 -Xaa ⁇ 5 - Cys-Xaa ⁇ -Xaa ⁇ 8 -Xaa ⁇ 9 (SEQ ID NO: 19).
  • the amino acids at position 1, 2, 3, 5, 6, 7, 8, 12, 13, 14, 15, 17, 18, and 19 in the template were varied to permit any amino acid except cysteine (Cys).
  • the Lin20 library was constructed to display a single linear peptide in a 20- amino acid template.
  • the amino acids at each position in the template were varied to permit any amino acid except cysteine (Cys).
  • binding polypeptides provided herein can include additions or truncations in the N- and/or C- termini. Such modified binding polypeptides are expected to bind KDR or VEGF/KDR complex.
  • the -GGGK linker present at the N-terminus of some of the binding polypeptides provided herein is an optional linker. Therefore, polypeptides having the same sequence, except without the terminal -GGGK sequences are also encompassed by the present invention, hi addition, binding polypeptides comprising the loop portion of the templates and sequences provided herein are expected to bind KDR and/or VEGF/KDR complex and are also encompassed by the present invention.
  • the loop portion of the templates and sequences includes the sequences between and including the two cysteine residues that are expected to form a disulfide bond, thereby generating a peptide loop structure.
  • the binding polypeptides of the present invention can include additional amino acid residues at the N- and/or C-termini.
  • the phage display libraries were created by making a" designed series" ⁇ 'f mutations or variations within a coding sequence for the polypeptide template, each mutant sequence encoding a peptide analogue corresponding in overall structure to the template except having one or more amino acid variations in the sequence of the template.
  • the novel variegated (mutated) DNA provides sequence diversity, and each transformant phage displays one variant of the initial template amino acid sequence encoded by the DNA, leading to a phage population (library) displaying a vast number of different but structurally related amino acid sequences.
  • the amino acid variations are expected to alter the binding properties of the binding peptide or domain without significantly altering its structure, at least for most substitutions. It is preferred that the amino acid positions that are selected for variation (variable amino acid positions) will be surface amino acid positions, that is, positions in the amino acid sequence of the domains which, when the domain is in its most stable confonnation, appear on the outer surface of the domain (i.e., the surface exposed to solution). Most preferably the amino acid positions to be varied will be adjacent or close together, so as to maximize the effect of substitutions.
  • Phage bearing a target-binding moiety form a complex with the target on the solid support whereas non-binding phage remain in solution and may be washed away with excess buffer. Bound phage are then liberated from the target by changing the buffer to an exfreme pH (pH 2 or pH 10), changing the ionic strength of the buffer, adding denaturants, or other known means.
  • a protein elution was performed, i.e., some phage were eluted from target using VEGF in solution (competitive elution); and also, very high affinity binding phage that could not be competed off incubating with VEGF overnight were captured by using the phage sti ⁇ boMd ' to ' s ⁇ 'l-)straLt , e' l f ⁇ - 1 ' ' infection of E. coli cells.
  • the recovered phage may then be amplified through infection of bacterial cells and the screening process repeated with the new pool that is now depleted in non-binders and enriched in binders.
  • the recovery of even a few binding phage is sufficient to carry the process to completion.
  • the gene sequences encoding the binding moieties derived from selected phage clones in the binding pool are determined by conventional methods, described below, revealing the peptide sequence that imparts binding affinity of the phage to the target.
  • the sequence diversity of the population falls with each round of selection until desirable binders remain. The sequences converge on a small number of related binders, typically 10-50 out of the more than 10 million original candidates from each library.
  • sequence infonnation may be used to design other secondary phage libraries, biased for members having additional desired properties.
  • disulfide binding loop is advantageous because it leads to increased affinity and specificity for such peptides.
  • the disulfide bond might be opened by free cysteines or other thiol-containing molecules.
  • the -CH 2 -S-S-CH 2 - cross-link has a prefened geometry in which the dihedral bond between sulfurs is close to 90 degrees, but the exact geometry is determined by the context of other side groups and the binding state of the molecule. Preferred modifications of the closing cross-link of the binding loop will preserve the overall bond lengths and angles as much as possible.
  • linear polypeptides derived from the foregoing sequences maybe readily prepared, e.g., by substitution of one or both cysteine residues, which may retain at least some of the KDR or VEGF/KDR binding activity of the original polypeptide containing the disulfide linkage, h making such substitutions for Cys, the amino acids Gly, Ser, and Ala are preferred, and it is also preferred to substitute both Cys residues, so as not to leave a single Cys that may cause the polypeptide to dimerize or react with other free thiol groups in a solution. All such linearized derivatives that retain KDR or VEGF/KDR binding properties are within the scope of this invention.
  • Direct synthesis of the polypeptides of the invention maybe accomplished using conventional techniques, including solid-phase peptide synthesis, solution- phase synthesis, etc.
  • Solid-phase synthesis is prefened. See Stewart et al, Solid- Phase Peptide Synthesis (W. H. Freeman Co., San Francisco, 1989); Merrifield, J. Am. Chem. Soc, 85:2149-2154 (1963); Bodanszky and Bodanszky, The Practice of Peptide Synthesis (Springer-Verlag, New York, 1984), inco ⁇ orated herein by reference.
  • Polypeptides according to the invention may also be prepared commercially by companies providing peptide synthesis as a service (e.g., BACHEM Bioscience, Inc., King of Prussia, PA; Quality Controlled Biochemicals, hie, Hopkinton, MA). Automated peptide synthesis machines, such as manufactured by Perkin-Elmer Applied Biosystems, also are available.
  • the polypeptide compound is preferably purified once it has been isolated or synthesized by either chemical or recombinant techniques.
  • purification pmposes there are many standard methods that may be employed, including reversed-phase high-pressure liquid chromatography (RP-HPLC) using an alkylated silica column such as C 4 -, C 8 - or C ⁇ 8 -silica.
  • RP-HPLC reversed-phase high-pressure liquid chromatography
  • a gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid.
  • Ion-exchange chromatography can also be used to separate peptides based on their charge.
  • the degree of purity of the polypeptide may be determined by various methods, including identification of a major large peak on HPLC.
  • a polypeptide that produces a single peak that is at least 95% of the input material on an HPLC column is prefened. Even more preferable is a polypeptide that produces a single peak that is at least 97%, at least 98%, at least 99% or even 99.5% or more of the input material on an HPLC column.
  • analysis of the peptide composition may be carried out. Such composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide.
  • the amino acid content of the peptide can be confirmed by hydrolyzing the peptide in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequenators, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine the sequence of the peptide.
  • KDR or VEGF/KDR complex binding polypeptides according to the present invention also may be produced using recombinant DNA techniques, utilizing nucleic acids (polynucleotides) encoding the polypeptides according to this invention and then expressing them recombinantly, i.e., by manipulating host cells by introduction of exogenous nucleic acid molecules in known ways to cause such host cells to produce the desired KDR or VEGF/KDR complex binding polypeptides.
  • nucleic acids polynucleotides
  • KDR or VEGF/KDR complex binding moiety of this invention is inco ⁇ orated in a hybrid polypeptide or fusion protein.
  • a determination of the affinity of the KDR or VEGF/KDR complex binding moiety for KDR or VEGF/KDR complex relative to another protein or target is a useful measure, and is referred to as specificity for KDR or VEGF/KDR complex.
  • Standard assays for quantitating binding and determining affinity include equilibrium dialysis, equilibrium binding, gel filtration, or the monitoring of numerous spectroscopic changes (such as a change in fluorescence polarization) that may result from the interaction of the binding moiety and its target. These techniques measure the concentration of bound and free ligand as a function of ligand (or protein) concentration.
  • concentration of bound polypeptide [Bound]) is related to the concentration of free polypeptide ([Free]) and the concentration of binding sites for the polypeptide, i.e., on KDR or VEGF/KDR complex, (N), as described in the following equation:
  • K a a quantitative measure of the binding affinity.
  • the association constant, K a is the reciprocal of the dissociation constant, K D .
  • the K D is more frequently reported in measurements of affinity.
  • Preferred KDR or VEGF/KDR complex binding polypeptides have a K D for KDR or VEGF/KDR complex in the range of 1 nanomolar (nM) to 100 micromolar ( ⁇ M), which includes K D values of less than 10 nM, less than 20 nM, less than 40 nM, less than 60 nM, less than 80 nM, less than 1 ⁇ M, less than 5 ⁇ M, less than 10 ⁇ M, less than 20 ⁇ M, less than 40 ⁇ M, less than 60 ⁇ M, and less than 80 ⁇ M.
  • Imaging agents operate in a dynamic system in that binding of the imaging agent to the target (KDR or VEGF/KDR complex, e.g., on activated endothelium) may not be in a stable equilibrium state throughout the imaging procedure. For example, when the imaging agent is initially injected, the concentration of imaging agent and of agent-target complex rapidly increases. Shortly after injection, however, the circulating (free) imaging agent starts to clear through the kidneys or liver, and the plasma concentration of imaging agent begins to drop. This drop in the concentration of free imaging agent in the plasma eventually causes the agent-target complex to dissociate.
  • the usefulness of an imaging agent depends on the difference in rate of agent-target dissociation relative to the clearing rate of the agent. Ideally, the dissociation rate will be slow compared to the clearing rate, resulting in a long imaging time during which there is a high concentration of agent- target complex and a low concentration of free imaging agent (background signal) in the plasma.
  • Quantitative measurement of dissociation rates maybe easily performed using several methods known in the art, such as fiber optic fluorimetry (see, e.g., Anderson & Miller, Clin. Chem., 34(7):1417-21 (1988)), surface plasmon resonance (see, Malmborg et al, J. Immunol. Methods, 198(l):51-7 (1996) and Schuck, Current Opinion in Biotechnology, 8:498-502 (1997)), resonant minor, and grating coupled planar waveguiding (see, e.g., Hutchinson, Molec. Biotechnology, 3:47-54 (1995)).
  • fiber optic fluorimetry see, e.g., Anderson & Miller, Clin. Chem., 34(7):1417-21 (1988)
  • surface plasmon resonance see, Malmborg et al, J. Immunol. Methods, 198(l):51-7 (1996) and Schuck, Current Opinion in Biotechnology, 8:498-502 (1997)
  • resonant minor
  • BIAcore surface plasmon resonance sensor Biacore AB, Uppsala SE
  • IAsys resonant mirror sensor Fiber Applied Sensor Technology, Cambridge GB
  • BIOS-1 grated coupled planar waveguiding sensor Articleificial Sensor Instruments, Zurich CH.
  • Methods of Screening Polypeptides Identified by Phage Display For Their ability To Bind To Cells Expressing The Target hi another aspect of the invention, methods of screening binding polypeptides identified by phage display for their ability to bind to cells expressing the target (and not to cells which do not express the target) are provided. These methods address a significant problem associated with screening peptides identified by phage display: frequently the peptides so identified do not have sufficient affinity for the target to be screened against target-expressing cells in conventional assays.
  • the method takes advantage of the increase in affinity and avidity associated with multivalent binding and permit screening of polypeptides with low affinities against target-expressing cells.
  • the method generally consists of preparation and screening of multimeric constructs including one or more binding polypeptides.
  • polypeptides identified by phage display as binding to a target are biotinylated and complexed with avidin, streptavidin or neutravidin to form tetrameric constructs. These tetrameric constructs are then incubated with cells that express the desired target and cells that do not, and binding of the tetrameric construct is detected. Binding may be detected using any method of detection known in the art.
  • the avidin, streptavidin, or neutravidin may be conjugated to a detectable marker (e.g., a radioactive label, a fluorescent label, or an enzymatic label which undergoes a color change, such as HRP (horse radish peroxidase), TMB (tetramethyl benzidine) or alkaline phosphatase).
  • a detectable marker e.g., a radioactive label, a fluorescent label, or an enzymatic label which undergoes a color change, such as HRP (horse radish peroxidase), TMB (tetramethyl benzidine) or alkaline phosphatase).
  • HRP horse radish peroxidase
  • TMB tetramethyl benzidine
  • alkaline phosphatase alkaline phosphatase
  • the tetrameric constructs may be screened against cells which naturally express the target or cells which have been engineered via recombinant DNA technologies to express the target (e.g., transfectants, transformants, etc.). If cells which have been transfected to express the target are used, mock transfected cells (i.e., cells transfected without the genetic material encoding the target) may be used as a control.
  • target e.g., transfectants, transformants, etc.
  • mock transfected cells i.e., cells transfected without the genetic material encoding the target
  • the tetrameric complexes may optionally be screened in the presence of serum.
  • the assay may also be used to rapidly evaluate the effect of serum on the binding of peptides to the target.
  • the methods disclosed herein are particularly useful in preparing and evaluating combinations of distinct binding polypeptides for use in dimeric or multimeric targeting contracts which contain two or more binding polypeptides.
  • Use of biotin/avidin complexes allows for relatively easy preparation of tetrameric constructs containing one to four different binding peptides.
  • affinity and avidity of a targeting construct may be increased by inclusion of two or more targeting moieties which bind to different epitopes on the same target.
  • the screening methods described herein are useful in identifying combinations of binding polypeptides which may have increased 5 affinity when included in such multimeric constructs.
  • the screening methods described herein may be used to screen KDR and VEGF/KDR complex binding polypeptides identified by phage display, such as those described herein. As described in more detail in Example 5 infra, these methods may be used to assess the specific binding of KDR binding polypeptides to cells which express KDR or have been engineered to express KDR.
  • Tetrameric complexes of biotinylated KDR binding polypeptides of the invention and neutravidin-HRP may be prepared and screened against cells transfected to express KDR as well as mock transfected cells (without any KDR).
  • the assay may be used to identify KDR binding polypeptides which bind specifically to KDR-expressing cells (and do not bind to cells that do not express KDR) even when the m nddeiit-t ' te ⁇ Tbtth'e"poiyp*sptlde” ⁇ s on the order of 200nM-300nM.
  • the assay may be used to screen homotetrameric constructs containing four copies of a single KDR binding polypeptide of the invention as well as heterotetrameric (constructs containing two or more different KDR binding polypeptides.
  • the methods described herein are particularly useful for assessing combinations of KDR binding polypeptides for use in multimeric constructs, particularly constructs containing two or more KDR binding polypeptides which bind to different epitopes of KDR.
  • the assay may also be used to assess the effect of serum on the KDR binding polypeptides. Indeed, using the screening methods disclosed herein, KDR binding polypeptides, such as SEQ ID NOS:264, 294, and 356, were identified whose binding is not significantly affected by serum.
  • KDR and VEGF/KDR complex binding polypeptides modification or optimization of KDR and VEGF/KDR complex binding polypeptides is within the scope of the invention and the modified or optimized polypeptides are included within the definition of "KDR and VEGF/KDR complex binding polypeptides".
  • a polypeptide sequence identified by phage display can be modified to optimize its potency, pharmacokinetic behavior, stability and/or other biological, physical and chemical properties.
  • alkyl-substituted hydrophobic amino acids Including alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from Cl-10 carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions.
  • Substitution of aromatic-substituted hydrophobic amino acids Including phenylalanine, tryptophan, tyrosine, biphenylalainneTl-riap Hy ⁇ alan ⁇ rie, 2- naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy (from Cl-C4)-substituted forms of the previous listed aromatic amino acids, illustrative examples of which are: 2-,3- or 4-aminophenylalanine, 2-,3- or 4- chlorophenylalanine, 2-,3- or 4-methylphenylalanine, 2-,3- or 4- methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'-,
  • Substitution of amino acids containing basic functions Including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or aryl- substituted (from C1-C10 branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example.
  • N- epsilon-isopropyl-lysine 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)- alanine, N,N-gamma, gamma' -diethyl-homoarginine.
  • amides formed from alkyl, aromatic, heteroaromatic where the heteroaromatic group has one or more nitrogens, oxygens or sulfur atoms singly or in combination
  • carboxylic acids or any of the many well- known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • lysine, ornithine, or 2,3-diaminopropionic acid any of the many well- known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • Substitution of acidic amino acids Including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids.
  • Substitution of side chain amide residues Including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.
  • Another type of modification within the scope of the patent is to substitute the amide bonds within the backbone of the polypeptide.
  • it is common to substitute amide bonds within the backbone of the peptides with functionality that mimics the existing conformation or alters the conformation in the manner desired.
  • Such modifications may produce increased binding affinity or improved phannacokinetic behavior.
  • D-alanine or another D-amino acid, distal or proximal to the labile peptide bond
  • D-amino acid substitutions can, and at times, must be made, with D-amino acids whose side chains are not conservative replacements for those of the L-amino acid being replaced.
  • D-amino acids whose side chains are not conservative replacements for those of the L-amino acid being replaced. This is because of the difference in chirality and hence side-chain orientation, which may result in the accessing of a previously unexplored region of the binding site of the target which has moieties of different charge, hydrophobicity, steric requirements etc. than that serviced by the side chain of the replaced L-amino acid.
  • KDR or VEGF/KDR complex binding polypeptide in a particular application may necessitate modifications " of the peptide ' or fonnulations of the peptide to improve pharmacokinetic and phannacodynamic behavior. It is expected that the properties of the peptide may be changed by attachment of moieties anticipated to bring about the desired physical or chemical properties.
  • Such moieties may be appended to the peptide using acids or amines, via amide bonds or urea bonds, respectively, to the N- or C-terminus of the peptide, or to the pendant amino group of a suitably located lysine or lysine derivative, 2, 3- diaminopropionic acid, ornithine, or other amino acid in the peptide that possesses a pendant amine group or a pendant alkoxyamine or hydrazine group.
  • the moieties introduced may be groups that are hydrophilic, basic, or nonpolar alkyl or aromatic groups depending on the peptide of interest and the extant requirements for modification of its properties.
  • glycosylated amino acid residues e.g. serine, threonine or asparagine residues
  • Glycosylation which may be carried out using standard conditions, can be used to enhance solubility, alter phannacokinetics and pharmacodynamics or to enhance binding via a specific or non-specific interaction involving the glycosidic moiety.
  • glycosylated amino acids such as 0-(2-acetamido-2- deoxy-3,4,6-tri-O-acetyl- ⁇ -D-glucopyranosyl) serine or the analogous threonine derivative (either the D- or L- amino acids) can be inco ⁇ orated into the peptide during manual or automated solid phase peptide synthesis, or in manual or automated solution phase peptide synthesis.
  • D- or L-N ⁇ -(2-acetamido-2- deoxy-3,4,6-tri-O-acetyl- ⁇ -D-glucopyranosyl)-asparagine can be employed.
  • linkage of the amino acid to the glycoside is not limited to the formation of a bond to the anomeric carbon of the carbohydrate function. Instead, linkage of the carbohydrate moiety to the amino acid could be through any suitable, sufficiently reactive oxygen atom, nitrogen atom, carbon atom or other pendant atom of the carbohydrate function via methods employed for formation of C-heteroatom, C-C or heteroatom-heteroatom (examples are S-S, O-N, N-N, P-O, P-N) bonds known in the art.
  • salts may increase the water solubility or the ease of fonnulation of these peptides.
  • These may include, but are not restricted to, N-methylglucamine (meglumine), acetate, oxalates, ascorbates, etc.
  • the shortened cyclic peptides can be formed using disulfide bonds or amide bonds of suitably located carboxylic acid groups and ammo groups.
  • D-amino acids can be added to the peptide sequence to stabilize turn features (especially in the case of glycine).
  • alpha, beta, gamma or delta dipeptide or turn mimics (such as ⁇ , ⁇ , ⁇ , or ⁇ turn mimics), some of which are shown in schematics 1, 2 and 3 as shown in FIG. 26, can be employed to mimic structural motifs and turn features in a peptide and simultaneously provide stability from proteolysis and enhance other properties such as, for example, conformational stability and solubility (structure 1: Hart et al, J. Org. Chem., 64, 2998-2999(1999); structure 2: Hanessian et al, "Synthesis of a Versatile Peptidomimetic Scaffold" in Methods in Molecular Medicine, Vol. 23:
  • Disulfide Mimetics Also within the scope of the invention is the substitution of disulfide mimetics for disulfide bonds within the KDR or VEGF/KDR complex binding peptides of the invention.
  • disulfide-containing peptides When disulfide-containing peptides are employed in generating 99m Tc-based radiopharmaceuticals, a significant problem is the presence of the disulfide bond.
  • the integrity of the disulfide bond is difficult to maintain during procedures designed to inco ⁇ orate 99m Tc via routes that are reliant upon the reduction of pertechnetate ion and subsequent inco ⁇ oration of the reduced Tc species into substances bearing Tc-specific chelating groups. This is because the disulfide bond is rather easily reduced by the reducing agents commonly used in kits devised for one-step preparation of radiopharmaceuticals. Therefore, the ease with which the disulfide bond can be reduced during Tc chelation may require substitution with mimetics of the disulfide bonds.
