Patent Publication Number: US-9408926-B2

Title: KDR and VEGF/KDR binding peptides and their use in diagnosis and therapy

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. application Ser. No. 12/898,119, filed Oct. 5, 2010, which is a continuation-in-part of U.S. application Ser. No. 12/480,578 filed on Jun. 8, 2009 which is a continuation of U.S. application Ser. No. 10/661,156 filed on Sep. 11, 2003, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 10/382,082 filed on Mar. 3, 2003, now abandoned, and a continuation-in-part of International Application No. PCT/US03/06731 filed on Mar. 3, 2003. U.S. application Ser. No. 10/382,082 and International Application No. PCT/US03/06731 both claim the benefit of U.S. Provisional Application No. 60/360,851 filed on Mar. 1, 2002 and U.S. Provisional Application No. 60/440,411 filed on Jan. 15, 2003. The contents of the above applications are incorporated herein by reference. 
     U.S. application Ser. No. 12/898,119 is also a continuation-in-part of U.S. application Ser. No. 11/954,130 filed on Dec. 11, 2007, now U.S. Pat. No. 7,985,402, which is a continuation-in-part of U.S. application Ser. No. 11/608,395, filed Dec. 8, 2006, now U.S. Pat. No. 7,794,693, which claims priority to and benefit of U.S. Provisional Application No. 60/833,342, filed Jul. 25, 2006 and U.S. Provisional Application No. 60/749,240, filed Dec. 9, 2005, and is a continuation-in-part of U.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 10/382,082, filed Mar. 3, 2003, now abandoned and a continuation-in-part of International Application No. PCT/US03/06731, filed Mar. 3, 2003, both of which claim priority to and benefit of U.S. Provisional Application No. 60/440,411, filed Jan. 15, 2003; and U.S. Provisional Application No. 60/360,851, filed Mar. 1, 2002. The contents of the above applications are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to polypeptides and compositions useful for detecting and targeting primary receptors on endothelial cells for vascular endothelial growth factor (VEGF), i.e., vascular endothelial growth factor receptor-2 (VEGFR-2, also known as kinase domain region (KDR) and fetal liver kinase-1 (Flk-1)), and for imaging and targeting complexes formed by VEGF and KDR. The involvement of VEGF and KDR in angiogenesis makes the 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. The present invention also relates to targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates, which target KDR and are useful in therapeutic and diagnostic compositions and methods of preparation of the same. The invention includes targeted ultrasound contrast agents, and particularly targeted microbubbles which include such targeting vector-phospholipid conjugates. 
     BACKGROUND OF THE INVENTION 
     In the developing embryo, the primary vascular network is established by in situ differentiation of meso-dermal cells in a process called vasculogenesis. After embryonic vasculogenesis however, it is believed that all subsequent generation of new blood vessels, in the embryo or in adults, is governed by the sprouting or splitting of new capillaries from the pre-existing vasculature in a process called angiogenesis (Pepper, M. et al., 1996 . Enzyme Protein,  49:138-162; Risau, W., 1997 . Nature,  386:671-674). Angiogenesis is not only involved in embryonic development and normal tissue growth and repair, it is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis that takes place in the normal individual, angiogenic events are involved in a 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 shown promise as a 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. The best characterized of these pro-angiogenic agents or growth factors is vascular endothelial growth factor (VEGF) (Cohen et al.,  FASEB J.,  13: 9-22 (1999)). 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 et al.,  Cell,  94: 715-725 (1998)). VEGF, also referred to as VEGF-A, is synthesized as five different splice isoforms of 121, 145, 165, 189, and 206 amino acids. VEGF121 and VEGF165 are the main forms produced, particularly in tumors (see Cohen et al. 1999, supra). VEGF121 lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does not bind to heparin or extracellular matrix, unlike VEGF165. Each of the references cited in this paragraph is incorporated by reference in its entirety. 
     VEGF family members act primarily by binding to receptor tyrosine kinases. In general, receptor tyrosine kinases are glycoproteins having an extracellular domain capable of binding one or more specific growth factors, a transmembrane domain (usually an alpha helix), a juxtamembrane domain (where the receptor may be regulated, e.g., by phosphorylation), a tyrosine kinase domain (the catalytic 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. There are three endothelial cell-specific receptor tyrosine kinases known to bind VEGF: VEGFR-1 (Flt-1), VEGFR-2 (KDR or Flk-1), and VEGFR-3 (Flt4). Flt-1 and KDR (also known as VEGFR-2 or Flk-1, which are used interchangeably herein) 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 et al.,  J. Cell. Physiol.,  149: 50-59 (1991)). Moreover, KDR is thought to dominate the angiogenic response and is therefore of greater therapeutic and diagnostic interest (see Cohen et al. 1999, supra). Expression of KDR is highly upregulated in angiogenic vessels, especially in tumors that induce a strong angiogenic response (Veikkola et al.,  Cancer Res.,  60: 203-212 (2000)). The critical role of KDR in angiogenesis is highlighted by the complete lack of vascular development in homozygous KDR knockout mouse embryos (Folkman et al.,  Cancer Medicine,  5th Edition (B. C. Decker Inc.; Ontario, Canada, 2000) pp. 132-152). 
     KDR (kinase domain region) is made up of 1336 amino acids in its mature form. The glycosylated form of KDR 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 (Cohen et al. 1999, supra). VEGF 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 VEGF. 
     Thus, not only is the VEGF binding activity of KDR in vivo critical to angiogenesis, but the ability to detect KDR upregulation on endothelial cells or to detect VEGF/KDR binding complexes would be extremely beneficial in detecting or monitoring angiogenesis with particular diagnostic applications such as detecting malignant tumor growth. It would also be beneficial in therapeutic applications such as targeting tumorcidal agents or angiogenesis inhibitors to a tumor site or targeting agonists of KDR, VEGF/KDR, or angiogenesis to a desired site. 
     It is well known that gas filled ultrasound contrast agents are exceptionally efficient ultrasound reflectors for echography. Such ultrasound contrast agents include, for example, gas-filled microvesicles such as gas-filled microbubbles and gas filled microballoons. Gas filled microbubbles are particularly preferred ultrasound contrast agents. (In this disclosure the term of “microbubble” specifically designates a gaseous bubble surrounded or stabilized by phospholipids). For instance injecting into the bloodstream of living bodies suspensions of air- or gas-filled microbubbles in a carrier liquid will strongly reinforce ultrasonic echography imaging, thus aiding in the visualization of internal anatomical structures. Imaging of vessels and internal organs can strongly help in medical diagnosis, for instance for the detection of neoplastic, cardiovascular and other diseases. 
     For both diagnostic and therapeutic purposes it would be particularly beneficial to incorporate into gas filled ultrasound contrast agents, targeting vector compositions which exhibit high binding affinity for a desired target (such as, for example, KDR or the VEGF/KDR complex). For example, targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates may be used to prepare targeted, gas filled ultrasound contrast agents. In addition, it would be particularly beneficial to have methods for large scale production of highly purified forms of such targeting vector-phospholipid conjugates. Such compositions and methods would allow for production of compositions for use in diagnostic or therapeutic applications such as, for example, precise targeting of reporter moieties, tumoricidal agents or angiogenesis inhibitors to the target site. 
     SUMMARY OF THE INVENTION 
     The present invention relates to polypeptides and compositions useful for detecting and targeting primary receptors on endothelial cells for vascular endothelial growth factor (VEGF), i.e., vascular endothelial growth factor receptor-2 (VEGFR-2, also known as kinase domain region (KDR) and fetal liver kinase-1 (Flk-1)), and for imaging and targeting complexes formed by VEGF and KDR. The involvement of VEGF and KDR in angiogenesis makes the 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. 
     A group of polypeptides has been discovered that bind to KDR or VEGF/KDR complex (referred to herein as “KDR binding polypeptides” or “KDR binding moieties” and homologues thereof). Such KDR and VEGF/KDR binding polypeptides will concentrate at the sites of angiogenesis, thus providing a means for detecting and imaging sites of active angiogenesis, which can include sites of neoplastic tumor growth. Such 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. 
     In answer to the need for improved materials and methods for detecting, localizing, measuring and possibly affecting (inhibiting or enhancing), e.g., angiogenesis, it has been surprisingly discovered that seven families of non-naturally occurring polypeptides bind specifically to KDR or VEGF/KDR complex. Appropriate labeling of such polypeptides provides detectable imaging agents that can bind, e.g., at high concentration, to KDR-expressing endothelial cells or cells exhibiting VEGF/KDR complexes, providing angiogenesis-specific imaging agents. The KDR and VEGF/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 angiogenesis, thereby providing 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. 
     Another advantageous use of the KDR and VEGF/KDR complex binding polypeptides disclosed herein is to target therapeutic agents (including compounds capable of providing a therapeutic, radiotherapeutic or cytotoxic effect), or delivery vehicles for therapeutics (including drugs, genetic material, etc.) to sites of angiogenesis or other tissue expressing KDR. 
     Constructs comprising two or more KDR or KDR/VEGF binding polypeptides show improved ability to bind the target molecule compared to the corresponding monomeric binding polypeptides. For example, as shown in Experiment D of Example 5, tetrameric constructs of KDR binding polypeptides provided herein showed improved ability to bind KDR-transfected 293H cells. Combining two or more binding polypeptides in a single molecular construct appears to improve the avidity of the construct over the monomeric binding polypeptides as shown by a decrease in K D . 
     In addition, as demonstrated herein, 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 No. 60/440,201, U.S. application Ser. No. 10/379,287, filed Mar. 3, 2003, and U.S. application Ser. No. 10/661,032 by Christophe Arbogast et al., filed Sep. 11, 2003, the contents of which are incorporated herein) were made. Constructs comprising two or more binding polypeptides provided herein are expected to bind to multiple sites on KDR or VEGF/KDR. The heteromeric constructs show superior binding ability over both the corresponding monomers and multimeric constructs comprising multiple copies of the same binding polypeptide. Furthermore, heteromeric constructs comprising two or more binding peptides specific for different epitopes, together with a control peptide, were also able to efficiently bind KDR-transfected 293H cells. Thus, inclusion of two or more binding polypeptides that recognize different epitopes further improves the avidity of the construct for the target molecule, as demonstrated by a decrease in K D . 
     Heteromeric constructs of the binding polypeptides provided herein show improved ability to inhibit receptor tyrosine kinase function. Based on experiments described herein, dimeric and other multimeric constructs of the present invention comprising at least two binding polypeptides specific for different epitopes of KDR and/or KDR/VEGF complex are expected to inhibit the function of receptor tyrosine kinases. In particular, such constructs are expected to inhibit the function of VEGFR-2/KDR, VEGFR-1/Flt-1 and VEGFR-3/Flt-4. 
     For the purposes of the present invention, receptor tyrosine kinase function can include any one of: oligomerization of the receptor, receptor phosphorylation, kinase activity of the receptor, recruitment of downstream signaling molecules, induction of genes, induction of cell proliferation, induction of cell migration, or combination thereof. For example, 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. In addition, heteromeric constructs of binding peptides provided herein inhibit VEGF-stimulated endothelial cell migration. As shown herein, targeting two or more distinct epitopes on KDR with a single binding construct greatly improves the ability of the construct to inhibit receptor function. Even binding peptides with weak ability to block receptor activity can be used to generate heteromeric constructs having improved ability to block VEGF-induced receptor function. 
     Therefore, the present invention also is drawn to constructs comprising two or more binding polypeptides. In one embodiment, the multimeric constructs comprise two or more copies of a single binding polypeptide. In another embodiment, the multimeric constructs of the present invention comprise two or more binding polypeptides, such that at least two of the binding polypeptides in the construct are specific for different epitopes of KDR and/or KDR/VEGF. These constructs are also referred to herein as “heteromeric constructs,” “heteromultimers,” etc. The constructs of the present invention can also include unrelated, or control peptide(s). 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. In specific embodiments, 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 the group consisting of Consensus Sequences 1-5 below: 
     Consensus Sequence 1: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -Cys-X 12 -X 13 -X 14  (TN8), wherein 
     X 1  is Ala, Arg, Asp, Gly, His, Leu, Lys, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 2  is Asn, Asp, Glu, Gly, Ile, Leu, Lys, Phe, Ser, Thr, Trp, Tyr or Val; 
     X 3  is Asn, Asp, Gln, Glu, Ile, Leu, Met, Thr, Trp or Val; 
     X 5  is Ala, Arg, Asn, Asp, Gln, Glu, His, Ile, Lys, Phe, Pro, Ser, Trp or Tyr; 
     X 6  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 7  is Ala, Asn, Asp, Glu, Gly, His, Ile, 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, Gln, Glu, Gly, Ile, Leu, Met, Pro, Thr, Trp, Tyr or Val; 
     X 10  is Ala, Arg, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr; 
     X 12  is Arg, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 13  is Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp or Tyr; and 
     X 14  is Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 2: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Cys-X 16 -X 17 -X 18  (TN12), wherein 
     X 1  is Ala, Asn, Asp, Gly, Leu, Pro, Ser, Trp or Tyr (preferably Asn, Asp, Pro or Tyr); 
     X 2  is Ala, Arg, Asn, Asp, Gly, His, Phe, Pro, Ser, Trp or Tyr (preferably Asp, Gly, Pro, Ser or Trp); 
     X 3  is Ala, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val (preferably Trp); 
     X 5  is Arg, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Thr, Trp, Tyr or Val (preferably Glu, Ile or Tyr); 
     X 6  is Ala, Arg, Asn, Cys, Glu, Ile, Leu, Met, Phe, Ser, Trp or Tyr (preferably Glu, Phe or Tyr); 
     X 7  is Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Pro, Ser, Thr, Trp, Tyr or Val (preferably Glu); 
     X 8  is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Phe, Pro, Ser, Trp, Tyr or Val (preferably Gln or Ser); 
     X 9  is Asp, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp or Tyr (preferably Asp); 
     X 10  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val (preferably Lys or Ser); 
     X 11  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Trp, Tyr or Val (preferably Gly or Tyr); 
     X 12  is Ala, Arg, Gln, Gly, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val (preferably Trp or Thr); 
     X 13  is Arg, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, Trp or Val (preferably Glu or Trp); 
     X 14  is Arg, Asn, Asp, Glu, His, Ile, Leu, Met, Phe, Pro, Thr, Trp, Tyr or Val (preferably Phe); 
     X 16  is Ala, Asn, Asp, Gln, Glu, Gly, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val (preferably Asp); 
     X 17  is Arg, Asn, Asp, Cys, Gly, His, Phe, Pro, Ser, Trp or Tyr (preferably Pro or Tyr); and 
     X 18  is Ala, Asn, Asp, Gly, His, Leu, Phe, Pro, Ser, Trp or Tyr (preferably Asn, Pro or Trp), 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 3: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -Gly-X 9 -Cys-X 11 -X 12 -X 13  (TN7), wherein 
     X 1  is Gly or Trp; 
     X 2  is Ile, Tyr or Val; 
     X 3  is Gln, Glu Thr or Trp; 
     X 5  is Asn, Asp or Glu; 
     X 6  is Glu, His, Lys or Phe; 
     X 7  is Asp, Gln, Leu, Lys Met or Tyr; 
     X 9  is Arg, Gln, Leu, Lys or Val; 
     X 11  is Arg, Phe, Ser, Trp or Val; 
     X 12  is Glu, His or Ser; and 
     X 13  is Glu, Gly, Trp or Tyr, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 4: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -Cys-X 13 -X 14 -X 15  (TN9), wherein 
     X 1  is Arg, Asp, Gly, Ile, Met, Pro or Tyr (preferably Tyr); 
     X 2  is Asp, Gly, His, Pro or Trp (preferably Gly or Trp); 
     X 3  is Gly, Pro, Phe, Thr or Trp (preferably Pro); 
     X 5  is Ala, Asp, Lys, Ser, Trp or Val (preferably Lys); 
     X 6  is Asn, Glu, Gly, His or Leu; 
     X 7  is Gln, 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 Trp (preferably Pro); 
     X 10  is Ala, Gly, His, Leu, Trp or Tyr (preferably His or Trp); 
     X 11  is Ala, Asp, Gln, Leu, Met, Thr or Trp; 
     X 13  is Ala, Lys, Ser, Trp or Tyr (preferably Trp); 
     X 14  is Asp, Gly, Leu, His, Met, Thr, Trp or Tyr (preferably His, Trp, or Tyr); and 
     X 15  is Asn, Gln, Glu, Leu, Met, Pro or Trp (preferably Glu, Met or Trp), 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 5: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -Ser-Gly-Pro-X 12 -X 13 -X 14 -X 15 -Cys-X 17 -X 18 -X 19  (MTN13; SEQ ID NO:1), wherein 
     X 1  is Arg, Glu, His, Ser or Trp; 
     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 Gln, Ile, Lys, Tyr or Val; 
     X 8  is Gln, Ile, Leu, Met or Phe; 
     X 12  is Asn, Asp, Gly, His or Tyr; 
     X 13  is Gln, Gly, Ser or Thr; 
     X 14  is Glu, Lys, Phe or Ser; 
     X 15  is Glu, Ile, Ser or Val; 
     X 17  is Glu, Gly, Lys, Phe, Ser or Val; 
     X 18  is Arg, Asn, Ser or Tyr; and 
     X 19  is Asp, Gln, Glu, Gly, Met or Tyr, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex. 
     Further analysis of the polypeptides isolated from the TN8 library (see Consensus Sequence 1) revealed sub-families of preferred binding polypeptides, which are described by the Consensus Sequences 6, 7 and 8 as follows: 
     Consensus Sequence 6: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -Tyr-Cys-X 12 -X 13 -X 14 , wherein 
     X 1  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 Ile, Leu or Trp; 
     X 5  is Ala, Arg, Glu, Lys or Ser (preferably Glu); 
     X 6  is Ala, Asp, Gln, Glu, Thr or Val (preferably Asp or Glu); 
     X 7  is Asp or Glu; 
     X 8  is Trp or Tyr; 
     X 9  is Thr or Tyr (preferably Tyr); 
     X 12  is Glu, Met, Phe, Trp or Tyr (preferably Trp, Phe, Met, or Tyr); 
     X 13  is Ile, Leu or Met; and 
     X 14  is Ile, Leu, Met, Phe or Thr (preferably Thr or Leu), 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 7: Trp-Tyr-Trp-Cys-X 5 -X 6 -X 7 -Gly-X 9 -X 10 -Cys-X 12 -X 13 -X 14  (SEQ ID NO:2), wherein 
     X 5  is Asp, Gln or His; 
     X 6  is His or Tyr (preferably Tyr); 
     X 7  is Ile, His or Tyr; 
     X 9  is Ile, Met or Val; 
     X 10  is Gly or Tyr; 
     X 12  is Asp, Lys or Pro; 
     X 13  is Gln, Gly or Trp; and 
     X 14  is Phe, Ser or Thr, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 8: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -Gly-X 10 -Cys-X 12 -X 13 -X 14 , wherein 
     X 1  is Gly, Leu, His, Thr, Trp or Tyr (preferably Trp, Tyr, Leu or His); 
     X 2  is Ile, Leu, Thr, Trp or Val (preferably Val, Ile or Leu); 
     X 3  is Asp, Glu, Gln, Trp or Thr, (preferably Glu, Asp or Gln); 
     X 5  is Ala, Arg, Asn, Asp, His, Phe, Trp or Tyr (preferably Tyr, Trp or Phe); 
     X 6  is Ala, Asp, Gln, His, Lys, Met, Ser, Thr, Trp, Tyr or Val; 
     X 7  is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr or Val; 
     X 8  is Asp, Phe, Ser, Thr, Trp or Tyr (preferably Thr, Ser or Asp); 
     X 10  is Ala, Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr (preferably Arg or Lys); 
     X 12  is Arg, Gln, His, Ile, Lys, Met, Phe, Thr, Trp, Tyr or Val (preferably Tyr, Trp, Phe, Ile or Val); 
     X 13  is Arg, Asn, Asp, Glu, His, Met, Pro, Ser or Thr; and 
     X 14  is Arg, Gln, Glu, Gly, Phe, Ser, Trp or Tyr, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex. 
     Further analysis of the polypeptides isolated from the TN12 library (see Consensus Sequence 2) revealed sub-families of preferred binding polypeptides, which are described by Consensus Sequences 9-12 and 9A as follows: 
     Consensus Sequence 9: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -Trp-Gly-Gly-X 12 -X 13 -Cys-X 15 -X 16 -X 17  (SEQ ID NO:3) (TN11, i.e., 11-mer binders isolated from the TN12 library), wherein 
     X 1  is Ser, Phe, Trp, Tyr or Gly (preferably Ser); 
     X 2  is Arg, Gly, Ser or Trp (preferably Arg); 
     X 3  is Ala, Glu, Ile or Val (preferably Val or Ile); 
     X 5  is Ala, Phe or Trp (preferably Trp or Phe); 
     X 6  is Glu or Lys (preferably Glu); 
     X 7  is Asp, Ser, Trp or Tyr (preferably Asp, Trp or Tyr); 
     X 8  is Phe, Pro or Ser (preferably Ser); 
     X 12  is Gln or Glu (preferably Glu); 
     X 13  is Ile, Phe or Val; 
     X 15  is Gln, Ile, Leu, Phe or Tyr (preferably Phe, Tyr or Leu); 
     X 16  is Arg, Gly or Pro (preferably Arg); and 
     X 17  is Gln, 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 9A: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -Cys-X 15 -X 16 -X 17  (TN11, i.e., 11-mer binders isolated from the TN12 library; SEQ ID NO:3), wherein 
     X 1  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 2  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val; 
     X 3  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr or Val; 
     X 5  is Ala, Arg, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Trp, Tyr or Val; 
     X 6  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr; 
     X 7  is Ala, Arg, Asp, Asn, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Ser, Thr, Trp, Tyr or Val; 
     X 8  is Ala, Arg, Asp, Asn, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 9  is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Phe, Pro, Ser, Trp or Tyr; 
     X 10  is Asp, Gln, Glu, Gly, His, Ile, Leu, Phe, Ser, Thr, Trp, Tyr or Val; 
     X 11  is Ala, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 12  is Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 13  is Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 15  is Ala, Asp, Asn, Glu, Gly, Ile, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 16  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; 
     X 17  is Ala, Arg, Asp, Asn, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 10: Tyr-Pro-X 3 -Cys-X 5 -Glu-X 7 -Ser-X 9 -Ser-X 11 -X 12 -X 13 -Phe-Cys-X 16 -X 17 -X 18  (TN12; SEQ ID NO:4), wherein 
     X 3  is Gly or Trp (preferably Trp); 
     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); 
     X 11  is Gly or Val (preferably Val); 
     X 12  is Thr or Val (preferably Thr); 
     X 13  is Arg or Trp (preferably Arg); 
     X 16  is Ala or Val (preferably Val); 
     X 17  is Asp or Pro (preferably Pro); and 
     X 18  is Gly or Trp (preferably Trp), 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 11: X 1 -X 2 -X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -Gly-X 12 -Trp-X 14 -Cys-X 16 -X 17 -X 18  (TN12; SEQ ID NO:5), wherein 
     X 1  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, Trp or Tyr (preferably Trp or Tyr); 
     X 5  is Glu, Met or Thr (preferably Glu); 
     X 6  is Ile, Leu, Met or Phe (preferably Met, Leu, or Phe); 
     X 7  is Arg, Asp, Glu, Met, Trp or Val; 
     X 8  is Asn, Gln, Gly, Ser or Val; 
     X 9  is Asp or Glu; 
     X 10  is Lys, Ser, Thr or Val (preferably Lys); 
     X 12  is Arg, Gln, Lys or Trp (preferably Trp, Arg, or Lys); 
     X 14  is Asn, Leu, Phe or Tyr (preferably Tyr, Phe, or Asn); 
     X 16  is Gly, Phe, Ser or Tyr (preferably Tyr or Phe); 
     X 17  is Gly, Leu, Pro or Ser (preferably Pro or Ser); and 
     X 18  is Ala, Asp, Pro, Ser, Trp or Tyr, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 12: Asn-Trp-X 3 -Cys-X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 -X 13 -X 14 -Cys-X 16 -X 17 -X 18  (TN12; SEQ ID NO:6), wherein 
     X 3  is Glu or Lys; 
     X 5  is Glu or Gly; 
     X 6  is Trp or Tyr; 
     X 7  is Ser or Thr; 
     X 8  is Asn or Gln; 
     X 9  is Gly or Met; 
     X 10  is Phe or Tyr; 
     X 11  is Asp or Gln; 
     X 12  is Lys or Tyr; 
     X 13  is Glu or Thr; 
     X 14  is Glu or Phe; 
     X 16  is Ala or Val; 
     X 17  is Arg or Tyr; and 
     X 18  is Leu or Pro, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex. 
     Analysis of the binding polypeptides isolated from a linear display library (Lin20) defined two families of preferred embodiments including the amino acid sequences of Consensus Sequences 13 and 14 as follows: 
     Consensus Sequence 13: Z 1 -X 1 -X 2 -X 3 -X 4 -X 5 -Z 2  (Lin20), wherein, 
     Z 1  is a polypeptide of at least one amino acid or is absent; 
     X 1  is Ala, Asp, Gln or Glu (preferably Gln or Glu); 
     X 2  is Ala, Asp, Gln, Glu Pro (preferably Asp, Glu or Gln); 
     X 3  is Ala, Leu, Lys, Phe, Pro, Trp or Tyr (preferably Trp, Tyr, Phe or Leu); 
     X 4  is Asp, Leu, Ser, Trp, Tyr or Val (preferably Tyr, Trp, Leu or Val); 
     X 5  is Ala, Arg, Asp, Glu, Gly, Leu, Trp or Tyr (preferably Trp, Tyr or Leu); and 
     Z 2  is a polypeptide of at least one amino acid or is absent, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Consensus Sequence 14: X 1 -X 2 -X 3 -Tyr-Trp-Glu-X 7 -X 8 -X 9 -Leu (Lin20; SEQ ID NO:7), wherein, the sequence can optionally have a N-terminal polypeptide, C-terminal polypeptide, or a polypeptide at both termini of at least one amino acid; and wherein 
     X 1  is Asp, Gly or Ser (preferably Gly); 
     X 2  is Ile, Phe or Tyr; 
     X 3  is Ala, Ser or Val; 
     X 7  is Gln, Glu, Ile or Val; 
     X 8  is Ala, Ile or Val (preferably Ile 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 Trp and the amino acid sequence of X 7 -X 10  is Asp-Trp-Tyr-Tyr (SEQ ID NO:8). More preferred structures include polypeptides comprising Consensus Sequence 1, wherein X 3  is Trp and the amino acid sequence of X 5 -X 10  is Glu-Glu-Asp-Trp-Tyr-Tyr (SEQ ID NO:9). Additional preferred polypeptides comprising Consensus Sequence 1 include polypeptides in which: X 3  is Trp and the amino acid sequence of X 5 -X 10  is Glu-Glu-Asp-Trp-Tyr-Tyr (SEQ ID NO: 9), and the peptide X 13 -X 14  is Ile-Thr. Of these preferred polypeptides, it is additionally preferred that X 1  will be Pro and X 12  will be one of Phe, Trp or Tyr. 
     Particular embodiments of the cyclic polypeptide families described above are disclosed in Tables 1, 2, 4, 5, 6 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 of ten amino acids, for example, as follows: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 10) 
               
               
                 Asn-Asn-Ser-Cys-Trp-Leu-Ser-Thr-Thr-Leu-Gly-Ser- 
               
               
                 Cys-Phe-Phe-Asp, 
               
               
                   
               
               
                 (SEQ ID NO: 11) 
               
               
                 Asp-His-His-Cys-Tyr-Leu-His-Asn-Gly-Gln-Trp-Ile- 
               
               
                 Cys-Tyr-Pro-Phe, 
               
               
                   
               
               
                 (SEQ ID NO: 12) 
               
               
                 Asn-Ser-His-Cys-Tyr-Ile-Trp-Asp-Gly-Met-Trp-Leu- 
               
               
                 Cys-Phe-Pro-Asp. 
               