  • the oxime moiety has been employed as a linker by investigators in a number of contexts. Of the most interest is the wortcby J l ⁇ t ⁇ er et" al". ' (W'ahT'a ⁇ d Mutter, Tetrahedron Lett., 37:6861-6864 (1996)).
  • the amino acids 4, containing an aminoalcohol function, and 5, containing an alkoxyamino function, are inco ⁇ orated into the peptide chain, not necessarily at the end of the peptide chain (FIG. 27). After fonnation of the peptide the sidechain protecting groups are removed. The aldehyde group is unmasked and an oxime linkage is formed.
  • Lanthionines are cyclic sulfides, wherein the disulfide linkage (S-S) is replaced by a carbon-sulfur (C-S) linkage. Thus, the lability to reduction is far lower. Lanthionines have been prepared by a number of methods since 1971.
  • Lanthionines are readily prepared using known methods. See, for example, Robey et al, Altai. Biochem., 177:373-377 (1989); Inman et al, Bioconjugate
  • Resin-bound serine can be employed to prepare the lanthionine ring on resin either using a bromination- dehydrobromination-thiol addition sequence or by dehydration with disuccinimidyl carbonate followed by thiol addition.
  • Ploinsky et al M. J. Med. Chem., 35:4185- 4194 (1992); Mayer et al, "Peptides, Frontiers of Peptide Science", in Proceedings of the 15 th American Peptide Symposium, Tarn & Kaumaya (Eds.), June 14-19, 1995, Nashville, Term. (Klumer Academic Pub., Boston), pp. 291-292.
  • Diaryl Ether or Diarylamine Linkage Diaryl Ether Linkage From Intramolecular Cyclization of Aryl Boronic Acids and Tyrosine Recently the reaction of arylboronic acids with phenols, amines and heterocyclic amines in the presence of cupric acetate, in air, at ambient temperature, in dichloromethane using either pyridine or triethylamine as a base to provide unsymmetrical diaryl ethers and the related amines in good yields (as high as 98%) has been reported. See, Evans et al, Tetrahedron Lett., 39:2937-2940 (1998); Chan et al, Tetrahedron Lett, 39:2933-2936 (1998); Lam et al, Tetrahedron Lett,
  • Another approach that may be employed involves intramolecular cyclization of suitably located vicinal amino mercaptan functions (usually derived from placement of a cysteine at a terminus of the linear sequence or tethered to the sequence via a side-chain nitrogen of a lysine, for example) and aldehyde functions to provide thiazolidines which result in the formation of a bicyclic peptide, one ring of which is that formed by the residues in the main chain, and the second ring being the thiazolidine ring.
  • Scheme 2 (FIG. 29) provides an example.
  • the required aldehyde function can be generated by sodium metaperiodate cleavage of a suitably located vicinal aminoalcohol function, which can be present as an unprotected serine tethered to the chain by appendage to a side chain amino group of a lysine moiety.
  • the required aldehyde function is generated by unmasking of a protected aldehyde derivative at the C-terminus or the N-terminus of the chain.
  • An example of this strategy is found in: Botti et al, J. Am. Chem. Soc, .. -.5:10018- 10034 (1996).
  • Lactams Based on Intramolecular Cyclization of Pendant Amino Groups with Carboxyl Groups on Resin Macrocyclic peptides have been prepared by lactam formation by either head to tail or by pendant group cyclization. The basic strategy is to prepare a fully protected peptide wherein it is possible to remove selectively an amine protecting group and a carboxy protecting group. Orthogonal protecting schemes have been developed. Of those that have been developed the allyl, trityl and Dde methods have been employed most. See, Mellor et al, "Synthesis of Modified Peptides", in Fmoc Solid Phase Synthesis: A Practical Approach, White and Chan (eds) (Oxford University Press, New York, 2000), Chapt.
  • the Dde approach is of interest because it utilizes similar protecting groups for both the carboxylic acid function (Dmab ester) and the amino group (Dde group). Both are removed with 2- 10% hydrazine in DMF at ambient temperature.
  • the Dde can be used for the amino group and the allyl group can be used for the carboxyl.
  • a lactam function available by intramolecular coupling via standard peptide coupling reagents (such as HATU, PyBOP etc), could act as a surrogate for the disulfide bond.
  • the Dde/Dmab approach is shown in Scheme 3a (FIG. 30).
  • a linear sequence containing, for example, the Dde-protected lysine and Dmab ester can be prepared on a Tentagel-based Rink amide resin at low load (-0J-0.2 mmol/g). Deprotection of both functions with hydrazine is then followed by on-resin cyclization to give the desired products.
  • amino acids such as trans-4-(iV-Dde)methylaminocyclohexane carboxylic acid, trans-4- (iV-Dde)methylaminobenzoic acid, or their alloc congeners can be employed.
  • Yet another approach is to employ the safety catch method to intramolecular lactam formation during cleavage from the resin.
  • a linear sequence containing, for example, the Dde-protected lysine and Dmab ester may be prepared on a Tentagel-based Rink amide resin at low load ( ⁇ 0J-0.2 mmol/g). Deprotection of both functions with hydrazine is then followed by on-resin cyclization to give the desired products. Subsequently cleavage from resin and purification may also be carried out.
  • diamino acids such as trans-4-(iv ⁇ Dde)methylaminocyclohexane carboxylic acid or trans-4-(iv-Dde)methylamino benzoic acid would be required.
  • An alternative scenario is to employ the safety catch method to intramolecular lactam formation during cleavage from the resin.
  • the Grubbs reaction involves the metathesis/cyclization of olefin bonds and is illustrated as shown below. See, Schuster et al., Angewandte. Chem. Int. Edn Engl, 36:2036-2056 (1997); Miller et al, J. Am. Chem. Soc, 118:9606-9614 (1996).
  • the lysine ⁇ -amino group is another option with appendage of the olefin- containing unit as part of an acylating moiety, for example. If instead the lysine side chain amino group is alkylated with an olefin containing tether, it can still function as a point of attachment for a reporter as well.
  • the use of 5-pentenoic acid as an acylating agent for the lysine, ornithine, or diaminopropionic side chain amino groups is another possibility.
  • the length of the olefm-containing tether can also be varied in order to explore structure activity relationships.
  • linkers or spacers between the targeting sequence of the KDR or VEGF/KDR complex binding peptide and the detectable label or therapeutic agent.
  • Use of such linkers/spacers may improve the relevant properties of the binding peptide (e.g., increase serum stability, etc.).
  • These linkers may include, but are not restricted to, substituted or unsubstituted alkyl chains, polyethylene glycol derivatives, amino acid spacers, sugars, or aliphatic or aromatic spacers common in the art.
  • linkers which are combinations of the moieties described above, can also be employed to confer special advantage to the properties of the peptide.
  • Lipid molecules with linkers may be attached to allow formulation of ultrasound bubbles, liposomes or other aggregation based constructs.
  • Such constructs could be employed as agents for targeting and delivery of a diagnostic reporter, a therapeutic agent (e.g., a chemical "warhead” for therapy) or a combination of these.
  • Polypeptides Constructs employing dimers, multimers or polymers of one or more VEGF or VEGF/KDR complex binding polypeptides of the invention are also contemplated. Indeed, there is ample literature evidence that the binding of low potency peptides or small molecules can be substantially increased by the formation of dimers and multimers. Thus, dimeric and multimeric constructs (both homogeneous and heterogeneous) are within the scope of the instant invention, h deed, as discussed in more detail in the Examples, it is within the scope of the present invention to include multiple KDR or VEGF/KDR complex binding polypeptide sequences in a dimeric or multimeric constract.
  • these constructs may exhibit improved binding compared to a monomeric construct.
  • the polypeptide sequences in the dimeric constructs may be attached at their N- or C- terminus or the N-epsilon nitrogen of a suitably placed lysine moiety (or another function bearing a selectively derivatizable group such as a pendant oxyamino or other nucleophihc group), or may be joined together via one or more linkers employing the appropriate attachment chemistry.
  • This coupling chemistry may include amide, urea, thiourea, oxime, or ammoacetylamide (from chloro- or bromoacetamide derivatives, but is not so limited.
  • any of the following methods may be utilized to prepare dimeric or multimeric constructs of KDR or VEGF/KDR complex binding polypeptides of the invention.
  • Fully protected KDR-binding peptides can be built up on Ellman-type safety catch resin using automated or manual Fmoc peptide synthesis protocols. Backes et al, J. Am. Chem. Soc, 118(12):3055-56 (1996).
  • a di-lysine derivative can be constructed on 2- chlorotrityl resin. See, for example, Fields et al, "Principles and Practice of Solid Phase Synthesis" in Synthetic Peptides, A Users Guide, Grant, Ed. (W.H. Freeman Co., New York, 1992), Chapt. 3, pp.
  • the prior-mentioned safety-catch resin is activated and the desired N-deprotected labeling group- functionalized di-lysine derivative is added to the activated safety-catch resin.
  • the pendant amino groups are acylated by the carboxy-terminus of the safety-catch resin- bound peptide which is now detached from the resin and an integral part of the di- lysine stracture.
  • An excess of the safety-catch resin-bound peptide can be employed to insure complete reaction of the amino groups of the di-lysine constract. Optimization of the ratio of the reacting partners in this scheme optimizes the yield.
  • the protecting groups on the KDR-binding peptides are removed employing trifluoroacetic acid based cleavage protocols.
  • KDR-binding peptides are present in one constract is easily accomplished.
  • Orthogonal protection schemes such as an allyloxycarbonyl group on one nitrogen and an Fmoc group on the other, or employing the Fmoc group in conjunction with the iV-Dde protecting group on the other, for example
  • Unmasking of one of the amino groups, followed by reaction of the resulting product with an activated safety-catch resin-bound KDR-binding peptide as described above provides a di-lysine construct having a single KDR-binding peptide attached. Removal of the second protecting group unmasks the remaining nitrogen. See, also, Mellor et al. , "Synthesis of Modified Peptides" in Fmoc Solid Phase Peptide
  • Method B A KDR-binding peptide is assembled on a Rink-amide resin by automated or manual peptide coupling methods, usually employing Fmoc peptide synthesis protocols.
  • the peptide may possess a C-terminus or N-terminus functionalized with a linker or a linker-labeling group constract that may possess an additional nucleophihc group such as the ⁇ -amino group of a lysine moiety, for example.
  • Cleavage of the protecting groups is accomplished employing trifluoroacetic acid with appropriate modifiers depending on the nature of the peptide.
  • the fully deprotected peptide is then reacted with a large excess of a bifunctional electrophile such as the commercially available glutaric acid bis-N-hydroxysuccinimide ester
  • Method C A modular scheme can be employed to prepare dimeric or higher multimeric constructs bearing suitable labeling groups as defined above, hi a simple illustration, finoc-lysine(iV-Dde) Rink amide resin is treated with piperidine to remove the fmoc moiety. Then a labeling function, such as biotin, 5-carboxyfluorescein or N,N-
  • Dimethyl-Gly-Ser(O-t-Bu)-Cys(Acm)-Gly-OH is coupled to the nitrogen atom.
  • the resin is next treated with hydrazine to remove the iV-Dde group.
  • the resin is treated with cyanuric chloride and a hindered base such as diisopropylethylamine in a suitable solvent such as DMF, NMP or dichloromethane to provide a monofunctionalized dichlorotriazine bound to the resm.
  • a suitable solvent such as DMF, NMP or dichloromethane
  • the incoming peptides may be protected or unprotected as the situation wanants. Cleavage of protecting groups is accomplished employing trifluoroacetic acid-based deprotection reagents as described above, and the desired materials are purified by high performance liquid chromatography.
  • lysine derivatives may be serially employed to increase the multiplicity of the multimers.
  • the use of related, more rigid molecules bearing the requisite number of masked, or orthogonally protected nitrogen atoms to act as scaffolds to vary the distance between the KDR- binding peptides, to increase the rigidity of the construct (by constraining the motion and relative positions of the KDR-binding peptides relative to each other and the reporter) is entirely within the scope of methods A-C and all other methods described herein.
  • the references cited above are inco ⁇ orated by reference herein in their entirety.
  • the KDR or NEGF/KDR complex binding moieties according to this invention will be extremely useful for detection and/or imaging of KDR or VEGF/KDR complex in vitro or in vivo, and particularly for detection and/or imaging of sites of angiogenesis, in which VEGF and KDR are intimately involved, as explained above. Any suitable method of assaying or imaging KDR or VEGF KDR complex may be employed.
  • the KDR and VEGF/KDR complex binding moieties of the invention also have utility in the treatment of a variety of disease states, including those associated with angiogenesis or those associated with a number of pathogens.
  • the KDR and VEGF/KDR complex binding moieties of the invention may themselves be used as therapeutics or may be used to localize one or more therapeutic agents (e.g. , a chemotherapeutic, a radiotherapeutic, genetic material, etc.) to KDR expressing cells, including sites of angiogenesis.
  • a binding polypeptide according to the invention can be detectably labeled, e.g., fluorescently labeled, enzymatically labeled, or labeled with a radioactive or paramagnetic metal, then contacted with the solution, and thereafter formation of a complex between the binding polypeptide and the KDR or VEGF/KDR complex target can be detected.
  • a fluorescently labeled KDR or VEGF/KDR complex binding peptide may be used for in vitro KDR or VEGF/KDR complex detection assays, wherein the peptide is added to a solution to be tested for KDR or VEGF/KDR complex under conditions allowing binding to occur.
  • the complex between the fluorescently labeled KDR or VEGF/KDR complex binding peptide and KDR or VEGF/KDR complex target can be detected and quantified by measuring the increased fluorescence polarization arising from the KDR or VEGF/KDR complex- bound peptide relative to that of the free peptide.
  • a sandwich-type "ELISA" assay may be used, wherein a KDR or VEGF/KDR complex binding polypeptide is immobilized on a solid support such as a plastic tube or well, then the solution suspected of containing KDR or VEGF/KDR complex target is contacted with the immobilized binding moiety, non- binding materials are washed away, and complexed polypeptide is detected using a suitable detection reagent, such as a monoclonal antibody recognizing KDR or VEGF/KDR complex.
  • a suitable detection reagent such as a monoclonal antibody recognizing KDR or VEGF/KDR complex.
  • the monoclonal antibody is detectable by conventional means known in the art, including being detectably labeled, e.g., radiolabeled, conjugated with an enzyme such as horseradish peroxidase and the like, or fluorescently labeled, etc.
  • binding polypeptides of the invention can be immobilized on a solid substrate such as a chromatographic support or other matrix material, then the immobilized binder can be loaded or contacted with the solution under conditions suitable for formation of a binding polypeptide:KDR complex or binding polypeptide: VEGF/KDR complex.
  • the non-binding portion of the solution can be removed and the complex may be detected, e.g., using an anti-KDR or anti-
  • VEGF/KDR complex antibody or an anti-binding polypeptide antibody, or the KDR or NEGF/KDR complex target may be released from the binding moiety at appropriate elution conditions.
  • the biology of angiogenesis and the roles " of NEGF and KDR in initiating' and maintaining it have been investigated by many researchers and continues to be an active field for research and development, hi furtherance of such research and development, a method of purifying bulk amounts of KDR or VEGF/KDR complex in pure form is desirable, and the binding polypeptides according to this invention are especially useful for that prupose, using the general purification methodology described above.
  • a particularly prefened use for the polypeptides according to the present invention is for creating visually readable images of KDR expressing tissue, such as, for example, neoplastic tumors, which require angiogenesis for survival and metastasis, or other sites of angiogenic activity.
  • the KDR and VEGF/KDR complex binding polypeptides disclosed herein may be converted to imaging reagents by conjugating the polypeptides with a label appropriate for diagnostic detection, optionally via a linker.
  • a peptide exhibiting much greater specificity for KDR or VEGF/KDR complex than for other serum proteins is conjugated or linked to a label appropriate for the detection methodology to be employed.
  • the KDR or VEGF/KDR complex binding polypeptide may be conjugated with or without a linker to a paramagnetic chelate suitable for magnetic resonance imaging (MRI), with a radiolabel suitable for x-ray, PET or scintigrapic imaging (including a chelator for a radioactive metal), with an ultrasound contrast agent (e.g., a stabilized microbubble, a ultrasound contrast agent, a microsphere or what has been referred to as a gas filled "liposome”) suitable for ultrasound detection, or with an optical imaging dye.
  • MRI magnetic resonance imaging
  • a radiolabel suitable for x-ray
  • PET or scintigrapic imaging including a chelator for a radioactive metal
  • an ultrasound contrast agent e.g., a stabilized microbubble, a ultrasound contrast agent, a microsphere or what has been referred to as a gas filled "liposome”
  • an optical imaging dye e.g., a stabilized microbubble, a ultrasound contrast agent, a
  • Suitable linkers can be substituted or unsubstituted alkyl chains, amino acid chains (e.g., polyglycine), polyethylene glycols, polyamides, and other simple polymeric linkers known in the art.
  • the technique of using a detectably labeled KDR or VEGF/KDR complex binding moiety is based on the premise that the label generates a signal that is detectable outside the patient's body.
  • the detectably labeled KDR or VEGF/KDR complex binding moiety is administered to the patient in which it is desirable to detect, e.g., angiogenesis
  • the high affinity of the KDR or VEGF/KDR complex binding moiety for KDR or VEGF/KDR complex causes the binding moiety to bind to the site of angiogenesis arid accuriiulate label at " the site " of "" angiogenesis.
  • Sufficient time is allowed for the labeled binding moiety to localize at the site of angiogenesis.
  • the signal generated by the labeled peptide is detected by a scanning device which will vary according to the type of label used, and the signal is then converted to an image of the site of angiogenesis.
  • the peptide(s) of the invention can be conjugated with for example, avidin, biotin, or an antibody or antibody fragment that will bind the detectable label or radiotherapeutic.
  • one or more KDR-binding peptides can be conjugated to sfreptavidin (potentially generating multivalent binding) for in vivo binding to KDR-expressing cells.
  • a biotinylated detectable label or radiotherapeutic construct e.g., a chelate molecule complexed with a radioactive metal
  • a biotinylated detectable label or radiotherapeutic construct can be infused which will rapidly concentrate at the site where the targeting construct is bound.
  • This approach in some situations can reduce the time required after administering the detectable label until imaging can take place. It can also increase signal to noise ratio in the target site, and decrease the dose of the detectable label or radiotherapeutic constract required. This is particularly useful when a radioactive label or radiotherapeutic is used as the dose of radiation that is delivered to normal but radiation-sensitive sites in the body, such as bone-manow, kidneys, and liver is decreased.
  • the KDR or VEGF/KDR complex binding moieties of the present invention can advantageously be conjugated with one or more paramagnetic metal chelates in order to form a contrast agent for use in MRI.
  • Preferred paramagnetic metal ions have atomic numbers 21-29, 42, 44, or 57-83. This includes ions of the transition metal or lanthanide series which have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1.1 Bohr magneton.
  • Preferred paramagnetic metals include, but are not limited to, chromium (TTT), manganese (TT), manganese (TTT), iron (TT), iron (TTT), cobalt (TT), nickel (TT), copper (TT), praseodymium (LT), neodymium (TTT), samarium (TTT), gadolinium (TTT), terbium (TTT), dysprosium (TTT), holmium (TTT), erbium (TTT), europium (TTT) and ytterbium ( l), chromium UUj, iron (HI), and gadolinium (LT).
  • the trivalent cation, Gd 3+ is particularly prefened for MRI contrast agents, due to its high relaxivity and low toxicity, with the further advantage that it exists in only one biologically accessible oxidation state, which minimizes undesired metabolysis of the metal by a patient.
  • Another useful metal is Cr 3+ , which is relatively inexpensive.
  • Gd(HI) chelates have been used for clinical and radiologic MR applications since 1988, and approximately 30% of MR exams currently employ a gadolinium-based contrast agent. Additionally, heteromultimers of the present invention also can be conjugated with one or more supe ⁇ aramagnetic particles.
  • the practitioner will select a metal according to dose required to detect angiogenesis and considering other factors such as toxicity of the metal to the subject (Tweedle et al, Magnetic Resonance Imaging (2nd ed.), vol. 1, Partain et al, Eds. (W.B. Saunders Co. 1988), pp. 796-797).
  • the desired dose for an individual metal will be proportional to its relaxivity, modified by the biodistribution, pharmacokinetics and metabolism of the metal.
  • the paramagnetic metal chelator(s) is a molecule having one or more polar groups that act as a ligand for, and complex with, a paramagnetic metal.
  • Suitable chelators are known in the art and include acids with methylene phosphonic acid groups, methylene carbohydroxamine acid groups, carboxyethyhdene groups, or carboxymethylene groups.
  • chelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTP A), 1, 4,7,10-tetraazacyclo- tetradecane-l,4,7,10-tetraacetic acid (DOT A), 1 -substituted 1,4,7,- tricarboxymethyl- 1 ,4,7, 10-teraazacyclododecane (DO3 A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetra-azacyclotetradecane- l,4,8,H-tetraacetic acid (TETA).
  • DTP A diethylenetriaminepentaacetic acid
  • DOT A 1, 4,7,10-tetraazacyclo- tetradecane-l,4,7,10-tetraacetic acid
  • DO3 A 1 -substituted 1,4,7,- tricarboxymethyl- 1 ,4,7, 10-teraazacyclododecane
  • Additional chelating ligands are ethylene bis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof, including 5-C1-EHPG, 5Br-EHPG, 5-Me-EHPG, 5t-Bu-EHPG, and 5sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTP A; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (O and/or N), which macrocyclic compounds can consist of one ring, or two or tliree rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo
  • TETMA 1,4,7,10-tetraazacyclotetradecane- 1,4,7, 10-tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is 1,4,8,11- tetraazacyclotetradecane-l,4,8Jl -(methyl tetraacetic acid); derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTHA); derivatives of l,5J0-N,N',N"-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and l,3,5-N,N',N"-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM).
  • TETMA 1,4,8,11- tetraazacyclotetradecane-l,4,8Jl -(methyl tetraacetic acid)
  • PDTA 1,3
  • a preferred chelator for use in the present invention is DTP A, and the use of DO3A is particularly prefened.
  • Examples of representative chelators and chelating groups contemplated by the present invention are described in WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473, PCT/US98/20182, and US 4,899,755, US 5,474,756, US 5,846,519 and US 6,143,274, all ofwhich are hereby inco ⁇ orated by reference.
  • the chelator of the MRI contrast agent is coupled to the KDR or VEGF/KDR complex binding polypeptide.
  • the positioning of the chelate(s) should be selected so as not to interfere with the binding affinity or specificity of the KDR or VEGF/KDR complex binding polypeptide.
  • the chelate(s) will be appended either to the N-terminus or the C- terminus, however the chelate(s) may also be attached anywhere within the sequence, hi prefened embodiments, a chelator having a free central carboxylic acid group (e.g., DTPA-Asp( ⁇ -COOH)-)OtBu) makes it easy to attach at the N-terminus of the peptide by formation of an amide bond.
  • the chelate(s) could also be attached at the C-terminus with the aid of a linker.
  • isothiocyanate conjugation chemistry could be employed as a way of linking the appropriate isothiocyanate group bearing DTPA to a free amino group anywhere within the peptide sequence, hi general, the KDR or VEGF/KDR complex binding moiety can be bound directly or covalently to the metal chelator (or other detectable label), or it may be coupled or conjugated to the metal " chelator using a linker, which may be, without limitation, amide, urea, acetal, ketal, double ester, carbonyl, carbamate, thiourea, sulfone, thioester, ester, ether, disulfide, lactone, imine, phosphoryl, or phosphodiester linkages; substituted or unsubstituted saturated or unsaturated alkyl chains; linear, branched, or cyclic amino acid chains or a smgie ammo acid or different amino acids (e.g., extensions of the N- or C- terminus of the K
  • the molecular weight of the linker can be tightly controlled.
  • the molecular weights can range in size from less than 100 to greater than 1000.
  • the molecular weight of the linker is less than 100.
  • biodegradable functionalities can include ester, double ester, amide, phosphoester, ether, acetal, and ketal functionalities.
  • known methods can be used to couple the metal chelate(s) and the KDR or VEGF/KDR complex binding moiety using such linkers. See, e.g., WO 95/28967, WO 98/18496, WO 98/18497 and discussion therein.
  • VEGF/KDR complex binding moiety can be linked through its N- or C-terminus via an amide bond, for example, to a metal coordinating backbone nitrogen of a metal chelate or to an acetate arm of the metal chelate itself.
  • the present invention contemplates linking of the chelate on any position, provided the metal chelate retains the ability to bind the metal tightly in order to minimize toxicity.
  • the KDR or VEGF/KDR complex binding moiety may be modified or elongated in order to generate a locus for attachment to a metal chelate, provided such modification or elongation does not eliminate its ability to bind KDR or VEGF/KDR complex.
  • MRI contrast reagents prepared according to the disclosures herein may be used in the same manner as conventional MRI contrast reagents.
  • certain MR techniques and pulse sequences may be prefened to enhance the contrast of the site to the background blood and tissues.
  • These techniques include (but are not limited to), for example, black blood angiography sequences that seek to make blood dark, such as fast spin echo sequences (see, e.g., Alexander et al, Magnetic Resonance in Medicine, 40(2): 298-310 (1998)) and flow-spoiled gradient echo sequences (see, e.g., Edelman et al, Radiology, 177(1): 45-50 (1990)).
  • the labeled reagent is administered to the patient in the form of an injectable composition.
  • the method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially, or intracavitarilly.
  • intravenous or intraarterial administration is prefened.
  • the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the site of angiogenesis at least 10%.
  • the patient is scanned in the MRI machine to determine the location of any sites of angiogenesis.
  • angiogenesis e.g., tumor
  • a tumorcidal agent or anti-angiogenic agent e.g., inhibitors of VEGF
  • the patient can be subsequently scanned to visualize tumor regression or anest of angiogenesis.
  • Ultrasound contrast agents are intense sound wave reflectors because of the acoustic differences between the agent and the surrounding tissue.
  • Gas containing or gas generating ultrasound contrast agents are particularly useful because of the acoustic difference between liquid (e.g., blood) and the gas-containing or gas generating ultrasound contrast agent.
  • ultrasound contrast agents comprising microbubbles, ultrasound contrast agents, and the like may remain for a longer time in the blood stream after injection than other detectable moieties; a targeted KDR or VEGF/KDR complex-specific ultrasound agent therefore may demonstrate supenor imaging ol" sites of angiogenesis.
  • the KDR or VEGF/KDR complex binding moiety may be linked to a material which is useful for ultrasound imaging.
  • the KDR or VEGF/KDR complex binding polypeptides may be linked to materials employed to form vesicles (e.g., microbubbles, ultrasound contrast agents, microspheres, etc.), or emulsions containing a liquid or gas which functions as the detectable label (e.g., an echogenic gas or material capable of generating an echogenic gas).
  • vesicles e.g., microbubbles, ultrasound contrast agents, microspheres, etc.
  • emulsions containing a liquid or gas which functions as the detectable label e.g., an echogenic gas or material capable of generating an echogenic gas.
  • Materials for the preparation of such vesicles include surfactants, lipids, sphingolipids, oligolipids, phospholipids, proteins, polypeptides, carbohydrates, and synthetic or natural polymeric materials.
  • phospholipids, and particularly saturated phospholipids are prefened.
  • the prefened gas-filled microbubbles of the invention can be prepared by means known in the art, such as, for example, by a method described in any one of the following patents: EP 554213, US 5,413,774, US 5,578,292, EP 744962, EP 682530, US 5,556,610, US 5,846,518, US 6,183,725, EP 474833, US 5,271,928, US 5,380,519, US 5,531,980, US 5,567,414, US 5,658,551, US 5,643,553, US
  • At least one of the phospholipid moieties has the structure 18 or 19 (FIG. 33) and described in US 5,686,060, which is herein inco ⁇ orated by reference.
  • Suitable phospholipids include esters of glycerol with one or two molecules of fatty acids (the same or different)and phosphoric acid, wherein the phosphoric acid residue is in turn bonded to a hydrophilic group, such as choline, serine, inositol, glycerol, ethanolamine, and the like groups.
  • Fatty acids present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22, that may be saturated or may contain one or more unsaturations.
  • Suitable fatty acids are lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid.
  • Mono esters of phospholipid are also known m the art as the "lyso" forms of the phospholipids.
  • phospholipids are phosphatidic acids, i.e. the diesters of glycerol-phosphoric acid with fatty acids, sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain, cardiolipins, i.e. the esters of 1,3- diphosphatidylglycerol with a fatty acid, gangliosides, cerebrosides, etc.
  • the term phospholipids includes either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.
  • Examples of naturally occun ⁇ ng phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.
  • PC phosphatidylcholine
  • semisynthetic phospholipids are the partially or fully hydro genated derivatives of the naturally occu ing lecithins.
  • Examples of synthetic phospholipids are e.g., dilauryloyl- phosphatidylcholine ("DLPC”), dimvristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”), diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine (“DSPC”), l-myristoyl-2- palmitoylphosphatidylcholine (“MPPC”), l-palmitoyl-2- myristoylphosphatidylcholine (“PMPC”), l-palmitoyl-2-stearoylphosphatid- ylcholine (“PSPC”), l-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”), dioleoylphosphatidylycholine (“DOPC”), 1,2 Distearoyl-sn-glycero-3- Ethy
  • compositions also may contain PEG-4000 and/or palmitic acid. Any of the gases disclosed herein or known to the skilled artisan may be employed; however, inert gases, such as SF 6 or fluorocarbons like CF 4) C 3 F 8 and C 4 F ⁇ o, are prefened.
  • gases such as SF 6 or fluorocarbons like CF 4
  • the prefened microbubble suspensions of the present invention may be prepared from phospholipids using known processes such as a freeze-drying or spray-drying solutions of the crude phospholipids in a suitable solvent or using the processes set forth in EP 554213; US 5,413,774; US 5,578,292; EP 744962; EP 682530; US 5,556,610; US 5,846,518; US 6,183,725; EP 474833; US 5,271,928; US 5,380,519; US 5,531,980; US 5,567,414; US 5,658,551; US 5,643,553; US
  • the phospholipids are dissolved in an organic solvent and the solution is dried without going through a liposome formation stage. This can be done by dissolving the phospholipids in a suitable organic solvent together with a hydrophilic stabilizer substance or a compound soluble both in the organic solvent and water and freeze-drying or spray-drying the solution.
  • the criteria used for selection of the hydrophilic stabilizer is its solubility in the organic solvent of choice.
  • hydrophilic stabilizer compounds soluble in water and the organic solvent are, e.g., a polymer, like polyvinyl pynolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc., malic acid, glycolic acid, maltol, and the like.
  • PVP polyvinyl pynolidone
  • PVA polyvinyl alcohol
  • PEG polyethylene glycol
  • Any suitable organic solvent may be used as long as its boiling point is sufficiently low and its melting point is sufficiently high to facilitate subsequent drying.
  • Typical organic solvents include, for example, dioxane, cyclohexanol, tertiary butanol, tetrachlorodifluoro ethylene (C 2 C1 4 F 2 ) or 2-methyl-2- butanol. 2-methyl-2-butanol and C 2 C1 F 2 are prefened.
  • the freeze dried or spray dried phospholipid powders Prior to formation of the suspension of microbubbles by dispersion in an aqueous carrier, the freeze dried or spray dried phospholipid powders are contacted with air or another gas. When contacted with the aqueous carrier the powdered phospholipids whose stracture has been disrupted will form lamellarized or laminarized segments that will stabilize the microbubbles of the gas dispersed therein.
  • This method pennits production of suspensions of microbubbles which are stable even when stored for prolonged periods and are obtained by simple dissolution of the dried laminarized phospholipids (which have been stored under a desired gas) without shaking or any violent agitation.
  • microbubbles can be prepared by suspending a gas into an aqueous solution at high agitation speed, as disclosed e.g. in WO 97/29783.
  • a further process for preparing microbubbles is disclosed in co-pending European patent application no. 03002373, herein inco ⁇ orated by reference, which comprises preparing an emulsion of an organic solvent in an aqueous medium in the presence of a phospholipid and subsequently lyophilizing said emulsion, after optional washing and/or filtration steps.
  • non-film forming surfactants including polyoxypropylene glycol and polyoxyethylene glycol and similar compounds, as well as various copolymers thereof; fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid or their derivatives, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene may be added.
  • the amount of these non-film forming surfactants is usually up to 50% by weight of the total amount of surfactants but preferably between 0 and 30%.
  • Other gas containing suspensions include those disclosed in, for example, US
  • microballoon refers to gas filled bodies with a material boundary or envelope. More on microballoon fonnulations and methods of preparation may be found in EP 324 938 (US 4,844,882); US 5,711,933; US 5,840,275; US 5,863,520; US 6,123,922; US 6,200,548; US 4,900,540; US 5,123,414; US 5,230,882; US 5,469,854; US 5,585,112; US 4,718,433; US 4,774,958; WO 95/01187; US 5,529,766; US 5,536,490; and US 5,990,263, the contents ot which are inco ⁇ orated herein by reference.
  • the prefened microballoons have an envelope including a biodegradable physiologically compatible polymer or, a biodegradable solid lipid.
  • the polymers useful for the preparation of the microballoons of the present invention can be selected from the biodegradable physiologically compatible polymers, such as any of those described in any of the following patents: EP 458745, US 5,711,933, US 5,840,275, EP 554213, US 5,413,774 and US 5,578,292, the entire contents of which are inco ⁇ orated herein by reference, hi particular, the polymer can be selected from biodegradable physiologically compatible polymers, such as polysaccharides of low water solubility, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as ⁇ -caprolactone, ⁇ -valerolactone and polypeptides.
  • poly(ortho)esters see e.g., US 4,093,709; US 4,131,648; US 4,138,344; US 4,180,646); polylactic and polyglycolic acid and their copolymers, for instance DEXON (see J. Heller, Biomaterials I (1980), 51; poly(DL-lactide-co- ⁇ -caprolactone), poly(DL-lactide-co- ⁇ -valerolactone), poly(DL-lactide-co- ⁇ -butyrolactone), polyalkylcyanoacrylates; polyamides, polyhydroxybutyrate; polydioxanone; poly- ⁇ -aminoketones (A. S. Angeloni, P.
  • microballoons of the present invention can also be prepared according to the methods of WO-A-96/15815, inco ⁇ orated herein by reference, where the microballoons are made from a biodegradable membrane comprising biodegradable lipids, preferably selected from mono- di-, tri-glycerides, fatty acids, sterols, waxes and mixtures thereof.
  • biodegradable lipids preferably selected from mono- di-, tri-glycerides, fatty acids, sterols, waxes and mixtures thereof.
  • Prefened lipids are di- or tri-glycerides, e.g., di- or tri-myristin, -palmityn or -stearin, in particular tripalmitin or tristearin.
  • microballoons may employ any of the gases disclosed herein of known to the skilled artisan; however, inert gases such as fluorinated gases are prefened.
  • the microballoons may be suspended in a pharmaceutically acceptable liquid carrier with optional additives known to those of ordinary skill in the art and stabilizers.
  • gas-containing contrast agent formulations include microparticles (especially aggregates of microparticles) having gas contained therein or otherwise associated therewith (for example being adsorbed on the surface thereof and/or contained within voids, cavities or pores therein).
  • Methods for the preparation of these agents are as described in EP 0122624; EP 0123235; EP 0365467; US 5,558,857; US 5,607,661; US 5,637,289; US 5,558,856; US 5,137,928; WO 95/21631 or WO 93/13809, inco ⁇ orated herein by reference in their entirety.
  • any of these ultrasound compositions should also be, as far as possible, isotonic with blood.
  • small amounts of isotonic agents may be added to any of above ultrasound contrast agent suspensions.
  • the isotonic agents are physiological solutions commonly used in medicine and they comprise aqueous saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose solution, etc.
  • the ultrasound compositions may include standard pharmaceutically acceptable additives, including, for example, emulsifying agents, viscosity modifiers, cryoprotectants, lyoprotectants, bulking agents etc. Any biocompatible gas may be used in the ultrasound contrast agents useful in the invention.
  • gas includes any substances (including mixtures) substantially in gaseous form at the normal human body temperature.
  • the gas may thus include, for example, air, nitrogen, oxygen, CO 2 , argon, xenon or krypton, fluorinated gases (including for example, perfluorocarbons, SF 6 , SeF 6 ) a low molecular weight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), for example, an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne and/or mixtures thereof.
  • fluorinated gases including for example, perfluorocarbons, SF 6 , SeF 6
  • Fluorinated gases include materials which contain at least one fluorine atom such as SF 6; freons (organic compounds containing one or more carbon atoms and fluorine, i.e., CF 4 , C 2 F 6; C 3 F 8; C 4 F 8 C 4 F ⁇ o CBrF 3; CCI 2 F2,C 2 CIF 5) and CBrClF 2 ) and perfluorocarbons.
  • freons organic compounds containing one or more carbon atoms and fluorine, i.e., CF 4 , C 2 F 6; C 3 F 8; C 4 F 8 C 4 F ⁇ o CBrF 3; CCI 2 F2,C 2 CIF 5) and CBrClF 2
  • perfluorocarbon refers to compounds containing only carbon and fluorine atoms and includes, in particular, saturated, unsaturated, and cyclic perfluorocarbons.
  • the saturated perfluorocarbons which are usually prefened, have the formula C n F n+2 , where n is from 1 to 12, preferably from 2 to 10, most preferably from 3 to 8 and even more preferably from 3 to 6.
  • Suitable perfluorocarbons include, for example, CF 4 , C 2 F 6 , C 3 F 8 C 4 F 8 , C 4 F 10 , C 5 F 12 , C 6 F 12 , C 7 F 14 , C 8 F 18 , and C 9 F 20 .
  • the gas or gas mixture comprises SF 6 or a perfluorocarbon selected from the group consisting of C 3 F 8 C 4 F 8 , C 4 F 10 , C 5 F 12 , C 6 F 12 , C 7 F 14 , C 8 F 18 , with C 4 F 10 being particularly prefened.
  • SF 6 or a perfluorocarbon selected from the group consisting of C 3 F 8 C 4 F 8 , C 4 F 10 , C 5 F 12 , C 6 F 12 , C 7 F 14 , C 8 F 18 , with C 4 F 10 being particularly prefened.
  • a precursor to a gaseous substance e.g., a material that is capable of being converted to a gas in vivo, often referred to as a "gas precursor"
  • gas precursor e.g., a material that is capable of being converted to a gas in vivo
  • the gas precursor and the gas it produces are physiologically acceptable.
  • the gas precursor may be pH-activated, photo-activated, temperature activated, etc.
  • certain perfluorocarbons may be used as temperature activated gas precursors. These perfluorocarbons, such as perfiuoropentane, have a liquid/gas phase transition temperature above room temperature (or the temperature at which the agents are produced and/or stored) but below body temperature; thus they undergo a phase shift and are converted to a gas within the human body.
  • the gas can comprise a mixture of gases.
  • the following combinations are particularly preferred gas mixtures: a mixture of gases (A) and (B) in which, at least one of the gases (B), present in an amount of between 0.5 - 41% by vol., has a molecular weight greater than 80 daltons and is a fluorinated gas and (A) is selected from the group consisting of air, oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance of the mixture being gas A.
  • ultrasound vesicles may be larger than the other detectable labels described herein, they may be linked or conjugated to a plurality of KDR or VEGF/KDR complex binding polypeptides in order to increase the targeting efficiency of the agent.
  • Attachment to the ultrasound contrast agents described above may be via direct covalent bond between the KDR or VEGF/KDR complex binding polypeptide and the material used to make the vesicle or via a linker, as described previously.
  • WO 98/53857 generally for a description of the attachment of a peptide to a bifunctional PEG linker, which is then reacted with a liposome composition. See also, Lanza et al, Ultrasound in Med. & Bio., 23(6):863-870 (1997).
  • a number of methods maybe used to prepare suspensions of microbubbles conjugated to KDR or VEGF/KDR complex binding polypeptides. For example, one may prepare maleimide-derivatized microbubbles by inco ⁇ orating 5 % (w/w) of N-MPB-PE (1 , 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido- phenyl butyramide), (Avanti Polar-Lipids, Inc) in the phospholipid formulation.
  • N-MPB-PE 1- , 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido- phenyl butyramide), (Avanti Polar-Lipids, Inc) in the phospholipid formulation.
  • solutions of mercaptoacetylated KDR-binding peptides (10 mg/ml in DMF), which have been incubated in deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) are added to the maleimide- activated microbubble suspension. After incubation in the dark, under gentle agitation, the peptide conjugated microbubbles may be purified by centrifugation.
  • Compounds that can be used for derivatization of microbubbles typically include the following components: (a) a hydrophobic portion, compatible with the material forming the envelope of the microbubble or of the microballoon, in order to allow an effective inco ⁇ oration of the compound in the envelope of the vesicel; said portion is represented typically by a lipid moiety (dipalmitin, distearoyl); and (b) a spacer (typically PEGs of different molecular weights), which may be optional in some cases (for example, microbubbles may for instance present difficulties to be freeze dried if the spacer is too long) or preferred in some others (e.g., peptides may be less active when conjugated to a microballoon with short spacers); and (c) a reactive group capable of reacting with a conesponding reacting moiety on the peptide to be conjugated (e.g., maleimido with the -SH group of cysteine).