            
           
         
       
     
     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 from the group of amino acid sequences set forth in Table 3, infra. 
     The polypeptides of the invention can optionally have additional amino acids attached at either or both of the N- and C-terminal ends. In preferred embodiments, binding polypeptides according to the invention can be prepared having N-terminal and/or C-terminal flanking peptides of one or more, preferably two, amino acids corresponding to the flanking peptides of the display construct of the phage selectant from which the binding polypeptides were isolated. Preferred amino-terminal flanking peptides include Ala-Gly- (most preferably for TN7, TN8 and TN9 sequences), Gly-Ser- (most preferably for TN10 sequences), Gly-Asp- (most preferably for TN12 sequences), Ala-Gln- (most preferably for linear sequences), and Ser-Gly- (most preferably for MTN13 sequences). Preferred carboxy-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). Single terminal amino acids can also be added to the binding polypeptides of the invention, and preferred terminal amino acids will preferably correspond to the parental phage display construct, e.g., most preferably, 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 Gln- (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). Conservative substitutions (i.e., substitute amino acids selected within the following groups: {Arg, His, Lys}, {Glu, Asp}, {Asn, Cys, Glu, Gly, Ser, Thr, Tyr}, {Ala, Ile, Leu, Met, Phe, Pro, Trp, Val}) for such flanking amino acids are also contemplated. 
     Examination of the sequence information and binding data from the isolates of libraries containing polypeptides with the potential to form loop structures (e.g., libraries designated TN7, TN8, TN9, TN10, TN12 and MTN13) identifies a series of KDR or VEGF/KDR complex binding polypeptides that may form loop structures. In specific embodiments, cyclic KDR- or VEGF/KDR-binding polypeptides of the invention comprise, or alternatively, consist of, an amino acid sequence selected from Loop Consensus Sequences 15-19 as follows: 
     Loop Consensus Sequence 15: Cys-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -Cys (TN8), wherein 
     X 2  is Ala, Arg, Asn, Asp, Gln, Glu, His, Ile, Lys, Phe, Pro, Ser, Trp or Tyr (preferably Asp, Glu or Tyr); 
     X 3  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val (preferably Glu, Met or Tyr); 
     X 4  is Ala, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val (preferably Asp); 
     X 5  is Ala, Asp, Glu, Gly, Leu, Phe, Pro, Ser, Thr, Trp or Tyr (preferably Trp or Thr); 
     X 6  is Arg, Gln, Glu, Gly, Ile, Leu, Met, Pro, Thr, Trp, Tyr or Val (preferably Gly or Tyr); and 
     X 7  is Ala, Arg, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Trp 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 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -Cys (TN 12), wherein 
     X 2  is Arg, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Thr, Trp, Tyr or Val (preferably Glu, Ile or Tyr); 
     X 3  is Ala, Arg, Asn, Cys, Glu, Ile, Leu, Met, Phe, Ser, Trp or Tyr (preferably Glu, Phe or Tyr); 
     X 4  is Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Pro, Ser, Thr, Trp, Tyr or Val (preferably Glu); 
     X 5  is Ala, Asn, Asp, Gln, Glu, Gly, His, Met, Phe, Pro, Ser, Trp, Tyr or Val (preferably Gln or Ser); 
     X 6  is Asp, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp or Tyr (preferably Asp); 
     X 7  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val (preferably Lys or Ser); 
     X 8  is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Trp, Tyr or Val (preferably Gly or Tyr); 
     X 9  is Ala, Arg, Gln, Gly, His, Ile, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val (preferably Trp or Thr); 
     X 10  is Arg, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Thr, Trp or Val (preferably Glu or Trp); and 
     X 11  is Arg, Asn, Asp, Glu, His, Ile, Leu, Met, Phe, Pro, Thr, Trp, Tyr or Val (preferably Phe), 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     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, Gln, Leu, Lys, Met or Tyr; and 
     X 6  is Arg, Gln, Leu, Lys or Val, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     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, Trp or Val (preferably Lys); 
     X 3  is Asn, Glu, Gly, His or Leu; 
     X 4  is Gln, 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 Trp (preferably Pro or Trp); 
     X 7  is Ala, Gly, His, Leu, Trp or Tyr (preferably Trp); and 
     X 8  is Ala, Asp, Gln, Leu, Met, Thr or Trp, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex; or 
     Loop Consensus Sequence 19: Cys-X 2 -X 3 -X 4 -X 5 -Ser-Gly-Pro-X 9 -X 10 -X 11 -X 12 -Cys (MTN13; SEQ ID NO:13), wherein 
     X 2  is Asp, Glu, His or Thr; 
     X 3  is Arg, His, Lys or Phe; 
     X 4  is Gln, Ile, Lys, Tyr or Val; 
     X 5  is Gln, Ile, Leu, Met or Phe; 
     X 9  is Asn, Asp, Gly, His or Tyr; 
     X 10  is Gln, Gly, Ser or Thr; 
     X 11  is Glu, Lys, Phe or Ser; and 
     X 12  is Glu, Ile, Ser or Val, 
     and wherein the polypeptide binds KDR or a VEGF/KDR complex. 
     Preferred embodiments of the cyclic peptides of Loop Consensus Sequence 15 include KDR and/or VEGF/KDR complex binding polypeptides comprising Loop Consensus Sequences 20-22 as follows: 
     Loop Consensus Sequence 20: Cys-X 2 -X 3 -X 4 -X 5 -X 6 -Tyr-Cys (TN8), wherein 
     X 2  is Ala, Arg, Glu, Lys or Ser (preferably Glu); 
     X 3  is Ala, Asp, Gln, Glu, Thr or Val (preferably Asp or Glu); 
     X 4  is Asp or Glu; 
     X 5  is Trp or Tyr; and 
     X 6  is Thr or Tyr (preferably Tyr); or 
     Loop Consensus Sequence 21: Cys-X 2 -X 3 -X 4 -Gly-X 6 -X 7 -Cys (TN8), wherein 
     X 2  is Asp, Gln or His; 
     X 3  is His or Tyr (preferably Tyr); 
     X 4  is His, Ile or Tyr; 
     X 6  is Ile, Met or Val; and 
     X 7  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, Trp or Tyr (preferably Tyr, Trp or Phe); 
     X 3  is Ala, Asp, Gln, His, Lys, Met, Ser, Thr, Trp, Tyr or Val; 
     X 4  is Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Pro, Ser, Thr or Val; 
     X 5  is Asp, Phe, Ser, Thr, Trp or Tyr (preferably Thr, Ser or Asp); and 
     X 7  is Ala, Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Trp or Tyr (preferably Arg or Lys). 
     Preferred embodiments of the cyclic peptides of Loop Consensus Sequence 16 include KDR and/or VEGF/KDR complex binding polypeptides comprising sequences of Loop Consensus Sequences 23-26 as follows: 
     Loop Consensus Sequence 23: Cys-X 2 -X 3 -X 4 -X 5 -Trp-Gly-Gly-X 9 -X 10 -Cys (TN11, i.e., 11-mers based on isolates of the TN12 library; SEQ ID NO:14), wherein 
     X 2  is Ala, Phe or Trp (preferably Trp or Phe); 
     X 3  is Glu or Lys (preferably Glu); 
     X 4  is Asp, Ser, Trp or Tyr (preferably Asp, Trp or Tyr); 
     X 5  is Phe, Pro or Ser (preferably Ser); 
     X 9  is Gln or Glu (preferably Glu); and 
     X 10  is Ile, Phe or Val; or 
     Loop Consensus Sequence 24: Cys-X 2 -Glu-X 4 -Ser-X 6 -Ser-X 8 -X 9 -X 10 -Phe-Cys (TN12; SEQ ID NO:15), wherein 
     X 2  is His or Tyr; 
     X 4  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 Thr); and 
     X 10  is Arg or Trp (preferably Arg); or 
     Loop Consensus Sequence 25: Cys-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -Gly-X 9 -Trp-X 11 -Cys (TN12; SEQ ID NO:16), wherein 
     X 2  is Glu, Met or Thr (preferably Glu); 
     X 3  is Ile, Leu, Met or Phe (preferably Met, Leu or Phe); 
     X 4  is Arg, Asp, Glu, Met, Trp or Val; 
     X 5  is Asn, Gln, Gly, Ser or Val; 
     X 6  is Glu or Asp; 
     X 7  is Lys, Ser, Thr or Val (preferably Lys); 
     X 9  is Arg, Gln, Lys or Trp (preferably Trp, Arg or Lys); and 
     X 11  is Asn, Leu, Phe or Tyr (preferably Tyr, Phe or Asn); or 
     Loop Consensus Sequence 26: Cys-X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -Cys (TN 12), wherein 
     X 2  is Glu or Gly; 
     X 3  is Trp or Tyr; 
     X 4  is Ser or Thr; 
     X 5  is Asn or Gln; 
     X 6  is Gly or Met; 
     X 7  is Phe or Tyr; 
     X 8  is Asp or Gln; 
     X 9  is Lys or Tyr; 
     X 10  is Glu or Thr; and 
     X 11  is Glu or Phe. 
     Preferred embodiments of the cyclic peptides of Loop Consensus Sequence 17 include KDR and/or VEGF/KDR complex binding polypeptides comprising sequences of Loop Consensus Sequence 27 as follows: 
     Loop Consensus Sequence 27: 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, Gln, Leu, Lys, Met or Tyr; and 
     X 6  is Arg, Gln, 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, Trp 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 Trp (preferably Pro); 
     X 7  is His, Leu, Trp or Tyr (preferably Trp or His); and 
     X 8  is Ala, Asp, Gln, Leu, Met, Thr or Trp. 
     Preferred embodiments of the cyclic peptides of Loop Consensus Sequence 19 include KDR and/or VEGF/KDR complex binding polypeptides comprising sequences of Loop Consensus Sequence 29 as follows: 
     Loop Consensus Sequence 29: Cys-X 2 -X 3 -X 4 -X 5 -Ser-Gly-Pro-X 9 -X 10 -X 11 -X 12 -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 Gln, Ile, Lys, Tyr or Val; 
     X 5  is Gln, Ile, Leu, Met or Phe; 
     X 9  is Asn, Asp, Gly, His or Tyr; 
     X 10  is Gln, Gly, Ser or Thr; 
     X 11  is Glu, Lys, Phe or Ser; and 
     X 12  is Glu, Ile, Ser or Val. 
     Chemical or physical modifications, as well as any sequence modifications, described herein are encompassed for use with any of the specific sequences disclosed herein and/or any specific sequences that conform to any of the consensus sequences described herein. 
     The KDR and VEGF/KDR binding polypeptides described above can optionally have additional amino acids attached at either or both of the N- and C-terminal ends and can be modified, optimized or employed in multimeric constructs. Further, the invention includes homologues of the KDR and VEGF/KDR complex binding peptides as defined herein. 
     Another aspect of the present invention relates to modifications of the foregoing polypeptides to provide specific angiogenesis imaging agents by detectably labeling a polypeptide according to the present invention. Such detectable labeling can involve radiolabeling, enzymatic labeling, or labeling with MRI paramagnetic chelates or microparticles or superparamagnetic particles; incorporation into ultrasound bubbles, microparticles, microspheres, emulsions, or liposomes; or conjugation with optical dyes. 
     In another aspect of the present invention, methods for isolating KDR or KDR-expressing cells using the present binding polypeptides are provided. 
     Additionally, 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 KDR or VEGF/KDR complex, angiogenesis or diseases associated with a number of pathogens, including, for example, malaria, HIV, SIV, Simian hemorrhagic fever, etc. 
     When the binding peptides disclosed herein are used as therapeutic agents, it may be advantageous to enhance the serum residence time of the peptides. This can be accomplished by: a) conjugating to the peptide a moiety, such as maleimide, that reacts with free sulfhydryl groups on serum proteins, such as serum albumin, b) conjugating to the peptide a moiety, such as a fatty acid, that binds non-covalently to serum proteins, especially serum albumin, c) conjugating to the peptide a polymer, such as PEG, that is known to enhance serum residence time, and/or d) fusing DNA that encodes the KDR-binding peptide to DNA that encodes a serum protein such as human serum albumin or an antibody and expressing the encoded fusion protein. 
     In another aspect of the invention, 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, these methods may be used to rapidly assess the stability of the peptides in the presence of serum. 
     In another embodiment of the invention, a multimeric polypeptide construct having the ability to bind to KDR or VEGF/KDR complex comprising at least one amino acid sequence selected from any of the polypeptides described above is envisioned. In a particular embodiment, the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NOS: 20-86, 87-136, 187-192, 193-203, 207-259 and 505-516. I a particular embodiment, the amino acid sequence selected from the group consisting of: SEQ ID NOS: 137-186. 
     In one embodiment, the amino acid sequence further comprises N-terminal and/or C-terminal flanking peptides of one or more amino acids. In another embodiment, the amino acid sequence comprises a modification selected from the group consisting of: an amino acid substitution, and amide bond substitution, a D-amino acid substitution, a glycosylated amino acid, a disulfide mimetic substitution, an amino acid translocation, a retroinverso peptide, a peptoid, a retro-inverso peptoid, and a synthetic peptide. 
     In another embodiment, the polypeptide can be conjugated to a detectable label or a therapeutic agent, optionally further comprising a linker or spacer between the polypeptide and the detectable label or the therapeutic agent. 
     In a particular embodiment, the detectable label or the therapeutic agent is selected from the group consisting of: an enzyme, a fluorescent compound, a liposome, an optical dye, a paramagnetic metal ion, a superparamagnetic particle, an ultrasound contrast agent and a radionuclide. In one embodiment, the therapeutic agent or detectable label comprises a radionuclide, including, for example,  18 F,  124 I,  125 I,  131 I,  123 I,  77 Br,  76 Br,  99m Tc,  51 Cr,  67 Ga,  68 Ga,  47 Sc,  51 Cr,  167 Tm,  141 Ce,  111 In,  168 Yb,  175 Yb,  140 La,  90 Y,  88 Y,  153 Sm,  166 Ho,  165 Dy,  166 Dy,  62 Co,  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,  117 mSn,  149 Pm,  161 Tb,  177 Lu,  198 Au or  199 Au. In a particular embodiment, the therapeutic agent or detectable label further comprises a chelator, such as, for example, a compound selected from the group consisting of: formula 20, 21, 22, 23a, 23b, 24a, 24b, and 25. In a particular embodiment, the detectable label comprises an ultrasound contrast agent that can comprise, for example, a phospholipid stabilized microbubble or a microballoon comprising a gas. Alternatively, the detectable label can comprise one or more paramagnetic metal ions or a superparamagnetic particle and one or more chelators. 
     In another embodiment, the invention is directed to an ultrasound contrast agent comprising at least one KDR or VEGF/KDR complex binding polypeptide comprising an amino acid sequence of one of the following and optionally further comprising N-terminal and/or C-terminal flanking peptides of one or more amino acids described herein. In a particular embodiment, the gas filled microvesicles comprise phospholipid stabilized microbubbles or microballoons. In one embodiment, the phospholipid stabilized microbubbles or microballoons further comprise a fluorinated gas. 
     In another embodiment, the invention is directed to a scintigraphic imaging agent comprising at least one KDR or VEGF/KDR complex binding polypeptide comprising an amino acid sequence of one of the following and optionally further comprising N-terminal and/or C-terminal flanking peptides of one or more amino acids described herein. In a particular embodiment, the scintigraphic imaging agent can comprise at least one radionuclide useful in scintigraphic imaging and at least one KDR or VEGF/KDR complex binding moiety comprising a polypeptide of the invention. In a particular embodiment, the scintigraphic imaging agent can comprise at least one chelator selected from the group consisting of: formula 20, 21, 22, 23a, 23b, 24a, 24b and 25. In one embodiment, the radionuclide is selected from the group consisting of  99m Tc and  111 In. 
     In another embodiment, the invention is directed to an agent useful in radiotherapy comprising at least one KDR or VEGF/KDR complex binding polypeptide comprising an amino acid sequence of one of the following and optionally further comprising N-terminal and/or C-terminal flanking peptides of one or more amino acids described herein. 
     In another embodiment, the invention is directed to an agent useful in radiotherapy comprising at least one radionuclide useful in radiotherapy and at least one KDR or VEGF/KDR complex binding moiety comprising a polypeptide of the invention. In a particular embodiment, the agent can comprise at least one chelator selected from the group consisting of: formula 20, 21, 22, 23a, 23b, 24a, 24b and 25. In a particular embodiment, the radionuclide is selected from the group consisting of:  177 Lu,  90 Y,  153 Sm and  166 Ho. 
     In another embodiment, the invention is directed to a method of synthesizing a polypeptide or a multimeric polypeptide construct having the ability to bind KDR or VEGF/KDR complex comprising a cyclic polypeptide formed by introducing an amide bond between two side chains. 
     In another embodiment, the invention is directed to a method of synthesizing a polypeptide or a multimeric polypeptide construct having the ability to bind KDR or VEGF/KDR complex comprising a polypeptide and a linker comprising at least one glycosylated amino acid selected from the group consisting or serine, threonine and homoserine. 
     In another embodiment, the invention is directed to a method of synthesizing a multimeric polypeptide construct having the ability to bind KDR or VEGF/KDR complex selected from the group consisting of D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30 and D31, comprising: a) treating a purified peptide monomer with glutaric acid bis-N-hydroxysuccinimidyl ester; and b) contacting the peptide monomer in (a) with a second peptide monomer in the presence of N,N-(Diisopropyl)aminomethylpolystyrene, thereby forming the multimeric polypeptide. 
     In another embodiment, the invention is directed to a multimeric polypeptide having the ability to bind to KDR or VEGF/KDR complex selected from the group consisting of: D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30 and D31. 
     In another embodiment, the invention is directed to a dimeric polypeptide construct having the ability to bind to KDR or VEGF/KDR, wherein each peptide of the dimer comprises a sequence of a polypeptide of the invention. In a particular embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of: SEQ ID NOS: 20-86, 87-136, 187-192, 193-203, 207-259 and 505-516. In a particular embodiment, the amino acid sequence of the polypeptide is selected from the group consisting of: SEQ ID NOS: 137-186. 
     Any of the dimers of the invention can comprise N-terminal and/or C-terminal flanking peptides of one or more amino acids, as well as a modification such as, for example, an amino acid substitution, and amide bond substitution, a D-amino acid substitution, a glycosylated amino acid, a disulfide mimetic substitution, an amino acid translocation, a retroinverso peptide, a peptoid, a retro-inverso peptoid or a synthetic peptide. The dimeric constructs of the invention can be conjugated to a detectable label or a therapeutic agent, optionally further comprising a linker or spacer between the polypeptide and the detectable label or the therapeutic agent. The detectable label or the therapeutic agent can be, for example, an enzyme, a fluorescent compound, a liposome, an optical dye, one or more paramagnetic metal ions or a superparamagnetic particle, an ultrasound contrast agent or one or more radionuclides. In a particular embodiment, the therapeutic agent or detectable label comprises one or more radionuclides. In a particular embodiment, a dimeric construct can be labeled with one or more radionuclides such as, for example,  18 F,  124 I,  125 I,  131 I,  123 I,  77 Br,  76 Br,  99m Tc,  51 Cr,  68 Ga,  47 Sc,  51 Cr,  167 Tm,  141 Ce,  111 In,  168 Yb,  175 Yb,  140 La,  90 Y,  88 Y,  153 Sm,  166 Ho,  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,  117 mSn,  149 Pm,  161 Tb,  177 Lu,  198 Au or  199 Au. In a particular embodiment, each peptide of the dimer is selected from an amino acid sequence selected from the group consisting of the sequences listed in Tables 1-11 and 27. 
     In another embodiment, the invention is directed to a multimeric polypeptide having the ability to bind to KDR or VEGF/KDR complex, wherein the multimeric polypeptide comprises at least one peptide monomer comprising an amino acid sequence selected from the group consisting of those sequences listed in Tables 1-11 and 27. 
     In another embodiment, the invention is directed to a method of inhibiting VEGF-induced vascular permeability comprising administering and agent comprising a peptide of the invention. In a particular embodiment, the agent comprises D10. 
     In yet another embodiment, the present invention includes methods for detecting, monitoring and/or evaluating a therapeutic response following administration of a contrast agent conjugated to KDR binding moieties described herein, including imaging before and after treatment. In a preferred embodiment imaging occurs before treatment and at one or more timepoints after treatment. In one embodiment treatment is for a condition associated with angiogenesis and the treatment comprises administration of an anti-angiogenic agent. In another embodiment treatment is for cancer or another hyperproliferative disease, particularly for KDR-expressing cancers such as prostate cancer. In one embodiment the treatment for cancer comprises administration of an anti-cancer agent. In another embodiment, the treatment of cancer comprises radiotherapy, RF ablation or focused ultrasound treatment. In a particular embodiment, the contrast agent is an ultrasound contrast agent. In a preferred embodiment the contrast agent comprises D5. 
     The present invention provides targeting vector-phospholipid conjugates and particularly targeting peptide-phospholipid conjugates which are useful in the preparation of gas filled ultrasound contrast agents. In a preferred embodiment the targeting peptide-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinity and thus are useful components of contrast agents for imaging and monitoring processes involving angiogenesis. 
     The present invention also provides monomeric and dimeric peptide phospholipid conjugates (also referred to herein as lipopeptides) which are useful in preparing gas filled ultrasound contrast agents, and particularly in preparing ultrasound contrast agents which target KDR and may be used for imaging and monitoring processes involving angiogenesis. 
     The present invention also provides methods and processes for the large scale production of highly pure monomeric and dimeric peptide phospholipid conjugates, particularly monomeric and dimeric peptide phospholipids conjugates having high KDR binding affinity. 
     The present invention also provides methods and processes for the large scale production of highly pure dimeric peptide phospholipid conjugates having minimal levels of trifluoroacetic acid (TFA). 
     The present invention also provides methods for synthesizing monomeric peptides in high purity and the construction of peptide phospholipid conjugates from multiple peptide subunits. 
     The present invention also provides monomeric peptides which bind KDR or the VEGF/KDR complex with high affinity, as well as methods of synthesizing and using such monomeric peptides. 
     The present invention also provides targeted ultrasound contrast agents prepared from such targeting vector-phospholipid conjugates. Such targeted ultrasound contrast agents are useful for imaging target-bearing tissue. In a preferred embodiment, the targeted ultrasound contrast agents are targeted microbubbles and the targeting vector-phospholipid conjugates include targeting peptides which exhibit high KDR binding affinity and thus are useful components of contrast agents for imaging KDR-bearing tissue and particularly for imaging and monitoring of tumors and angiogenesis processes. Methods of preparing and using such targeted ultrasound contrast agents are also provided. 
     These and other aspects of the present invention will become apparent with reference to the following detailed description. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A and 1B  are graphs illustrating the saturation binding curves of binding peptide/neutravidin-HRP complexes.  FIG. 1A  illustrates the saturation binding curve for SEQ ID NO:264 and SEQ ID NO:294.  FIG. 1B  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  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). 
         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 (f) 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 for (c), and 2.22 nM for (d). All peptides had a C-terminal biotin and JJ spacer. 
         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 peptide comprising SEQ ID NO:294, and a biotinylated SEQ ID 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. Increasing concentrations (as indicated in the X axis) of uncomplexed peptides were added to the assay as indicated. Free SEQ ID NO:264 was able to decrease the binding of the SEQ ID NO:264 complex to KDR-transfected cells. 
         FIG. 8  illustrates structures of binding polypeptide sequences tested in Example 6: SEQ ID NOS:294, 368, 369, 337, 371 and 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 ligands attached were tested for binding to KDR-expressing and non-expressing 293H cells. 
         FIG. 10  is a bar graph illustrating percent inhibition of  125 I-labeled VEGF binding by binding polypeptides (a) acetylated SEQ ID NO:294 (without the modified C-terminus, GDSRVCWEDSWGGEVCFRYDP; SEQ ID NO:374); (b) SEQ ID NO:263 (without the modified C-terminus, AGDSWCSTEYTYCEMIGT; SEQ ID NO:375); (c) biotinylated SEQ ID NO:264 with a JJ spacer; and (d) SEQ ID NO:277 (biotinylated with the JJ spacer), at two concentrations (30 μM and 0.3 μM), to KDR-expressing 293H transfectants. 
         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 μg/mL; indicated as “α-KDR”) 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 μM) 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 SEQ ID NO:277, D2, D1, D3, and AQDWYYDEILSMADQLRHAFLSGG (SEQ ID NO:376) at three different concentrations (10 μM, 0.3 μM, and 0.03 μM) 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 D1 to completely block the VEGF-induced phosphorylation of KDR in HUVECs at 10 nM and the majority of phosphorylation at 1 nM. Reprobing the blot for total KDR (lower panel) demonstrated that the effects of the tested compounds was not due to reduced sample loading. Homodimers (D2 and D3) composed of the two binding sequences contained in D1 did not interfere with the phosphorylation at up to 100 nM. 
         FIG. 17  is a graph showing that D1 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 0 min, 30 min, and 90 min. 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. A: D6; B: SEQ ID NO:277; C: SEQ ID NO:377 (AEGTGDLHCYFPWVCSLDPGPEGGGK; negative control); F: SEQ ID NO:377; negative control. 
         FIGS. 23A and 23B  show the kinetic analysis of D1 (see  FIG. 36 ), binding to murine KDR-Fc. All sensograms are fit to the bivalent analyte model. 