  • a hydrophobic portion
  • KDR-binding polypeptide conjugated microbubbles may be prepared using biotin/avidin.
  • avidin-conjugated microbubbles may be prepared using a maleimide-activated phospholipid microbubble suspension, prepared as described above, which is added to mercaptoacetylated-avidin (which has been incubated with deacetylation solution).
  • Biotinylated KDR or VEGF/KDR complex-binding peptides (prepared as described herein ) are then added to the suspension of avidin-conjugated microbubbles, yielding a suspension of microbubbles conjugated to KDR or VEGF/KDR complex-binding peptides.
  • the lyophilized residue may be stored and transported without need of temperature control of its environment and in particular it may be supplied to hospitals and physicians for on site formulation into a ready-to-use administrable suspension without requiring such users to have special storage facilities.
  • a two-component kit which can include two separate containers or a dual-chamber container, hi the former case preferably the container is a conventional septum-sealed vial, wherein the vial containing the lyophilized residue of step b) is sealed with a septum through which the carrier liquid may be injected using an optionally prefilled syringe.
  • the syringe used as the container of the second component is also used then for injecting the contrast agent.
  • the dual-chamber container is a dual-chamber syringe and once the lyophilizate has been reconstituted and then suitably mixed or gently shaken, the container can be used directly for injecting the contrast agent, hi both cases means for directing or permitting application of sufficient bubble forming energy into the contents of the container are provided.
  • the size of the gas microbubbles is substantially independent of the amount of agitation energy applied to the reconstituted dried product. Accordingly, no more than gentle hand shaking is generally required to give reproducible products with consistent microbubble size.
  • aqueous phase can be inte ⁇ osed between the water-insoluble gas and the environment, to increase shelf life of the product.
  • a material necessary for fonning the contrast agent is not already present in the container (e.g. a targeting ligand to be linked to the phospholipid during reconstitution)
  • it can be packaged with the other components of the kit, preferably in a form or container adapted to facilitate ready combination with the other components of the kit.
  • the present invention may use conventional containers, vials and adapters.
  • the only requirement is a good seal between the stopper and the container.
  • the quality of the seal therefore, becomes a matter of primary concern; any degradation of seal integrity could allow undesirable substances to enter the vial, h addition to assuring sterility, vacuum retention is essential for products stoppered at ambient or reduced pressures to assure safe and proper reconstitution.
  • the stopper it may be a compound or multicomponent formulation based on an elastomer, such as poly(isobutylene) or butyl rubber.
  • Ultrasound imaging techniques which may be used in accordance with the present invention include known techniques, such as color Doppler, power Doppler, Doppler amplitude, stimulated acoustic imaging, and two- or three-dimensional imaging techniques. fundamental modes, with the second harmonic prefened.
  • the contrast agents formed by phospholipid stabilized microbubbles may, for example, be administered in doses such that the amount of phospholipid injected is in the range OJ to 200 ⁇ g/kg body weight, preferably from about 0J to 30 ⁇ g/kg.
  • Microballoons-containing contrast agents are typically administered in doses such that the amount of wall-forming polymer or lipid is from about 10 ⁇ g/kg to about 20 mg/kg of body weight.
  • a number of optical parameters may be employed to determine the location of KDR or VEGF/KDR complex with in vivo light imaging after injection of the subject with an optically-labeled KDR or VEGF/KDR complex binding polypeptide.
  • Optical parameters to be detected in the preparation of an image may include transmitted radiation, abso ⁇ tion, fluorescent or phosphorescent emission, light reflection, changes in absorbance amplitude or maxima, and elastically scattered radiation.
  • biological tissue is relatively translucent to light in the near infrared (NT ) wavelength range of 650- 1000 nm. NTR radiation can penetrate tissue up to several centimeters, pennitting the use of the KDR or VEGF/KDR complex binding polypeptides of the present invention for optical imaging of KDR or VEGF/KDR complex in vivo.
  • NT near infrared
  • the KDR or VEGF/KDR complex binding polypeptides may be conjugated with photolabels, such as optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having abso ⁇ tion or emission maxima in the range of 400-1500 nm.
  • photolabels such as optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having abso ⁇ tion or emission maxima in the range of 400-1500 nm.
  • the KDR or VEGF/KDR complex binding polypeptide may alternatively be derivatized with a bioluminescent molecule.
  • the preferced range of abso ⁇ tion maxima for photolabels is between 600 and 1000 nm to minimize interference with the signal from hemoglobin.
  • photoabso ⁇ tion labels have large molar abso ⁇ tivities, e.g., > 10 5 cm ⁇ M "1 , while fluorescent optical dyes will have high quannim yields.
  • optical dyes include, but are not limited to those described in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and references cited therein.
  • the photolabels maybe covalently linked directly to the KDR or VEGF/KDR complex binding peptide or linked to the KDR or VEGF/KDR complex binding p ' eptide'V ⁇ a"a"iin ⁇ er, as es tfe - previously.
  • the patient is scanned with one or more light sources (e.g., a laser) in the wavelength range appropriate for the photolabel employed in the agent.
  • the light used may be monochromatic or polychromatic and continuous or pulsed. Transmitted, scattered, or reflected light is detected via a photodetector tuned to one or multiple wavelengths to detennine the location of KDR or VEGF/KDR complex in the subject. Changes in the optical parameter may be monitored over time to detect accumulation of the optically-labeled reagent at the site of angiogenesis. Standard image processing and detecting devices may be used in conjunction with the optical imaging reagents of the present invention.
  • optical imaging reagents described above may also be used for acousto- optical or sonoluminescent imaging performed with optically-labeled imaging agents (see, US 5,171,298, WO 98/57666, and references cited therein).
  • acousto-optical imaging ultrasound radiation is applied to the subject and affects the optical parameters of the transmitted, emitted, or reflected light.
  • sonoluminescent imaging the applied ultrasound actually generates the light detected. Suitable imaging methods using such techniques are described in WO 98/57666.
  • the KDR or VEGF/KDR complex binding moieties may be conjugated with a radionuclide reporter appropriate for scintigraphy, SPECT, or PET imaging and/or with a radionuclide appropriate for radiotherapy.
  • Constructs in which the KDR or VEGF/KDR complex binding moieties are conjugated with both a chelator for a radionuclide useful for diagnostic imaging and a chelator useful for radiotherapy are within the scope of the invention.
  • a peptide is complexed with one of the various positron emitting metal ions, such as 51 Mn, 52 Fe, 60 Cu, 68 Ga, 72 As, 94 Tc, or 110 h ⁇ .
  • the binding moieties of the invention can also be labeled by halogenation using radionuclides such as 1S F, 124 1, 125 1, 131 1, 123 1, 77 Br , and 76 Br.
  • Prefened metal radionuclides for scintigraphy or radiotherapy include 99m Tc, 51 Cr, 67 Ga, 68 Ga, 47 Sc, 51 Cr, 167 Tm, 141 Ce , h ⁇ , 168 Yb, 175 Yb, 140 La, 90 Y, 88 Y, 153 Sm, 16 1Ho, 165 Dy, 166 Dy, 62 Cu, 64 Cu, 67 Cu, 97 Ru, 103 Ru, 186 Re, 188 Re, 203 Pb, 211 Bi, 212 Bi, 213 Bi, 214 Bi, 105 Rh, 109 Pd, 117m Sn, 149 Pm, 161 Tb, 177 Lu, 198 Au and 199 Au.
  • the prefened radionuclides include Cu, 7 Ga, Ga, 99m Tc, and ⁇ In.
  • the preferred radionuclides include 64 Cu, 90 Y, 105 Rh, ⁇ ⁇ h ⁇ , 117m Sn, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 188 Re, and 199 Au.
  • 99m Tc is particularly prefened for diagnostic applications because of its low cost, availability, imaging properties, and high specific activity.
  • This isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is readily available from a 99 Mo- 99m Tc generator.
  • the metal radionuclides may be chelated by, for example, linear, macrocyclic, te ⁇ yridine, and N 3 S, N 2 S 2 , orN 4 chelants (see also, US 5,367,080, US 5,364,613, US 5,021,556, US 5,075,099, US 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTP A, EDTA, DOTA, DO3 A, TETA, and bisamino bistbiol (BAT) chelators (see also US 5,720,934).
  • N 4 chelators are described in US 6,143,274; US 6,093,382; US 5,608,110; US 5,665,329; US 5,656,254; and US 5,688,487.
  • Certain N 3 S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in US5,662,885; US 5,976,495; and US 5,780,006.
  • the chelator may also include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N 3 S, and N 2 S 2 systems such as MAMA
  • the chelator may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate array.
  • complexes containing ligand atoms that are not donated to the metal in a tetradentate array include the boronic acid adducts of technetium and rhenium dioximes, such as are described in US 5,183,653; US 5,387,409; and US 5,118,797, the disclosures of which are inco ⁇ orated by reference herein, in their entirety.
  • disulfide bonds of a KDR or VEGF/KDR complex binding polypeptide of the invention are used as two ligands for chelation of a radionuclide such as 99 Tc. h this way the peptide loop is expanded by the introduction of Tc (peptide-S-S-peptide changed to peptide-S-Tc-S-peptide).
  • Tc peptide-S-S-peptide changed to peptide-S-Tc-S-peptide.
  • the other chelating groups for Tc can be supplied by amide nitrogens of the backbone, another cystine amino acid or other modifications of amino acids.
  • Particularly preferred metal chelators include those of Formula 20, 21, 22, 23a, 23b, 24a, 24b and 25 (FIGS. 34A-F).
  • Formulas 20-22 are particularly useful for lanthanides such as paramagnetic Gd 3+ and radioactive lanthanides such as 177 Lu, 90 Y, 153 Sm, m h ⁇ , or 166 Ho.
  • Formulas 23a-24b are particularly useful for radionuclides 99m Tc, 186 Re, or 188 Re.
  • Forumula 25 (FIG. 34F) is particularly useful for 99m Tc.
  • the chelators may be covalently linked directly to the KDR or VEGF/KDR complex binding moiety or linked to the KDR or VEGF/KDR complex binding polypeptide via a linker, as described previously, and then directly labeled with the radioactive metal of choice (see, WO 98/52618, US 5,879,658, and US 5,849,261).
  • Radioactive technetium are particularly useful for diagnostic imaging and complexes of radioactive rhenium are particularly useful for radiotherapy, h forming a complex of radioactive technetium with the reagents of this invention, the technetium complex, preferably a salt of Tc-99mgnacclmetate, is reacted with the reagent in the presence of a reducing agent.
  • a reducing agent Prefened reducing agents are dithionite, stannous and fenous ions; the most preferred reducing agent is stannous chloride.
  • Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a reagent of the invention to be labeled and a sufficient amount of reducing agent to label the reagent with Tc-99m.
  • the complex may be formed by reacting a peptide of this invention conjugated with an appropriate chelator with a pre- formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art.
  • the labile complex may be fonned using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example.
  • the Tc-99m pertechnetate salts useful with the present invention are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts.
  • Preparation of the complexes of the present invention where the metal is radioactive rhenium may be accomplished using rhenium starting materials in the +5 or +7 oxidation state.
  • rhenium starting materials in the +5 or +7 oxidation state examples include NH 4 ReO 4 or KReO 4 .
  • Re(V) is available as, for example, [ReOCl 4 ](NBu 4 ), [ReOCl 4 ](AsPh 4 ), ReOCl 3 (PPh 3 ) 2 and as ReO 2 ( ⁇ yridine) + , where Ph is phenyl and Bu is n-butyl.
  • Other rhenium reagents capable of forming a rhenium complex may also be used.
  • Radioactively-labeled scintigraphic imaging agents provided by the present invention are provided having a suitable amount of radioactivity.
  • it is generally prefened to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 mCi to 100 mCi per mL.
  • the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi.
  • the solution to be injected at unit dosage is from about 0.01 ml to about 10 mL.
  • Typical doses of a radionuclide-labeled KDR or VEGF/KDR complex binding imaging agents according to the invention provide 10-20 mCi.
  • a gamma camera calibrated for the gamma ray energy of the nuclide inco ⁇ orated in the imaging agent is used to image areas of uptake of the agent and quantify the amount of radioactivity present in the site. Imaging of the site in vivo can take place in a matter of a few minutes.
  • imaging can take place, if , desired, in hours or even longer, after the radiolabeled peptide is injected into a patient, hi most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking ofscintiphotos.
  • the compounds can be administered using many methods which include, but are not limited to, a single or multiple IV or IP injections, using a quantity of radioactivity that is sufficient to cause damage or ablation of the targeted KDR-expressing tissue, but not so much that substantive damage is caused to non-target (normal tissue).
  • the quantity and dose required is different for different constracts, depending on the energy and half-life of the isotope used, the degree of uptake and clearance of the agent from the body and the mass of the tumor.
  • doses can range from a single dose of about 30-50 mCi to a cumulative dose of up to about 3 Curies.
  • the radiotherapeutic compositions of the invention can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection.
  • Radiation stabilizers are known to those skilled in the art, and may include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.
  • a single, or multi-vial kit that contains all of the components needed to prepare the complexes of this invention, other than the radionuclide, is an integral part of this invention.
  • a single- vial kit preferably contains a chelating ligand, a source of stannous salt, or other pharmaceutically acceptable reducing agent, and is appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9.
  • the quantity and type of reducing agent used would depend highly on the nature of the exchange complex to be formed. The proper conditions are well known to those that are skilled in the art. It is preferred that the kit contents be in lyophilized form.
  • Such a single vial kit may optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine- pentaacetic acid (DPTA), ethylenediamine tetraacetic acid (EDTA), or ⁇ , ⁇ ,or ⁇ cyclodextrin that serve to improve the radiochemical purity and stability of the final product.
  • the kit may also contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.
  • a multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical.
  • one vial may contain all of the ingredients that are required to form a labile Tc(V) complex on addition of pertechnetate (e.g., the stannous source or other reducing agent).
  • pertechnetate e.g., the stannous source or other reducing agent.
  • Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the ligand, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized.
  • reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present in either or both vials.
  • the KDR or NEGF/KDR complex binding polypeptides of the present invention can be used to improve the activity of therapeutic agents such as anti- angiogenic or tumorcidal agents against undesired angiogenesis such as occurs in neoplastic tumors, by providing or improving their affinity for KDR or NEGF/KDR complex and their residence time at a KDR or VEGF/KDR complex on endothelium undergoing angiogenesis.
  • therapeutic agents are provided by conjugating a KDR or VEGF/KDR complex binding polypeptide according to the invention with a therapeutic agent.
  • the therapeutic agent may be a radiotherapeutic, discussed above, a drug, chemotherapeutic or tumorcidal agent, genetic material or a gene delivery vehicle, etc.
  • the KDR or VEGF/KDR complex binding polypeptide portion of the conjugate causes the therapeutic to "home" to the sites of KDR or VEGF/KDR complex (i.e., activated endothelium), and to improve the affinity of the conjugate for the endothelium, so that the therapeutic activity of the conjugate is more localized and concentrated at the sites of angiogenesis.
  • Such conjugates will be useful in treating angiogenesis associated diseases, especially neoplastic tumor growth and metastasis, in mammals, including humans, which method comprises administering to a mammal in need thereof an effective amount of a KDR or
  • VEGF/KDR complex binding polypeptide according to the invention conjugated with a therapeutic agent conjugated with a therapeutic agent.
  • the invention also provides the use of such conjugates in the manufacture of a medicament for the treatment of angiogenesis associated diseases in mammals, including humans.
  • Suitable therapeutic agents for use in this aspect of the invention include, but are not limited to: antineoplastic agents, such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L- PAM, or phenylalanine mustard), mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin (actino
  • the KDR or VEGF/KDR complex binding polypeptides of the present invention may also be used to target genetic material to KDR-expressing cells. Thus, they may be useful in gene therapy, particularly for treatment of diseases associated with angiogenesis. h this embodiment, genetic material or one or more delivery vehicles containing genetic material useful in treating an angiogenesis- related disease may be conjugated to one or more KDR binding moieties of the invention and administered to a patient.
  • the genetic material may include nucleic acids, such as RNA or DNA, of either natural or synthetic origin, including recombinant RNA and DNA and antisense RNA and DNA.
  • Types of genetic material that may be used include, for example, genes carried on expression vectors such as plasmids, phagemids, cosmids, yeast artificiarchromosomes (YAC's) arid defective or "helper” viruses, antigene nucleic acids, both single and double stranded RNA and DNA and analogs thereof, such as phosphorothioate and phosphorodithioate oligodeoxynucleotides. Additionally, the genetic material may be combined, for example, with lipids, proteins or other polymers.
  • Delivery vehicles for genetic material may include, for example, a virus particle, a retroviral or other gene therapy vector, a liposome, a complex of lipids (especially cationic lipids) and genetic material, a complex of dextran derivatives and genetic material, etc.
  • the constracts of the invention are utilized in gene therapy for treaunent of diseases associated with angiogenesis.
  • genetic material, or one or more delivery vehicles containing genetic material, e.g., useful in treating an angiogenesis-related disease can be conjugated to one or more KDR or VEGF/KDR complex binding polypeptides or heteromultimers of the invention and administered to a patient.
  • Constructs including genetic material and the KDR-binding polypeptides of the invention may be used, in particular, to selectively introduce genes into angiogenic endothelial cells, which may be useful not only to treat cancer, but also after angioplasty, where inhibition of angiogenesis may inhibit restenosis.
  • Therapeutic agents and the KDR or VEGF/KDR complex binding moieties of the invention can be linked or fused in known ways, using the same type of linkers discussed elsewhere in this application.
  • Preferred linkers will be substituted or unsubstituted alkyl chains, amino acid chains, polyethylene glycol chains, and other simple polymeric linkers known in the art.
  • the therapeutic agent is itself a protein, for which the encoding DNA sequence is known, the therapeutic protein and KDR or VEGF/KDR complex binding polypeptide may be coexpressed from the same synthetic gene, created using recombinant DNA techniques, as described above.
  • the coding sequence for the KDR or VEGF/KDR complex binding polypeptide maybe fused in frame with that of the therapeutic protein, such that the peptide is expressed at the amino- or carboxy-terminus of the therapeutic protein, or at a place between the termini, if it is determined that such placement would not destroy the required biological function of either the therapeutic protein or the KDR or VEGF/KDR complex binding polypeptide.
  • a particular advantage of this general approach is that concatamerization of multiple, tandemly ananged KDR or VEGF/KDR complex binding polypeptides is possible, thereby increasing the number and concentration of KDR or VEGF/KDR complex binding sites associated with each therapeutic protein, hi this manner KDR or VEGF/KDR complex binding avidity is increased which would be expected to improve the efficacy of the recombinant therapeutic fusion protein.
  • KDR and VEGF/KDR complex binding polypeptide may be useful in imaging or therapeutic applications.
  • the coding sequence for a KDR or VEGF/KDR complex binding peptide may be fused in frame to a sequence encoding an antibody (or an antibody fragment or recombinant DNA constract including an antibody, etc.) which, for example, binds to a chelator for a radionuclide (or another detectable label).
  • the antibody expressing the KDR or VEGF/KDR complex binding polypeptide is then administered to a patient and allowed to localize and bind to KDR-expressing tissue.
  • the chelator-radionuclide complex (or other detectable label), which the antibody recognizes is administered, permitting imaging of or radiotherapy to the KDR-expressing tissues.
  • the coding sequence for a KDR or VEGF/KDR complex binding peptide may be fused in frame to a sequence encoding, for example, serum proteins or other proteins that produce biological effects (such as apoptosis, coagulation, internalization, differentiation, cellular stasis, immune system stimulation or suppression, or combinations thereof).
  • the resulting recombinant proteins are useful in imaging, radiotherapy, and therapies directed against cancer and other diseases that involve angiogenesis or diseases associated with the pathogens discussed herein.
  • constructs including KDR or KDR/VEGF complex binding polypeptides of the present invention can themselves be used as therapeutics to treat a number of diseases.
  • a protein or other molecule e.g., a growth factor, hormone etc.
  • constructs including such binding moieties could be useful as therapeutics.
  • binding of a binding moiety itself inhibits a disease process constracts containing such binding moieties could also be useful as therapeutics.
  • constructs including KDR complex binding polypeptides that inhibit the binding of VEGF to KDR may be used as anti-angiogenic agents.
  • Some peptides of the invention that inhibit activation of KDR are discussed in Example 9 infra.
  • Certain constructs of the invention including multimers and heteromultimers that inhibit activation of KDR are also discussed in the Examples.
  • a particularly prefened heteromultimer is the heterodimer-containing construct Dl (structures provided by the examples).