         FIGS. 24A and 24B  show 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. 
         FIGS. 25A and 25B  show the 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 or delta dipeptide or turn mimics (such as α, β, γ, or δ turn mimics), shown in panels 1, 2 and 3. 
         FIG. 27  shows an oxime linker. The amino acids containing an aminoalcohol function (4), and containing an alkoxyamino function (5), are incorporated 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 (D1; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277)[(Biotin-JJK-(O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337))-NH)CONH 2 ]—NH 2 ). 
         FIG. 37  shows dimer 2 (D2; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277) [(Biotin-JJK-(O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-AGPTWCEDDWYYCWLFGTJK (SEQ ID NO:493))-NH)CONH 2 ]—NH 2 ). 
         FIG. 38  shows dimer 3 (D3; Ac-VC WEDS WGGEVCFRYDPGGGK (SEQ ID NO:337)[(Biotin-JJK-(O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337))-NH)CONH 2 ]—NH 2 ). 
         FIG. 39  shows dimer 4 (D4; Ac-AGPTWCEDDWYYCWLFGTJK (SEQ ID NO:338)[DOTA-JJK-(O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337))-NH)CONH 2 ]—NH 2 ). 
         FIG. 40  shows dimer 5 (D5; Ac-VC WEDS WGGEVCFRYDPGGGK (SEQ ID NO:337) (JJ-C(═O)(CH 2 ) 3 C(═O)—K—NH(CH 2 ) 4 —(S)—CH((Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277))-NH)CONH 2 )—NH 2 ). 
         FIG. 41  shows dimer 8 (D8; Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK (SEQ ID NO:356) {Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK (SEQ ID 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 (D10Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294) (JJ-Glut-NH(CH 2 ) 4 —(S)—CH(PnAO6-Glut-NH) (C═O—)]—NH 2 }—NH 2 ). 
         FIG. 44  shows dimer 11 (D11; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277) {Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337)[JJ-Glut-NH(CH 2 ) 4 —(S)—CH(DOTA-JJ-NH—) (C═O)—]—NH 2 }—NH 2 ). 
         FIG. 45  shows dimer 12 (D12; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){[PnAO6-Glut-K(Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337) (—C(═O)CH 2 (OCH 2 CH 2 ) 2 OCH 2 C(═O)—)—NH 2 ]}—NH 2 ). 
         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-AQDWYYDEILSMADQLRHAFLSGGGGGK (SEQ ID NO:356){PnAO6-Glut-K[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:477) (JJ-Glut)-NH 2 ]}—NH 2 ). 
         FIG. 48  shows dimer 15 (D15; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){[Ac-GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294)[JJ-Glut]-NH 2 ]-K(PnAO6-Glut)}—NH 2 ). 
         FIG. 49  shows dimer 16 (D16; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){PnAO6-Glut-K [Ac-GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294)[—C(═O)CH 2 O(CH 2 CH 2 O) 2 CH 2 C(═O)NH(CH 2 ) 3 O(CH 2 CH 2 O) 2 (CH 2 ) 3 NH C(═O)CH 2 O(CH 2 CH 2 O) 2 CH 2 C(═O)—]—NH 2 ]}—NH 2 ). 
         FIG. 50  shows dimer 17 (D17; Ac-AQDWYYDEILJGRGGRGGRGGK (SEQ ID NO:478) {K[Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337) (JJ-Glut)-NH 2 ]}—NH 2 ). 
         FIG. 51  shows dimer 18 (D18; Ac-APGTWCDYDWEYCWLGTFGGGK (SEQ ID NO:497) {PnAO6-Glut-K[Ac-GVDFRCEWSDWGEVGCRSPDYGGGK (SEQ ID NO:489) (JJ-Glut)-NH 2 ]}—NH 2 ). 
         FIG. 52  shows dimer 19 (D19; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){Biotin-K[Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337) (JJ-Glut)-NH 2 ]}—NH 2 ). 
         FIG. 53  shows dimer 20 (D20; (((-JJ)-AGPTWCEDDWYYCWLFGTGGGGK (SEQ ID NO:480)-NH 2 )-Glut-JJ)VCWEDSWGGEVCFRYDPGGG (SEQ ID NO:370)-NH 2 ). 
         FIG. 54  shows dimer 21 (D21; [(-JJ)-AGPTWCEDDWYYCWLFGTGGGGK (SEQ ID NO:480) (PnAO6-Glut)-NH 2 ]-Glut-(JJ)-VCWEDSWGGEVCFRYDPGGG (SEQ ID NO:370)-NH 2 ). 
         FIG. 55  shows dimer 22 (D22; Ac-GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294) {JJ-Glut-JJ-AGPTWCEDDWYYCWLFTGGGK (SEQ ID NO:481)-NH 2 }—NH 2 ). 
         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 dimer 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-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){Ac-GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294)[JJ-Glut-NH(CH 2 )4-(S)—CH(NH 2 )C(═O)—]—NH 2 }—NH 2 ). 
         FIG. 59  shows dimer 26 (D26; AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){(-Glut-JJ-VCWEDSWGGEVCFRYDPGGG (SEQ ID NO:370)-NH 2 )—K}—NH 2 ). 
         FIG. 60  shows dimer 27 (D27; Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277){Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337)[S(GalNAc-alpha-D)-G-S(GalNAc-alpha-D)-Glut-S(GalNAc-alpha-D)-G-S(GalNAc-alpha-D)-NH(CH 2 ) 4 —(S)—CH(Biotin-JJNH—)C(═O)—]—NH 2 }—NH 2 ). 
         FIG. 61  shows dimer 28 (D28; comprising AQEPEGYAYWEVITLYHEEDGDGGK (SEQ ID NO:305) and AQAFPRFGGDDYWIQQYLRYTDGGK (SEQ ID NO:306)). 
         FIG. 62  shows dimer 29 (D29; comprising AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277) and VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337)). 
         FIG. 63  shows dimer 6 (D6; comprising GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294) and AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277)). 
         FIG. 64  shows dimer 7 (D7; comprising GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294) and AGPKWCEEDWYYCMITGTGGGK (SEQ ID NO:264)). 
         FIG. 65  is a graph showing the inhibition of tumor growth by D6 as a function of D6 concentration. 
         FIG. 66  shows that D27 (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 D25 (diamonds), which lacks those chemical modifications. 
         FIGS. 67A and 67B  show that Adjunct A enhances the potency of D6 in blocking the biological effects of VEGF in a migration assay with cultured HUVECs.  FIG. 67A : Diamonds: D6 alone at the indicated concentrations. Squares: D6 at the indicated concentrations plus 100 nM Adjunct A (constant).  FIG. 67B  shows the structure of Adjunct A. 
         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 D27). 
         FIG. 71  depicts % inhibition±s.d. of specific  125 I-VEGF binding to KDR-transfected cells by SEQ ID NO:504 (squares) and D1 (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) or D1 (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. 74  is a graphical representation showing specific binding of complexes of peptide (SEQ ID NO:321) and  125 I-streptavidin (in the absence and presence of VEGF) to KDR-transfected cells at various concentrations (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. 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. 81  shows uptake and retention of bubble contrast in the tumor up to 30 minutes post injection for suspensions of microbubbles conjugated to SEQ ID NO:356. In 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 (see  FIGS. 82 and 83 ). 
         FIG. 82  shows uptake and retention of bubble contrast in the tumor up to 30 minutes post injection for suspensions of microbubbles conjugated to a SATA-modified peptide comprising SEQ ID NO:356. In contrast, the same bubbles showed only transient (no more than 10 minutes) visualization/bubble contrast in the AOI situated outside the matrigel. 
         FIG. 83  shows uptake and retention of bubble contrast in the matrigel up to 30 minutes post injection for suspensions of microbubbles conjugated to a SATA-modified peptide comprising SEQ ID NO:294. In contrast, the same bubbles showed only transient (no more than 10 minutes) visualization/bubble contrast in the AOI situated outside the matrigel. 
         FIG. 84  is a graph showing the results of in vitro binding assays. Microvascular endothelial cells (MVECs, Cascade Biologics, Portland, Oreg.) were used to assess the in vitro efficacy of D6 and related analogues for their ability to inhibit VEGF-stimulated proliferation. 
         FIG. 85  shows a typical example of peptide-conjugated ultrasound contrast agents bound to KDR- or mock-transfected cells in presence of 10% human serum (magnification: 100×). 
         FIG. 86  is a schematic representation of the synthesis scheme used to prepare 4-{2-(2-Hydroxyimino-1,1-dimethylpropylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyric acid, N-hydroxysuccinimide ester (Compound B) using 4-{2-(2-Hydroxyimino-1,1-dimethyl-propylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyric acid as a starting reagent. 
         FIGS. 87A-C  are schematic representations depicting synthesis schemes and structures for Dimer D30.  FIG. 87A  shows the synthesis scheme for the preparation of Compound 3.  FIG. 87B  shows the synthesis scheme for dimer D30: Preparation of Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337){[PnAO6-Glut-K(-Glut-JJ-NH(CH 2 ) 4 —(S)—CH(Ac-AQDWYYDEILJGRGGRGGRGG (SEQ ID NO:478)-NH)C(═O)NH 2 ]—NH 2 }—NH 2 : D30 from Compound 3 and Compound 4.  FIG. 87C  shows the structure of dimer D30. 
         FIGS. 88A-D  are schematic representations depicting synthesis schemes and structures for dimer D31.  FIG. 88A  shows the synthesis scheme for the preparation of Compound 2. 
         FIG. 88B  shows the synthesis scheme for the preparation of Compound 4 (a peptide related to SEQ ID NO:374).  FIG. 88C  depicts the synthesis scheme and structure for dimer D31 (i.e., Preparation of Ac-AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO:277)[Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:337)[SGS-Glut-SGS-(S)—NH(CH 2 ) 4 —CH(Biotin-JJ-NH)—C(═O)]—NH 2 ]—NH 2 ).  FIG. 88D  shows the structure of D31. 
         FIG. 89  is a graph that shows competition of targeted bubbles by corresponding free peptide. 
         FIG. 90  is a graph that shows competition of targeted bubbles conjugate to D23 by corresponding free peptide. 
         FIG. 91  is a graph that shows competition of targeted bubbles with free dimer. 
         FIG. 92  is a graph showing binding values obtained with the suspensions of microbubbles conjugated to the D23, SATA-modified SEQ ID NO:480, SATA-modified SEQ ID NO:294 or SEQ ID NO:294/SEQ ID NO:480 (50/50) are equivalent (see Example 43). 
         FIG. 93  is a graph showing dimer D10 blocks the increased peritoneal vascular permeability induced by VEGF injected intraperitoneally. Solutions containing the indicated additions were injected intraperitoneally, and their effect on peritoneal permeability was assessed by measuring the O.D. of the injected fluid at 590 nm after administering Evan&#39;s Blue dye i.v. as described in Example 44. 
         FIG. 94  is a graph showing uptake and retention of bubble contrast in the tumor up to 30 minutes post injection for suspensions of phospholipid stabilized microbubbles conjugated to a heteromultimeric construct (D23). 
         FIG. 95  is a graph depicting the binding of Lu-D13 to KDR-transfected 293H cells. Mock=mock-transfected. Trans=KDR-transfected. MS=mouse serum. 
         FIG. 96  is a graph showing the specific binding of a Tc-labeled polypeptide (SEQ ID NO:339) to KDR-transfected 293H cells after subtracting the binding to mock-transfected 293H cells. 
         FIG. 97  is a bar graph demonstrating that Tc-labeled SEQ ID NO:277 with Tc-chelate binding to KDR-transfected 293H cells is inhibited by about 80% in the presence of 40% rat serum. 
         FIG. 98  is a graph showing the ability of a contrast agent comprising D5 to detect early anti-angiogenic therapeutic effect of treatment with a therapeutic agent. Imax, LPO and area of a tumor in an OFA rat are shown. 
         FIG. 99  is a graph showing the ability of a contrast agent comprising D5 to detect early anti-angiogenic therapeutic effect of treatment with a therapeutic agent. Imax, LPO and area of a tumor in a second rat are shown. 
         FIG. 100  is a graph showing the ability of a contrast agent comprising D5 to detect early anti-angiogenic therapeutic effect of treatment with a therapeutic agent. Imax, LPO and area of a tumor in a third rat are shown. 
         FIG. 101  is a graph showing the Imax, LPO and area of a tumor in a fourth rat which did not receive a therapeutic agent. 
         FIG. 102  is a graph showing variation in echographic parameters 48 hours after sunitinib treatment in the rats referenced in  FIGS. 98, 99 and 100 . 
         FIG. 103  provides immunohistochemistry images of a mammary tumor in the rat referenced in  FIG. 98 . 
         FIG. 104  provides immunohistochemistry images of a mammary tumor in the rat referenced in  FIG. 101 . 
         FIG. 105  illustrates a method for the production of a monomeric peptide phospholipid conjugate (1) from a linear peptide monomer (2). 
         FIG. 106  illustrates a monomeric peptide phospholipid conjugate (1) including a peptide with high binding affinity for KDR. 
         FIG. 107  illustrates a method for the production of a precursor dimer peptide (16) from peptide monomers. 
         FIG. 108  illustrates a method for the conjugation of the precursor dimer peptide shown in  FIG. 105  to DSPE-PEG2000-NH 2  to form a dimeric peptide phospholipid conjugate (11) containing peptides which bind with high affinity to KDR. 
         FIG. 109  illustrates a dimeric peptide-phospholipid conjugate (11) containing peptides which bind with high affinity to KDR. 
         FIG. 110  illustrates a method for the production of dimer peptide-phospholipid conjugates (such as (21)) having minimal levels of TFA. 
         FIG. 111  illustrates another method for the production of dimer peptide-phospholipid conjugates (such as (21)) having minimal levels of TFA. 
         FIG. 112  illustrates another method for the production of dimer peptide-phospholipid conjugates having minimal levels of TFA. 
         FIG. 113  illustrates another representative monomeric peptide (32) having a high binding affinity for KDR. 
         FIG. 114  illustrates another monomeric peptide-phospholipid conjugate (31) which includes the monomeric peptide shown in  FIG. 113 . 
         FIGS. 115A-C  show images obtained by using the dimer peptide-phospholipid conjugate (11) (shown in  FIG. 109 ) in a contrast agent at: 1) baseline ( FIG. 115A ); 2) after 25 minutes ( FIG. 115B ); and 3) after subtraction of the baseline and free circulating bubbles ( FIG. 115C ). 
         FIGS. 116A-C  show images obtained by using the monomeric phospholipid peptide conjugate (1) (shown in  FIG. 106 ) in a contrast agent at baseline ( FIG. 116A ); after 25 minutes ( FIG. 116B ); and after subtraction of the baseline and free circulating bubbles ( FIG. 116C ). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A description of preferred embodiments of the invention follows. 
     While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 
     Definitions 
     In the following sections, 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 that 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. 
     The term “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. The terms “bacteriophage” and “phage” are used herein interchangeably. 
     The term “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—). The term “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. 
     The term “binding polypeptide” as used herein 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. Specific examples of 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 incorporating such peptides. Also included within the definition of KDR and VEGF/KDR complex binding polypeptides are polypeptides that are modified or optimized as disclosed herein. 
     Specific examples of such modifications are discussed in detail infra, but 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 purpose 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 peptides according to this invention on solid supports or for attachment of fluorescent dyes; pharmacokinetic modifications, structural modifications to retain structural features, formation of salts to increase water solubility or ease of formulation, and the like. 
     In addition to the detectable labels described further herein, other 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. Moreover, dimers or multimers of one or more KDR or VEGF/KDR binding polypeptides can 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. 
     “Homologues” of the binding polypeptides described herein can 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. The term “homologous”, as used herein, refers to the degree of sequence similarity between two polymers (i.e., polypeptide molecules or nucleic acid molecules). Where the same nucleotide or amino acid residue or one with substantially similar properties (i.e., a conservative substitution) occupies a sequence position in the two polymers under comparison, then the 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. 
     The term “binding” refers to the determination by standard assays, including those described herein, that a binding polypeptide recognizes and binds reversibly to a given target. Such standard assays include, but are not limited to equilibrium dialysis, gel filtration, and the monitoring of spectroscopic changes that result from binding. 
     The term “specificity” refers to a binding polypeptide having a higher binding affinity for one target over another. The term “KDR specificity” refers to a KDR binding moiety having a higher affinity for KDR than for an irrelevant target. The term “VEGF/KDR specificity” refers to a VEGF/KDR complex binding moiety having a higher affinity for a VEGF/KDR complex than for another given target. Binding specificity can 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 10 μM, more preferably less than 1.0 μM, most preferably less than 0.5 μM or even lower. 
     The term “patient” as used herein refers to any mammal, especially humans. 
     The term “pharmaceutically acceptable” carrier or excipient refers to a non-toxic carrier or excipient that can be administered to a patient, together with a compound of this invention, such that it does not destroy the biological or pharmacological activity thereof. The following common abbreviations are used throughout this specification: 9-fluorenylmethyloxycarbonyl (fmoc or Fmoc), 1-hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), acetic anhydride (Ac 2 O), (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), trifluoroacetic acid (TFA), Reagent B (TFA:H 2 O:phenol:triisopropylsilane, 88:5:5:2), N,N-diisopropylethylamine (DIEA), O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorphosphate (HATU), N-hydroxysuccinimide (NHS), solid phase peptide synthesis (SPPS), dimethyl sulfoxide (DMSO), dichloromethane (DCM), dimethylformamide (DMF), and N-methylpyrrolidinone (NMP). 
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides novel binding moieties that bind KDR or a complex of VEGF and KDR. Such 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. In particular, the binding polypeptides of this invention, when appropriately labeled, are useful for detecting, imaging and localizing tumor-induced angiogenesis. Thus, 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. 
     In addition, the binding polypeptides can themselves be used as therapeutic agents. Specific 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 transformed to express an exogenous peptide on their surface. In 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., 5×10 9 ) of potential binders can be tested and successful binders isolated in a short period of time. 
     In order to prepare a phage library of displaying polypeptides to screen for binding polypeptides such as KDR or VEGF/KDR complex binding polypeptides, 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. In 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 purpose 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). 
     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. In 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 Laboratory Manual  (Academic Press, Inc., San Diego, 1996) and U.S. Pat. No. 5,223,409 (Ladner et al.), incorporated herein by reference. 
     In isolating the specific polypeptides according to this invention, seven cyclic peptide (or “loop”) libraries, designated TN6/VI, TN7/IV, TN8/IX, TN9/IV, TN10/IX, TN12/I, and MTN13/I, and a linear library, designated Lin20, were used. Each library was constructed for expression of diversified polypeptides on M13 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 M13 phage, at the amino terminus of protein III. The libraries are designated TN6/VI (having a potential 3.3×10 12  amino acid sequence diversity), TN7/IV (having a potential 1.2×10 14  amino acid sequence diversity), TN8/IX (having a potential 2.2×10 15  amino acid sequence diversity), TN9/IV (having a potential 4.2×10 16  amino acid sequence diversity), TN10/IX (having a potential 3.0×10 16  amino acid sequence diversity), TN12/I (having a sequence diversity of 4.6×10 19 ), MTN13/I (having a potential 8.0×10 17  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 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Cys-Xaa 10 -Xaa 11 -Xaa 12 . 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 (Ile), 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 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Cys-Xaa 11 -Xaa 12 -Xaa 13 . 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/IX 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 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Cys-Xaa 12 -Xaa 13 -Xaa 14 . 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/IV library utilized a template sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Cys-Xaa 13 -Xaa 14 -Xaa 15 . 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 TN10/IX library utilized a template sequence Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Xaa 12 -Cys-Xaa 14 -Xaa 1s -Xaa 16 . 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 TN12/I 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 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Xaa 9 -Xaa 10 -Xaa 11 -Xaa 12 -Xaa 13 -Xaa 14 -Cys-Xaa 16 -Xaa r -Xaa 18 . 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 permit 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 Xaa 1 -Xaa 2 -Xaa 3 -Cys-Xaa 5 -Xaa 6 -Xaa 7 -Xaa 8 -Ser-Gly-Pro-Xaa 12 -Xaa 13 -Xaa 14 -Xaa 15 -Cys-Xaa r -Xaa 18 -Xaa 19  (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). 
     The 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. For example, 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 sequence, are also encompassed by the present invention. In 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. Furthermore, 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 of 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 that, when the domain is in its most stable conformation, 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. 
     As indicated previously, the techniques discussed in Kay et al.,  Phage Display of Peptides and Proteins: A Laboratory Manual  (Academic Press, Inc., San Diego, 1996) and U.S. Pat. No. 5,223,409 are particularly useful in preparing a library of potential binders corresponding to the selected parental template. The seven libraries discussed above were prepared according to such techniques, and they were screened for KDR or VEGF/KDR complex binding polypeptides against an immobilized target, as explained in the examples to follow. 
     In a typical screen, a phage library is contacted with and allowed to bind the target, or a particular subcomponent thereof. To facilitate separation of binders and non-binders, it is convenient to immobilize the target on a solid support. 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 extreme pH (pH 2 or pH 10), changing the ionic strength of the buffer, adding denaturants, or other known means. To isolate the binding phage exhibiting the polypeptides of the present invention, 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 still bound to substrate for 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. After a few rounds of selection, 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. When the selection process works, 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. An increase in the number of phage recovered at each round of selection, and of course, the recovery of closely related sequences are good indications that convergence of the library has occurred in a screen. After a set of binding polypeptides is identified, the sequence information may be used to design other secondary phage libraries, biased for members having additional desired properties. 
     Formation of the disulfide binding loop is advantageous because it leads to increased affinity and specificity for such peptides. However, in serum, the disulfide bond might be opened by free cysteines or other thiol-containing molecules. Thus, it may be useful to modify the cysteine residues to replace the disulfide cross-link with another less reactive linkage. The —CH 2 —S—S—CH 2 — cross-link has a preferred 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. Suitable such alternative cross-links include thioether linkages such as —CH 2 —S—CH 2 —CH 2 —, —CH 2 —CH 2 —S—CH 2 —, —CH 2 —CH 2 —S—CH 2 —CH 2 —; lactam linkages such as —CH 2 —NH—CO—CH 2 — and —CH 2 —CO—NH—CH 2 —; ether linkages such as —CH 2 —CH 2 —O—CH 2 —CH 2 —; alkylene bridges such as —(CH 2 ) n — (where n=4, 5, or 6); the linkage —CH 2 —NH—CO—NH—CH 2 —, and similar groups known in the art. 
     Although polypeptides containing a stable disulfide-linked binding loop are most preferred, linear polypeptides derived from the foregoing sequences may be 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. In 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 may be accomplished using conventional techniques, including solid-phase peptide synthesis, solution-phase synthesis, etc. Solid-phase synthesis is preferred. 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), incorporated 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, Inc., Hopkinton, Mass.). 
     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. For purification purposes, 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 18 -silica. 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 preferred. 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. 
     In order to ensure that the peptide obtained using any of the techniques described above is the desired peptide for use in compositions of the present invention, 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. Alternatively, 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. Such procedures are within the capability of those skilled in the art (see Davis et al.,  Basic Methods in Molecular Biology , (1986)), incorporated by reference. Recombinant production of short peptides such as those described herein may not be practical in comparison to direct synthesis, however recombinant means of production may be very advantageous where a KDR or VEGF/KDR complex binding moiety of this invention is incorporated in a hybrid polypeptide or fusion protein. 
     In the practice of the present invention, 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. The 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:
 
[Bound]= N ×[Free]/((1 /K   a )+[Free]).
 
     A solution of the data to this equation yields the association constant, 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 (μM), 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 μM, less than 5 μM, less than 10 μM, less than 20 μM, less than 40 μM, less than 60 μM, and less than 80 μM. 
     Where KDR or VEGF/KDR complex binding moieties are employed as imaging agents, other aspects of binding specificity may become more important. 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 may be easily performed using several methods known in the art, such as fiber optic fluorimetry (see, e.g., Anderson &amp; Miller,  Clin. Chem.,  34(7):1417-21 (1988)), surface plasmon resonance (see, Malmborg et al.,  J. Immunol. Methods,  198(1):51-7 (1996) and Schuck, Current Opinion in  Biotechnology,  8:498-502 (1997)), resonant mirror, and grating coupled planar waveguiding (see, e.g., Hutchinson,  Molec. Biotechnology,  3:47-54 (1995)). Automated biosensors are commercially available for measuring binding kinetics: BIAcore surface plasmon resonance sensor (Biacore AB, Uppsala SE), IAsys resonant mirror sensor (Fisons Applied Sensor Technology, Cambridge GB), BIOS-1 grated coupled planar waveguiding sensor (Artificial Sensor Instruments, Zurich CH). 
     Methods of Screening Polypeptides Identified by Phage Display for their Ability to Bind to Cells Expressing the Target: 
     In 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 that 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. However, ascertaining that a particular phage-identified peptide binds to cells that express the target (and does not bind to cells that do not) is a critical piece of information in identifying binding peptides that are potential in vivo targeting moieties. The method takes advantage of the increase in affinity and avidity associated with multivalent binding and permits 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. For example, 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. For example, to detect binding the avidin, streptavidin, or neutravidin may be conjugated to a detectable marker (e.g., a radioactive label, a fluorescent label, or an enzymatic label that undergoes a color change, such as HRP (horse radish peroxidase), TMB (tetramethyl benzidine) or alkaline phosphatase). 
     The biotinylated peptides are preferably complexed with neutravidin-HRP. Neutravidin exhibits lower non-specific binding to molecules than the other alternatives due to the absence of lectin binding carbohydrate moieties and cell adhesion receptor-binding RYD domain in neutravidin. See, Hiller et al.,  Biochem. J.,  248:167-171 (1987); Alon et al.,  Biochem. Biophys. Res. Commun.,  170:1236-41 (1990). 
     The tetrameric constructs can be screened against cells that naturally express the target or cells that have been engineered via recombinant DNA technologies to express the target (e.g., transfectants, transformants, etc.). If cells that 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. 
     The tetrameric complexes may optionally be screened in the presence of serum. Thus, 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 contructs that 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. Furthermore, it has now been found that affinity and avidity of a targeting construct may be increased by inclusion of two or more targeting moieties that bind to different epitopes on the same target. The screening methods described herein are useful in identifying combinations of binding polypeptides that may have increased affinity when included in such multimeric constructs. 
     In a preferred embodiment, 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 that 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). 
     As shown in Example 5, the assay can be used to identify KDR binding polypeptides that bind specifically to KDR-expressing cells (and do not bind to cells that do not express KDR) even when the monodentate K D  of the polypeptide is on the order of 200 nM-300 nM. 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 (e.g., 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 that 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. 
     Modification or Optimization of KDR and VEGF/KDR Complex Binding Polypeptides 
     As discussed, 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”. Specifically, 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. 
     Substitution of Amino Acid Residues 
     For example, one can make the following isosteric and/or conservative amino acid changes in the parent polypeptide sequence with the expectation that the resulting polypeptides would have a similar or improved profile of the properties described above: 
     Substitution of 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 C1-10 carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions. 
     Substitution of aromatic-substituted hydrophobic amino acids: Including phenylalanine, tryptophan, tyrosine, biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 2-benzothienylalanine, 3-benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy (from C1-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′-, 3′-, or 4′-amino-, 2′-, 3′-, or 4′-chloro-, 2,3, or 4-biphenylalanine, 2′,-3′,- or 4′-methyl-2, 3 or 4-biphenylalanine, and 2- or 3-pyridylalanine 
     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. Compounds that serve as illustrative examples include: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-gamma,gamma′-diethyl-homoarginine. Included also are compounds such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl ornithine where alkyl group occupies the pro-R position of the alpha carbon. Also included are the 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) and lysine, ornithine, or 2,3-diaminopropionic acid. 
     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. 
     Substitution of hydroxyl containing amino acids: Including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine or threonine. It is also understood that the amino acids within each of the categories listed above may be substituted for another of the same group. 
     Substitution of Amide Bonds 
     Another type of modification within the scope of the patent is to substitute the amide bonds within the backbone of the polypeptide. For example, to reduce or eliminate undesired proteolysis, or other degradation pathways that diminish serum stability, resulting in reduced or abolished bioactivity, or to restrict or increase conformational flexibility, 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 pharmacokinetic behavior. It is understood that those knowledgeable in the art of peptide synthesis can make the following amide bond-changes for any amide bond connecting two amino acids with the expectation that the resulting peptides could have the same or improved activity: insertion of alpha-N-methylamides or peptide amide backbone thioamides, removal of the carbonyl to produce the cognate secondary amines, replacement of one amino acid with an aza-aminoacid to produce semicarbazone derivatives, and use of E-olefins and substituted E-olefins as amide bond surrogates. 
     Introduction of D-Amino Acids 
     Another approach within the scope of the patent is the introduction of D-alanine, or another D-amino acid, distal or proximal to the labile peptide bond. In this case it is also understood to those skilled in the art that such 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. 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 that has moieties of different charge, hydrophobicity, steric requirements etc. than that serviced by the side chain of the replaced L-amino acid. 
     Modifications to Improve Pharmacokinetic or Pharmacodynamic Properties 
     It is also understood that use of the KDR or VEGF/KDR complex binding polypeptide in a particular application may necessitate modifications of the peptide or formulations of the peptide to improve pharmacokinetic and pharmacodynamic 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. 
     Glycosylation of Amino Acid Residues 
     Yet another modification within the scope of the invention is to employ glycosylated amino acid residues (e.g., serine, threonine or asparagine residues), singly or in combination in the either the binding moiety (or moieties) or the linker moiety or both. Glycosylation, which may be carried out using standard conditions, can be used to enhance solubility, alter pharmacokinetics and pharmacodynamics or to enhance binding via a specific or non-specific interaction involving the glycosidic moiety. In another approach glycosylated amino acids such as O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl) serine or the analogous threonine derivative (either the D- or L-amino acids) can be incorporated into the peptide during manual or automated solid phase peptide synthesis, or in manual or automated solution phase peptide synthesis. Similarly D- or L-N γ -(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β-D-glucopyranosyl)-asparagine can be employed. The use of amino acids glycosylated on a pendant oxygen, nitrogen or sulfur function by the agency of suitably functionalized and activated carbohydrate moieties that can be employed in glycosylation is anticipated. Such carbohydrate functions could be monosaccharides, disaccharides or even larger assemblies of oligosaccharides (Kihlberg, January (2000) Glycopeptide synthesis. In: Fmoc Solid Phase Peptide Synthesis—A Practical Approach (Chan, W. C. and White, P. D. Eds) Oxford University Press, New York, N.Y. Chap. 8, pp 195-213). 
     Also anticipated is the appendage of carbohydrate functions to amino acids by means other than glycosylation via activation of a leaving group at the anomeric carbon. 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. 
     Formation of Salts 
     It is also within the scope of the invention to form different salts that may increase the water solubility or the ease of formulation of these peptides. These may include, but are not restricted to, N-methylglucamine (meglumine), acetate, oxalates, ascorbates, etc. 
     Structural Modifications that Retain Structural Features 
     Yet another modification within the scope of the invention is truncation of cyclic polypeptides. The cyclic nature of many polypeptides of the invention limits the conformational space available to the peptide sequence, particularly within the cycle. Therefore truncation of the peptide by one or more residues distal or even proximal to the cycle, at either the N-terminal or C-terminal region may provide truncated peptides with similar or improved biological activity. A unique sequence of amino acids, even as small as three amino acids, which is responsible for the binding activity, may be identified, as noted for RGD peptides (see, e.g., Plow et al.,  Blood,  70(1): 110-5 (1987); Oldberg et al.,  Journal of Biological Chemistry,  263(36):19433-19436 (1988); Taub et al.,  Journal of Biological Chemistry,  264(1):259-65 (1989); Andrieux et al.,  Journal of Biological Chemistry,  264(16):9258-65 (1989); and U.S. Pat. No. 5,773,412 and U.S. Pat. No. 5,759,996, each of which is incorporated herein by reference). 
     It has also been shown in the literature that large peptide cycles can be substantially shortened, eliminating extraneous amino acids, but substantially including the critical binding residues. See, U.S. Pat. No. 5,556,939, incorporated by reference herein. 
     The shortened cyclic peptides can be formed using disulfide bonds or amide bonds of suitably located carboxylic acid groups and amino groups. 
     Furthermore, D-amino acids can be added to the peptide sequence to stabilize turn features (especially in the case of glycine). In another approach alpha, beta, gamma or delta dipeptide or turn mimics (such as α, β, γ, or δ 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 : Peptidomimetics Protocols , W. M. Kazmierski, Ed. (Humana Press Inc., Totowa, N.J., 1999), Chapter 10, pp. 161-174; structure 3: WO 01/16135). 
     Substitution of 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. 
     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 incorporate  99m Tc via routes that are reliant upon the reduction of pertechnetate ion and subsequent incorporation 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. Accordingly, another modification within the scope of the invention is to substitute the disulfide moiety with mimetics utilizing the methods disclosed herein or known to those skilled in the art, while retaining the activity and other desired properties of the KDR-binding polypeptides of the invention: 
     1) Oxime Linker 
     The oxime moiety has been employed as a linker by investigators in a number of contexts. Of the most interest is the work by Mutter et al. (Wahl and Mutter,  Tetrahedron Lett.,  37:6861-6864 (1996)). The amino acids 4, containing an aminoalcohol function, and 5, containing an alkoxyamino function, are incorporated into the peptide chain, not necessarily at the end of the peptide chain ( FIG. 27 ). After formation of the peptide the sidechain protecting groups are removed. The aldehyde group is unmasked and an oxime linkage is formed. 
     2) Lanthionine Linker 
     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. 
     Preparation of Lanthionines using Bromoacetylated Peptides 
     Lanthionines are readily prepared using known methods. See, for example, Robey et al.,  Anal. Biochem.,  177:373-377 (1989); Inman et al.,  Bioconjugate Chem.,  2:458-463 (1991); Ploinsky et al.,  Med. Chem.,  35:4185-4194 (1992); Mayer et al., “Peptides, Frontiers of Peptide Science”, in Proceedings of the 15 th  American Peptide Symposium, Tam &amp; Kaumaya (Eds.), Jun. 14-19, 1995, Nashville, Term. (Klumer Academic Pub., Boston), pp. 291-292; Wakao et al.,  Jpn. Kokai Tokyo Koho , JP 07300452 A2 (1995). Preparation of peptides using Boc automated peptide synthesis followed by coupling the peptide terminus with bromoacetic acid gives bromoacetylated peptides in good yield. Cleavage and deprotection of the peptides is accomplished using HF/anisole. If the peptide contains a cysteine group its reactivity can be controlled with low pH. If the pH of the medium is raised to 6-7 then either polymerization or cyclization of the peptide takes place. Polymerization is favored at high (100 mg/mL) concentration whereas cyclization is favored at lower concentrations (1 mg/mL), e.g., 6 cyclizes to 7 (Scheme 1;  FIG. 28 ). 
     Inman et al. demonstrated the use of N α -(Boc)-N ε -[N-(bromoacetyl)-β-alanyl]-lysine as a carrier of the bromoacetyl group that could be employed in Boc peptide synthesis thus allowing placement of a bromoacetyl bearing moiety anywhere in a sequence. In preliminary experiments they found that peptides with 4-6 amino acids separating the bromoacetyl-lysine derivative from a cysteine tend to cyclize, indicating the potential utility of this strategy. 
     Preparation of Lanthionines via Cysteine Thiol Addition to Acrylamides 
     Several variants of this strategy may be implemented. 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, Tam &amp; Kaumaya (Eds.), Jun. 14-19, 1995, Nashville, Term. (Klumer Academic Pub., Boston), pp. 291-292). Conjugate addition of thiols to acrylamides has also been amply demonstrated and a reference to the addition of 2-mercaptoethanol to acrylamide is provided (Wakao et al.,  Jpn. Kokai Tokyo Koho , JP 07300452 A2 (1995)). 
     3) 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.,  39:2941-2944 (1998). In the case of N-protected tyrosine derivatives as the phenol component the yields were also as high as 98%. This demonstrates that amino acid amides (peptides) are expected to be stable to the transformation and that yields are high. Precedent for an intramolecular reaction exists in view of the facile intramolecular cyclizations of peptides to lactams, intramolecular biaryl ether formation based on the S N Ar reaction and the generality of intramolecular cyclization reactions under high dilution conditions or on resin, wherein the pseudo-dilution effect mimics high dilution conditions. 
     4) Formation of Cyclic Peptides with a Thiazolidine Linkage Via Intramolecular Reaction of Peptide Aldehydes with Cysteine Moieties 
     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 that 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. In some cases 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.,  118:10018-10034 (1996). 
     5) 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. 6, pp. 169-178. 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. Alternatively, 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  FIG. 30 . 
     Thus, 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 (˜0.1-0.2 mmol/g). Deprotection of both functions with hydrazine is then followed by on-resin cyclization to give the desired products. 
     In the allyl approach, shown in  FIG. 31 , the pendant carboxyl that is to undergo cyclization is protected as an allyl ester and the pendant amino group is protected as an alloc group. On resin, both are selectively unmasked by treatment with palladium tris-triphenylphosphine in the presence of N-methylmorpholine and acetic acid in DMF. Residual palladium salts are removed using sodium diethyldithiocarbamate in the presence of DIEA in DMF, followed by subsequent washings with DMF. The lactam ring is then formed employing HATU/HOAt in the presence of N-methylmorpholine. Other coupling agents can be employed as described above. The processing of the peptide is then carried out as described above to provide the desired peptide lactam. 
     Subsequently cleavage from resin and purification can also be carried out. For functionalization of the N-terminus of the peptide, it is understood that 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. 
     Thus, 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 (˜0.1-0.2 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. For functionalization of the N-terminus of the peptide it is understood that 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. 
     6) Cyclic Peptides Based on Olefin Metathesis 
     The Grubbs reaction ( FIG. 32 ) 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). 
     It is readily seen ( FIG. 32 ) that if the starting material is a diolefin (1) that the resulting product will be cyclic compound 17. The reaction has in fact been applied to creation of cycles from olefin-functionalized peptides. See, e.g., Pernerstorfer et al.,  Chem. Commun.,  20:1949-50 (1997); see, also, Covalent capture and stabilization of cylindrical 13-sheet peptide assemblies, Clark et al.,  Chem. Eur. J.,  5(2):782-792 (1999); Highly efficient synthesis of covalently cross-linked peptide helices by ring-closing metathesis, Blackwell et al.,  Angew. Chem., Int. Ed.,  37(23):3281-3284 (1998); Synthesis of novel cyclic protease inhibitors using Grubbs olefin metathesis, Ripka et al.,  Med. Chem. Lett.,  8(4):357-360 (1998); Application of Ring-Closing Metathesis to the Synthesis of Rigidified Amino Acids and Peptides, Miller et al.,  J. Am. Chem. Soc.,  118(40):9606-9614 (1996); Supramolecular Design by Covalent Capture, Design of a Peptide Cylinder via Hydrogen-Bond-Promoted Intermolecular Olefin Metathesis, Clark et al.,  J. Am. Chem. Soc.,  117(49):12364-12365 (1995); Synthesis of Conformationally Restricted Amino Acids and Peptides Employing Olefin Metathesis, Miller et al.,  J. Am. Chem. Soc.,  117(21):5855-5856 (1995). One can prepare either C-allylated amino acids or possibly N-allylated amino acids and employ them in this reaction in order to prepare carba-bridged cyclic peptides as surrogates for disulfide bond containing peptides. 
     One may also prepare novel compounds with olefinic groups. Functionalization of the tyrosine hydroxyl with an olefin-containing tether is one option. The lysine ε-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 olefin-containing tether can also be varied in order to explore structure activity relationships. 
     Manipulation of Peptide Sequences 
     Other modifications within the scope of the invention include common manipulations of peptide sequences, which can be expected to yield peptides with similar or improved biological properties. These include amino acid translocations (swapping amino acids in the sequence), use of retroinverso peptides in place of the original sequence or a modified original sequence, peptoids and retro-inverso peptoid sequences. Structures wherein specific residues are peptoid instead of peptidic, which result in hybrid molecules, neither completely peptidic nor completely peptoid, are anticipated as well. 
     Linkers 
     Additional modifications within the scope of the invention include introduction of 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. Furthermore, linkers that 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. 
     Multimeric Constructs of KDR and VEGF/KDR Complex Binding 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. Indeed, 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 construct. Moreover, as shown in Example 4 infra, these constructs can 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 nucleophilic 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 aminoacetylamide (from chloro- or bromoacetamide derivatives, but is not so limited. For example, 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. Modified polypeptides and peptide-derived molecules are shown, for example, in  FIGS. 79A-79G . 
     Method A 
     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). Separately, using standard methods known in the art of peptide synthesis, 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. 77-183; Barlos et al., “Convergent Peptide Synthesis” in  Fmoc Solid Phase Peptide Synthesis , Chan, W. C. and White, P. D., Eds. (Oxford University Press, New York, 2000), Chapt. 9, pp. 215-228. Liberation of this from the 2-chlorotrityl resin without removal of the side-chain protecting groups, activation of the carboxyl group and coupling to any amine-functionalized labeling group provides a di-lysine derivative whose protected pendant nitrogen atoms may be unmasked to give two free amino groups. 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 structure. An excess of the safety-catch resin-bound peptide can be employed to insure complete reaction of the amino groups of the di-lysine construct. 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. 
     The synthesis of dimeric and multimeric constructs wherein two or more KDR-binding peptides are present in one construct 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) can be employed to distinguish the pendant nitrogen atoms of the di-lysine derivatives described above. 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 Synthesis , Chan, W. C. and White, P. D., Eds. (Oxford University Press, New York, 2000), Chapt. 6, pp. 169-176. The resulting product may be reacted with a second safety-catch resin bearing another KDR-binding peptide to provide a fully-protected homodimeric construct, which after removal of protecting groups with trifluoroacetic acid, provides the desired material. 
     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 construct that may possess an additional nucleophilic group such as the ε-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 (Tyger Scientific, Inc.). The resulting monoamidated, mono-N-hydroxysuccinimidyl ester of glutaric acid is then treated with an additional equivalent of the same peptide, or an equivalent of a different KDR-binding peptide. Purification of the resulting material by HPLC affords the desired homodimeric construct bearing a suitable labeling group. 
     Method C 
     A modular scheme can be employed to prepare dimeric or higher multimeric constructs bearing suitable labeling groups as defined above. In a simple illustration, fmoc-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. After thorough washing, 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 resin. Subsequent successive displacement of the remaining chlorine atoms by two equivalents of a KDR-binding peptide provides a resin-bound homo-dimeric labeling group-functionalized construct. Falorni et al.,  Tetrahedron Lett.,  39(41):7607-7610 (1998); Johnson et al.,  Tetrahedron Lett.,  54(16):4097-4106 (1998); Stankova et al.,  Mol. Diversity,  2(1/2):75-80 (1996). The incoming peptides may be protected or unprotected as the situation warrants. 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. 
     It is understood that in each of these methods 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 incorporated by reference herein in their entirety. 
     Uses for KDR or VEGF/KDR Complex Binding Polypeptides: 
     The KDR or VEGF/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. 
     In vitro: 
     For detection of KDR or VEGF/KDR complex in solution, 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. As an example, 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. 
     Alternatively, 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. 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. 
     For detection or purification of soluble KDR or VEGF/KDR complex in or from a solution, 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 VEGF/KDR complex target may be released from the binding moiety at appropriate elution conditions. 
     The biology of angiogenesis and the roles of VEGF and KDR in initiating and maintaining it have been investigated by many researchers and continues to be an active field for research and development. In 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 purpose, using the general purification methodology described above. 
     In Vivo: 
     Diagnostic Imaging 
     A particularly preferred 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. Preferably, 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. For example, 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. 
     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. 
     In general, 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&#39;s body. For example, when 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 and accumulate 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 that will vary according to the type of label used, and the signal is then converted to an image of the site of angiogenesis. 
     In another embodiment, rather than directly labeling a KDR or VEGF/KDR complex binding polypeptide with a detectable label or radiotherapeutic construct, 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. For example, one or more KDR-binding peptides can be conjugated to streptavidin (potentially generating multivalent binding) for in vivo binding to KDR-expressing cells. After the unbound targeting construct has cleared from the body, a biotinylated detectable label or radiotherapeutic construct (e.g., a chelate molecule complexed with a radioactive metal) can be infused and 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 construct 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-marrow, kidneys, and liver is decreased. This approach, sometimes referred to as pre-targeting or two-step, or three-step approaches was reviewed by S.F. Rosebrough in  Q. J. Nucl. Med.,  40:234-251 (1996), which is incorporated by reference herein. 
     The present invention also includes methods for detecting, monitoring and/or evaluating therapeutic responses by imaging following administration of one or more KDR binding moieties described herein conjugated to a detectable label. Such methods are especially useful for monitoring the therapeutic response of disorders related to angiogenesis and/or hyperproliferative diseases, especially diseases associated with endothelial cell hyperproliferation, such as cancer, particularly prostate cancer, mammary cancer, ovarian cancer, liver cancer, colon cancer, renal cancer, bone cancer, bladder cancer, pancreatic cancer, lung cancer, uterine cancer and testicular cancer. 
     Such methods may include the steps of (a) administering a contrast agent comprising one or more KDR-binding moieties conjugated to a detectable label to a subject with cancer or another disease related to angiogenesis or cell (particularly endothelial cell) hyperproliferation; (b) obtaining an initial image of the disease; (c) administering a therapeutic agent appropriate for the treatment of the disease; (d) obtaining a subsequent image of the hyperproliferative disease; and (e) comparing the initial and the subsequent images to evaluate the effectiveness of the therapy. 
     In certain embodiments the subject is a mammal. In certain embodiments the subject is a human, and the disease is cancer. The therapeutic agent is an agent appropriate to treat a disorder associated with angiogesis or cell hyperproliferation. In one embodiment the therapeutic agent is an anti-cancer agent. Such agents are known to those skilled in the art and are discussed infra. In another embodiment, the anti-cancer treatment may include treatment of the patient with radiotherapy, RF ablation or focused ultrasound. 
     The therapeutic response may be any type of therapeutic response known to those of ordinary skill in the art. In certain embodiments, the therapeutic response is reduction in tumor size and/or vascularity. Thus, the present invention provides medical personnel with the ability to adjust treatment strategies based on the adequacy of the therapeutic response. If the therapeutic response is insufficient, a different therapeutic agent or a higher dose of the same therapeutic agent may be warranted. 
     A. Magnetic Resonance Imaging (MRI) 
     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 that have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1.7 Bohr magneton. Preferred paramagnetic metals include, but are not limited to, chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III) and ytterbium (III), chromium (III), iron (III), and gadolinium (III). The trivalent cation, Gd 3+ , is particularly preferred 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(III) 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 superparamagnetic 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). Generally, 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, carboxyethylidene groups, or carboxymethylene groups. Examples of chelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclo-tetradecane-1,4,7,10-tetraacetic acid (DOTA), 1-substituted 1,4,7,-tricarboxymethyl-1,4,7,10-teraazacyclododecane (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). 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 DTPA; 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 three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is 1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid); derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and 1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). A preferred chelator for use in the present invention is DTPA, and the use of DO3A is particularly preferred. 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 U.S. Pat. No. 4,899,755, U.S. Pat. No. 5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6,143,274, all of which are hereby incorporated by reference. 
     In accordance with the present invention, 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. Preferably, 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. In preferred embodiments, a chelator having a free central carboxylic acid group (e.g., DTPA-Asp(β-COOH)—)OtBu) makes it easy to attach at the N-terminus of the peptide by formation of an amide bond. The chelate(s) can also be attached at the C-terminus with the aid of a linker. Alternatively, isothiocyanate conjugation chemistry can be employed as a way of linking the appropriate isothiocyanate group bearing DTPA to a free amino group anywhere within the peptide sequence. 
     In 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 of a single amino acid or different amino acids (e.g., extensions of the N- or C-terminus of the KDR or VEGF/KDR complex binding moiety); derivatized or underivatized polyethylene glycol, polyoxyethylene, or polyvinylpyridine chains; substituted or unsubstituted polyimide chains; derivatized or underivatized polyamine, polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol), polyglycerol, or oligosaccharide (e.g., dextran) chains; alternating block copolymers; malonic, succinic, glutaric, adipic and pimelic acids; caproic acid; simple diamines and dialcohols; any of the other linkers disclosed herein; or any other simple polymeric linkers known in the art (see, e.g., WO 98/18497, WO 98/18496). Preferably 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. Preferably the molecular weight of the linker is less than 100. In addition, it may be desirable to utilize a linker that is biodegradable in vivo to provide efficient routes of excretion for the imaging reagents of the present invention. Depending on their location within the linker, such biodegradable functionalities can include ester, double ester, amide, phosphoester, ether, acetal, and ketal functionalities. 
     In general, known methods can be used to couple the metal chelate(s) and the KDR or VEGF/KDR complex binding moiety using linkers. See, e.g., WO 95/28967, WO 98/18496, WO 98/18497 and discussion therein. The KDR or 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. Similarly, 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. When imaging a site of angiogenesis, certain MR techniques and pulse sequences may be preferred 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)). These methods also include flow independent techniques that enhance the difference in contrast, such as inversion-recovery prepared or saturation-recovery prepared sequences that will increase the contrast between angiogenic tumor and background tissues. Finally, magnetization transfer preparations may also improve contrast with these agents (see, e.g., Goodrich et al.,  Investigative Radiology,  31(6): 323-32 (1996)). 
     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. For imaging active angiogenesis, intravenous or intraarterial administration is preferred. For MRI, it is contemplated that the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the site of angiogenesis at least 10%. After injection with the KDR or VEGF/KDR complex binding moiety-containing MRI reagent, the patient is scanned in the MRI machine to determine the location of any sites of angiogenesis. In therapeutic settings, upon angiogenesis (e.g., tumor) localization, a tumorcidal agent or anti-angiogenic agent (e.g., inhibitors of VEGF) can be immediately administered, if necessary, and the patient can be subsequently scanned to visualize tumor regression or arrest of angiogenesis. 
     B. Ultrasound Imaging 
     When ultrasound is transmitted through a substance, the acoustic properties of the substance will depend upon the velocity of the transmissions and the density of the substance. Changes in the acoustic properties will be most prominent at the interface of different substances (solids, liquids, gases). 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. Because of their size, 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 superior imaging of sites of angiogenesis. 
     In this aspect of the invention, the KDR or VEGF/KDR complex binding moiety may be linked to a material that is useful for ultrasound imaging. For example, 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 that 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. See, e.g., WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18497, WO 98/18496, and WO 98/18501, incorporated herein by reference in their entirety. 
     For contrast agents comprising suspensions of stabilized microbubbles (a preferred embodiment), phospholipids, and particularly saturated phospholipids are preferred. The preferred 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, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat. No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entirety. In a preferred embodiment, at least one of the phospholipid moieties has the structure 18 or 19 ( FIG. 33 ) and described in U.S. Pat. No. 5,686,060, which is herein incorporated by reference. In ultrasound applications the contrast agents formed by phospholipid stabilized microbubbles can be administered, for example, in doses such that the amount of phospholipid injected is in the range 0.1 to 200 μg/kg body weight, preferably from about 0.1 to 30 μg/kg. 
     Examples of 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. Examples of 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 in the art as the “lyso” forms of the phospholipids. 
     Further examples of 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. As used herein, the term phospholipids includes either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures. Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins. 
     Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins. 
     Examples of synthetic phospholipids are e.g., dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”), diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoylphosphatid-ylcholine (“PSPC”), 1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”), dioleoylphosphatidylycholine (“DOPC”), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts, diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts, dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts, dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts, distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts, dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts, dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts, dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts, distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid (“DAPA”) and its alkali metal salts, dimyristoyl phosphatidyl-ethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), distearoyl phosphatidyl-ethanolamine (“DSPE”), dimyristoyl phosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin (“DPSP”), and distearoyl sphingomyelin (“DSSP”). 
     Other preferred phospholipids include dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid and dipalmitoylphosphatidylserine. The 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 10 , are preferred. 