  • Other preferred heterodimer constracts include D4, D5, and D6 (structures provided in Examples 12 and 18 below).
  • the binding polypeptides and constracts thereof of the present invention are useful as therapeutic agents for treating conditions that involve endothelial cells. Because an important function of endothelial cells is angiogenesis, or the fonnation of blood vessels, the polypeptides and constracts thereof are particularly useful for treating conditions that involve angiogenesis.
  • Conditions that involve angiogenesis include, for example, solid tumors, tumor metastases and benign tumors. Such tumors and related disorders are well known in the art and include, for example, melanoma, central nervous system tumors, neuroendocrine tumors, sarcoma, multiple myeloma as wells as cancer of the breast, lung, prostate, colon, head & neck, and ovaries.
  • Benign tumors include, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas.
  • rheumatoid arthritis rheumatoid arthritis
  • psoriasis ocular diseases
  • ocular diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rebeosis, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma and wound granulation.
  • Other relevant diseases or conditions that involve blood vessel growth include intestinal adhesions, atherosclerosis, scleroderma, and hypertropic scars, and ulcers.
  • binding polypeptides and constracts thereof of the present invention can be used to reduce or prevent uterine neovascularization required for embryo implantation, for example, as a birth control agent.
  • Heteromultimers of this invention can also be useful for treating vascular permeability events that can result when VEGF binds KDR.
  • hi renal failure for example, it has been shown that anti- VEGF antibodies can reverse damage.
  • the compounds of the present invention can reverse renal permeability pathogenesis in, for example, diabetes.
  • the KDR or VEGF/KDR complex binding polypeptides of the present invention may be useful in treating diseases associated with certain pathogens, including, for example, malaria, HIV, SIN, Simian hemorrhagic fever virus, etc.
  • Sequence homology searches of KDR-binding peptides identified by phage display using the BLAST program at ⁇ CBI has identified a number of homologous proteins known or expected to be present on the surface of pathogenic organisms. Homologies were noted between the polypeptides of the invention and proteins from various malaria strains, HIN, SIN, simian hemonhagic fever virus, and an enterohemonhagic E. coli strain.
  • homologous proteins such as PfEMPl and EBL-1
  • hypermutable adhesion proteins known to play roles in virulence. These proteins possess multiple binding sites that are capable of binding to more than one target molecule on the host's surface. Their high mutation and recombination rates allow them to quickly develop new binding sites to promote survival and/or invasion.
  • proteins such as gpl20 of HIN which also has homology to some of the KDR-binding peptides disclosed herein
  • KDR-binding peptide sequences disclosed herein may have usefulness in blocking infection with the pathogen species that possesses the homology. Indeed, a similar strategy is being employed to block HIV infection by trying to prevent virus envelope proteins from binding to their known cellular surface targets such as CD4. See, Howie et al, "Synthetic peptides representing discontinuous CD4 binding epitopes of HIV- 1 gpl20 that induce T cell apoptosis and block cell death induced by gpl20", FASEB J, 12(11):991-998 (1998).
  • KDR may represent a previously unknown target for a number of pathogens, and the KDR binding peptides of the invention maybe useful in treating the diseases associated with those pathogens.
  • the binding polypeptides and constracts thereof can be administered to an individual over a suitable time course depending on the nature of the condition and the desired outcome.
  • the binding polypeptides and constracts thereof can be administered prophylactically, e.g., before the condition is diagnosed or to an individual predisposed to a condition.
  • the binding polypeptides and constructs thereof can be administered while the individual exhibits symptoms of the condition or after the symptoms have passed or otherwise been relieved (such as after removal of a tumor).
  • the binding polypeptides and constracts thereof of the present invention can be administered a part of a maintenance regimen, for example to prevent or lessen the recunence or the symptoms or condition.
  • the binding polypeptides and constracts thereof of the present invention can be administered systemically or locally.
  • the quantity of material administered will depend on the seriousness of the condition. For example, for treatment of an angiogenic condition, e.g., in the case of neoplastic tumor growth, the position and size of the tumor will affect the quantity of material to be administered.
  • the precise dose to be employed and mode of administration must per force in view of the nature of the complaint be decided according to the circumstances by the physician supervising treatment, hi general, dosages of the agent conjugate of the present invention will follow the dosages that are routine for the therapeutic agent alone, although the improved affinity of a binding polypeptide or heteromultimer of the invention for its target may allow a decrease in the standard dosage.
  • Such conjugate phannaceutical compositions are preferably formulated for parenteral administration, and most preferably for intravenous or intra-arterial administration.
  • phannaceutical compositions may be given as a bolus, as two or more doses separated in time, or as a constant or non-linear flow infusion.
  • the term "therapeutic” includes at least partial alleviation of symptoms of a given condition.
  • the binding polypeptides and constructs thereof of the present invention do not have to produce a complete alleviation of symptoms to be useful.
  • treatment of an individual can result in a decrease in the size of a tumor or diseased area, or prevention of an increase in size of the tumor or diseased area.
  • Treatment can result in reduction in the number of blood vessels in an area of interest or can prevent an increase in the number of blood vessels in an area of interest.
  • Treatment can also prevent or lessen the number or size of metastatic outgrowths of the main tumor(s).
  • Symptoms that can be alleviated include physiological characteristics such as VEGF receptor activity and migration ability of endothelial cells.
  • the binding polypeptides and constracts thereof of the present invention can inhibit activity of VEGF receptors, including VEGF-2/KDR, VEGF-l/Flt-1 and VEGF-3/Flt-4. Such inhibition can be detected, for example, by measuring the phosphorylation state of the receptor in the presence of or after treatment with the binding polypeptides or constracts thereof. Such inhibition can also be detected by measuring the ability of endothelial cells to migrate in the presence of or after treatment with the binding polypeptides or constracts thereof.
  • the size of the area of interest e.g., the tumor or lesion
  • the phosphorylation state of the relevant receptor, or the migration ability of endothelial in an area of interest can be measured in samples taken from the individual.
  • the VEGF receptors or endothelial cells can be isolated from the sample and used in assays described herein.
  • the dosage of the polypeptides and constracts thereof may depend on the age, sex, health, and weight of the individual, as well as the nature of the condition and overall treatment regimen.
  • the biological effects of the polypeptides and constructs thereof are described herein. Therefore, based on the biological effects of the binding polypeptides and constracts provided herein, and the desired outcome of treatment, the prefened dosage is determinable by one of ordinary skill in the art through routine optimization procedures.
  • the daily regimen is in the range of about 0J ⁇ g/kg to about 1 mg/kg.
  • binding polypeptides and constracts thereof provided herein can be administered as the sole active ingredient together with a pharmaceutically acceptable excipient, or can be administered together with other binding polypeptides and constracts thereof, other therapeutic agents, or combination thereof, hi addition, the binding polypeptides and constracts thereof can be conjugated to therapeutic agents, for example, to improve specificity, residence time in the body, or therapeutic effect.
  • therapeutic agents include, for example, other anti-angiogenic compounds, and tumoricidal compounds.
  • the therapeutic agent can also include antibodies.
  • the binding polypeptide or constructs thereof of the present invention can be used as an endothelial cell homing device.
  • the binding polypeptide or constructs thereof can be conjugated to nucleic acid encoding, for example, a therapeutic polypeptide, in order to target the nucleic acid to endothelial cells.
  • the endothelial can internalize and express the conjugated nucleic acid, thereby delivering the therapeutic peptide to the target cells.
  • the therapeutic agent can be associated with an ultrasound contrast agent composition, said ultrasound contrast agent including the KDR or VEGF complex binding peptides of the invention linked to the material employed to fonn the vesicles (particularly microbubbles or microballoons) comprised in the contrast agent, as previously described.
  • said contrast agent/therapeutic agent association can be carried out as described in US 6,258,378, herein inco ⁇ orated by reference.
  • the pathogenic site can be inadiated with an energy beam (preferably ultrasonic, e.g. with a frequency of from 0.3 to 3 MHz), to cause the bursting of microvesicles, as disclosed for instance in the above cited U.S. Patent No. 6,258,378.
  • the therapeutic effect of the therapeutic agent can thus be advantageously enlianced by the energy released by the burst of the microvesicles, in particular causing an effective delivery of the therapeutic agent to the targeted pathogenic site.
  • the binding polypeptides and constracts thereof can be administered by any suitable route.
  • Suitable routes of administration include, but are not limited to, topical application, transdermal, parenteral, gastrointestinal, intravaginal, and transalveolar.
  • Compositions for the desired route of administration can be prepared by any of the methods well known in the pharmaceutical arts, for example, as described in Remington: The Science and Practice of Pharmacy, 20* ⁇ ed., Lippincott, Williams and Wilkins, 2000.
  • the binding polypeptides can be suspended, for example, in a cream, gel or rinse which allows the polypeptides or constructs to penetrate the skin and enter the blood stream, for systemic delivery, or contact the area of interest, for localized delivery.
  • compositions suitable for topical application include any pharmaceutically acceptable base in which the polypeptides are at least minimally soluble.
  • the polypeptides can be applied in pharmaceutically acceptable suspension together with a suitable transdermal device or "patch."
  • suitable transdermal devices for administration of the polypeptides of the present invention are described, for example, in U.S. Patent No. 6,165,458, issued December 26, 2000 to Foldvari, et al, and U.S. Patent No. 6,214,166 1, issued August 4, 2001 to Sintov, et al, the teachings of which are inco ⁇ orated herein by reference.
  • the polypeptides can be injected intravenously, intramuscularly, intraperitoneally, or subcutaneously.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • compositions include, but are not limited to, sterile water, saline solution, and buffered saline (including buffers like phosphate or acetate), alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, paraffin, etc.
  • the composition may also include a solubilizing agent and a local anaesthetic such as lidocaine to ease pain at the site of the injection, preservatives, stabilizers, wetting agents, emulsifiers, salts, lubricants, etc. as long as they do not react deleteriously with the active compounds.
  • the composition may comprise conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds.
  • the ingredients will be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hennetically sealed container such as an ampoule or sachette indicating the quantity of active agent in activity units.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade "water for injection" or saline.
  • an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.
  • the polypeptides can be inco ⁇ orated into pharmaceutically acceptable powders, pills or liquids for ingestion, and suppositories for rectal or vaginal administration.
  • the polypeptides can be suspended in a pharmaceutically acceptable excipient suitable for aerosolization and inhalation or as a mouthwash.
  • Devices suitable for transalveolar administration such as atomizers and vaporizers are also included within the scope of the invention.
  • Suitable formulations for aerosol delivery of polypeptides using buccal or pulmonary routes can be found, for example in U.S. Patent No.
  • polypeptides of the present invention can be administered nasally or ocularly, where the polypeptide is suspended in a liquid pharmaceutically acceptable agent suitable for drop wise dosing.
  • the polypeptides of the present invention can be administered such that the polypeptide is released in the individual over an extended period of time (sustained or controlled release).
  • the polypeptide can be formulated into a composition such that a single administration provides delivery of the polypeptide for at least one week, or over the period of a year or more.
  • Controlled release systems include monolithic or reservoir-type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches and iontophoretic devices.
  • the polypeptides of the present invention are encapsulated or admixed in a slowly degrading, non-toxic polymer. Additional formulations suitable for controlled release of the polypeptides provided herein are described in U.S.
  • Patent No. 4,391,797 issued July 5, 1983, to Folkman, et al, the teachings of which are inco ⁇ orated herein by reference.
  • Another suitable method for delivering the polypeptides of the present to an individual is via in vivo production of the polypeptide.
  • a gene encoding the polypeptide can be administered to the individual such that the encoded polypeptide is expressed.
  • the gene can be transiently expressed.
  • the gene encoding the polypeptide is transfected into cells that have been obtained from the patient, a method refened to as ex vivo gene therapy. Cells expressing the polypeptide are then returned to the patient's body. Methods of ex vivo gene therapy are well known in the art and are described, for example, in U.S. Patent No.
  • Protein A Magnetic Beads (#100.02) were purchased from Dynal (Oslo, Norway). Heparin (#H-3393) was purchased from Sigma Chemical Company (St. Louis, MO). A 2-component tetramethyl benzidine (TMB) system was purchased from KPL (Gaithersburg, MD).
  • microtiter plates were washed with a Bio-Tek 404 plate washer (Winooski, VT).
  • ELISA signals were read with a Bio-Tek plate reader (Winooski, VT).
  • Agitation of 96-well plates was on a LabQuake shaker (Labindustries, Berkeley, CA).
  • Ml 3 phage display libraries were prepared for screening against immobilized KDR and VEGF/KDR targets: Cyclic peptide display libraries TN6/VI, TN7/TV, TN8/IX, TN9/IV, TNIO/D , TNI 2/1, and MTN13/I, and a linear display library, Lin20. The design of these libraries has been described, supra.
  • the DNA encoding the library was synthesized with constant DNA on either side so that the DNA can be PCR amplified using Taq DNA polymerase (Perkin- Ehner, Wellesley, MA), cleaved with Ncol and Pst ⁇ , and ligated to similarly cleaved phage display vector.
  • XLl-Blue MFR' E. coli cells were transformed with the ligated D ⁇ A. All of the libraries were constructed in same manner.
  • Protein A Magnetic Beads were blocked once with IX PBS (pH 7.5), 0.01% Tween-20, 0.1% HSA (Blocking Buffer) for 30 minutes at room temperature and then washed five times with IX PBS (pH 7.5), 0.01% Tween-20, 5 ⁇ g/ml heparin (PBSTH Buffer).
  • the cyclic peptide, or "constrained loop", libraries were pooled for the initial screening into two pools: TN6/VL TN7/IV and TN8/IX were in one pool; TN9/IV, TN10/IX and TN12/I were in the second pool.
  • the two pooled libraries and the linear library (Lin20) were depleted against Trail R4 Fc fusion (an inelevant Fc fusion) and then selected against KDR Fc fusion.
  • 10 11 plaque fonning units (pfu) from each library per 100 ⁇ l PBSTH were pooled together, e.g., 3 pooled libraries would result in a total volume of ⁇ 350 ⁇ l in PBSTH.
  • Trail R4-Fc fusion beads 500 ⁇ l of Trail R4-Fc fusion (OJ ⁇ g/ ⁇ l stock in PBST (no heparin)) were added to 1000 ⁇ l of washed, blocked protein A magnetic beads. The fusion was allowed to bind to the beads overnight with agitation at 4°C. The next day, the magnetic beads were washed 5 times with PBSTH. Each phage pool was incubated with 50 ⁇ l of Trail R4 Fc fusion beads on a Labquake shaker for 1 hour at room temperature (RT). After incubation, the phage supernatant was removed and incubated with another 50 ⁇ l of Trail R4 beads. This was repeated for a total of 5 rounds of depletion, to remove non-specific Fc fusion and bead binding phage from the libraries.
  • KDR target beads 500 ⁇ l of KDR-Fc fusion (0J ⁇ g/ ⁇ l stock in PBST (no heparin)) were added to 500 ⁇ l of washed, blocked beads. The KDR- Fc fusion was allowed to bind overnight with agitation at 4°C. The next day, the beads were washed 5 times with PBSTH. Each depleted library pool was added to 100 ⁇ l of KDR-Fc beads and allowed to incubate on a LabQuake shaker for 1 hour at RT. Beads were then washed as rapidly as possible with 5 X 1 ml PBSTH using a magnetic stand (Promega) to separate the beads from the wash buffer.
  • Phage still bound to beads after the washing were eluted once with 250 ⁇ l of VEGF (50 ⁇ g/ml, ⁇ 1 ⁇ M) in PBSTH for 1 hour at RT on a LabQuake shaker. The 1-hour elution was removed and saved. After the first elution, the beads were incubated again with 250 ⁇ l of VEGF (50 ⁇ g/ml, ⁇ l ⁇ M) overnight at RT on a LabQuake shaker. The two VEGF elutions were kept separate and a small aliquot taken from each for titering. Each elution was mixed with an aliquot of XLl-Blue MRF' (or other F' cell line) E.
  • coli cells which had been chilled on ice after having been grown to mid-logarithmic phase.
  • the remaining beads after VEGF elution were also mixed with cells to amplify the phage still bound to the beads, i.e., KDR-binding phage that had not been competed off by the two NEGF incubations (T-hbur and overnight (O/ ⁇ ) elutions).
  • T-hbur and overnight (O/ ⁇ ) elutions After approximately 15 minutes at room temperature, the phage/cell mixtures were spread onto Bio-Assay Dishes (243 X 243 X 18 mm, ⁇ alge ⁇ unc) containing 250 ml of ⁇ ZCYM agar with 50 ⁇ g/ml of ampicillin. The plate was incubated overnight at 37°C.
  • each pool yielded three amplified eluates. These eluates were panned for 2-3 more additional rounds of selection using ⁇ 10 ° input phage/round according to the same protocol as described above. For each additional round, the KDR-Fc beads were prepared the night before the round was initiated.
  • the amplified elution re-screen on KDR- Fc beads was always eluted in the same manner and all other elutions were treated as washes.
  • the amplified elution recovered by using the still-bound beads to infect E. coli the 1-hour and overnight VEGF elutions were performed and then discarded as washes. Then the beads were used to again infect E. coli and produce the next round amplified elution.
  • each library pool only yielded three final elutions at the end of the selection. Two pools and one linear library, therefore, yielded a total of 9 final elutions at the end of the selection.
  • This selection procedure was repeated for all libraries in- the absence of heparin in all binding buffers, i.e., substituting PBST (PBS (pH 7.5), 0.01% Tween- 20) for PBSTH in all steps.
  • Protein A magnetic beads were blocked once with Blocking Buffer for 30 minutes at room temperature and then washed five times with PBSTH.
  • KDR-Fc fusion depletion beads 1 mL of KDR-Fc fusion (0J ⁇ g/ ⁇ l stock in PBST (no heparin)) was added to 1 mL of washed, blocked beads.
  • VEGF vascular endothelial growth factor
  • the beads were washed 5 times with PBSTH. Each depleted library pool was added to 100 ⁇ l of KDR: VEGF complex beads and allowed to incubate on a LabQuake shaker for 1 hour at RT. Beads were then washed as rapidly as possible with 5 ⁇ 1 mL PBSTH using a magnetic stand (Promega) to separate the beads from the wash buffer. To elute the phage still bound after washing, the beads were mixed with cells to amplify the phage still bound to the beads.
  • the phage/cell mixtures were spread onto Bio-Assay Dishes (243 x 243 x 18 mm, Nalge Nuiic) containing 250 ml of NZCYM agar with 50 ⁇ g/ml of ampicillin. The plate was incubated overnight at 37°C. The next day, each amplified phage culture was harvested from its respective plate. Over the next day, the input, output and amplified phage cultures were titered for FOI. This selection protocol was repeated for two additional rounds using 10 10 input phage from each amplified elution.
  • VEGF complex To assess binding to KDR: VEGF complex, another set of KDR plates was prepared as above and then 100 ⁇ l of VEGF (1 ⁇ g ml) in PBST was added to each KDR well and allowed to incubate at RT for 30 minutes. Each plate was then washed with PBST (PBS, 0.05% Tween-20).
  • PBST PBS, 0.05% Tween-20
  • each overnight phage culture was diluted 1 : 1 (or to 10 10 pfu if using purified phage stock) with PBS, 0.05% Tween-20, 1% BSA. 100 ⁇ l of each diluted culture was added and allowed to incubate at RT for 2-3 hours. Each plate was washed 5 times with PBST. The binding phage were visualized by adding 100 ⁇ l of a 1:10,000 dilution of HRP-anti-M13 antibody conjugate (Pharmacia), diluted in PBST, to each well, then incubating at room temperature for 1 hr. Each plate was washed 7 times with PBST (PBS, 0.05% Tween-20), then the plates were developed with HRP substrate ( ⁇ 10 minutes) and the absorbance signal (630 nm) detected with plate reader.
  • HRP-anti-M13 antibody conjugate Pharmacia
  • KDR and VEGF/KDR complex binding phage were recovered, amplified, and the sequences of the display peptides responsible for the binding were determined by standard DNA sequencing methods.
  • the binding peptides of the phage isolates are set forth in Tables 1-7, infra.
  • phage isolate sequence PKWCEEDWYYCMI T (SEQ ID NO:21) was used as a template to constract a library that allowed one-, two-, and three-base mutations to the parent sequence at each variable codon.
  • phage isolate sequence SRVCWEDSWGGEVCFRY (SEQ ID NO:88) was used as a template to construct a library that allowed one-, two-, and three-base mutations to the parent sequence at each variable codon.
  • a recunent motif from the initial TN8 sequences was kept constant (WVEC— TG-C— ; SEQ ID NO:260) and all of the other codon positions (i.e., at "-") were allowed to vary (all possible 20 amino acids) using NNK codon substitution, where N stands for any nucleotide and K stands for any keto nucleotide (G or T).