     The preferred 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; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entirety. Most preferably, 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. In this embodiment the criteria used for selection of the hydrophilic stabilizer is its solubility in the organic solvent of choice. Examples of hydrophilic stabilizer compounds soluble in water and the organic solvent are, e.g., a polymer, like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc., malic acid, glycolic acid, maltol, and the like. Such hydrophilic compounds also aid in homogenizing the microbubbles size distribution and enhance stability under storage. 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 Cl 4 F 2 ) or 2-methyl-2-butanol. 2-methyl-2-butanol and C 2 Cl 4 F 2  are preferred. 
     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 structure has been disrupted will form lamellarized or laminarized segments that will stabilize the microbubbles of the gas dispersed therein. This method permits production of suspensions of microbubbles that 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. 
     Alternatively, 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 incorporated 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. 
     Additives known to those of ordinary skill in the art can be included in the suspensions of stabilized microbubbles. For instance, 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, U.S. Pat. No. 5,798,091, WO 97/29783, also EP 881 915, incorporated herein by reference in their entirety. These agents may be prepared as described in U.S. Pat. No. 5,798,091 or WO97/29783. 
     Another preferred ultrasound contrast agent comprises ultrasound contrast agents. The term “microballoon” refers to gas filled bodies with a material boundary or envelope. More on microballoon formulations and methods of preparation may be found in EP 324 938 (U.S. Pat. No. 4,844,882); U.S. Pat. No. 5,711,933; U.S. Pat. No. 5,840,275; U.S. Pat. No. 5,863,520; U.S. Pat. No. 6,123,922; U.S. Pat. No. 6,200,548; U.S. Pat. No. 4,900,540; U.S. Pat. No. 5,123,414; U.S. Pat. No. 5,230,882; U.S. Pat. No. 5,469,854; U.S. Pat. No. 5,585,112; U.S. Pat. No. 4,718,433; U.S. Pat. No. 4,774,958; WO 95/01187; U.S. Pat. No. 5,529,766; U.S. Pat. No. 5,536,490; and U.S. Pat. No. 5,990,263, the contents of which are incorporated herein by reference. 
     The preferred 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, U.S. Pat. No. 5,711,933, U.S. Pat. No. 5,840,275, EP 554213, U.S. Pat. No. 5,413,774 and U.S. Pat. No. 5,578,292, the entire contents of which are incorporated herein by reference. In 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 ε-caprolactone, γ-valerolactone and polypeptides. Other suitable polymers include poly(ortho)esters (see e.g., U.S. Pat. No. 4,093,709; U.S. Pat. No. 4,131,648; U.S. Pat. No. 4,138,344; U.S. Pat. No. 4,180,646); polylactic and polyglycolic acid and their copolymers, for instance DEXON (see J. Heller, Biomaterials 1 (1980), 51; poly(DL-lactide-co-ε-caprolactone), poly(DL-lactide-co-γ-valerolactone), poly(DL-lactide-co-γ-butyrolactone), polyalkylcyano-acrylates; polyamides, polyhydroxybutyrate; polydioxanone; poly-β-aminoketones (A. S. Angeloni, P. Ferruti, M. Tramontini and M. Casolaro, The Mannich bases in polymer synthesis: 3. Reduction of poly(beta-aminoketone)s to poly(gamma-aminoalcohol)s and their N-alkylation to poly(gamma-hydroxyquaternary ammonium salt)s, Polymer 23, pp 1693-1697, 1982.); polyphosphazenes (Allcock, Harry R. Polyphosphazenes: new polymers with inorganic backbone atoms (Science 193:1214-19 (1976)) and polyanhydrides. The microballoons of the present invention can also be prepared according to the methods of WO-A-96/15815, incorporated 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. Preferred lipids are di- or tri-glycerides, e.g., di- or tri-myristin, -palmityn or -stearin, in particular tripalmitin or tristearin. The microballoons may employ any of the gases disclosed herein of known to the skilled artisan; however, inert gases such as fluorinated gases are preferred. 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. 
     Other 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; U.S. Pat. No. 5,558,857; U.S. Pat. No. 5,607,661; U.S. Pat. No. 5,637,289; U.S. Pat. No. 5,558,856; U.S. Pat. No. 5,137,928; WO 95/21631 or WO 93/13809, incorporated herein by reference in their entirety. 
     Any of these ultrasound compositions should also be, as far as possible, isotonic with blood. Hence, before injection, 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. Additionally, 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. The term “gas” as used herein 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. However, fluorinated gases are preferred. Fluorinated gases include materials that 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 10 , CBrF 3 , CCl 2 F 2 , C 2 CIF 5 , and CBrClF 2 ) and perfluorocarbons. The term 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 preferred, 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 . Most preferably 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 preferred. See also WO 97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO 98/05364, WO 98/17324. 
     In certain circumstances it may be desirable to include 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”). Preferably the gas precursor and the gas it produces are physiologically acceptable. The gas precursor may be pH-activated, photo-activated, temperature activated, etc. For example, certain perfluorocarbons may be used as temperature activated gas precursors. These perfluorocarbons, such as perfluoropentane, 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. 
     As discussed, 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. 
     Since 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 (or known to those skilled in the art) 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. For example, see 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 . &amp;  Bio.,  23(6):863-870 (1997). 
     A number of methods may be used to prepare suspensions of microbubbles conjugated to KDR or VEGF/KDR complex binding polypeptides. For example, one may prepare maleimide-derivatized microbubbles by incorporating 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. Then, 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 incorporation 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 corresponding reacting moiety on the peptide to be conjugated (e.g., maleimido with the —SH group of cysteine). 
     Alternatively, KDR-binding polypeptide conjugated microbubbles may be prepared using biotin/avidin. For example, 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. 
     Unless it contains a hyperpolarized gas, known to require special storage conditions, 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. Preferably in such a case it can be supplied in the form of a two-component kit, which can include two separate containers or a dual-chamber container. In 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. In such a case the syringe used as the container of the second component is also used then for injecting the contrast agent. In the latter case, preferably 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. In both cases means for directing or permitting application of sufficient bubble forming energy into the contents of the container are provided. 
     However, as noted above, in the stabilised contrast agents according to the invention 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. 
     It can be appreciated by one of ordinary skilled in the art that other two-chamber reconstitution systems capable of combining the dried powder with the aqueous solution in a sterile manner are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed between the water-insoluble gas and the environment, to increase shelf life of the product. Where a material necessary for forming 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. 
     No specific containers, vial or connection systems are required; 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. In addition to assuring sterility, vacuum retention is essential for products stoppered at ambient or reduced pressures to assure safe and proper reconstitution. As to the stopper, it may be a compound or multicomponent formulation based on an elastomer, such as poly(isobutylene) or butyl rubber. 
     Ultrasound imaging techniques that can 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. Preferably, the ultrasound imaging is effected in a contrast-specific imaging mode so as to substantially remove, or at least reduce, the dominant (linear) contribution of tissue in the echo signals, with respect to the (non-linear) contribution of the contrast agent; examples of contrast-specific imaging modes include harmonic imaging (HI), pulse inversion (PI), power modulation (PM) and contrast pulse sequencing (CPS) techniques, as described, for example, in “Rafter et al., Imaging technologies and techniques, Cardiology Clinics 22 (2004), pp. 181-197” (the entire disclosure of which is herewith incorporated by reference). 
     In ultrasound applications 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 0.1 to 200 μg/kg body weight, preferably from about 0.1 to 30 μg/kg. Microballoons-containing contrast agents are typically administered in doses such that the amount of wall-forming polymer or lipid is from about 10 μg/kg to about 20 mg/kg of body weight. 
     As shown in the Examples, ultrasound contrast agents conjugated to KDR binding moieties of the invention, such as, for example, those comprising SEQ ID NOS:356, 294 and 480 and the dimer D23, are able to bind to KDR-expressing tissue and thus are useful in providing an image of such tissue. Indeed, compounds of the invention, such as phospholipid stabilized microbubbles conjugated to the heterodimer D23, can be used to image angiogenic tissue in vivo. 
     The present invention also includes methods for detecting, monitoring and/or evaluating therapeutic responses following administration of an ultrasound contrast agent conjugated to KDR binding moieties described herein. Such methods are especially useful for monitoring the therapeutic response of disorders related angiogenesis and/or hyperproliferative diseases, especially diseases associated with endothelial cell hyperproliferation. The disease may include inflammatory diseases, such as, for instance, rheumatoid diseases and inflammatory bowel diseases, and cancers such as prostate cancer, mammary cancer, ovarian cancer, liver cancer, colon cancer, renal cancer, bone cancer, bladder cancer, pancreatic cancer, lung cancer, uterine cancer and testicular cancer. 
     Such methods may include the steps of (a) administering an ultrasound contrast agent described above to a subject with cancer or another disease related to angiogesis or endothelial cell hyper proliferation; (b) obtaining an initial image of the disease; (c) administering a therapeutic agent appropriate for the treatment of the disease; (d) obtaining a subsequent image of the hyperproliferative disease; and (e) comparing the initial and the subsequent images to evaluate the effectiveness of the therapy. 
     In certain embodiments the ultrasound contrast agent comprises a dimer, such as D5. In certain embodiments the subject is a mammal. In certain embodiments the subject is a human, and the disease is cancer. In a preferred embodiment the disease is prostate cancer. 
     The therapeutic agent is an agent appropriate to treat the disorder. For disorders associated with angiogesis or (endothelial) cell hyperproliferation, the therapeutic agent is an anti-angiogenesis agent or an anti-hyperproliferation agent. In one embodiment the therapeutic agent is an anti-cancer agent. Such agents are known to those skilled in the art and include, for example, sunitinib, the therapeutic agents discussed infra, imatinib, sorafenib and bevacizumab. 
     The therapeutic response may be any type of therapeutic response known to those of ordinary skill in the art. In certain embodiments, the therapeutic response is reduction (or increase) in tumor size and/or vascularity. Tumor size is typically assessed by visual observation of the image (or sequence of images) obtained by subjecting the patient to a suitable imaging technique, in particular ultrasound imaging (e.g. fundamental B-mode ultrasound imaging). Vascularity can be assessed by, for example, analyzing the time-intensity curves of ultrasound signal, to determine suitable quantification parameters (in particular indicative of the vascularization) such as Imax (maximal peak enhancement), AUC (area under the curve), WIR (Wash-in-rate) or TTP (Time-to Peak); KDR expression can instead be assessed by LPO (late phase opacification). Thus, the present invention provides medical personnel with the ability to assess and if necessary adjust treatment strategies based on the adequacy of the therapeutic response. If the therapeutic response is insufficient, a different therapeutic agent or a higher dose of the same therapeutic agent may be warranted. If the therapeutic response is sufficient the current protocol will proceed unadjusted. Evaluation of the therapeutic response following adjustments to the treatment protocol would proceed in the same manner. 
     C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging 
     In accordance with the present invention, 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, absorption, fluorescent or phosphorescent emission, light reflection, changes in absorbance amplitude or maxima, and elastically scattered radiation. For example, biological tissue is relatively translucent to light in the near infrared (NIR) wavelength range of 650-1000 nm. NIR radiation can penetrate tissue up to several centimeters, permitting 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. 
     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 absorption 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 preferred range of absorption maxima for photolabels is between 600 and 1000 nm to minimize interference with the signal from hemoglobin. Preferably, photoabsorption labels have large molar absorptivities, e.g., &gt;10 5  cm −1  M −1 , while fluorescent optical dyes will have high quantum yields. Examples of 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 may be covalently linked directly to the KDR or VEGF/KDR complex binding peptide or linked to the KDR or VEGF/KDR complex binding peptide via a linker, as described previously. 
     After injection of the optically-labeled KDR or VEGF/KDR complex binding moiety, 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 determine 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. 
     The optical imaging reagents described above may also be used for acousto-optical or sonoluminescent imaging performed with optically-labeled imaging agents (see, U.S. Pat. No. 5,171,298, WO 98/57666, and references cited therein). In acousto-optical imaging, ultrasound radiation is applied to the subject and affects the optical parameters of the transmitted, emitted, or reflected light. In sonoluminescent imaging, the applied ultrasound actually generates the light detected. Suitable imaging methods using such techniques are described in WO 98/57666. 
     D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy 
     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. 
     For use as a PET agent 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,  94m Tc, or  110 In. The binding moieties of the invention can also be labeled by halogenation using radionuclides such as  18 F,  124 I,  125 I,  131 I,  123 I,  77 Br, and  76 Br. Preferred metal radionuclides for scintigraphy or radiotherapy include  99m Tc,  51 Cr,  67 Ga,  68 Ga,  47 Sc,  51 Cr,  167 Tm,  141 Ce,  111 In,  168 Yb,  175 Yb,  140 La,  90 Y,  88 Y,  153 Sm,  166 H,  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 choice of metal will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes the preferred radionuclides include  64 Cu,  67 Ga,  68 Ga,  99m Tc, and  111 In. For therapeutic purposes, the preferred radionuclides include  64 Cu,  90 Y,  105 Rh,  111 In,  117m Sn,  149 Pm,  153 Sm,  161 Tb,  166 Dy,  166 Dy,  175 Yb,  177 Lu,  186/188 Re, and  199 Au.  99m Tc is particularly preferred for diagnostic applications because of its low cost, availability, imaging properties, and high specific activity. The nuclear and radioactive properties of Tc-99m make this isotope an ideal scintigraphic imaging agent. 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, terpyridine, and N 3 S, N 2 S 2 , or N 4  chelants (see also, U.S. Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, DO3A, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. Pat. No. 5,720,934). For example, N 4  chelators are described in U.S. Pat. No. 6,143,274; U.S. Pat. No. 6,093,382; U.S. Pat. No. 5,608,110; U.S. Pat. No. 5,665,329; U.S. Pat. No. 5,656,254; and U.S. Pat. No. 5,688,487. Certain N 3 S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,976,495; and U.S. Pat. No. 5,780,006. The chelator may also include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N 3 5, and N 2 S 2  systems such as MAMA (monoamidemonoaminedithiols), DADS (N 2 5 diaminedithiols), CODADS and the like. These ligand systems and a variety of others are described in Liu and Edwards,  Chem. Rev.,  99:2235-2268 (1999) and references therein. 
     The chelator may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate array. These include the boronic acid adducts of technetium and rhenium dioximes, such as are described in U.S. Pat. No. 5,183,653; U.S. Pat. No. 5,387,409; and U.S. Pat. No. 5,118,797, the disclosures of which are incorporated by reference herein, in their entirety. 
     In another embodiment, 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  99m Tc. In this way the peptide loop is expanded by the introduction of Tc (peptide-S—S-peptide changed to peptide-S—Tc—S-peptide). This has also been used in other disulfide containing peptides in the literature (Chen et al.,  J. Nucl. Med.,  42:1847-1855 (2001)) while maintaining biological activity. 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 ) and  FIG. 35 . Formulas 20-22 ( FIGS. 34A-C ) are particularly useful for lanthanides such as paramagnetic Gd 3+  and radioactive lanthanides such as  177 Lu,  90 Y,  153 Sm,  111 In, or  166 Ho. Formulas 23a-24b ( FIGS. 34D  and F) and  FIG. 35  are particularly useful for radionuclides  99m Tc,  186 Re, or  188 Re. Formula 25 ( FIG. 34F ) and the structure shown in  FIG. 35  are particularly useful for  99m Tc. These and other metal chelating groups are described in U.S. Pat. No. 6,093,382 and U.S. Pat. No. 5,608,110, which are incorporated by reference herein in their entirety. Additionally, the chelating group of formula 22 ( FIG. 34C ) is described in, for example, U.S. Pat. No. 6,143,274; the chelating group of formula 24 is described in, for example, U.S. Pat. No. 5,627,286 and U.S. Pat. No. 6,093,382, and the chelating groups of formula 25 and  FIG. 35  are described in, for example, U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,780,006; and U.S. Pat. No. 5,976,495. 
     In the above Formulas 24a and 24b ( FIG. 34E ), X is either CH 2  or O; Y is C 1 -C 10  branched or unbranched alky, aryl, aryloxy, arylamino, arylaminoacyl, or arylalkyl comprising C 1 -C 10  branched or unbranched alkyl groups, hydroxy or C 1 -C 10  branched or unbranched polyhydroxyalkyl groups, C 1 -C 10  branched or unbranched hydroxy or polyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups; J is C(═O)—, OC(═O)—, SO 2 —, NC(═O)—, NC(═S)—, N(Y), NC(═NCH 3 )—, NC(═NH)—, N═N—, homopolyamides or heteropolyamines derived from synthetic or naturally occurring amino acids; and n is 1-100. Other variants of these structures are described, for example, in U.S. Pat. No. 6,093,382. The disclosures of each of the foregoing patents, applications and references are incorporated by reference herein, in their entirety. 
     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, U.S. Pat. No. 5,879,658, and U.S. Pat. No. 5,849,261). 
     Complexes of radioactive technetium are particularly useful for diagnostic imaging and complexes of radioactive rhenium are particularly useful for radiotherapy. In forming a complex of radioactive technetium with the reagents of this invention, the technetium complex, preferably a salt of Tc-99m pertechnetate, is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous and ferrous 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. Alternatively, 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 formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example. Among 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. Examples of compounds in which rhenium is in the Re(VII) state are 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 (pyridine) 4   + , 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. In forming Tc-99m radioactive complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 mCi to 100 mCi per mL. 
     Generally, 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. After injection of the KDR or VEGF/KDR complex-specific radionuclide imaging agent into the patient, a gamma camera calibrated for the gamma ray energy of the nuclide incorporated 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. However, imaging can take place, if desired, hours or even longer, after the radiolabeled peptide is injected into a patient. In 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 of scintiphotos. 
     Proper dose schedules for the radiotherapeutic compounds of the present invention are known to those skilled in the art. The compounds can be administered using many methods that 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 constructs, 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. In general, 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 α,β, or γ 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. For example, 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 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. As above, reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present in either or both vials. 
     As shown in the Examples, compounds of the invention comprising a radionuclide, particularly heteromultimers such as D10 conjugated to a radionuclide (optionally via a chelator), are useful in imaging KDR or VEGF/KDR complex expressing tissue (such as angiogenic tissue). 
     Additionally, the Examples establish that compounds of the invention conjugated to a therapeutic radionuclide, particularly heteromultimers such as D13 conjugated to a chelator and complexed with a therapeutic radionuclide, are useful in radiotherapy of tumors expressing KDR. 
     Other Therapeutic Applications 
     The KDR or VEGF/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 VEGF/KDR complex and their residence time at a KDR or VEGF/KDR complex on endothelium undergoing angiogenesis. In this aspect of the invention, hybrid 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. 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 (actinomycin D), daunorubcin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testoiactone, trilostane, amsacrine (m-AMSA), aparaginase (L-aparaginase), Erwina aparaginase, etoposide (VP-16), interferon cx-2a, Interferon cx-2b, teniposide (VM-26, vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate, adriamycin, and arabinosyl; anti-angiogenic agents such as tyrosine kinase inhibitors with activity toward signaling molecules important in angiogenesis and/or tumor growth such as SU5416 and SU6668 (Sugen/Pharmacia &amp; Upjohn), endostatin (EntreMed), angiostatin (EntreMed), Combrestatin (Oxigene), cyclosporine, 5-fluorouracil, vinblastine, doxorubicin, paclitaxel, daunorubcin, immunotoxins; coagulation factors; antivirals such as acyclovir, amantadine azidothymidine (AZT or Zidovudine), ribavirin and vidarabine monohydrate (adenine arahinoside, ara-A); antibiotics, antimalarials, antiprotozoans such as chloroquine, hydroxychloroquine, metroidazole, quinine and meglumine antimonate; anti-inflammatories such as diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates. 
     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. In 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 artificial chromosomes (YAC&#39;s) and 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. 
     In a preferred embodiment the constructs of the invention are utilized in gene therapy for treatment of diseases associated with angiogenesis. In this embodiment, 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 multimers (e.g., homomultimers 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. More preferably, if 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 may be 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 arranged 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. In 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. 
     Similar recombinant proteins containing one or more coding sequences for a KDR and VEGF/KDR complex binding polypeptide may be useful in imaging or therapeutic applications. For example, in a variation of the pre-targeting applications discussed infra, the coding sequence for a KDR or VEGF/KDR complex binding peptide can be fused in frame to a sequence encoding an antibody (or an antibody fragment or recombinant DNA construct including an antibody, etc.) that, 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. After the non-binding antibodies have been allowed to clear, the chelator-radionuclide complex (or other detectable label), which the antibody recognizes is administered, permitting imaging of or radiotherapy to the KDR-expressing tissues. 
     Additionally, 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. 
     Additionally, 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. For example, where binding of a protein or other molecule (e.g., a growth factor, hormone etc.) is necessary for or contributes to a disease process and a binding moiety inhibits such binding, constructs including such binding moieties could be useful as therapeutics. Similarly, where binding of a binding moiety itself inhibits a disease process, constructs containing such binding moieties could also be useful as therapeutics. 
     As binding of VEGF and activation of KDR is necessary for angiogenic activity, in one embodiment constructs including KDR complex binding polypeptides that inhibit the binding of VEGF to KDR (or otherwise inhibit activation of 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 preferred heteromultimer is the heterodimer-containing construct D1 (structures provided by the examples). Other preferred heterodimer constructs include D4, D5, D6, D10, D13, D17, D23, D27, D30 and D31 (structures provided in the Examples below). The binding polypeptides and constructs 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 formation of blood vessels, the polypeptides and constructs 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 &amp; neck, and ovaries. Additional tumors and related disorders are listed in Table I of U.S. Pat. No. 6,025,331, issued Feb. 15, 2000 to Moses, et al., the teachings of which are incorporated herein by reference. Benign tumors include, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas. As shown in Example 15, compounds of the invention, including heteromultimers such as D6, are useful in treating and/or slowing the growth of certain tumors. 
     Other relevant diseases that involve angiogenesis include for example, rheumatoid arthritis, psoriasis, and ocular diseases, such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rebeosis, Osler-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. Furthermore, the binding polypeptides and constructs 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. In renal failure, for example, it has been shown that anti-VEGF antibodies can reverse damage. In a similar way, the compounds of the present invention can reverse renal permeability pathogenesis in, for example, diabetes. 
     Furthermore, 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, SIV, Simian hemorrhagic fever virus, etc. Sequence homology searches of KDR-binding peptides identified by phage display using the BLAST program at NCBI 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, HIV, SIV, simian hemorrhagic fever virus, and an enterohemorrhagic  E. coli  strain. Some of the homologous proteins, such as PfEMP1 and EBL-1, are 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&#39;s surface. Their high mutation and recombination rates allow them to quickly develop new binding sites to promote survival and/or invasion. Similarly, proteins such as gp120 of HIV (which also has homology to some of the KDR-binding peptides disclosed herein) play critical roles in the adhesion of pathogens to their hosts. Although not reported previously, it is possible that many of the pathogen proteins with homology to the KDR-binding peptides disclosed herein also bind to KDR. Comparison of the pathogen protein sequences with the corresponding peptide sequences may suggest changes in the peptide sequence or other modifications that will enhance its binding properties. Additionally, the 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 gp120 that induce T cell apoptosis and block cell death induced by gp120 ”, FASEB J,  12(11):991-998 (1998). Thus, KDR may represent a previously unknown target for a number of pathogens, and the KDR binding peptides of the invention may be useful in treating the diseases associated with those pathogens. 
     The binding polypeptides and constructs 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 constructs 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). In addition, the binding polypeptides and constructs thereof of the present invention can be administered a part of a maintenance regimen, for example to prevent or lessen the recurrence or the symptoms or condition. As described below, the binding polypeptides and constructs 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. In 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 pharmaceutical compositions are preferably formulated for parenteral administration, and most preferably for intravenous or intra-arterial administration. Generally, and particularly when administration is intravenous or intra-arterial, pharmaceutical compositions may be given as a bolus, as two or more doses separated in time, or as a constant or non-linear flow infusion. 
     As used herein 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. For example, 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 constructs thereof of the present invention can inhibit activity of VEGF receptors, including VEGFR-2/KDR, VEGFR-1/Flt-1 and VEGFR-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 constructs 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 constructs thereof. Based on the teachings provided herein, one of ordinary skill in the art would know how and be able to administer a suitable dose of binding polypeptide or construct thereof as provided herein, and measure the effect of treatment on the parameter of interest. For example, the size of the area of interest (e.g., the tumor or lesion) can be measured before and after treatment. In another embodiment, 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 constructs 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 constructs provided herein, and the desired outcome of treatment, the preferred dosage is determinable by one of ordinary skill in the art through routine optimization procedures. Typically, the daily regimen is in the range of about 0.1 μg/kg to about 1 mg/kg. 
     The binding polypeptides and constructs 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 constructs thereof, other therapeutic agents, or combination thereof. In addition, the binding polypeptides and constructs thereof can be conjugated to therapeutic agents, for example, to improve specificity, residence time in the body, or therapeutic effect. Such other therapeutic agents include, for example, other anti-angiogenic compounds, and tumoricidal compounds. The therapeutic agent can also include antibodies. 
     Furthermore, the binding polypeptide or constructs thereof of the present invention can be used as an endothelial cell homing device. Therefore, 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. Once exposed to the nucleic acid conjugated binding polypeptide, the endothelial cell can internalize and express the conjugated nucleic acid, thereby delivering the therapeutic peptide to the target cells. 
     In another embodiment of the invention, the therapeutic agent can be associated with an ultrasound contrast agent composition, said ultrasound contrast agent including the KDR or VEGF/KDR complex binding peptides of the invention linked to the material employed to form the vesicles (particularly microbubbles or microballoons) comprising the contrast agent. For example, the therapeutic agent can be associated with the contrast agent and delivered as described in U.S. Pat. No. 6,258,378, herein incorporated by reference. Thus, after administration of the ultrasound contrast agent and the optional imaging of the contrast agent bound to the pathogenic site expressing the KDR or VEGF/KDR complex, the pathogenic site can be irradiated with an energy beam (preferably ultrasonic, e.g., with a frequency of from 0.3 to 3 MHz), to rupture or burst of microvesicles. The therapeutic effect of the therapeutic agent can thus be advantageously enhanced by the energy released by the rupture of the microvesicles, in particular causing an effective deliver of the therapeutic agent to the targeted pathogenic site. 
     The binding polypeptides and constructs 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 th  ed., Lippincott, Williams and Wilkins, 2000. 
     For topical application, the binding polypeptides can be suspended, for example, in a cream, gel or rinse that 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. 
     For transdermal administration, the polypeptides can be applied in pharmaceutically acceptable suspension together with a suitable transdermal device or “patch.” Examples of suitable transdermal devices for administration of the polypeptides of the present invention are described, for example, in U.S. Pat. No. 6,165,458, issued Dec. 26, 2000 to Foldvari, et al., and U.S. Pat. No. 6,274,166B1, issued Aug. 4, 2001 to Sintov, et al., the teachings of which are incorporated herein by reference. 
     For parenteral administration, the polypeptides can be injected intravenously, intramuscularly, intraperitoneally, or subcutaneously. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Other pharmaceutically acceptable carriers 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. Where necessary, 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. Similarly, the composition may comprise conventional excipients, i.e. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application that do not deleteriously react with the active compounds. Generally, 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 hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent in activity units. Where 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. Where the composition is to be administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration. 
     For gastrointestinal and intravaginal administration, the polypeptides can be incorporated into pharmaceutically acceptable powders, pills or liquids for ingestion, and suppositories for rectal or vaginal administration. 
     For transalveolar, buccal or pulmonary 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. Pat. No. 6,312,665B1, issued Nov. 6, 2001 to Pankaj Modi, the teachings of which are incorporated herein by reference. 
     In addition, the polypeptides of the present invention can be administered nasally or ocularly, where the polypeptide is suspended in a liquid pharmaceutically acceptable agent suitable for dropwise 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). For example, 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. In one embodiment, 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. Pat. No. 4,391,797, issued Jul. 5, 1983, to Folkman, et al., the teachings of which are incorporated 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. In a particular embodiment, the gene encoding the polypeptide is transfected into cells that have been obtained from the patient, a method referred to as ex vivo gene therapy. Cells expressing the polypeptide are then returned to the patient&#39;s body. Methods of ex vivo gene therapy are well known in the art and are described, for example, in U.S. Pat. No. 4,391,797, issued Mar. 21, 1998 to Anderson, et al., the teachings of which are incorporated herein by reference. 
     Isolation, formulation and use of KDR or VEGF/KDR complex binding moieties in accordance with this invention will be further illustrated in the following Examples to follow. 
     As previously mentioned, the present invention also provides monomeric peptide phospholipid conjugates having a linear peptide monomer which binds with high affinity to KDR and dimeric peptide phospholipid conjugates having two distinct monomer subunits, each binding to KDR. In addition, highly efficient methods for large scale production of purified forms of these conjugates and precursor materials are provided. Such methods include the production of dimeric peptide phospholipid conjugates having minimal levels of TFA. 
     The phospholipid may be selected from the group consisting of: phosphatidylethanolamines and modified phosphatidylethanolamines. Particularly preferred phospholipids include phosphatidylethanolamines modified by linking a hydrophilic polymer thereto. Examples of modified phosphatidylethanolamines are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG. DSPE-PEG2000, DSPE-PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are preferred, with DSPE-PEG2000 particularly preferred. Note that a salt form of the phospholipid may be used, such as, for example, the trimethyl ammonium salt, the tetramethylammonium salt, the triethylammonium salt, sodium salt, etc. 
     These compounds may be incorporated into gas-filled ultrasound contrast agents, such as, for example, gas filled microbubbles to form contrast agents that provide excellent imaging of target-bearing tissue. In a preferred embodiment, targeting vector-phospholipid conjugates which include targeting peptides which bind with high affinity to KDR are incorporated into targeted microbubbles. As shown herein, such targeted microbubbles selectively localize at KDR-bearing tissue, permitting imaging of such tissue, and, in particular imaging of tumors and angiogenic processes, including those processes associated with neoplastic development. 
     Monomer Conjugates 
     Generally 
     Table 1-A provides a description for the identification labels shown in  FIGS. 105, 106, 113 and 114 . 
     
       
         
           
               
               
             
               
                 TABLE 1-A 
               
               
                   
               
             
            
               
                 1 
                 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK 
               
               
                   
                 (DSPE-PEG2000-NH-Glut)-NH 2  (SEQ ID NO. 1) 
               
               
                   
               
               
                 2 
                 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH 2   
               
               
                   
                  (SEQ ID NO. 2) 
               
               
                   
               
               
                 3 
                 mono-NHS ester of glutaryl-peptide monomer (2) 
               
               
                   
                 Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(NHS-Glut)-NH 2   
               
               
                   
                  (SEQ ID NO. 3) 
               
               
                   
               
               
                 4 
                 DSPE-PEG2000-NH 2  phospholipid 1,2-distearoyl- 
               
               
                   
                 sn-glycero-3-phosphoethanolaminocarbonyloxy- 
               
               
                   
                 (PEG2000)-amine 
               
               
                   
               
               
                 31 
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK 
               
               
                   
                 (DSPE-PEG2000-NH-Glut)-NH 2  (SEQ ID NO. 4) 
               
               
                   
               
               
                 32 
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH 2    
               
               
                   
                 (SEQ ID NO. 5) 
               
               
                   
               
            
           
         
       
     
     As shown if  FIGS. 105 and 106  the monomeric peptide phospholipid conjugate (1) N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L- methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L1-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysinamide, is a phospholipid conjugate. This conjugate is also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH 2  (SEQ ID NO. 1) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH2. It comprises a 29 amino acid linear peptide monomer (2) N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L- alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L1-arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide, also referred to as Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2 (SEQ ID NO. 2) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2. This novel peptide monomer binds with high affinity to KDR. It should be understood that analogs and derivatives of the monomeric peptide phospholipid conjugate (1) and the linear peptide monomer (2) are intended to be included within the scope of the present invention. 
       FIG. 114  provides the structure of another monomeric peptide phospholipid conjugate (31), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl- L-phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-{N6-[1,2-distearoyl-sn-glycero-3-phosphoethanolaminocarbonyloxy-(PEG2000)-aminoglutaryl]}-L-lysine-amide, a phospholipid conjugate. This conjugate is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH 2  (SEQ ID NO. 4) and Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys(DSPE-PEG2000-NH-Glut)-NH 2 . As shown in  FIG. 113 , the conjugate comprises a 28 amino acid linear peptide monomer (32), N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L- phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide, which is also referred to as Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK-NH 2  (SEQ ID NO. 5) and Ac-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH 2 . This peptide monomer has been shown to bind with high affinity to KDR (See U.S. application Ser. No. 10/661,156). It should be understood that analogs and derivatives of the monomeric peptide phospholipid conjugate and the linear peptide monomer are intended to be included within the scope of the present invention. 
     Additional monomeric peptide phospholip conjugates according to the present invention include Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH 2  (SEQ. ID NO. 6) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH 2  (SEQ. ID NO. 7). These peptide monomers bind with high affinity to KDR. It should be understood that analogs and derivatives of these monomeric peptide monomers and peptide phospholipids conjugates comprising these monomers are intended to be included within the scope of the present invention. 
     As shown in the Examples, ultrasound contrast agents such as gas filled microbubbles formulated with the monomeric peptide phospholipid conjugates (1) and (31) displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits. 
     Methods of Preparation of Monomer Peptide-Phospholipid Conjugates 
     Ideally, to facilitate production of a monomeric peptide phospholipid conjugate, the linear peptide monomer should be prepared in bulk. Then conjugation of the purified linear peptide monomer to the phospholipid, such as, for example, a pegylated phospholipid in salt form, e.g., DSPE-PEG2000-NH 2  phospholipid ammonium salt via the linker disuccinimidyl glutarate (DSG), may be used to provide monomeric peptide phospholipid conjugates. 
     In preparing monomeric peptide phospholipid conjugates, methods according to the present invention provide at least the following advantages: increased yield of peptide synthesis; reduced extent of racemization; avoidance of previously observed piperidine amide formation during synthesis, efficient purification of peptide monomers, development of a procedure for conjugation of peptide monomers on larger scale; and development of purification protocols that would allow the ready separation of the monomeric peptide phospholipid conjugates from the starting DSPE-PEG2000-NH 2  phospholipid ammonium salt. 
     With respect to the methods described below (including the Examples section of this specification), it should be understood that while such methods may specifically reference the use of linear peptide monomers (2) and (32) to form monomeric peptide phospholipids conjugates (1) and (31), these methods also may be used with peptide monomers Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH 2  (SEQ. ID NO. 6) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH 2  (SEQ. ID NO. 7), as well as analogs and derivatives of any of the above. It also should be understood that the numerical values referred to in the synthesis of monomeric peptide phospholipid conjugates are representative. 
     Linear peptide monomers may be prepared by SPPS. The sequence of the linear peptide monomers may be constructed as a C-terminal carboxamide on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Peptide synthesis may be accomplished using Fmoc chemistry on a SONATA®/Pilot Peptide Synthesizer. Problems previously observed with this process have been racemization, incomplete couplings and piperidine amide formation, each of which contribute to suboptimal yield and purity. A dramatic decrease in the formation of the piperidine amide may be attained by the use of 25% piperidine in DMF containing HOBt (0.1M) as the reagent for Fmoc removal. Racemization may be considerably reduced by using DIC/HOBt as the activator for most couplings; a 3 h coupling time using a four-fold excess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF (6×). N a -Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution. 
     To optimize the yield, the problem of aggregation of the resin during the synthesis of longer peptides, which can be devastating even when optimal coupling reagents are employed, may be addressed. To reduce aggregation during peptide assembly the strategy of using pseudoproline dipeptides to incorporate X-Thr or X-Ser as dipeptides instead of sequential couplings of X and Thr or X and Ser, may be employed. For linear peptide monomers sequential couplings of Leu11-Ser12 and Leu22-Ser23 may be replaced by the single coupling of the pseudoproline dipeptide, Fmoc-Leu-Ser(ψMe,Mepro)-OH. Additional optimization may be accomplished by reducing the number of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OH segments may lead to cyclization of the activated acid function with the distal amide function to produce an inactive diketopiperazine; this may reduce coupling yields in a time dependant manner. This problem may be avoided by addition of Fmoc-Glyn-OH (n=2, 3) to the reaction vessel and sequential addition of HOBt and DIC; the activated Fmoc-Glyn-OH may be intercepted by the resin-bound amino group before appreciable cyclization to the diketopiperazine takes place. With these improvements, the synthesis of linear peptide monomers may be completed on the Sonata Peptide Synthesizer on a 10 mmol synthesis scale. 
     After chain elongation, the Fmoc may be removed from the N-terminus. The peptide and the free amino group may be acetylated. Then the peptide sequence may be cleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction the crude peptide may be isolated as a solid by evaporation of the volatiles, trituration of the residue with diethyl ether and washing of the solid thus obtained using the same solvent. In another variation the peptide may be precipitated from the reaction mixture by addition of diethyl ether to the reaction mixture, collecting the solid thus formed and washing with the same solvent. 
     Linear peptide monomers may be purified as described below. Again, the numerical references are representative. Crude linear peptide monomers (0.5 g) may be dissolved in CH3 CN (40 mL/g) and this solution may be diluted to a final volume of 100 mL with water. The solution may then be filtered. The filtered solution may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm) equilibrated with 10% CH3 CN in water (0.1% TFA). After loading, the composition of the eluent may then be ramped to 20% CH3 CN-water (0.1% TFA) over 1 min, and a linear gradient may be initiated at a rate of 0.6%/min of CH3 CN (0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may be checked for purity on an analytical reversed phase C18 column (Waters XTerra MS-C18, 10, 120 Å, 4.6×50 mm) and fractions containing the product in &gt;95% purity may be combined and freeze-dried. For each purification of 0.5 g of crude peptide 0.12 g (24%) of linear peptide monomer may be consistently isolated and will provide the peptide in the same yield and purity. 
     Synthesis of monomeric peptide phospholipid conjugates may be performed as described below. The numerical references are again representative. The last step in the synthesis may be the conjugation of the phospholipid, such as, for example, a pegylated phospholipid such as DSPE-PEG2000-NH 2  phospholipid ammonium salt to a linear peptide monomer. The PEG2000 moiety of DSPE-PEG2000-NH 2  phospholipid ammonium salt (4) is nominally comprised of 45 ethylene glycol units. It should be understood, however, that this material is a distribution of PEG containing species whose centroid is the nominal compound containing 45 ethylenoxy units. The conjugation of a linear peptide monomer with DSPE-PEG2000-NH2 phospholipid ammonium salt may be accomplished by preparation of the glutaric acid monoamide mono NHS ester of a linear peptide monomer and reaction of this with the free amino group of the phospholipid ammonium salt. Thus a linear peptide monomer may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate, which may result in precipitation of the peptide glutaric acid monoamide mono-NHS ester. The supernatant containing un-reacted DSG may be decanted and the intermediate peptide mono-NHS ester may be washed several times with ethyl acetate to remove traces of DSG. Mass spectral data confirms the formation of the peptide mono-NHS ester as a clean product. The solid mono-NHS ester may be dissolved in DMF and reacted with DSPE-PEG2000-NH 2  phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h. The linear peptide monomer glutaric acid monoamide mono-NHS ester may be used in excess to maximize the consumption of the phospholipid ammonium salt because free phospholipid ammonium salt may complicate the isolation of monomeric peptide phospholipid conjugates in highly pure form. 
     The reaction mixture may be diluted with a 1:1 mixture of water (0.1% TFA) and CH 3 CN—CH 3 OH (1:1, v/v) (0.1% TFA) (˜100 mL), applied to a reversed phase C2 column (Kromasil® Prep C2, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) and the column may be eluted with a 3:1 mixture of water (0.1% TFA) and CH 3 CN—CH 3 OH (1:1, v/v) (0.1% TFA) to remove hydrophilic impurities. Then the product may be eluted using a gradient of CH 3 CN—CH 3 OH (1:1) (0.1% TFA) into water (0.1% TFA) (see Experimental Section for details). The collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult-to-separate DSPE-PEG2000-NH 2  phospholipid which has very little UV absorbance. This indicates the clear separation of the monomeric peptide phospholipid conjugates and DSPE-PEG2000-NH 2  phospholipid. The pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide monomeric peptide phospholipid conjugates as a colorless solid. In order to prepare the required quantity of the monomeric peptide phospholipid conjugates, several runs may be conducted employing 0.5 g to 1.0 g of linear peptide monomer. In all cases the target monomeric peptide phospholipid conjugates may be were isolated in high yield and purity (e.g., 57-60% yield and &gt;99% purity). 
     Dimer Conjugate 
     Generally 
     Table 2-A provides a description for the identification labels shown in  FIGS. 107, 108 and 109 . 
     
       
         
           
               
               
             
               
                 TABLE 2-A 
               
               
                   
               
             
            
               
                 11 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDP- 
               
               
                   
                 GGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]- 
               
               
                   
                 NH 2  cyclic (2-12) disulfide}- 
               
               
                   
                 NH 2  cyclic (6-13) disulfide 
               
               
                   
               
               
                 12 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH 2  cyclic 
               
               
                   
                 (6-13) disulfide 
               
               
                   
               
               
                 13 
                 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH 2  cyclic 
               
               
                   
                 (2-12) disulfide 
               
               
                   
               
               
                 14 
                 mono-NHS ester of glutaryl-peptide 12 
               
               
                   
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]- 
               
               
                   
                 NH 2  cyclic (6-13)disulfide 
               
               
                   
               
               
                 15 
                 ivDde-bearing dimer 
               
               
                   
                 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYD- 
               
               
                   
                 PGGGK[-Adoa-Adoa-Glut-K(ivDde)]-NH 2  cyclic 
               
               
                   
                 (2-12) disulfide}-NH 2  cyclic (6-13) disulfide 
               
               
                   
               
               
                 16 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVC 
               
               
                   
                 FRYDPGGGK(-Adoa-Adoa-Glut-K)-NH 2  cyclic 
               
               
                   
                 (2-12) disulfide]-NH 2  cyclic (6-13) disulfide 
               
               
                   
               
               
                 17 
                 Mono-NHS ester of glutaryl-peptide 16 
               
               
                   
                 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVC 
               
               
                   
                 FRYDPGGGK[-Adoa-Adoa-Glut-K(NHS-Glut)]-NH 2  cyclic 
               
               
                   
                 (2-12) disulfide}-NH 2  cyclic (6-13)disulfide 
               
               
                   
               
               
                 18 
                 DSPE-PEG2000-NH 2  phospholipid 
               
               
                   
               
            
           
         
       
     
     As shown in those figures the dimeric peptide phospholipid conjugate (11) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L- cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L- lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide, consists of two monomeric peptide chains which bind KDR: a 21 amino acid cyclic disulfide peptide monomer (13) Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl- glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl)amide cyclic (2-12) disulfide, and a 22 amino acid cyclic disulfide peptide monomer (12) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L- threonyl-glycyl-glycyl-glycyl-L-lysinamide cyclic 6-13 disulfide tethered by a glutaryl linker. It should be understood that analogs and derivatives of the dimeric peptide phospholipid conjugate (11) and the cyclic disulfide peptide monomers (12) and (13) are intended to be included within the scope of the present invention. 
     Ultrasound contrast agents (e.g. gas filled microbubbles) formulated with the dimeric peptide phospholipid conjugate (11) displayed high KDR binding which was confirmed using echographic examination of VX2 tumors in rabbits. 
     Methods of Preparation of Dimer-Phospholipid Conjugates 
     To accomplish synthesis of the dimeric peptide phospholipid conjugate (11), the monomers used for this purpose optimally should be prepared in bulk. Then the monomers may be tethered to each other using di-succinimidyl glutarate as a linker to form the precursor dimer peptide (16), Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-cystinyl-L-tryptophyl-L-leucyl-L-phenylalanyl-glycyl-L- threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L-lysyl(8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl)amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide. Then conjugation of the purified precursor dimer peptide (16) to a DSPE-PEG2000-NH 2  phospholipid ammonium salt (18) again via disuccinimidyl glutarate may be used in order to provide the target dimeric peptide phospholipid conjugate (11). 
     In preparing dimeric peptide phospholipid conjugate (11), methods according to the present invention provide at least the following advantages: increased yield of automated chain elongation of the peptide sequences; reduced extent of racemization encountered during synthesis; avoidance of previously observed piperidine amide formation during synthesis of peptide monomer (13); cyclization of linear di-cysteine containing peptide precursors of (12) and (13) using procedures amenable to multigram scale yet allowing efficient and practical sample handling; efficient purification of monomer peptides (12) and (13); maximized yield and purity of precursor dimer peptide (16); development of a procedure for conjugation of the precursor dimer peptide (16) on larger scale; and development of purification protocols that would allow the ready separation of the target dimeric peptide phospholipid conjugate (11) from phospholipid ammonium salt (18). 
     The dimeric peptide phospholipid conjugate (11) may be prepared by automated synthesis of the peptide monomers (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, and (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH 2  cyclic (2-12) disulfide, their efficient coupling using disuccinimidyl glutarate (DSG) to give an ivDde-protected dimer, its deprotection and subsequent coupling to DSPE-PEG2000-NH 2 , also via a glutaryl linkage. Using procedures according to the present invention, monomer peptides may be synthesized on a 10 mmol scale without complication and after HPLC purification may be obtained in about 20% yield and &gt;95% purity. Such methods allow dimer formation reactions and the subsequent conjugation to the phospholipid component providing formation of dimeric peptide phospholipid conjugate (11) to be carried out on a gram scale. The precursor dimer peptide (16) may be obtained from the monomer peptides routinely in about 32% yield and &gt;95% purity. The dimeric peptide phospholipid conjugate (11) may be produced from the precursor dimer peptide (16) in 57-60% yield and &gt;99% purity. 
     Dimeric peptide phospholipid conjugates may be prepared as described below. It should be appreciated that the numerical values referred to in this representative description of the synthesis of dimeric peptide phospholipid conjugates are representative. 
     Described below is a representative method for the solid phase synthesis and disulfide cyclization of a peptide monomer (12) Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, and a peptide monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide. 
     The peptides may be constructed as their C-terminal carboxamides on Pal-Peg-PS-resin (substitution level: 0.2 mmol/g). Chain elongation may be accomplished using Fmoc chemistry employing optimized deprotection and coupling protocols on a SONATA®/Pilot Peptide Synthesizer on a 10 mmol synthesis scale. The optimized synthesis of the peptides by automated SPSS may be developed by study of peptide impurities and the effect of changes of particular elements of the protocols on the overall yield and purity of the peptides obtained. 
     Analysis of the impurities obtained from nonoptimized syntheses of the monomer peptides indicates that the major problems are racemization, incomplete couplings and piperidine amide formation (presumably via an intermediate aspartamide or glutarimide intermediate), each of which contributes to suboptimal yield and purity. A dramatic decrease in formation of the piperidine amide may be attained by the use of 25% piperidine in DMF containing HOBt (0.1M) as the reagent for fmoc removal. Racemization may be considerably reduced by using DIC/HOBt as the activator for most couplings; and a 3 h coupling time using a four-fold excess of pre-activated Fmoc-amino acid with an intervening wash with anhydrous DMF (6×). N- α Fmoc amino acids may be dissolved just before their coupling turn and pre-activated with DIC/HOBt in DMF for 4 min and transferred to the reaction vessel. This may be accomplished on the Sonata instrument by loading the solid Fmoc-amino acids into the amino acid vessels of the instrument and then programming the instrument to add DMF, HOBt/DMF and DIC/DMF sequentially with bubbling of the solution after each addition. 
     To optimize the yield, the problem of aggregation of the resin during the synthesis of longer peptides, which can be devastating even when optimal coupling reagents are employed, may be addressed. To reduce aggregation during peptide assembly the strategy of using pseudoproline dipeptides to incorporate X-Thr or X-Ser (X refers to the n-1 amino acid of the sequence) as dipeptides instead of sequential couplings of X and Thr or X and Ser, may be employed. Thus, for the monomer (12), Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys(ivDde)-NH2 cyclic (6-13) disulfide, sequential coupling of suitably protected Thr and Gly (shown in bold above) may be replaced by the single coupling of the pseudoproline dipeptide, Fmoc-Gly-Thr(ψ Me,Me pro)-OH. Similarly, in the synthesis of the monomer (13), Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH2 cyclic (2-12) disulfide, the pseudoproline dipeptide, Fmoc-Asp(OtBu)-Ser(ψ Me,Me pro)-OH may be employed to replace the sequential coupling of suitably protected Ser and Asp (shown in bold font above). Further optimization may be accomplished by reducing the number of couplings by using Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, in lieu of serial coupling of Fmoc-Gly-OH. Activation of -Gly-Gly-OH segments can lead to cyclization of the activated acid function with the distal amide function to produce an inactive diketopiperazine; this may reduce coupling yields in a time dependant manner. This problem may be avoided by addition of Fmoc-Glyn-OH (n=2, 3) to the reaction vessel and sequential addition of HOBt and DIC; the activated Fmoc-Glyn-OH may be intercepted by the resin-bound amino group before appreciable cyclization to the diketopiperazine takes place. After chain elongation is completed the N-terminal Fmoc protecting group may be removed from each of the peptides and the free amino group may be acetylated. 
     The pseudo-orthogonally protected derivative, Fmoc-Lys(ivDde)-OH may be used to enable the selective unmasking of the ε-amine of the C-terminal lysine of the monomer and dimer peptides and their subsequent functionalization, which also may be optimized. The ivDde group on the ε-amine of the C-terminal lysine of each of the peptide monomers may be removed using 10% hydrazine in DMF. Then Fmoc-Adoa, for monomer (13) or Lys(ivDde) for monomer (12) may be appended to the exposed lysine ε-amino group using 4 equivalents of the Fmoc amino acid and 4 equivalents each of DIC and HOBt in DMF for 10 h. After completion of the synthesis, the peptide sequence may be cleaved from the resin and deprotected using “Reagent B” (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) for 4 h. After the cleavage reaction was complete the peptide may be precipitated, washed with diethyl ether and dried. 
     The following procedures for cyclization of the linear di-cysteine containing peptides may be used to provide optimal scale-up of monomer peptides. Generally the aerial oxidation of linear di-cysteine peptides may be carried out at ca 0.5-5 mg/mL (for the disclosed peptide monomers ˜0.18-1.8 mM in peptide, ˜0.36-3.6 mM in cysteine thiol). In order to work at significantly higher concentrations DMSO-assisted cyclization of di-cysteine peptides allows the cyclization of ˜10 g of the linear peptides in good yields in as little as ˜50 mL of solution. Therefore the crude linear di-cysteine peptides may be cyclized in 95% DMSO-H2O (5 mL/g) at pH 8.5 at ambient temperature. The progress of the cyclization may be routinely followed by mass spectroscopy and HPLC. Although cyclization may be essentially complete in ˜36 h, the reaction mixture may be generally stirred for up to 48 h. The cyclic disulfide peptides may be precipitated from the reaction mixture by dilution with CH 3 CN and the resulting off-white crude solid peptides may be collected by filtration. This is a convenient method for removing DMSO from the crude cyclic peptide. 
     Purification and isolation of monomer peptide (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]—NH 2  may be accomplished as described below. Note that as used herein the designation “C*” refers to a cysteine residue that contributes to a disulfide bond. Attempts to dissolve 0.5 g of the crude peptide in up to 300 mL of 30% CH 3 CN in water (0.1% TFA) have been unsuccessful. Therefore, as an alternative, the crude peptide, (0.5 g) may be dissolved in DMSO (5 mL/g) and this solution may be diluted to a final volume of 100 mL with 20% CH 3 CN-water. The solution may be filtered. The filtered solution may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm) equilibrated with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA), and the column may be eluted with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA) to wash DMSO from the column. The composition of the eluent then may be ramped to 35% CH 3 CN-water (0.1% TFA) over 1 min, and a linear gradient may be initiated at a rate of 0.5%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA) and run for 50 min. Eluted fractions may be checked for purity on an analytical reversed phase C18 column (Waters XTerra® MS-C18, 10μ, 120 Å, 4.6×50 mm) and fractions containing the product in &gt;95% purity may be combined and freeze-dried. For each purification of 0.5 g of crude peptide 0.1 g (20%) for (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK [K(ivDde)]—NH2 may be isolated. Repeat purifications have been found to provide the peptide consistently in the same yield and purity. 
     The peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2  may be purified and isolated as described for peptide monomer (12) except that the subject peptide may be dissolved in 20% CH 3 CN (0.1% TFA) in 0.1% aqueous TFA (0.5 g peptide/100 mL) instead of a DMSO-containing diluent. The resulting solution of crude peptide may be loaded onto the preparative HPLC column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) equilibrated with 10% CH 3 CN in water (0.1% TFA). The column may be eluted with 10% CH 3 CN (0.1% TFA)/water (0.1% TFA) at 100 mL/min for 5 min. Then the composition of the eluent may be ramped to 30% CH 3 CN (0.1% TFA)/water (0.1% TFA) over 1 min and a linear gradient rate of 0.5%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA) may be initiated, and maintained until the desired peptide is completely eluted from the column. Product-containing fractions may be analyzed on a Waters XTerra® analytical reversed phase C-18 column (10μ, 120 Å) and fractions containing the product in &gt;95% purity may be pooled and freeze-dried to afford the cyclic disulfide peptide monomer (13) (0.12 g, 24% yield) in &gt;95% purity. The 10 g of crude peptide monomer may be purified serially in this manner. 
     Described below is a representative method for preparing the precursor dimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[—NH 2  cyclic (2-12) disulfide]-NH 2  cyclic (6-13) disulfide. The preparation of the precursor dimer peptide may be accomplished by the tethering of the monomer peptides in a two step procedure. First, Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]-NH 2  (12) may be reacted with disuccinimidyl glutarate (DSG, 5 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. The reaction mixture may be diluted with ethyl acetate, which results in precipitation of the glutaric acid monoamide mono-NHS ester of the peptide. The supernatant, containing unreacted DSG, may be decanted and the mono-NHS ester may be washed several times with ethyl acetate to remove traces of DSG. Mass spectral data confirms the formation of the mono-NHS ester as a clean product. This may be redissolved in DMF and reacted with monomer peptide Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2  (13) in the presence of DIEA (5 eq). HPLC and MS results indicate the formation of the ivDde-bearing dimer, as a single major product. The ivDde protecting group on the ε-amine of Lys of the dimer may be removed by stirring the reaction mixture with hydrazine (10%) in DMF for 20 min. The solution then may be acidified with TFA and diluted with 10% CH3 CN (0.1% TFA)-water (0.1% TFA), applied to a preparative reversed phase C18 HPLC column and purified by a gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueous TFA. In order to provide the needed quantity of the precursor dimer peptide, the reaction may be conducted employing from 0.5 g to as much as 1 g of each of the monomer peptides. In every case the required precursor dimer peptide may be isolated in ˜32% yield and &gt;95% purity confirming the reproducibility and scalability of the procedures. 
     The final step in the synthesis may be the conjugation of DSPE-PEG2000-NH 2  phospholipid ammonium salt (18) to the precursor dimer peptide. As mentioned previously, the PEG2000 moiety of DSPE-PEG2000-NH2 is nominally comprised of 45 ethylene glycol units. It should be understood, however, that this material is a distribution of PEG containing species whose centroid is the nominal compound containing 45 ethylenoxy units. 
     Conjugation of the DSPE-PEG2000-NH 2  to the precursor dimer peptide may be accomplished by preparation of a glutaric acid monoamide mono NHS ester of the precursor dimer and reaction of this with the free amino group of the phospholipid ammonium salt. Thus the ivDde bearing precursor dimer peptide (16) may be reacted with DSG (4 eq.) in DMF in the presence of DIEA (5 eq.) for 30 min. As in the preparation of the precursor dimer peptide the solution may be diluted with ethyl acetate to precipitate the glutaric acid monoamide mono-NHS ester of the dimer (17), as a solid. The supernatant may be decanted to remove the un-reacted DSG. The solid glutaric acid monoamide mono-NHS ester of the dimer peptide (17) may then be washed several times with ethyl acetate to remove traces of DSG. Mass spectral results confirm the formation of the glutaric acid monoamide mono-NHS ester of the peptide dimer as a clean product. 
     The dimer glutaric acid monoamide mono-NHS ester (17) may be dissolved in DMF-CH 2 Cl 2  (8:2) and reacted with DSPE-PEG2000-NH 2  phospholipid ammonium salt (0.9 eq.) in the presence of DIEA (4 eq.) for 24 h. The NHS ester (17) may be used in excess to maximize the consumption of the phospholipid ammonium salt because any free phospholipid may complicate the purification and isolation of the final product. The reaction mixture may be diluted with water (0.1% TFA)-CH3 CN—CH 3 OH (1:1) (0.1% TFA) (˜100 mL), applied to a reversed phase C4 column (Kromasil® Prep C4, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) and the column may be eluted with water (0.1% TFA)-CH 3 CN—CH 3 OH (1:1) (0.1% TFA) solvent mixture to remove hydrophilic impurities. Then the product may be eluted using a gradient of CH 3 CN—CH 3 OH (1:1) (0.1% TFA) into water (0.1% TFA). The collected fractions may be analyzed by reversed phase HPLC using an ELS detector which allows the detection of the desired product and the often difficult to separate DSPE-PEG2000-NH 2  phospholipid ammonium salt which has no strong UV chromophore. This indicates the clear separation of dimeric peptide phospholipid conjugate and DSPE-PEG2000-NH 2  phospholipid ammonium salt. The pure product-containing fractions may be collected, concentrated on a rotary evaporator (to reduce the content of methanol) and freeze-dried to provide the dimer peptide phospholipid conjugate as a colorless solid. 
     In order to prepare the required quantity of the dimer peptide phospholipid conjugate, several runs may be conducted employing 0.5 g to 1.0 g of the precursor dimer peptide. In all cases the target dimer peptide phospholipid conjugate may be isolated in 57-60% yield and in &gt;99% purity. The bulk quantity of dimer peptide phospholipid conjugate, obtained from the serial runs described above may be obtained by dissolution of the product from the individual runs in t-butanol-acetonitrile-water (1:1:3) followed by lyophilization. The procedure of Ellman for quantitative estimation of free thiol may be applied to the bulk sample of the dimeric peptide phospholipid conjugate; free thiol, if present will be below the limit of detection. Amino acid composition analysis gives results within the acceptable limits, supporting the assigned structure of the peptide derivative. MALDI-TOF mass spectral analysis also supports the presumed structure of the dimeric peptide phospholipid conjugate. 
     Methods of Preparation of Dimer-Phospholipid Conjugates Having Low or Negligible Levels of TFA 
     The present invention also provides methods for producing dimeric peptide-phospholipid conjugates having very low levels of TFA. While certain methods provide for the synthesis and purification of such conjugates on a gram scale, formation of a lyso-version of the conjugates has been observed upon storage of lyophilized material at 5° C. or upon storage of aqueous solutions of the conjugates. It is believed that the lyso-compound is formed by TFA-promoted acid hydrolysis of one of the phospholipid fatty acid esters in dimer peptide-phospholipid conjugates. 
     To obtain the phospholipid peptide as a stable material bearing a pharmaceutically acceptable counterion, highly efficient methods for obtaining dimer peptide-phospholipid conjugates were discovered which convert the TFA salts of the dimer peptide-phospholipid conjugate, or any suitable precursor(s), to analogous pharmaceutical acetate salt(s). Representative embodiments of these methods are provided below. 
     Table 3-A provides a description for the identification labels shown in  FIGS. 110, 111 and 112 . 
     