  • N stands for any nucleotide
  • K stands for any keto nucleotide
  • each nucleotide within a particular codon was allowed to evolve by adding fixed amounts of the other three nucleotides that did not conespond to the nucleotide of the parent codon. This nucleotide mixing is accomplished in the synthesis of the template DNA used to make the library.
  • the parent nucleotide within each codon was maintained at 64% for SEQ ID NO:21 and 67% for SEQ ID NO:88, whereas the other nucleotides were added at the remainder frequency divided by three. Since the parent nucleotides are in the majority, the overall consensus sequence for the whole library should still contain the parental sequence.
  • the TN8 motif described above was kept constant and all of the other positions in were allowed to vary with NNK substitution in the template oligonucleotide. To extend the substitution, NNK diversity was also permitted in the two flanking amino acid positions, thus adding variable amino acid positions N-terminal and C-tenninal to the display peptide.
  • the secondary library template therefore, encoded a display peptide of the following sequence: Xaa-Xaa- T ⁇ -Val-Glu-Cys-Xaa-Xaa-Xaa-Thr-Gly-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:261), where Xaa can be any amino acid.
  • SEQ ID NO:261 a display peptide of the following sequence: Xaa-Xaa- T ⁇ -Val-Glu-Cys-Xaa-Xaa-Xaa-Thr-Gly-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa (SEQ ID NO:261), where Xaa can be any amino acid.
  • this library was quite diverse in all allowed positions and only resembled the parent motif in the residues that were held constant.
  • Binding phage were eluted through three steps: (1) elution for 1 hour at room temperature, the eluted phage being used to infect cells for amplification, (2) elution overnight, wherein fresh competition elution peptide was added to the bound phage and incubated at 4°C overnight with mixing, the eluted phage being then used to infect cells for amplification, and (3) the remaining beads (bearing uneluted binding phage) were used to infect cells directly. Three rounds of selections were performed.
  • Plaques were picked from rounds 2 and 3 and analyzed by ELISA and sequencing. KDR positive isolates were assayed further for competition with 50 ⁇ M free parent peptide. Those peptides that showed minimal competition with the parent peptide were deemed higher affinity binders and were synthesized. These sequences are listed in the following table as SEQ TD NOS:22-33 for the TN8 secondary library and SEQ TD NOS:89-95 for the TN12 secondary library.
  • Class I peptides only bind KDR in the absence of heparin, and therefore presumably target the heparin binding domain of KDR; Class H peptides bind in the presence or absence of heparin or VEGF, and therefore presumably bind at a non-involved site on KDR; Class IH peptides exhibit binding characteristics that are not affected by heparin but are perturbed in the presence of VEGF, and therefore presumably these bind either to VEGF or the VEGF binding domain of KDR.
  • NA signifies data not available, hi the elution column, 1 HR, O/N, and Cell stand for 1 hour VEGF, overnight VEGF, and bead infection elutions, respectively. In some cases, a particular isolate sequence was observed in two different elutions. For the isolates identified by second generation library, VEGF elutions were substituted with peptide elutions (see below).
  • peptides with selected lysine residues these were protected with l-(4,4- dimethyl-2,6-dioxocyclohex-l-ylidene)-3-methylbutyl (ivDde), which allows bcicuuvc - , . , and can be removed after coupling with 2% hydrazine in DMF or 0.5 M hydroxylamine, pH 8, in water.
  • ivDde l-(4,4- dimethyl-2,6-dioxocyclohex-l-ylidene)-3-methylbutyl
  • Each peptide was labeled with fluorescein on the C-terminal lysine using fluorescein (N-hydroxysuccinimide ester derivative) or fluorescein isothiocyanate (FITC) in DMF, 2% diisopropylethylamine (DIPEA). If the peptide contained an ivDde protected lysine, the reaction was quenched by the addition of 2% hydrazine, which reacts with all free NHS-fluorescein and removes the internal protecting group. For all other peptides, the reaction was quenched by the addition of an equal volume of 0.5 M hydroxylamine, pH 8.
  • the quenched reactions were then diluted with water to less than 10% DMF and then purified using C18 reverse phase chromatography.
  • the peptides were characterized for purity and correct mass on an LC-MS system (HP1100 HPLC with in-line SCIEX AP150 single quadrapole mass spectrometer).
  • Fluorescence anisotropy measurements were performed in 384-well microplates in a volume of 10 ⁇ l in binding buffer (PBS, 0.01% Tween-20, pH 7.5) using a Tecan Polarion fluorescence polarization plate reader. In some cases, heparin (0.5 ⁇ g/ml) or 10% ⁇ human serum was added to the binding buffer (data not shown). The concentration of fluorescein labeled peptide was held constant (20 nM) and the concentration of KDR-Fc (or similar target) was varied. Binding mixtures were equilibrated for 10 minutes in the microplate at 30°C before measurement.
  • r 0bS is the observed anisotropy
  • r t - ree is the anisotropy of the free peptide
  • r b0Und is the anisotropy of the bound peptide
  • K D is the apparent dissociation constant
  • KDR is the total KDR concentration
  • P is the total fluorescein-labeled peptide concen ra ion. D was ca cu a e m a irec therefore these values represent KDR binding to the fluorescein labeled peptide.
  • peptides were injected at 20 ⁇ l/min. for 1 minute using the kinject program. Following a 1 -minute dissociation, any remaining peptide was stripped from the target surface with a quick injection of IM NaCl for 25 sec. at 50 ⁇ l/min. All samples were injected in duplicate. Between each peptide series a buffer injection and a non-target binding peptide injection served as additional controls. Sensorgrams were analyzed using the simultaneous k a /k d fitting program in the BIAevaluation software 3J. Apparent K D by this method is set forth as BiaKo in Table 8.
  • Binding affinities determined for the synthesized polypeptides are set forth in Table 8, below.
  • the putative disulfide- constrained cyclic peptide moieties of the polypeptides are underlined.
  • each peptide was tested for binding to the complex in both assays (fluorescence anisotropy/Biacore) as above.
  • KDR- VEGF complex was formed by mixing together a two fold molar excess of VEGF with KDR-Fc. This mixture was then used in the direct binding titration using a fluorescein labeled peptide as done previously.
  • fluorescein labeled peptide was also tested for binding to KDR and VEGF alone to assess their specificity for complex.
  • Reagent B (TFA: H 2 O: phenol: triisopropylsilane 88:5:5:2), diisopropylethylamine (DIEA), O-(lH-benzotriazole-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-l-yl)-lJ,3,3- tetramethyluronium hexafluorophosphate (HATU), N-hydroxysuccinimide (NHS), solid phase peptide synthesis (SPPS), dimethyl sulfoxide (DMSO), dichloromethane (DCM), dimethylformamide (DMF), human serum albumin (HSA), and radiochemical purity (RCP).
  • DIEA diisopropylethylamine
  • HBTU O-(lH-benzotriazole-l-yl)-N,N,N',N
  • the concentration of the amino acid was 0.25M, and the concentrations for HOBt and DIC respectively were 0.5 M.
  • a typical amino acid coupling cycle (not including wash steps) was to dispense piperidine solution (2.4 mL) to each well and mix for 4 min, then empty all wells. NMP (320 ⁇ L), HOBt solution (320 ⁇ L, 4 eq), amino acid (640 ⁇ L, 4 eq) and DIC (320 ⁇ L, 4 eq) solutions were dispensed to each well. The coupling time was 3h; then the resin was washed. The cycle was repeated for each amino acid.
  • the resin-bound peptide was treated with 25%> piperidine to remove the Fmoc protecting group. After washing, the resin bound peptide was capped with 1.0M Ac 2 O (1.2 ml per well) and diisopropylethylamine in DMF, optionally including varying amounts of HOBt in the mixture for 30 min. The resin was washed with methanol and then dichloromethane and dried. Cleavage of the peptides from the resin and side-chain deprotection was accomplished using Reagent B for 4.5 h. The cleavage solutions were collected and the resins were washed with an additional aliquot of Reagent B. The combined solutions were concentrated to dryness.
  • the purified linear di-cysteine containing peptides were dissolved in water, mixtures of water-acetonitrile, or mixtures of water-DMSO at concentrations between 0J mg/ml and 2.0 mg/ml.
  • the choice of solvent was a function of the solubility of the crude peptide in the solvent.
  • the pH of the solution was adjusted to pH 7.5-8.5 with aqueous ammoma, aqueous ammonium carbonate or aqueous ammonium bicarbonate.
  • the mixture was stined vigorously in air for 24- 48 hrs.
  • the pH of the solution was adjusted to pH 2 with aqueous trifluoroacetic acid.
  • the mixture was lyophilized to provide the crude cyclic disulfide containing peptide.
  • the cyclic disulfide peptide was then dissolved to a volume of 1-2 ml in aqueous (0J%> TFA) containing a minimum of acetonitrile (0J%> TFA).
  • the resulting solution was loaded onto a reverse phase column and the desired compound obtained by a gradient elution of acetonitrile into water, employing a C18, or C8 reverse phase semipreparative or preparative HPLC column, h the case of the DMSO-containing solutions, the solution was diluted until the DMSO concentration was minimal without precipitation of the peptide.
  • the resulting mixture was quickly acidified to pH 2 with dilute trifluoroacetic acid and loaded onto the reverse phase HPLC system and purified as described. Fractions containing the desired materials were pooled and the peptides isolated by lyophilization.
  • Method 2 for the ACT 357 MPS and ACT 496 MOS Synthesizers The peptides were synthesized as in Method 1 with the following changes. HBTU/HOBt/DIEA were used as the coupling reagent and NMP as the solvent. A low load (-0.2 mmol/g) Fmoc-GGGK(Boc)-NovSyn-TGR-resin-prepared from the above-described Nova-Syn TGR resin was employed for peptide synthesis on 0.01 mmol scale.
  • the crude ether-precipitated linear di-cysteine containing peptides were cyclized by dissolution in water, mixtures of aqueous acetonitrile (0.1% TFA), or aqueous DMSO and adjustment of the pH of the solution to pH 7.5 - 8.5 by addition of aqueous ammonia, aqueous ammonium carbonate, or aqueous ammonium bicarbonate solution.
  • the peptide concentration was between 0J and 2.0 mg/ml.
  • the mixture was stirred in air for 24-48 hrs., acidified to a pH 2 with aqueous trifluoroacetic acid, and then purified by preparative reverse phase HPLC employing a gradient of acetonitrile into water. Fractions containing the desired material were pooled and the peptides were isolated by lyophilization.
  • MOS Synthesizer as in method 1.
  • the low load (-0.2 mmol/g) GGGK(Boc)- NovaSyn-TGR resin was employed for peptide synthesis.
  • the coupling solvent was NMP/DMSO 8:2.
  • the synthesis was performed at a 0.02 mmol scale using a coupling time of 3h.
  • the crude linear peptides were further processed as described for Method 1.
  • TGR resin (-0.2 mmol/g) was used for peptide synthesis. The coupling time was 30 minutes. The crude linear peptides were further processed as described for Method 1. Method 5 for the ABI 433 A Synthesizer Synthesis of peptides was carried out on a 0.25 mmol scale using the FastMoc protocol (Applied Biosystems Inc). hi each cycle of this protocol, 1.0 mmol of a dry protected amino acid in a cartridge was dissolved in a solution of 0.9 mmol of HBTU, 2 mmol of DIEA, and 0.9 mmol of HOBt in DMF with additional NMP added.
  • the peptides were made using 0J mmol of NovaSyn TGR (Rink amide) resin (resin substitution 0.2 mmol/g). The coupling time in this protocol was 21 min. Fmoc deprotection was carried out with 20% piperidine in NMP. At the end of the last cycle, the synthesized peptide was acetylated using acetic anhydride/DIEA/HOBt/NMP. The peptide resin was washed and dried for further manipulations or cleaved from the resin (using reagent B). Generally, the cleaved peptides were cyclized as in Method 1 before purification.
  • Method 6 Biotinylation of Resin-Bound Peptides
  • the peptides were prepared using Method 5.
  • the ivDde protecting group on the C-terminal lysine was selectively removed by treatment with 10% hydrazine in DMF.
  • the resin was then treated with a solution of Biotin-N-hydroxysuccinimidyl ester in DMF in the presence of DIEA. After washing, the resin was dried and cleavage was performed using with Reagent B.
  • the resin was filtered off and the filtrate concentrated to dryness.
  • the biotinylated peptide was dissolved in neat DMSO and treated with DIEA and stined for 4-6 hours to effect disulfide cyclization.
  • the crude mixture was purified by preparative HPLC.
  • the resin was filtered off, Reagent B was removed in vacuo and the peptide was precipitated by addition of anhydrous ether.
  • the solid fonned was collected, washed with ether and dried.
  • the solid was dissolved in anhydrous DMSO and the mixture was adjusted to pH 7.5 with DIEA and stined for 4-6 h to effect disulfide cyclization.
  • the disulfide cyclization reaction was monitored by analytical HPLC. After completion of the cyclization, the mixture solution was diluted with 25% acetonitnle m water and directly purified by HPLC on a reverse phase CI 8 column using a gradient of acetonitrile into water (both containing OJ %> TFA). Fractions were analyzed by analytical HPLC and those containing the pure product were collected and lyophilized to obtain the required biotinylated peptide.
  • Method 7 Biotinylation of Purified Peptides
  • the purified peptide (10 mg, prepared by methods 1-5) containing a free amino group was dissolved in anhydrous DMF or DMSO (1 ml) and Biotin-NHS ester (5 equivalents) and DIEA (5 equivalents) were added.
  • the reaction was monitored by HPLC and after the completion of the reaction (1-2 h.), the crude reaction mixture was directly purified by preparative HPLC. Fractions were analyzed by analytical HPLC and those containing the pure product were collected and lyophilized to obtain the required biotinylated peptide.
  • Method 8 Biotinylation of Resin-Bound Peptides Containing Linkers
  • 400 mg of the resin- containing peptide (made using the ABI 433 A Synthesizer and bearing an ivDde-protected lysine) was treated with 10% hydrazine in DMF (2 x 20 ml).
  • the resin was washed with DMF (2 x 20 ml) and DCM (1 x 20 ml).
  • the resin was resuspended in DMF (10 ml) and treated with Fmoc-aminodioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC (0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. After the reaction, the resin was washed with DMF (2 x 10 ml) and with DCM (1 x 10 ml). The resin was then treated with 20% piperidine in DMF (2 x 15 ml) for 10 min. each time. The resin was washed and the coupling with Fmoc-diaminodioxaoctanoic acid and removal of the Fmoc protecting group were repeated once more.
  • the resulting resin containing a peptide with a free amino group, was treated with a solution of Biotin-NHS ester (0.4 mmol, 5 equivalents) and DIEA (0.4 mmol, 5 equivalents) in DMF for 2 hours.
  • the peptide- resin was washed and dried as described previously and then treated with reagent B (20 mL) for 4h. The mixture was filtered, and the filtrate concentrated to dryness. The residue was stined with ether to produce a solid that was collected, washed with ether and dried. The solid was dissolved in anhydrous DMSO and the pH adjusted to 7.5 with DIEA.
  • the mixture was stined for 4-6 hr to effect the disulfide cyclization reaction, which was monitored by analytical HPLC.
  • the DMSO solution was iluted' tn"'25%'"' ⁇ c'et ⁇ nltril'e-' in water and applied directly to a reverse phase C-18 column. Purification was effected using a gradient of acetonitrile into water (both containing OJ % TFA). Fractions were analyzed by analytical HPLC and those containing the pure product were collected and lyophilized to provide the required biotinylated peptide.
  • Method 9 Formation of 5-Carboxyfluorescein-Labeled Peptides Peptide-resin obtained via Method 5, containing an ivDde protecting group on the epsilon nitrogen of lysine, was mixed with a solution of hydrazine in DMF (10%) hydrazine/DMF, 2 x 10 ml, 10 min) to remove the ivDde group.
  • the epsilon nitrogen of the lysine was labeled with fluorescein-5-isothiocyanate (0J2 mmol) and diisopropylethylamine (0J2 mmol) in DMF. The mixture was agitated for 12 h (fluorescein-containing compounds were protected from light).
  • the resin was then washed with DMF (3 x 10 mL) and twice with CH 2 C1 2 (10 mL) and dried under nitrogen for lh.
  • the peptide was cleaved from the resin using reagent B for 4h and the solution collected by filtration. The volatiles were removed under reduced pressure and the residue was dried under vacuum.
  • the peptide was precipitated with ether, collected and the precipitate was dried under a stream of nitrogen.
  • the precipitate was added to water (1 mg/ml) and the pH of the mixture was adjusted to 8 with 10% aqueous meglumine. Cyclization of the peptide was carried out for 48 h and the solution was freeze-dried.
  • the crude cyclic peptide was dissolved in water and purified by RP-HPLC on a C 18 column with a linear gradient of acetonitrile into water (both phases contained 0J%TFA). Fractions containing the pure product were collected and freeze-dried. The peptides were characterized by ES-MS and the purity was determined by RP-HPLC (linear gradient of acetonitrile into water/0.1 % TFA).
  • the Fmoc-protected peptide loaded resin was then treated with 20% piperidine in DMF (2 x 10 mL, 10 min.) and washed with DMF (3 x 10 mL).
  • a solution of N,N- dimethylglycine (0.11 mmol), HATU (1 mmol), and DIEA (0.11 mmol) in DMF (10 mL) was then added to the peptide loaded resin and the manual coupling was continued for 5 h. After the reaction the resin was washed with DMF (3 x 10 mL) and CH C1 2 (3 x 10 mL) and dried under vacuum.
  • Method 11 Formation of Mercaptoacetylated Peptides Using S-Acetylthioglycolic acid N-Hydoxysuccinimide Ester S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA) (0.0055mmol) was added to a solution of a peptide (0.005 mmol, obtained from Methods 1-5 with a free amine) in DMF (0.25 mL) and the reaction mixture was stined at ambient temperature for 6 h. The volatiles were removed under vacuum and the residue was purified by preparative HPLC using acetonitrile-water containing 0J%TFA.
  • Method 12 Formation of Mercaptoacetylated Peptides using S-Acetylthioglycolic acid
  • the mixture was then purified by preparative HPLC; the fractions containing pure peptide were combined and lyophilized.
  • the deprotection reaction employed 2% hydrazine in DMSO for 3h at room temperature. Purification of the reaction mixture afforded pure peptide.
  • Fmoc aminodioxaoctanoic acid was coupled twice successively to the peptide (produced by method 5) followed by Fmoc removal and coupling to S- acetylthioglycolic acid.
  • Method 13 Preparation of Homo and Heterodimers The required purified peptides were prepared by SPPS using Method 5. To prepare homodimers, half of the peptide needed to prepare the dimer was dissolved in DMF and treated with 10 equivalents of glutaric acid bis N-hydoxysuccinimidyl ester. The progress of the reaction was monitored by HPLC analysis and mass spectroscopy. At completion of the reaction, The volatiles were removed in vacuo and the residue was washed with ethyl acetate to remove unreacted bis-NHS ester. The residue was dried, re-dissolved in anhydrous DMF and treated with another half portion of the peptide in the presence of 2 equivalents of DIEA.
  • Example 4 Preparation of KDR and VEGF/KDR Complex Binding Polypeptides Utilizing the methods set forth above, biotinylated versions the KDR and VEGF/KDR complex binding polypeptides set forth in Table 10 were prepared.
  • the letter "j" in the peptide sequences refers to a spacer or linker group, 8-amino-3,6- dioxaoctanoyl.
  • biotinylated polypeptides (with the JJ spacer) to bind to KDR was assessed using the assay set forth in Example 5, following the procedures disclosed therein.
  • Several biotinylated peptides bound well to the KDR-expressing cells: SEQ ID NO:356 (K D 1.81 nM +/- 0.27), SEQ ID NO:264 (K D 14.87+/- 5.0 nM, four experiment average), SEQ ID NO:294"+ spacer ( b ⁇ .
  • the putative disulfide constrained cyclic peptide is indicated by underlining.
  • tetrameric complexes of KDR-binding peptides SEQ ID NO:294, SEQ ID NO:264, SEQ ID NO:277 and SEQ ID NO:356 and a control peptide, which does not bind to KDR were prepared and tested for their ability to bind 293H cells that were transie ⁇ tly-trari ' sfected with KDR:"" ⁇ 11 fdu ' tetrameric complexes of KDR-binding peptides were niotinylated and contained the JJ spacer, and bound to the KDR-expressing cells; however, SEQ ID NO:356 exhibited the best K D (1.81nM).