       
         
           
               
               
             
               
                 TABLE 3-A 
               
               
                   
               
             
            
               
                 21 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac- 
               
               
                   
                 VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH- 
               
               
                   
                 Glut)]-NH 2  cyclic (2-12) disulfide}-NH 2  cyclic (6-13) disulfide 
               
               
                   
               
               
                 22 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH 2  cyclic (2-12) disulfide•nTFA 
               
               
                   
               
               
                 23 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH 2  cyclic (2-12) disulfide•xHOAc 
               
               
                   
               
               
                 24 
                 mono-NHS ester of glutaryl-peptide 23 
               
               
                   
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[NHS-Glut-K(ivDde)]-NH 2  cyclic (2- 
               
               
                   
                 12) disulfide 
               
               
                   
               
               
                 25 
                 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH 2  cyclic (2-12) disulfide•yTFA 
               
               
                   
               
               
                 26 
                 Ac-VCWEDSWGGEVCFRYDPGGGK(Adoa-Adoa)-NH 2  cyclic (2-12) disulfide•zHOAc 
               
               
                   
               
               
                 27 
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac- 
               
               
                   
                 VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)-NH 2  cyclic (2-12) 
               
               
                   
                 disulfide]-NH 2  cyclic (6-13) disulfide•X HOAc 
               
               
                   
               
               
                 28 
                 Mono-NHS ester of glutaryl-peptide 27 
               
               
                   
                 Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac- 
               
               
                   
                 VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K(NHS-Glut)]-NH 2  cyclic 
               
               
                   
                 (2-12) disulfide}-NH 2  cyclic (6-13) disulfide 
               
               
                   
               
               
                 29 
                 DSPE-PEG2000-NH 2   
               
               
                   
               
            
           
         
       
     
     Where m, n, x, y, z are variable depending on lyophilization conditions. 
     Referring now to  FIGS. 110 and 111 , in certain embodiments monomer peptide components of heterodimer peptide (27), namely TFA salts compounds (22) and (25), are subjected to ion exchange chromatography on the macroporous sulfonic acid cation exchange resin AG MP-50 using a step gradient of ammonium acetate to convert them to their acetate salts. Then the two peptide monomer acetates (23) and (26) may be tethered through a glutaryl linker to form the dimer (27) as an acetate salt. Purification of the crude dimer acetate salt of (27), by C-18 preparative HPLC using a linear gradient method employing CH 3 CN H 2 O each containing 10 mM NH4OAc provides the pure dimer acetate (27). Conjugation of this dimer to DSPE-PEG2000-NH 2  (29) and final purification of the crude mixture by C-3 preparative HPLC using CH 3 CN/H 2 O/NH 4 OAc provides compound (21) as the acetate salt. 
     More specifically, compounds (22), (25) and (27) all bear side-chain carboxylic acid and amino groups. AG MP-50, a macroporous cation-exchange resin, may be used to allow full penetration of the resin by the peptides and to exploit the immobilization of the peptides via their basic (amino and guanidine groups). TFA salts of the peptides may be adsorbed to an AG MP-50 column (sulfonic acid form) and the column may be washed with water and then eluted with a step gradient of NH 4 OAc in 0 or 30% CH 3 CN/H 2 O, depending on the solubility of the peptides. The peptides may be eluted at about 600 mM NH 4 OAc and the acetate form of the peptides then may be obtained in pure form. Both IC fluorine analysis and CE TFA counter-ion analysis consistently show very low TFA content of the peptides. 
     Preferred methods also include redissolution/relyophilization of the final peptides several times to remove residual NH4OAc. Otherwise, residual traces of NH 4 OAc present in the peptides may give rise to free ammonia in the presence of DIEA. This may result in the formation of unwanted peptide-Glut-amide as a major product in subsequent preparation of (27) from the monomers (23) and (26) or final phospholipid-peptide conjugate (21) from the acetate salt of (27). 
     Referring now to  FIG. 111 , another embodiment provides the conversion of the TFA salt of dimer (27) to its analogous acetate salt by ion exchange chromatography on the macroporous sulfonic acid cation exchange resin AG MP-50. This dimer acetate then may be conjugated with DSPE-PEG2000-NH 2  followed by purification of the crude material by C-3 preparative column using CH 3 CN/H 2 O/NH 4 OAc to give the final compound (21) as an acetate salt. 
     While the methods described above and in  FIGS. 110 and 111  provide excellent results, the second approach has the advantage of requiring fewer steps. Additional details are provided below in the Examples section. 
     Turning to  FIG. 112 , another embodiment provides methods for providing dimeric conjugates having minimal amounts of TFA utilizing the size differential between the phospholipid-peptide conjugate (21) and TFA ions. In this embodiment 21●nTFA adduct may be eluted down a size exclusion column in the presence of ammonium bicarbonate buffer. The crude 21●nTFA initially may be freed of the lyso-compound by preparative HPLC on a Zorbax C-3 column using a linear gradient of acetonitrile into water. Both phases may be buffered with 10 mM ammonium acetate. This provides separation of the lyso-compound as indicated by analytical HPLC. 
     To further reduce the amount of TFA, the material may be applied to a Sephadex G-25 column and eluted with aqueous ammonium bicarbonate solution. The eluate may be monitored by HPLC. Product-containing fractions may be pooled and lyophilized to afford the desired material (21) essentially free of TFA and with high recovery rates. Additional detail is provided below in the Examples section. 
     Both the monomeric and dimeric peptide phospholipid conjugates described herein may be incorporated into ultrasound contrast agents such as, for example, gas filled microvesicles. Such gas filled microvesicles include, for example, gas filled microbubbles, gas filled microballoons, gas filled microcapsules, etc. In a preferred embodiment, the peptide phospholipid conjugates may be incorporated into ultrasound contrast agents comprising gas filled microbubbles. Methods of preparation of gas filled microbubbles from phospholipids and phospholipid conjugates are known to those skilled in the art. For example, microbubbles according to the present invention can be prepared by methods described in any one of the following patents: EP 554213, WO 04/069284, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,183,725, EP 474833, U.S. Pat. No. 5,271,928, U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551, U.S. Pat. No. 5,643,553, U.S. Pat. No. 5,911,972, U.S. Pat. No. 6,110,443, U.S. Pat. No. 6,136,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entirety. The methods disclosed in WO 04/069284 are particularly preferred. 
     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. Examples of 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 phospholipids are known in the art as the “lyso” forms of the phospholipid. 
     Further examples of 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. 
     As used herein, the term phospholipids includes either naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures. 
     Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins. Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins. 
     Examples of synthetic phospholipids are e.g., dilauryloyl-phosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoyl-phosphatidylcholine (“DPPC”), diarachidoylphosphatidylcholine (“DAPC”), distearoyl-phosphatidylcholine (“DSPC”), 1-myristoyl-2-palmitoylphosphatidylcholine (“MPPC”), 1-palmitoyl-2-myristoylphosphatidylcholine (“PMPC”), 1-palmitoyl-2-stearoylphosphatidylcholine (“PSPC”), 1-stearoyl-2-palmitoyl-phosphatidylcholine (“SPPC”), dioleoylphosphatidylycholine (“DOPC”), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dilauryloyl-phosphatidylglycerol (“DLPG”) and its alkali metal salts, diarachidoylphosphatidylglycerol (“DAPG”) and its alkali metal salts, dimyristoylphosphatidylglycerol (“DMPG”) and its alkali metal salts, dipalmitoyl-phosphatidylglycerol (“DPPG”) and its alkali metal salts, distearolyphosphatidylglycerol (“DSPG”) and its alkali metal salts, dioleoylphosphatidylglycerol (“DOPG”) and its alkali metal salts, dimyristoyl phosphatidic acid (“DMPA”) and its alkali metal salts, dipalmitoyl phosphatidic acid (“DPPA”) and its alkali metal salts, distearoyl phosphatidic acid (“DSPA”), diarachidoyl phosphatidic acid (“DAPA”) and its alkali metal salts, dimyristoyl phosphatidyl-ethanolamine (“DMPE”), dipalmitoyl phosphatidylethanolamine (“DPPE”), distearoyl phosphatidyl-ethanolamine (“DSPE”), dimyristoyl phosphatidylserine (“DMPS”), diarachidoyl phosphatidylserine (“DAPS”), dipalmitoyl phosphatidylserine (“DPPS”), distearoylphosphatidylserine (“DSPS”), dioleoylphosphatidylserine (“DOPS”), dipalmitoyl sphingomyelin (“DPSP”), and distearoyl sphingomyelin (“DSSP”). 
     Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer thereto. Examples of modified phospholipids are phosphatidylethanolamines (PE) modified with polyethylenglycol (PEG), in brief “PE-PEGs”, i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g. from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE-PEG or DAPE-PEG (where DAPE is 1,2-diarachidoyl-sn-glycero-3-phosphoethanolamine). The compositions also may contain other amphiphilic compounds including, for instance, fatty acids, such as palmitic acid, stearic acid, arachidonic acid or oleic acid; sterols, such as cholesterol, or esters of sterols with fatty acids or with sugar acids; glycerol or glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate; tertiary or quaternary alkyl-ammonium salts, such as 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and mixtures or combinations thereof. 
     Preferably, the formulation comprises at least one component bearing an overall net charge, such as, for instance, phosphatidic acid, PE-PEG, palmitic acid, stearic acid, Ethyl-DSPC or DSTAP, preferably in a molar amount of less than about 50%. Particularly preferred formulations may include mixtures of two or more of the following components: DSPC, DPPG, DPPA, DSPE-PEG1000, DSPE-PEG2000, palmitic acid and stearic acid. Some preferred phospholipids and formulations are set forth in the examples. Any of the gases disclosed herein or known to the skilled artisan may be employed; however, inert gases, such as SF6 or perfluorocarbons like CF 4 , C 3 F 8  and C 4 F 10 , are preferred, optionally in admixture with other gases such as air, nitrogen, oxygen or carbon dioxide 
     The preferred 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; WO 04/069284; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; U.S. Pat. No. 5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S. Pat. No. 6,136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entirety. Preferably, as disclosed in International patent application WO 04/069284, a microemulsion can be prepared which contains the phospholipids (e.g. DSPC and/or DSPA) in admixture with a lyoprotecting agent (such as, for instance, carbohydrates, sugar alcohols, polyglycols and mixtures thereof, as indicated in detail hereinafter) and optionally other amphiphilic materials (such as stearic acid), dispersed in an emulsion of water and of a water immiscible organic solvent. Preferred organic solvents are those having solubility in water of 1.0 g/l or lower, preferably lower about 0.01 g/l, and include, for instance, pentane, hexane, heptane, octane, nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopentane, cyclohexane, cyclooctane, 1-methyl-cyclohexane, benzene, toluene, ethylbenzene, 1,2-dimethylbenzene, 1,3-dimethylbenzene, di-butyl ether and di-isopropylketone, chloroform, carbon tetrachloride, 2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane), 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane), tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane, perfluoroheptane, perfluorononane, perfluorobenzene, perfluorodecalin, methylperfluorobutylether, methylperfluoroisobutylether, ethylperfluorobutylether, ethylperfluoroisobutylether and mixtures thereof. The peptide-phospholipid conjugate of the invention can be admixed together with the phospholipid forming the microvesicle&#39;s envelope, in the microemulsion. Preferably, an aqueous suspension of the peptide-phospholipid conjugate and of a PE-PEG (e.g. DSPE-PEG2000) is first prepared, which is then admixed together with an aqueous-organic emulsion comprising the phospholipid and the lyoprotecting agent. Preferably said mixing is effected under heating, e.g. form about 40° C. to 80° C. 
     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 structure has been disrupted will form lamellarized or laminarized segments that will stabilize the microbubbles of the gas dispersed therein. This method permits production of suspensions of microbubbles that 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. 
     Alternatively, 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 WO 2004/069284, herein incorporated 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. Some preferred preparation methods are disclosed in the examples. 
     The formulation for the preparation of the gas-filled microbubbles may advantageously further comprise a lyophilization additive, such as an agent with cryoprotective and/or lyoprotective effect and/or a bulking agent, for example an amino-acid such as glycine; a carbohydrate, e.g. a sugar such as sucrose, mannitol, maltose, trehalose, glucose, lactose or a cyclodextrin, or a polysaccharide such as dextran; or a polyglycol such as polyethylene glycol (e.g. PEG-4000). 
     Any of these ultrasound compositions should also be, as far as possible, isotonic with blood. Hence, before injection, 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. Additionally, 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 of the invention. The term “gas” as used herein 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. However, fluorinated gases are preferred. Fluorinated gases include materials that 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 10 , CBrF 3 , CCl 2 F 2 , C 2 ClF 5 , and CBrClF 2 ) and perfluorocarbons. The term 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 preferred, have the formula CnFn+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 2 , C 7 F 14 , C 8 F 18 , and C 9 F 20 . Most preferably 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 2 , C 6 F 12 , C 7 F 14 , C 8 F 18 , with C 4 F 10  being particularly preferred. See also WO 97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501, WO 98/05364, WO 98/17324. In a preferred embodiment the gas comprises C 4 F 10  or SF 6 , optionally in admixture with air, nitrogen, oxygen or carbon dioxide. 
     In certain circumstances it may be desirable to include 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”). Preferably the gas precursor and the gas it produces are physiologically acceptable. The gas precursor may be pH-activated, photo-activated, temperature activated, etc. For example, certain perfluorocarbons may be used as temperature activated gas precursors. These perfluorocarbons, such as perfluoropentane, 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. 
     As discussed above, 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. 
     Unless it contains a hyperpolarized gas, known to require special storage conditions, the lyophilized product 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. Preferably in such a case it can be supplied in the form of a two-component kit, which can include two separate containers or a dual-chamber container. In 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. In such a case the syringe used as the container of the second component is also used then for injecting the contrast agent. In the latter case, preferably 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. In both cases means for directing or permitting application of sufficient bubble forming energy into the contents of the container are provided. 
     However, as noted above, in the stabilised contrast agents according to the invention 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. 
     It can be appreciated by one of ordinary skilled in the art that other two-chamber reconstitution systems capable of combining the dried powder with the aqueous solution in a sterile manner are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed between the water-insoluble gas and the environment, to increase shelf life of the product. Where a material necessary for forming 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. 
     No specific containers, vial or connection systems are required; 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. In addition to assuring sterility, vacuum retention is essential for products stoppered at ambient or reduced pressures to assure safe and proper reconstitution. The stopper may be a compound or multicomponent formulation based on an elastomer, such as poly(isobutylene) or butyl rubber. 
     In ultrasound applications the contrast agents formed by phospholipid stabilized microbubbles can be administered, for example, in doses such that the amount of phospholipid injected is in the range 0.1 to 200 μg/kg body weight, preferably from about 0.1 to 30 μg/kg. 
     Ultrasound imaging techniques that can 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. Imaging may be done in harmonic (resonant frequency) or fundamental modes, with the second harmonic preferred. 
     The ultrasound contrast agents of the present invention may further be used in a variety of therapeutic imaging methods. The term therapeutic imaging includes within its meaning any method for the treatment of a disease in a patient which comprises the use of a contrast imaging agent (e.g. for the delivery of a therapeutic agent to a selected receptor or tissue), and which is capable of exerting or is responsible to exert a biological effect in vitro and/or in vivo. Therapeutic imaging may advantageously be associated with the controlled localized destruction of the gas-filled microvesicles, e.g. by means of an ultrasound burst at high acoustic pressure (typically higher than the one generally employed in non-destructive diagnostic imaging methods). This controlled destruction may be used, for instance, for the treatment of blood clots (a technique also known as sonothrombolysis), optionally in combination with the localized release of a suitable therapeutic agent. Alternatively, said therapeutic imaging may include the delivery of a therapeutic agent into cells, as a result of a transient membrane permeabilization at the cellular level induced by the localized burst of the microvesicles. This technique can be used, for instance, for an effective delivery of genetic material into the cells; optionally, a drug can be locally delivered in combination with genetic material, thus allowing a combined pharmaceutical/genetic therapy of the patient (e.g. in case of tumor treatment). 
     The term “therapeutic agent” includes within its meaning any substance, composition or particle which may be used in any therapeutic application, such as in methods for the treatment of a disease in a patient, as well as any substance which is capable of exerting or responsible to exert a biological effect in vitro and/or in vivo. Therapeutic agents thus include any compound or material capable of being used in the treatment (including diagnosis, prevention, alleviation, pain relief or cure) of any pathological status in a patient (including malady, affliction, disease lesion or injury). Examples of therapeutic agents are drugs, pharmaceuticals, bioactive agents, cytotoxic agents, chemotherapy agents, radiotherapeutic agents, proteins, natural or synthetic peptides, including oligopeptides and polypeptides, vitamins, steroids and genetic material, including nucleosides, nucleotides, oligonucleotides, polynucleotides and plasmids. 
     EXAMPLES 
     Methods for the Examples 
     The following methods were employed in Examples 4-10. The following common abbreviations are used: 9-fluorenylmethyloxycarbonyl (Fmoc), 1-hydroxybenzotriazole (HOBt), N,N′-diisopropylcarbodiimide (DIC), N-methylpyrrolidinone (NMP), acetic anhydride (Ac 2 O), (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), trifluoroacetic acid (TFA), Reagent B (TFA: H 2 O: phenol:triisopropylsilane 88:5:5:2), diisopropylethylamine (DIEA), O-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-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). 
     Method 1 for the ACT 357 MPS and ACT 496 MOS Synthesizers 
     The peptides were synthesized on NovaSyn TGR (Rink amide) resin (0.2 mmol/g) using the Advanced ChemTech ACT 357 or ACT 496 Synthesizers employing Fmoc peptide synthesis protocols, specifically using HOBt/DIC as the coupling reagents and NMP as the solvent. The Fmoc was removed by treating the Nova-Syn TGR (Rink amide-available from NovaBiochem, San Diego Calif.) resin-bound peptide with 25% piperidine in DMF twice (4 min and 10 min). All amino acids were dissolved in NMP (DMF was added when the amino acid was not soluble in pure NMP). The concentration of the amino acid was 0.25M, and the concentrations for HOBt and DIC respectively were 0.5 M. 
     For a 0.04 mmol scale synthesis: 
     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 μL), HOBt solution (320 μL, 4 eq), amino acid (640 μL, 4 eq) and DIC (320 μL, 4 eq) solutions were dispensed to each well. The coupling time was 3 h; then the resin was washed. The cycle was repeated for each amino acid. After the last amino acid coupling, 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. Ether was added to the residue with swirling or stirring to precipitate the peptides. The ether was decanted, and solid was collected. This procedure was repeated 2-3 times to remove impurities. The crude linear peptides were dissolved in DMSO and water mixtures, and purified by HPLC (column: Waters Associates Xterra C18, 19×50 mm; solvents: H 2 O with 0.1% TFA and CH 3 CN with 0.1% TFA; UV 220 μm; Flow rate: 50-60 mL/min). The solutions containing the peptide were lyophilized to give the desired peptides as white fluffy lyophilizates (&gt;90% purity). The purified linear di-cysteine containing peptides were dissolved in water, mixtures of water-acetonitrile, or mixtures of water-DMSO at concentrations between 0.1 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 ammonia, aqueous ammonium carbonate or aqueous ammonium bicarbonate. The mixture was stirred vigorously in air for 24-48 hrs. In the case of non-DMSO containing solvent systems, 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 (0.1% TFA) containing a minimum of acetonitrile (0.1% 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. In 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. 
     For a 0.01 mmol scale synthesis: 
     After the Fmoc group was removed, a standard coupling procedure used a solution of HOBt (720 μl, 6 eq), amino acid (804 μl, 6.6 eq), HBTU (720 μl, 6 eq) and DIEA (798 μl, 13.3 eq). The mixture was agitated for 15 min., emptied and the resin washed. After all couplings and after cleavage and purification as above, the solutions containing desired linear peptides were lyophilized to give the peptides (&gt;90% purity) as white fluffy solids. 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 0.1 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. 
     Method 3 for the ACT 496 MOS Synthesizer 
     The peptides were synthesized by using an Advanced ChemTech ACT 496 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 3 h. The crude linear peptides were further processed as described for Method 1. 
     Method 4 for the ACT 496 MOS Synthesizer 
     The peptides were synthesized using method 3 on the ACT 496 with HBTU/DIEA as the coupling reagents, and NMP as the solvent. 2,4,6-collidine as a 1 M solution was used as the base. The low load Fmoc-GGGK(ivDde)-Novsyn-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 433A Synthesizer 
     Synthesis of peptides was carried out on a 0.25 mmol scale using the FastMoc protocol (Applied Biosystems Inc). In 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 0.1 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 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 stirred for 4-6 hours to effect disulfide cyclization. The crude mixture was purified by preparative HPLC. 
     In a typical experiment, 200 mg of the resin-bound peptide was treated with 10% hydrazine in DMF (2×20 mL) and washed with DMF (2×20 mL) and then with dichloromethane (1×20 mL). The resin was resuspended in DMF (10 mL) and treated with a solution of Biotin-NHS ester (0.2 mmol, 5 equivalents) and DIEA (0.2 mmol), and the resin was mixed with the reagents for 4 h. The completion of the reaction was checked by the ninhydrin test. The peptide was then released from the resin by treatment with Reagent B (10 mL) for 4 h. The resin was filtered off, Reagent B was removed in vacuo and the peptide was precipitated by addition of anhydrous ether. The solid formed 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 stirred 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% acetonitrile in water and directly purified by HPLC on a reverse phase C18 column using a gradient of acetonitrile into water (both containing 0.1% 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 
     In a typical experiment, 400 mg of the resin-containing peptide (made using the ABI 433A Synthesizer and bearing an ivDde-protected lysine) was treated with 10% hydrazine in DMF (2×20 mL). The resin was washed with DMF (2×20 mL) and DCM (1×20 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 (2×10 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-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 4 h. The mixture was filtered, and the filtrate concentrated to dryness. The residue was stirred 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 stirred for 4-6 hr to effect the disulfide cyclization reaction, which was monitored by analytical HPLC. After the completion of the cyclization, the DMSO solution 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 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, 2×10 mL, 10 min) to remove the ivDde group. The epsilon nitrogen of the lysine was labeled with fluorescein-5-isothiocyanate (0.12 mmol) and diisopropylethylamine (0.12 mmol) in DMF. The mixture was agitated for 12 h (fluorescein-containing compounds were protected from light). The resin was then washed with DMF (3×10 mL) and twice with CH 2 Cl 2  (10 mL) and dried under nitrogen for 1 h. The peptide was cleaved from the resin using reagent B for 4 h 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 0.1% 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). 
     Method 10A: Preparation of Peptidic Chelate for Binding to Tc by Coupling of Single Amino Acids 
     Peptides were synthesized starting with 0.1 mmol of NovaSyn-TGR resin (0.2 mmol/g substitution). Deprotected (ivDde) resin was then treated according to the protocol A for the incorporation of Fmoc-Gly-OH, Fmoc-Cys(Acm)-OH and Fmoc-Ser(tBu)-OH. 
     Protocol A for manual coupling of single amino acid: 
     1. Treat with 4 equivalents of corresponding Fmoc-amino acid and 4.1 equivalents of HOBt and 4.1 equivalents of DIC for 5 h. 
     2. Wash with DMF (3×10 mL) 
     3. Treat with 20% piperidine in DMF (2×10 mL, 10 min.) 
     4. Wash with DMF (3×10 mL) 
     The Fmoc-protected peptide loaded resin was then treated with 20% piperidine in DMF (2×10 mL, 10 min.) and washed with DMF (3×10 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 (3×10 mL) and CH 2 Cl 2  (3×10 mL) and dried under vacuum. 
     Method 10B: Preparation of Peptidic Chelate for Binding to Tc by Appendage of the Glutaryl-PnAO6 Chelator to the Peptide 
     Preparation of 4-{2-(2-Hydroxyimino-1,1-dimethylpropylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyric acid, N-hydroxysuccinimide ester (Compound B, FIG.  86 ) 
     4-{2-(2-Hydroxyimino-1,1-dimethyl-propylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyric acid (Compound A,  FIG. 86 ) (40 mg) was dissolved in DMF (700 μL). N-Hydroxysuccinimide (1.5 equiv, 17.2 mg) and 1,3-diisopropylcarbodiimide (1.5 equiv, 24 μL) were added. The progress of the reaction was monitored by mass spectroscopy. After 17 h, the reaction was complete. The volatiles were removed in vacuo and the residue was washed with ether (5×) to remove the unreacted NHS. The residue was dried to provide compound B, which was used directly without further treatment or purification. See  FIG. 86  for reaction scheme. 
     Functionalization of Peptides with 4-{2-(2-Hydroxyimino-1,1-dimethylpropylamino)-1-[(2-hydroxyimino-1,1-dimethyl-propylamino)-methyl]-ethylcarbamoyl}-butyric acid, N-hydroxysuccinimide ester—(Compound B) 
     The peptide (prepared, for example, by Methods 1-13) is dissolved in DMF and treated with compound B and DIEA sufficient to maintain the basicity of the mixture. The progress of the reaction is monitored by HPLC and mass spectroscopy. At completion of the reaction the volatiles are removed in vacuo and the residue is either purified by reverse phase HPLC or processed further by selective removal of side chain protecting groups or subjected to cleavage of all remaining protecting groups as required by the next steps in the synthesis scheme. 
     Method 11: Formation of Mercaptoacetylated Peptides 
     Using S-Acetylthioglycolic acid N-Hydroxysuccinimide Ester 
     S-acetylthioglycolic acid N-hydroxysuccinimide ester (SATA) (0.0055 mmol) 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 stirred at ambient temperature for 6 h. The volatiles were removed under vacuum and the residue was purified by preparative HPLC using acetonitrile-water containing 0.1% TFA. Fractions containing the pure product were collected and freeze-dried to yield the mercaptoacetylated peptide. The mercaptoacetylated peptide was characterized by ESI-MS and the purity was determined by reverse phase HPLC analysis employing a linear gradient of acetonitrile into water (both containing 0.1% TFA). Examples of SATA-modified peptides include, but are not limited to: 
     