  • the tetrameric complexes of KDR-binding peptides SEQ ID NO:294, SEQ ID NO:264 exhibited improved binding over monomers of the same peptides. Moreover, inclusion of a spacer between the KDR- binding peptide and the biotin was shown to improve binding in Experiment B. hi Experiment C, it was shown that this assay can be used to assess the effect of serum on binding of peptides of the invention to KDR and VEGF/KDR complex.
  • the binding of SEQ ID NO:264, SEQ ID NO:294, and SEQ ID NO:356 was not significantly affected by the presence of serum, while the binding of SEQ ID NO:277 was reduced more than 50% in the presence of serum.
  • HUVEC cells were grown to almost 80%> confluence in 175 cm 2 tissue culture flasks (Becton Dickinson, Biocoat, cat # 6478) and then 10 ng/ml of bFGF (Oncogene, cat # PF003) was added for 24 h to induce expression of KDR.
  • mRNA was isolated using the micro-fast track 2.0 kit from Invitrogen (cat. # Kl 520-02). 12 ⁇ g of mRNA (measured by absorbance at 260 nM) was obtained from two flasks (about 30 million cells) following the kit instructions.
  • Reverse transcription to generate cDNA was performed with 2 ⁇ g of mRNA, oligo dT primer (5'-(T) 5 GC- 3 ') and/or smart H oligo (5 ⁇ AGCAGTGGTAACAACGCAGAGTACGCGGG-3 ') (SEQ ID NO:357) using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase.
  • MMLV Moloney Murine Leukemia Virus
  • the reaction was performed in a total volume of 20 ⁇ l and the reaction mix contained 2 ⁇ l of RNA, 1 ⁇ l smart H oligo, 1 ⁇ l of oligo dT primer, 4 ⁇ l of 5X first-strand buffer (250 mM Tris HCl pH 8.3, 375 mM KCl, 30 mM MgCl 2 ) 1 ⁇ l DTT (20 mM, also supplied with reverse fr ⁇ ns , cr ⁇ tase'j; F ⁇ TdNTP ' miX (lO'mM each of dATP, dCTP, dGTP, and dTTP in ddH 2 O, Stratagene, cat.
  • the PCR reaction was done in total volume of 50 ⁇ l and the reaction mix contained 2 ⁇ l 5' RACE ready cDNA library, 1 ⁇ l 5' oligo (10 ⁇ M), 1 ⁇ l 3' oligo (10 ⁇ M), 5 ⁇ l 10X PCR buffer [PCR buffer (200 mM Tris-HCl pH 8.8, 20 mM MgSO 4 , 100 mM KCl, 100 mM (NH 4 ) 2 SO 4 ) supplied with pfu enzyme plus 1% DMSO and 8% glycerol], 1 ⁇ l dNTP mix (10 mM) and 40 ⁇ l ddH 2 0.
  • PCR buffer 200 mM Tris-HCl pH 8.8, 20 mM MgSO 4 , 100 mM KCl, 100 mM (NH 4 ) 2 SO 4
  • the PCR reaction was performed by using a program set for 40 cycles of 1 minute at 94C, 1 minute at 68C and 4 minutes at 72C.
  • the PCR product was purified by extraction with 1 volume of phenol, followed by extraction with 1 volume of chloroform and precipitated using 3 volume of ethanol and 1/10 volume of 3M sodium acetate.
  • the PCR product was resuspended in 17 ⁇ l of ddH 2 O, the 2 ⁇ l of 10X Taq polymerase buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl 2 , 0.01%) gelatin) and 1 ⁇ l of Taq polymerase (Stratagene, cat.
  • the TOPO vector allows easy cloning of PCR products because of the A- overhang in Taq (PCR enzyme)-treated PCR products.
  • the extra-cellular domain and the cytoplasmic domain were amplified by PCR separately from TOPO-sKDR and TOPO-CYTO respectively and ligated later to create the full-length receptor.
  • An oligo with a Notl site at the 5' end of the extracellular domain (A TAA GAA TGC GGC CGC AGG ATG GAG AGC AAG GTG CTG CTG G) (SEQ ID NO:362) and an oligo complimentary to the 3' end of the extracellular domain (TTC CAA GTT CGT CTT TTC CTG GGC ACC) (SEQ ID NO: 363) were used to amplify by PCR the extracellular domain from TOPO- sKDR.
  • the 5' oligo (ATC ATT ATT CTA GTA GGC ACG GCG GTG) (SEQ ID NO:364) and the 3' oligo, with a Notl site (A TAA GAA TGC GGC CGC AAC AGG AGG AGA GCT CAG TGT GGT C) (SEQ ID ⁇ O:365), were used to amplify by PCR the cytoplasmic domain of KDR (with transmembrane domain) from TOPO-CYTO. Both PCR products were digested with Notl and ligated together to create the full-length receptor.
  • the cDNA encoding the full-length receptor was purified on an agarose gel and ligated into the Not I site of the pcDNA6/V5-HisC vector. Purification of DNA and ligation was done as described earlier for psKDR. The ligation reaction was used to transform a culture of DH5 ⁇ bacteria and a number of individual clones were analyzed for the presence and orientation of insert by restriction analysis of purified plasmid from each clone with EcoRI enzyme.
  • E. coli bacteria DH5 ⁇ containing pf-KDR was streaked onto LB with 50 ⁇ g/ml ampicillin (LB agar from US biologicals, cat. # 75851 and ampicillin from Sigma, cat. #A2804) plates from a glycerol stock and plates were left in a 37°C incubator to grow overnight. Next morning, a single colony was picked from the plate and grown in 3 ml of LB/ampicillin media (LB from US biologicals, cat. # US75852) at 37°C. After 8 hours, 100 ⁇ l of bacterial culture from the 3 ml tube was transfened to 250 ml of LB/ampicillin media for overnight incubation at 37°C.
  • Bacteria were grown up with circular agitation in a 500 ml bottle (Beckman, cat. # 355605) at 220 rpm in a Lab-Line incubator shalcer. The next day, the bacterial culture was processed using maxi-prep kit (QIAG ⁇ N, cat. # 12163). Generally, about lmg of plasmid DNA (as quantitated by absorbance at 260 nm) was obtained from 250 ml of bacterial culture.
  • Transfection was done as recommended in the lipofectamine 2000 protocol (Invitrogen, cat# 11668-019) using a poly-D-lysine-coated 96 well plate. 320 ng of KDR DNA (pc-DNA6-fKDR)/per well in 0J ml was used for 96 well transfection. Transfection was done in serum-containing media, the transfection reagent mix was removed from cells after 6-8 hours and replaced with regular serum-containing medium. Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. # 354640). The left half of the plate (48 wells) were mock-transfected (with no DNA) and the right half of the plate was transfected with KDR cDNA. The cells were 80-90% confluent at the time of transfection and completely confluent next day, at the time of the assay, otherwise the assay was aborted.
  • M199 media In order to prepare M199 media for the assay, one M199 medium packet (GH3CO, cat. # 31100-035), 20 ml of 1 mM H ⁇ P ⁇ S (GIBCO, cat. #15630-080) and 2 gm of DIFCO Gelatin (DIFCO, cat. # 0143-15-1 were added to 950 ml of ddH 2 0" and the pH of the solution was adjusted to 7.4 by adding approximately 4 ml of IN NaOH. After pH adjustment, the M199 media was warmed to 37°C in a water bath for 2 hours to dissolve the gelatin, then filter sterilized using 0.2 ⁇ m filters (Corning, cat. # 43109), and stored at 4°C to be used later in the assay.
  • GH3CO mM H ⁇ P ⁇ S
  • DIFCO DIFCO, cat. # 0143-15-1
  • SoftLink soft release avidin-sepharose was prepared by centrifuging the sepharose obtained from Promega (cat. # V2011) at 12,000 rpm for 2 minutes, washing twice with ice cold water (centrifuging in-between the washes) and resuspending the pellet in ice cold water to make a 50%> slurry in ddH O. A fresh 50%) slurry of avidin-sepharose was prepared for each experiment.
  • Biotinylated peptides SEQ ID NOS :294, 264, 277, 356, and the non-binding biotinylated control peptide were used to prepare 250 ⁇ M stock solutions in 50%> DMSO and a 33 ⁇ M stock solution of neutravidin-HRP was prepared by dissolving 2 mg of neutravidin-HRP (Pierce, cat. # 31001) in 1 mL of ddH O (all polypeptides contained the JJ spacer). Peptide stock solutions were stored at -20°C, whereas the Neutravidin HRP stock solution was stored at -80°C.
  • peptide/neutravidin-HRP complexes 10 ⁇ l of 250 ⁇ M biotinylated peptide stock solution and 10 ⁇ l of 33 ⁇ M neutravidin-HRP were added to 1 ml of M199 medium. This mixture was incubated on a rotator at 4°C for 60 minutes, followed by addition of 50 ⁇ l of soft release avidin-sepharose (50% slurry in ddH 2 0) to remove excess peptides and another incubation for 30 minutes on a rotator at 4°C.
  • Blocking solution was prepared by adding 20 ml of M199 medium to 10 mg of lyophilized unlabeled neutravidin (Pierce, cat. # 31000). Fresh blocking solution was used for each experiment.
  • each well of the 293H cells was washed once with 100 ⁇ l of M199 medium and incubated with 80 ⁇ l of blocking solution at 37°C. After one hour, cells were washed twice with 100 ⁇ l of M199 media and incubated with 70 ⁇ l of peptide/neutravidin-HRP dilutions of control peptide, SEQ ID NO:264, SEQ ID NO:294, SEQ ID NO:277, and SEQ ID NO:356 for two and half hours at room temperature. Each dilution was added to three separate wells of mock as well as KDR-transfected 293H cells (two plates were used for each saturation binding experiment).
  • binding constants are, as expected, lower than those measured by FP against the KDRFc construct for the related monodentate peptides SEQ ID NO:294 (69 nM), SEQ ID NO:264 (280 nM), SEQ ID NO:310 (51 nM), but similar to monodentate peptide SEQ ID NO:277 (3 nM).
  • SEQ ID NO:294 69 nM
  • SEQ ID NO:264 280 nM
  • SEQ ID NO:310 51 nM
  • monodentate peptide SEQ ID NO:277 3 nM.
  • the binding of peptide/neutravidin HRP complexes (FIG. 2) at a single concentration (5.55 nM) was plotted to demonstrate that a single concentration experiment can be used to differentiate between a KDR binding peptide (SEQ ID NOS:264, 295 and 277) from a non-binding peptide.
  • Experiment B was designed to look at the requirement of spacer (JJ, Table 10) between the KDR binding sequences (SEQ ID NOS :294 and 264) and biotin.
  • biotinylated peptides with and without spacer JJ were tested (e.g., biotinylated SEQ ID NO:264 with the JJ spacer, biotinylated SEQ ID NO:264 without the JJ spacer, SEQ ID NO:294 with a spacer, and biotinylated SEQ ID NO:
  • Experiment C was designed to look at the serum effect on the binding of SEQ ID NOS: 294, 264, 277 and 356.
  • biotinylated peptide/avidin HRP complexes of SEQ ID NOS:294, 264, 277 and 356 were tested in M199 media (as described above in Experiment A) with and without 40% rat serum.
  • This experiment was performed as described for Experiment A except that it was only done at single concentration of 6.66 nM for SEQ ID NOS: 294 and 264, 3.33 nM for SEQ ID NO:277 and 2.22 nM for SEQ ID NO:356.
  • Each of the polypeptides were biotinylated and had the JJ spacer.
  • Results in FIG. 5 indicate that binding of SEQ ID NO:264, SEQ ID NO:294, and SEQ ID NO:356 was not significantly affected by 40% rat serum, whereas binding of SEQ ID NO:277 was more than 50%> lower in presence of 40%> rat serum. More than an 80% drop in the binding of Tc-labeled SEQ ID NO:277 with Tc-chelate was observed in the presence of 40% rat serum (FIG. 27). Since the serum effect on the binding of Tc-labeled SEQ ID NO:277 is mimicked in the avidin HRP assay disclosed herein, this assay may be used to rapidly evaluate the serum effect on the binding of peptide(s) to KDR.
  • Experiment D was designed to evaluate the binding of tetrameric complexes of KDR and VEGF/KDR complex-binding polypeptides SEQ TD NO:294 and SEQ ID NO:264, particularly where the constructs included at least two KDR binding polypeptides.
  • the KDR binding peptides and control binding peptide were prepared as described above. This experiment was performed using the protocol set forth for Experiment A, except the procedures set forth below were unique to this expenment.
  • peptide/neutravidin HRP complexes To prepare peptide/neutravidin HRP complexes, a total 5.36 ⁇ L of 250 ⁇ M biotinylated peptide stock solution (or a mixture of peptide solutions, to give peptide molecules four times the number of avidin HRP molecules) and 10 ⁇ L of 33 ⁇ M Neutravidin HRP were added to 1 mL of Ml 99 medium. This mixture was incubated on a rotator at 4C for 60 minutes, followed by addition of 50 ⁇ L of soft release avidin-sepharose (50%> slurry in ddH 2 0) to remove excess peptides and another incubation for 30 minutes on a rotator at 4C.
  • soft release avidin-sepharose 50%> slurry in ddH 2 0
  • a peptide mixture composed of 50%> control peptide with 25% SEQ ID NO:294 and 25% SEQ ID NO:264 bound quite well to KDR-transfected cells relative to mock-transfected cells, indicating that there is a great advantage to targeting two sites or epitopes on the same target molecule. Furthermore, it was noted that tetrameric complexes containing different ratios of SEQ ID NO:294 and SEQ TD NO:264 (3:1, 2:2, and 1:3) all bound much better to KDR-transfected cells than pure tetramers of either peptide, in agreement with the idea that targeting two distinct sites on a single target molecule is superior to multimeric binding to a single site.
  • multimeric binding to a single target requires that the multimeric binding entity span two or more separate target molecules which are close enough together for it to bind them simultaneously, whereas a multimeric binder which can bind two or more distinct sites on a single target molecule does not depend on finding another target molecule within its reach to achieve multimeric binding.
  • Experiment E was designed to confirm that SEQ ID NO:294 and SEQ ID NO:264 bind to distinct sites (epitopes) on KDR. If these peptides bind to the same site on KDR, then they should be able to compete with each other; however, if they bind to different sites they should not compete. This experiment was performed using a single concentration of SEQ ID NO:264/avidin HRP (3.33 nM) solution in each well and adding a varying concentration (0-2.5 ⁇ M) of biotinylated control peptide with spacer, SEQ ID NO:264 and SEQ ID NO:294, none of which were complexed with avidin.
  • Example 6 Binding of Analogs of a KDR-binding Peptide to KDR-expressing Cells N-terminal and C-terminal truncations of a KDR binding polypeptide were made and the truncated polypeptides tested for binding to KDR-expressing cells. The synthesized polypeptides are shown in FIG. 8. Binding of the polypeptides to KDR-expressing cells was determined following the procedures of Example 3. All of the peptides were N-terminally acetylated and fluoresceinated for determining apparent K D according to the method described above (Example 3). The results indicate that, for the SEQ ID NO:294 (FIG. 8) polypeptide, the C- terminal residues outside the disulfide-constrained loop contribute to KDR binding.
  • SEQ ID NO:294 FIG. 8
  • Example 7 Bead-binding Assay to Confirm Ability of Peptides Identified by Phage Display to Bind KDR-expressing Cells
  • KDR-binding peptides containing SEQ ID NOS:264, 337, 363, and 373 were conjugated to fluorescent beads and their ability to bind to KDR-expressing 293H cells was assessed.
  • the experiments show these peptides can be used to bind particles such as beads to KDR-expressing sites. The results indicate that the binding of both KDR binding sequences improved with the addition of a spacer.
  • Biotinylation of an anti-KDR antibody Anti-KDR from Sigma (V-9134), as ascites fluid, was biotinylated using a kit from Molecular Probes (F-6347) according to the manufacturer's instructions.
  • peptide-conjugated fluorescent beads 0J mL of a 0.2 mM stock solution of each biotinylated peptide (prepared as set forth above, in 50%> DMSO) was incubated with 0J ml of Neutravidin-coated red fluorescent microspheres (2 micron diameter, custom-ordered from Molecular Probes) and 0.2 ml of 50mM MES (Sigma M-8250) buffer, pH 6.0 for 1 hour at room temperature on a rotator. As a positive control, biotinylated anti-KDR antibody was incubated with the
  • Neutravidin-coated beads as above, except that 0.03 mg of the biotinylated antibody preparation in PBS (Gibco #14190-136) was used instead of peptide solution. Beads can be stored at 4°C until needed for up to 1 week.
  • Binding Assay From the above bead preparations, 0J2 mL was spun for 10 minutes at 2000 rpm in a microcentrifuge at room temperature. The supernatant was removed and 0.06 ml of MES pH 6.0 was added. Each bead solution was then vortexed and sonicated in a water bath 15 min. To 1.47 ml of DMEM, high glucose (GIBCO #11965-084) with IX MEM Non-Essential Amino Acids Solution (NEAA) (GD3CO 11140-050) and 40% FBS (Hyclone SH30070.02) 0.03 ml of the somcated bead preparations was added.
  • Biotinylated SEQ ID NO:294 beads showed greater binding to KDR-transfected cells, and adding a hydrophilic spacer between the peptide portion and the biotin of the molecule (biotinylated SEQ TD NO:294 with the JJ spacer) significantly improved the specific binding to KDR in the transfected cells.
  • the peptide sequences of both SEQ ID NO:264 and SEQ ID NO:294 can be used to bind particles such as beads to KDR expressing sites. Addition of a hydrophilic spacer between the peptide and the group used for attachment to the particle should routinely be tested with new targeting molecules as it improved the binding for both of the peptides evaluated here.
  • Example 8 Competition of KDR binding peptides and 5 I-tabete ⁇ EGF or binding to KDR-transfected 293H cells
  • KDR-binding polypeptides were next assessed for their ability to compete with 125 I-labeled VEGF for binding to KDR expressed by transfected 293H cells. The results indicate that KDR-binding polypeptide SEQ ID NO:263 (Ac-
  • Transfection of293H cells 293H cells were transfected using the protocol described in Example 5. Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. # 354640). The left half of the plates (48 wells) were mock- transfected (with no DNA) and the right half of the plates were transfected with KDR cDNA. The cells were 80-90%) confluent at the time of transfection and completely confluent the next day, at the time of the assay; otherwise the assay was aborted.
  • M199 medium was prepared as described in Example 5.
  • 125 I-labeled VEGF solution for the assay: 25 ⁇ Ci of lyophilized 125 I- labeled VEGF (Amersham, cat. # 274) was reconstituted with 250 ⁇ l of ddH 2 O to create a stock solution, which was stored at -80C for later use. For each assay, a 300 pM solution of 125 I-labeled VEGF was made fresh by diluting the above stock solution in M199 medium. The concentration of 125 I-labeled VEGF was calculated daily based on the specific activity of the material on that day.
  • Preparation of 30 ⁇ M and 0.3 ⁇ M peptide solution in 300 pM 125 I-labeled VEGF For each 96 well plate, 10 ml of 300 pM 125 I-labeled VEGF in M199 medium was prepared at 4°C. Each peptide solution (3 mM, prepared as described above) was diluted 1:100 and 1:10000 in 300 ⁇ l ofM199 media with 300 pM 125 I-labeled VEGF to prepare 30 ⁇ M and 0.3 ⁇ M peptide solutions containing 300 pM of 125 I-labeled VEGF. Once prepared, the solutions were kept on ice until ready to use. The dilution of peptides in Ml 99 media containing 300 pM 125 I-labeled VEGF was done freshly for each experiment.
  • Assay to detect competition with 125 I-labeled VEGF in 293H cells Cells were used 24 hours after transfection, and to prepare the cells for the assay, they were washed 3 times with room temperature M199 medium and placed in the refrigerator. After 15 minutes, the Ml 99 medium was removed from the plate and replaced with 75 ⁇ l of 300 pM 125 I-labeled VEGF in Ml 99 medium (prepared as above) with the polypeptides. Each dilution was added to tliree separate wells of mock and KDR transfected cells. After incubating at 4°C for 2 hours, the plates were washed 5 times with cold binding buffer, gently blotted dry and checked under a microscope for cell loss.
  • solubilizing solution 2% Triton X-100, 10% Glycerol, 0.1% BSA
  • solubilizing solution 2% Triton X-100, 10% Glycerol, 0.1% BSA
  • the solubilizing solution in each well was mixed by pipeting up and down, and transfened to 1.2 ml tubes.
  • Each well was washed twice with 100 ⁇ l of solubilizing solution and the washes were added to the conesponding 1.2 ml tube.
  • Each 1.2 ml tube was then transfened to a 15.7 x 100 cm tube to be counted in an LKB Gamma Counter using program 54 ( 125 I window for 1 minute).
  • I- labeled VEGF to sites other than KDR (which may or may not be present m 293H cells) is not included when calculating the inhibition of 125 I-labeled VEGF binding to 293H cells by KDR-binding peptides.
  • Percentage inhibition was calculated using formula [(Y1-Y2)*100/Y1], wli ' ere ' ⁇ i is specific binding to KDR- transfected 293H cells in the absence peptides, and Y2 is specific binding to KDR- transfected 293H cells in the presence of peptides or DMSO. Specific binding to KDR-transfected 293H cells was calculated by subtracting binding to mock- transfected 293H cells from binding to KDR-transfected 293H cells.
  • SEQ ID NO:263 which due to its relatively high K d (>2 ⁇ M) was used as a negative control, did not compete significantly with 125 I-labeled VEGF, 12.69 ⁇ 7J8% at 30 ⁇ M and -5.45 ⁇ 9.37% at 0.3 ⁇ M (FIG. 10).
  • SEQ ID NOS:294 and 277 competed very well with 125 I-labeled VEGF, inhibiting 96.29 ⁇ 2.97% and 104.48 ⁇ 2.074% of 125 I-labeled VEGF binding at 30 ⁇ M and 52.27 ⁇ 3.78% and 80.96 ⁇ 3.8% at 0.3 ⁇ M (FIG. 10) respectively.
  • the percentage inhibition with SEQ TD NO:264 was 47.95 ⁇ 5.09% of 125 I-labeled VEGF binding at 30 ⁇ M and 24.41 ⁇ 8.43% at 0.3 ⁇ M (FIG. 10).
  • the three strongly KDR-binding polypeptides did compete with VEGF, and their potency increased with their binding affinity.
  • This assay will be useful for identifying peptides that bind tightly to KDR but do not compete with VEGF, a feature that may be useful for imaging KDR in tumors, where there is frequently a high local concentration of VEGF that would otherwise block the binding of KDR- targeting molecules.
  • Example 9 Inhibition of VEGF-induced KDR receptor activation by peptides identified by phage display
  • VEGF induced activation (phosphorylation) of KDR was assessed using the following assay.
  • a number of peptides of the invention were shown to inhibit activation of KDR in monomeric and/or tetrameric constructs. As discussed supra, peptides that inhibit activation of KDR may be useful as anti-angiogenic agents.
  • EGM-MV medium Biowhittalcer Catalog #CC-3125
  • Cells seeded into 100 mm dishes were allowed to become confluent, then cultured overnight in basal EBM medium lacking serum (Biowhittalcer Catalog #CC-3121).
  • the medium in the dishes was replaced with 10 ml fresh EBM medium at 37C containing either no additives (negative control), 5 ng/ml VEGF (Calbiochem Catalog #676472 or Peprotech Catalog #100-20) (positive control), or 5 ng/ml VEGF plus the indicated concentration of the KDR-binding peptide (prepared as described above), hi some cases, a neutralizing anti-KDR antibody (Catalog #AF357, R&D Systems) was used as a positive confrol inhibitor of activation, hi such cases, the antibody was pre-incubated with the test cells for 30 min at 37°C prior to the addition of fresh medium containing both VEGF and the antibody. After incubating the dishes 5 min.
  • D- PBS Delbecco's phosphate buffered saline
  • the first dish of a set was drained and 0.5 ml of Triton lysis buffer was added (20 mM Tris base pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA (ethylenediaminetetraacetic acid), 1 mM PMSF(phenyhnethylsulfonylfluoride), 1 mM sodium orthovanadate, 100 mM NaF, 50 mM sodium pyrophosphate, 10 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin).
  • the cells were quickly scraped into the lysis buffer using a cell scraper (Falcon, Cat No.
  • the lysates freshly prepared or frozen and thawed, were precleared by adding 20 ⁇ l of protein A-sepharose beads (Sigma 3391, preswollen in D-PBS, washed three times with a large excess of D-PBS, and reconstituted with 6 ml D- PBS to generate a 50% 0 slurry) and rocking at 4° C for 30 min.
  • the beads were pelleted by centrifugation for 2 min. in a Picofuge (Stratgene, Catalog #400550) at 2000 xg and the supernatants transfened to new 1.5 ml tubes.
  • Detection of phosphorylated KDR as well as total KDR in the immunoprecipitates was carried out by immunoblot analysis. Half (20 ⁇ L) of each immunoprecipitate was resolved on a 7.5% precast Ready Gel (Bio-Rad, Catalog #161-1154) by SDS-PAGE according to the method of Laemmli (Nature, 227:680- 685 (1970)).
  • Blots were blocked at room temperature in 5% Blotto- TBS (Pierce Catalog #37530) pre-warmed to 37° C for 2 hr. The blots were first probed with an anti-phosphotyrosine antibody (Transduction Labs, Catalog #P11120), diluted 1 :200 in 5% Blotto-TBS with 0.1% Tween 20 added for 2 hr. at room temp. The unbound antibody was removed by washing the blots four times with D-PBS containing 0.1% Tween 20 (D-PBST), 5 min. per wash.
  • D-PBST D-PBS containing 0.1% Tween 20
  • blots were probed with an HRP-conjugated sheep anti-mouse antibody (Amersham Biosciences Catalog #NA931) diluted 1:25,000 in 5% Blotto-TBS with 0.1% Tween 20 added for 1 hr. at room temp., and washed four times with D-PBST. Finally, the blots were incubated with 2 ml of a chemiluminescent substrate (ECL Plus, Amersham Catalog #RPN2132) spread on top for 2 min., drip-drained well, placed in plastic sheet protector (C-Line Products, Catalog #62038), and exposed to X-ray film (Kodak BioMax ML, Cat No. 1139435) for varying lengths of time to achieve optimal contrast.
  • chemiluminescent substrate ECL Plus, Amersham Catalog #RPN2132
  • the blots were stripped by incubating for 30 min. at 37° C in TBST with its pH adjusted to 2.4 with HCl, blocked for 1 hr. at room temp, with 5% Blotto-TBS with 0.1% Tween 20 (Blotto-TBST), and reprobed with an anti-Flic- 1 polyclonal antibody (Catalog #sc-315 from Santa Cruz Biotech), 1:200 in 5% Blotto-TBST with 1% nonnal goat serum (Life Tech Catalog #16210064) for 2 hr. at room temp. The unbound antibody was removed by washing the blots four times with D-PBST, 5 min. per wash.
  • the blots were probed with an HRP-conjugated donkey anti-rabbit secondary antibody (Amersham Biosciences Catalog #NA934) diluted 1:10,000 in 5% Blotto-TBST for 1 hr. at room temp., and washed four times with D-PBST. Finally, the blots were incubated with 2 ml of chemiluminescent substrate and exposed to X-ray film as described above.
  • this assay to detect agents capable of blocking the VEGF activation of KDR was assessed by adding a series of compounds to HUVECs in combination with VEGF and measuring KDR phosphorylation with the immunoblot assay described above. As negative and positive controls, immunoprecipitates from unstimulated HUVECs and from HUVECs stimulated with VEGF in the absence of any test compounds were also tested in every assay. When a neutralizing anti-KDR antibody (Catalog #AF-357 from R&D Systems) was combined with the VEGF, the extent of KDR phosphorylation was greatly reduced (FIG. 12, upper panel), indicating that the antibody was able to interfere with the ability of VEGF to bind to and activate KDR.
  • a neutralizing anti-KDR antibody Catalog #AF-357 from R&D Systems
  • AFPRFGGDDYWIQQYLRYTD a linear KDR-binding peptide identified by phage display
  • the assay was repeated with a synthetic peptide containing the KDR-binding sequence, Ac-AQAFPRFGGDDYWIQQYLRYTDGGK-NH 2 (SEQ ID NO:306) in the presence of VEGF.
  • SEQ ID NO:306 was able to inhibit the VEGF-induced phosphorylation of KDR.
  • SEQ ID NOS: 269 and 294 were the most potent compounds in the assay, producing at least a 50% inhibition of VEGF-induced KDR phosphorylation at 1 ⁇ M.
  • NOS:294 and 277 (prepared as described above) produced at least a 50% inhibition of VEGF-induced KDR phosphorylation at 10 nM.
  • Example 10 Binding of Tc-labeled SEQ ID NO:339 to KDR-transfected 293H cells The ability of Tc-labeled SEQ ID NO:339 to bind KDR was assessed using
  • Tc-labeled SEQ ID NO:277 i.e., Ac- AGPTWCEDDWYYCWLFGT-GGGK(N,N-dimethyl-Gly-Ser-Cys-Gly- di(aminodioxaocta-))-NH ) bound significantly better to KDR transfected 293H cells than to mock transfected 293H cells and binding increased with concentration of Tc-labeled SEQ ID NO:339 in a linear manner.
  • a stannous gluconate solution was prepared by adding 2 ml of a 20 ⁇ g/ml SnCl 2 - 2H 2 O solution in nitrogen-purged IN HCl to 1.0 ml of nitrogen-purged water containing 13 mg of sodium glucoheptonate. To a 4 ml autosampler vial was added 20-40 ⁇ l (20 - 40 ⁇ g) of SEQ ID NO:339 ligand dissolved in 50/50 ethanol/H 2 O, " 6-
  • reaction mixture was purified by HPLC on a Vydac C18 column (4.6 mm x 250 mm) at a flow rate of 1 ml/min., using 0.1% TFA in water as aqueous phase and 0.085%) TFA in acetonitrile as the organic phase. The following gradient was used: 29.5%> org. for 35 min., ramp to 85%> over 5 min., hold for 10 min.
  • the fraction containing 99m Tc SEQ ID NO:339 was collected into 500 ⁇ l of a stabilizing buffer containing 5 mg/ml ascorbic acid and 16 mg/ml hydroxypropyl- ⁇ - cyclodextrin in 50 mM phosphate buffer.
  • the mixture was concentrated using a speed vacuum apparatus to remove acetonitrile, and 200 ⁇ l of 0.1% HSA in 50 mM pH 5 citrate buffer was added.
  • the resulting product had an RCP of 100%.
  • the compound Prior to injection into animals, the compound was diluted to the desired radioconcentration with nonnal saline.
  • 293H cells were transfected using the protocol described above. Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. # 354640). The left half of the plates (48 wells) were mock-transfected (with no DNA) and the right half of the plate was transfected with KDR cDNA. The cells were 80-90%> confluent at the time of transfection and completely confluent the next day, at the time of the assay; otherwise the assay was aborted.
  • Opti-MEMI was obtained from Invitrogen (cat. # 11058-021) and human serum albumin (HSA) was obtained from Sigma (cat. # A-3782).
  • HSA human serum albumin
  • Tc-labeled SEQ ID NO:339 (117 ⁇ Ci/ml) was diluted 1:100, 1 :50, 1:25 and 1 : 10 in opti-MEMI with 0.1% HSA to provide solutions with final concentration of 1.17, 2.34, 4.68 and 11.7 ⁇ ' Ci/ml ' of Tc-labeled SEQ ID NO:339.
  • Tc-labeled SEQ ID NO:339 The ability of Tc-labeled SEQ ID NO:339 to specifically bind to KDR was assessed using transiently transfected 293H cells.
  • Tc-labeled SEQ ID NO:339 bound significantly better to KDR transfected 293H cells as compared to mock transfected 293H cells.
  • the binding of Tc-labeled SEQ ID NO:339 polypeptide to mock-transfected cells was subtracted from the binding to KDR- transfected cells.
  • a linear increase in the specific binding of Tc-labeled SEQ ID NO:339 to KDR was observed with increasing concentration of Tc-labeled SEQ ID NO:339 (FIG. 26).
  • the crude ether-precipitated linear di-cysteine containing peptides were cyclized by dissolution in water, mixtures of aqueous acetonitrile (0.1% TFA), aqueous DMSO or 100%) DMSO and adjustment of the pH of the solution to 7.5 - 8.5 by addition of aqueous ammonia, aqueous ammonium carbonate, aqueous ammonium bicarbonate solution or DIEA.
  • the mixture was stined in air for 16-48 h, acidified to pH 2 with aqueous trifluoroacetic acid and then purified by preparative reverse phase HPLC employing a gradient of acetonitrile into water. Fractions containing the desired material were pooled and the purified peptides were isolated by lyophilization.
  • the resin was washed with DMF (2 x 10 mL) and with DCM (1 10 mL). The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc- 8-amino-3,6-dioxaoctanoic acid and Fmoc protecting group removal were repeated once more. The resulting resin-bound peptide with a free amino group was washed and dried and then treated with reagent B (20 mL) for 4 h. The mixture was filtered and the filtrate concentrated to dryness. The residue was stined with ether to produce a solid, which was washed with ether and dried.
  • the solid was dissolved in anhydrous DMSO and the pH adjusted to 7.5 with DIEA.
  • the mixture was stined for 16h to effect the disulfide cyclization and the reaction was monitored by analytical HPLC.
  • the reaction mixture was diluted with 25%) acetonitrile in water and applied directly to a reverse phase C-18 column. Purification was effected using a gradient of acetonitrile into water (both containing 0.1%) TFA). Fractions were analyzed by HPLC and those containing the pure r product were combined and lyophilized to provide the required peptide.
  • the resin was washed with DMF (2 x 10 mL) and with DCM (lx 10 mL). The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc-8- amino-3,6-dioxaoctanoic acid and removal of the Fmoc protecting group were repeated once more.
  • the resulting resin-bound peptide with a free amino group was treated with a solution of Biotin-NHS ester (0.4 mmol, 5 equiv.) and DIEA (0.4 mmol, 5 equiv.) in DMF for 2 h.
  • the resin was washed and dried as described previously and then treated with Reagent B (20 mL) for 4 h.
  • the mixture was filtered and the filtrate concentrated to dryness.
  • the residue was stined with ether to produce a solid that was collected, washed with ether, and dried.
  • the solid was dissolved in anhydrous DMSO and the pH adjusted to 7.5 with DIEA.
  • the mixture was stined for 4-6 h to effect the disulfide cyclization which was monitored by HPLC.
  • the reaction mixture was diluted with 25% acetonitrile in water and applied directly to a reverse phase C-18 column. Purification was effected using a gradient of acetonitrile into water (both containing 0J%> TFA). Fractions were analyzed by HPLC and those containing the pure product were collected and lyophilized to provide the required biotinylated peptide.
  • the resin was resuspended in DMF (10 mL) and treated with Fmoc-8-amino-3.6-dioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC (0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. After the reaction, the resin was washed with DMF (2 10 mL) and with DCM (1 10 mL). The resin was then treated with 20% piperidine in DMF (2 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc-8- amino-3,6-dioxaoctanoic acid and removal of the Fmoc protecting group were repeated once.
  • the resulting resin-bound peptide with a free amino group was resuspended in DMF (10 mL) and treated with a solution of 1,4,7, 10- tetraazacyclododecane-1, 4,7,10-tetraacetic acid,-l,4,7-tris-t-butyl ester (DOTA-tris- t-butyl ester, 0.4 mmol, 5 equiv.), HOBt (0.4 mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) in DMF (10 mL) with mixing for 4 h.
  • DMF 1,4,7, 10- tetraazacyclododecane-1, 4,7,10-tetraacetic acid,-l,4,7-tris-t-butyl ester
  • HOBt 0.4 mmol
  • DIC 0.4 mmol
  • DIEA 0.8 mmol
  • the resin was washed with DMF (2 x 10 mL) and with DCM (1 x 10 mL) and treated with Reagent B (20 mL) for 4 h. The mixture was filtered and the filtrate concentrated to dryness. The residue was stined in ether to produce a solid that was collected, washed with ether, and dried. The solid was dissolved in anhydrous DMSO and the pH adjusted to 7.5 with DIEA. The mixture was stined for 16 h to effect the disulfide cyclization, which was monitored by HPLC. Upon completion of the cyclization, the mixture was diluted with 25% acetonitrile in water and applied directly to a reverse phase C-18 HPLC column.
  • PnA06 refers to 3-(2-amino-3-(2-hydroxyimino-lJ- dimethyl-propylamino)-propylamino)-3-methyl-butan-2-one oxime.
  • Example 8 The purified peptide monomers mentioned above in Example 8 were used in the preparation of various homodimeric and heterodimeric constructs.
  • dimer D5 For the preparation of the dimer D5, after the coupling reaction of the individual peptides, 50 ⁇ L of hydrazine was added to the reaction mixture (to expose the lysine N ⁇ -amino group) and the solution was stined for 2 min. The reaction mixture was diluted with water (1.0 mL) and the pH was adjusted to 2 with TFA. This was then purified by the method described above.
  • HPLC analysis data and mass spectral data for the dimeric peptides are given in Table 12 below.
  • System B Column: YMC C-4 (4.6 x 250 mm); Eluents: A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 25 % B, linear gradient 25- 60% B in 20 min; flow rate: 2.0 mL/min; detection: UN @ 220 nm.
  • System E Column: Waters XTena, 4.6 x 50 mm; Eluents:A: water (0.1%TFA), B: AC ⁇ (0.1%TFA); Elution: initial condition, 10 % B, linear gradient 10-60 % B in 10 min; flow rate: 3.0 mL/min; detection: UN @ 220 mn.
  • System F Column: Waters XTena, 4.6 x 50 mm; Eluents:A: water
  • System G Column: Waters XTerra, 4.6 x 50 mm; Eluents:A: water (O.P/oTFA), B: AC ⁇ (0.1%TFA); Elution: initial condition, 30 % B, linear gradient 30-75 % B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm.
  • System H Column: Waters XTe ⁇ a, 4.6 x 50 mm; Eluents:A: water (0J%TFA), B: AC ⁇ (O.P/oTFA); Elution: initial condition, 20 % B, linear gradient 20-52 % B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 mn.
  • System J Column: Waters XTena, 4.6 x 50 mm; Eluents: A: water (O.P/oTFA), B: AC ⁇ (O.P/oTFA); Elution: initial condition, 20 % B, linear gradient 20-60 % B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 n.
  • System K Column: Waters XTerra, 4.6 x 50 mm; Eluents: A: water
  • System L Column: Waters XTena, 4.6 x 50 mm; Eluents: A: water (O.P/oTFA), B: AC ⁇ (O.P/oTFA); Elution: initial condition, 5 % B, linear gradient 5-65 % B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm.
  • System M Column: Waters XTerra, 4.6 x 50 mm; Eluents: A: water (O.P/oTFA), B: ACN (O.P/oTFA); Elution: initial condition, 15 % B, linear gradient 15-50 % B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm.
  • System N Column: Waters XTena, 4.6 x 50 mm; Eluents:A: water
  • System R Column: YMC-C18, 4.6 x 250 mm; Eluents:A: water (O.P/oTFA), B: AC ⁇ (O.P/oTFA); Elution: initial condition, 25 % B, linear gradient 25-60 % B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm.
  • System S Column: YMC-C18, 4.6 x 100 mm; Eluents:A: water (O.P/oTFA),
  • the following experiment assessed the ability of E-DR-binding peptides to compete with 125 I-labeled VEGF for binding to KDR expressed by transfected 293H cells.
  • 293H cells were transfected with the KDR cD ⁇ A or mock- transfected by standard techniques. The cells were incubated with 125 I-VEGF in the presence or absence of competing compounds (at 10 ⁇ M, 0.3 ⁇ M, and 0.03 ⁇ M). After washing the cells, the bound radioactivity was quantitated on a gamma counter. The percentage inhibition of VEGF binding was calculated using the formula [(Yl - Y2) x 100/Yl], where Yl is specific binding to KDR-transfected 293H cells in the absence peptides, and Y2 is specific binding to KDR-transfected 293H cells in the presence of peptide competitors. Specific binding to KDR-transfected 293H cells was calculated by subtracting the binding to mock-transfected 293H cells from the binding to KDR-transfected 293H cells.

Abstract

L'invention concerne des polypeptides liants pour le complexe KDR ou VEGF/KDR, qui peuvent être utilisés de façon variée à chaque fois qu'il avantageux de traiter, détecter, isoler ou localiser une angiogenèse. Elle concerne, en particulier, des polypeptides synthétiques, isolés, pouvant se lier au complexe KDR ou VEGF/KDR avec une grande affinité (c'est à dire avec un KD<1 ñM).
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AU2009201114B2 (en) 2012-01-19
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EP2014310B1 (fr) 2012-10-24
CA2666005A1 (fr) 2003-09-12
ES2398393T3 (es) 2013-03-15
EP2014310A2 (fr) 2009-01-14
EP2301587A3 (fr) 2011-04-06
CA2477836A1 (fr) 2003-09-12
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JP2009161549A (ja) 2009-07-23
EP2301587A2 (fr) 2011-03-30
WO2003074005A2 (fr) 2003-09-12
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EP2014310A3 (fr) 2009-09-09
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