       
         
           
               
               
            
               
                   
                 SATA-modified 
               
            
           
           
               
            
               
                 SEQ ID NO: 480 
               
            
           
           
               
               
            
               
                   
                 Ac-AGPTWCEDDWYYCWLFGTGGGGK(SATA-JJ)-NH 2   
               
               
                   
               
               
                   
                 SATA-modified 
               
            
           
           
               
            
               
                 SEQ ID NO: 356 
               
            
           
           
               
               
            
               
                   
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SATA)-NH 2   
               
               
                   
               
               
                   
                 SATA-modified 
               
            
           
           
               
            
               
                 SEQ ID NO: 356 
               
            
           
           
               
               
            
               
                   
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(SATA-JJ)-NH 2   
               
            
           
         
       
     
     Method 12: Formation of Mercaptoacetylated Peptides Using S-Acetylthioglycolic Acid 
     Purified peptides from method 5, after disulfide cyclization, was coupled with S-acetylthioglycolic acid (1.5-10 eq.)/HOBt (1.5-10 eq.)/DIC (1.5-10 eq.) in NMP for 2-16 hours at room temperature. The mixture was then purified by preparative HPLC; the fractions containing pure peptide were combined and lyophilized. In the case of compounds with another lysine protected by an ivDde group, the deprotection reaction employed 2% hydrazine in DMSO for 3 h at room temperature. Purification of the reaction mixture afforded pure peptide. 
     In the case when preparing a compound with S-acetylthioglycolic acid coupled to two aminodioxaoctanoic acid groups and the peptide, the purified peptide from method 5 (having a free amino group), was coupled to AcSCH 2 CO—(NH—CH 2 —CH 2 —O—CH 2 —CH 2 —O—CH 2 —CO) 2 —OH (30 eq.)/HOBt (30 eq.)/DIC (30 eq.) in NMP for 40 hours at room temperature. The mixture was purified, and the ivDde group was removed. A second purification gave the final product as a white lyophilizate. 
     Alternatively 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 Homodimers 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-hydroxysuccinimidyl 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. The reaction was allowed to proceed for 24 h. This mixture was applied directly to a Waters Associates C-18 XTerra reverse phase HPLC column and purified by elution with a linear gradient of acetonitrile into water (both containing 0.1% TFA). 
     In the case of heterodimers, one of the monomers was reacted with the bis NHS ester of glutaric acid and after washing off the excess of bis NHS ester, the second peptide was added in the presence of DIEA. After the reaction, the mixture was purified by preparative HPLC. 
     Example 1 
     Library Screening Against KDR and KDR/VEGF Complex Targets 
     Chimeric fusions of 1 g Fc region with human KDR (#357-KD-050), murine KDR (#443-KD-050), human VEGFR-1 (#321-FL-050), human VEGFR-3 (#349-F4-050), and human Trail R4 (#633-TR-100) were purchased in carrier-free form (no BSA) from R &amp; D Systems (Minneapolis, Minn.). Trail R4 Fc is an irrelevant Fc fusion protein with the same Fc fusion region as the target Fc fusion (KDR Fc) and is used to deplete the libraries of Fc binders. VEGF 165  (#100-20) was purchased in carrier-free form from Peprotech (Rocky Hill, N.J.). 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.). 
     In the following procedures, 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, Calif.). 
     Eight M13 phage display libraries were prepared for screening against immobilized KDR and VEGF/KDR targets: Cyclic peptide display libraries TN6/VI, TN7/IV, TN8/IX, TN9/IV, TN10/IX, TN12/I, 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-Elmer, Wellesley, Mass.), cleaved with NcoI and PstI, and ligated to similarly cleaved phage display vector. XL1-Blue MFR′  E. coli  cells were transformed with the ligated DNA. All of the libraries were constructed in same manner. 
     KDR Selection Protocol in the Presence of Heparin 
     Protein A Magnetic Beads were blocked once with 1×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 1×PBS (pH 7.5), 0.01% Tween-20, 5 μg/mL heparin (PBSTH Buffer). 
     The cyclic peptide, or “constrained loop”, libraries were pooled for the initial screening into two pools: TN6/VI, 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 irrelevant Fc fusion) and then selected against KDR Fc fusion. 10 11  plaque forming units (pfu) from each library per 100 μL PBSTH were pooled together, e.g., 3 pooled libraries would result in a total volume of ˜350 μl in PBSTH. 
     To prepare the irrelevant Fc fusion beads, 500 μl of Trail R4-Fc fusion (0.1 μg/μl stock in PBST (no heparin)) were added to 1000 μl 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 μl 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 μL 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. 
     To prepare the KDR target beads, 500 μl of KDR-Fc fusion (0.1 μg/μl stock in PBST (no heparin)) were added to 500 μL 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 μL 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 5×1 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 μl of VEGF (50 μg/mL, ˜1 μM) 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 μl of VEGF (50 μg/mL, ˜1 μM) 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 XL1-Blue MRF′ (or other F′ cell line)  E. coli  cells that 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 VEGF incubations (1-hour and overnight (O/N) elutions). After approximately 15 minutes at room temperature, the phage/cell mixtures were spread onto Bio-Assay Dishes (243×243×18 mm, Nalge Nunc) containing 250 mL of NZCYM agar with 50 μg/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 (i.e., Fraction of Input=phage output divided by phage input). 
     In the first round, each pool yielded three amplified eluates. These eluates were panned for 2-3 more additional rounds of selection using ˜10 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. For the elution step in subsequent rounds, the amplified elution re-screen on KDR-Fc beads was always eluted in the same manner, and all other elutions were treated as washes. For example, for 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. Using this procedure, 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. 
     KDR Selection Protocol in the Absence of Heparin 
     ++A true TN11/1 library was used to screen for KDR binders. The same selection protocol as above (KDR Selection Protocol in the Presence of Heparin) was used, except heparin was omitted. The three elution conditions were VEGF elution (1 uM; 1 hr; same as original protocol), Dimer D6 elution (0.1 uM; 1 hr), and then bead elution (same as above). TN11/1 alone was used in the selection and screening. For selected peptides, see Table 27 and Consensus Sequence 9A. 
     KDR:VEGF Complex Selection Protocol in the Presence of Heparin 
     Protein A magnetic beads were blocked once with Blocking Buffer for 30 minutes at room temperature and then washed five times with PBSTH. 
     Two pools of constrained loop libraries and a linear library (Lin20) were prepared as before and then depleted against KDR Fc fusion alone, instead of Trail-R4 Fc fusion, to remove binders to the receptor without bound VEGF. Once depleted, the libraries were selected against the KDR:VEGF 165  complex. 
     To prepare KDR-Fc fusion depletion beads, 1 mL of KDR-Fc fusion (0.1 μg/μL stock in PBST (no heparin)) was added to 1 mL of washed, blocked beads. The fusion was allowed to bind overnight with agitation at 4° C. The next day, the beads were washed 5 times with PBSTH. Each phage pool was incubated with 50 μl of KDR-Fc fusion beads on a LabQuake shaker for 1 hour at RT. After incubation, the phage supernatant was removed and incubated with another 50 μL of KDR-Fc beads. This was repeated for a total of 5 rounds of depletion. 
     To prepare the KDR:VEGF complex beads, 300 μL of KDR-Fc fusion beads from above were incubated with 15 μL of VEGF (1 mg/mL). VEGF was allowed to bind for 1 hour at RT. The beads were washed 5 times with PBSTH. Each depleted library pool was added to 100 μl 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. After approximately 15 minutes at room temperature, the phage/cell mixtures were spread onto Bio-Assay Dishes (243×243×18 mm, Nalge Nunc) containing 250 mL of NZCYM agar with 50 μg/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. 
     KDR and KDR/VEGF Screening Assay 
     100 μl of KDR-Fc fusion or Trail R4-Fc fusion (1 μg/mL) were added to duplicate Immulon II plates, to every well, and allowed to incubate at 4° C. overnight. Each plate was washed twice with PBST (PBS, 0.05% Tween-20). The wells were filled to the top with 1×PBS, 1% BSA and allowed to incubate at RT for 2 hours. Each plate was washed once with PBST (PBS, 0.05% Tween-20). 
     To assess binding to KDR:VEGF complex, another set of KDR plates was prepared as above and then 100 μL of VEGF (1 μg/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). 
     Once the plates were prepared, 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 μl 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 μl 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. 
     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. 
     After isolation of KDR and VEGF/KDR complex isolates in initial selection rounds, certain isolates were selected to act as templates for the construction of secondary libraries, from which additional high affinity binding polypeptides were isolated. In a secondary TN8 library, the phage isolate sequence PKWCEEDWYYCMIT (SEQ ID NO:21) 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. In a secondary TN12 library, the 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. In a another TN8 secondary library, a recurrent 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). 
     Using a method of peptide optimization by soft randomization as described by Fairbrother et al.,  Biochemistry,  37(51):17754-17764 (1998), two libraries were prepared based on the SEQ ID NO:21 and SEQ ID NO:88 sequences. At each residue position, each nucleotide within a particular codon was allowed to evolve by adding fixed amounts of the other three nucleotides that did not correspond to the nucleotide of the parent codon. This nucleotide mixing is accomplished in the synthesis of the template DNA used to make the library. For these libraries, 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. Inspection of individual isolates, however, shows that multiple mutations are possible, thus allowing selection of peptides with improved binding ability compared to the parent sequence. 
     For the third library, 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-terminal to the display peptide. The secondary library template, therefore, encoded a display peptide of the following sequence: Xaa-Xaa-Trp-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. Unlike the previous two libraries, where the consensus sequence remains the parental sequence, this library was quite diverse in all allowed positions and only resembled the parent motif in the residues that were held constant. 
     A total of 2×10 11  pfu from each library was used as before, except the elution strategy was changed. Competition elution of bound phage was performed using the parental peptide (50 μM) that was used to make the particular secondary library (i.e., peptides of SEQ ID NOS:21, 88, and 40, respectively). 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 μM 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 ID NOS: 22-33 for the TN8 secondary library and SEQ ID NOS:89-95 for the TN12 secondary library. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 TN8/IXLibrary Isolates 
               
            
           
           
               
               
               
               
            
               
                 Sequence 
                 SEQ ID NO: 
                 Elution 
                 Class 
               
               
                   
               
               
                 DSWCSTEYTYCEMI 
                 20 
                 1 HR 
                 NA 
               
               
                   
               
               
                 PKWCEEDWYYCMIT 
                 21 
                 1 HR 
                 (III) 
               
               
                   
               
               
                 SDWCRVDWYYCWLM 
                 22 
                 O/N 
                 III 
               
               
                   
               
               
                 ANWCEEDWYYCFIT 
                 23 
                 O/N 
                 III 
               
               
                   
               
               
                 ANWCEEDWYYCWIT 
                 24 
                 O/N 
                 III 
               
               
                   
               
               
                 PDWCEEDWYYCWIT 
                 25 
                 O/N 
                 III 
               
               
                   
               
               
                 SNWCEEDWYYCYIT 
                 26 
                 O/N 
                 III 
               
               
                   
               
               
                 PDWCAADWYYCYIT 
                 27 
                 O/N 
                 III 
               
               
                   
               
               
                 PEWCEVDWYYCWLL 
                 28 
                 CELL 
                 III 
               
               
                   
               
               
                 PTWCEDDWYYCWLF 
                 29 
                 O/N 
                 III 
               
               
                   
               
               
                 SKWCEQDWYYCWLL 
                 30 
                 CELL 
                 III 
               
               
                   
               
               
                 RNWCEEDWYYCFIT 
                 31 
                 O/N 
                 III 
               
               
                   
               
               
                 VNWCEEDWYYCWIT 
                 32 
                 O/N 
                 III 
               
               
                   
               
               
                 ANWCEEDWYYCYIT 
                 33 
                 O/N 
                 III 
               
               
                   
               
               
                 VWECAKTFPFCHWF 
                 34 
                 1 HR 
                 I 
               
               
                   
               
               
                 VTVCYEGTRICEWH 
                 35 
                 1 HR 
                 NA 
               
               
                   
               
               
                 WVECRYSTGLCINY 
                 36 
                 O/N 
                 NA 
               
               
                   
               
               
                 WYWCDYYGIGCKWT 
                 37 
                 1 HR 
                 NA 
               
               
                   
               
               
                 WVECWWKSGQCYEF 
                 38 
                 1 HR, CELL 
                 (II) 
               
               
                   
               
               
                 WIQCDMETGLCTHG 
                 39 
                 1 HR, CELL 
                 II 
               
               
                   
               
               
                 WVECFMDTGACYTF 
                 40 
                 CELL, O/N 
                 II 
               
               
                   
               
               
                 WLECYAEFGHCYNF 
                 41 
                 CELL, O/N 
                 II 
               
               
                   
               
               
                 WIECDMLTGMCKHG 
                 42 
                 CELL 
                 NA 
               
               
                   
               
               
                 SVECFMDTGACYTF 
                 43 
                 CELL 
                 I 
               
               
                   
               
               
                 WIQCNSITGHCTSG 
                 44 
                 CELL 
                 II 
               
               
                   
               
               
                 WIECYHPDGICYHF 
                 45 
                 CELL 
                 (III) 
               
               
                   
               
               
                 QAWVECYAETGYCWPRSW 
                 46 
                 NA 
                 NA 
               
               
                   
               
               
                 VGWVECYQSTGFCYHSRD 
                 47 
                 NA 
                 NA 
               
               
                   
               
               
                 FTWVECHQATGRCVEWTT 
                 48 
                 NA 
                 NA 
               
               
                   
               
               
                 DWWVECRVGTGLCYRYDT 
                 49 
                 NA 
                 NA 
               
               
                   
               
               
                 DSWVECDAQTGFCYSFLY 
                 50 
                 NA 
                 NA 
               
               
                   
               
               
                 GGWVECYWATGRCIEFAG 
                 51 
                 NA 
                 NA 
               
               
                   
               
               
                 ERWVECRAETGFCYTWVS 
                 52 
                 NA 
                 NA 
               
               
                   
               
               
                 GGWVECRAETGHCQEYRL 
                 53 
                 NA 
                 NA 
               
               
                   
               
               
                 VAWVECYQTTGKCYTFRG 
                 54 
                 NA 
                 NA 
               
               
                   
               
               
                 EGWVECFANTGACFTYPR 
                 55 
                 NA 
                 NA 
               
               
                   
               
               
                 GVECYKHSGMCRSW 
                 56 
                 O/N 
                 II 
               
               
                   
               
               
                 GVWCDMVTGWCYHG 
                 57 
                 CELL 
                 II 
               
               
                   
               
               
                 WIECHYKTGHCIHS 
                 58 
                 CELL 
                 II 
               
               
                   
               
               
                 DFNCKMIDGFCLLK 
                 59 
                 1 HR 
                 II 
               
               
                   
               
               
                 WIQCDRKAGRCSRG 
                 60 
                 CELL 
                 II 
               
               
                   
               
               
                 TITCWMDTGHCMHE 
                 61 
                 CELL 
                 II 
               
               
                   
               
               
                 GINCYPATGKCQMG 
                 62 
                 CELL 
                 II 
               
               
                   
               
               
                 WTECHYATGKCHSF 
                 63 
                 CELL 
                 II 
               
               
                   
               
               
                 LNICKEDWYYCFLL 
                 64 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 GITCYSATGKCQMW 
                 65 
                 CELL 
                 II 
               
               
                   
               
               
                 WVQCASDTGKCIMG 
                 66 
                 CELL 
                 II 
               
               
                   
               
               
                 TGNCQEDWYYCWYF 
                 67 
                 CELL 
                 II 
               
               
                   
               
               
                 KELCEDDWYYCYLM 
                 68 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 HWECYSDTGKCWFF 
                 69 
                 O/N 
                 II 
               
               
                   
               
               
                 GITCYSDTGKCFSF 
                 70 
                 CELL 
                 II 
               
               
                   
               
               
                 AVTCWALTGHCVEE 
                 71 
                 O/N 
                 II 
               
               
                   
               
               
                 YVDCYYDTGRCYHQ 
                 72 
                 CELL 
                 II 
               
               
                   
               
               
                 WYWCQYHGVCPQS* 
                 73 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 LVMCISPEGYCYEI 
                 74 
                 O/N 
                 II 
               
               
                   
               
               
                 LIECYAHTGLCFDF 
                 75 
                 O/N 
                 II 
               
               
                   
               
               
                 HWWCAFQPQECEYW 
                 76 
                 1 HR 
                 III 
               
               
                   
               
               
                 HYECWYPEGKCYFY 
                 77 
                 CELL 
                 II 
               
               
                   
               
               
                 WYWCHHIGMYCDGF 
                 78 
                 1 HR 
                 III 
               
               
                   
               
               
                 WEWCPIDAWECIML 
                 79 
                 1 HR 
                 II 
               
               
                   
               
               
                 WLECYTEFGHCYNF 
                 80 
                 1 HR 
                 II 
               
               
                   
               
               
                 WVECWWKYGQCYEF 
                 81 
                 1 HR 
                 II 
               
               
                   
               
               
                 PNTCETFDLYCWWI 
                 82 
                 1 HR 
                 II 
               
               
                   
               
               
                 WIICDGNLGWCWEG 
                 83 
                 O/N 
                 II 
               
               
                   
               
               
                 GEQCSNLAVACCST 
                 84 
                 O/N 
                 II 
               
               
                   
               
               
                 WVECYDPWGWCWEW 
                 85 
                 CELL 
                 NA 
               
               
                   
               
               
                 WYWCMHYGLGCPYR 
                 86 
                 CELL 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 TN12/I Library Isolates* 
               
            
           
           
               
               
               
               
            
               
                 Sequence 
                 SEQ ID NO: 
                 Elution 
                 Class 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 YPWCHELSDSVTRFCVPW 
                 87 
                 1 HR 
                 (III) 
               
               
                   
               
               
                 SRVCWEDSWGGEVCFRY 
                 88 
                 1 HR 
                 (III) 
               
               
                   
               
               
                 SRVCWEYSWGGEVCYRV 
                 89 
                 O/N 
                 III 
               
               
                   
               
               
                 FGECWEYFWGGEFCLRV 
                 90 
                 CELL 
                 III 
               
               
                   
               
               
                 WRICWESSWGGEVCIGH 
                 91 
                 CELL 
                 III 
               
               
                   
               
               
                 YGVCWEYSWGGEVCLRF 
                 92 
                 CELL 
                 III 
               
               
                   
               
               
                 SSVCFEYSWGGEVCFRY 
                 93 
                 CELL 
                 III 
               
               
                   
               
               
                 SRVCWEYSWGGQICLGY 
                 94 
                 CELL 
                 III 
               
               
                   
               
               
                 FSVCWEYSWGGEVCLRQ 
                 95 
                 CELL 
                 III 
               
               
                   
               
               
                 DHMCRSPDYQDHVFCMYW 
                 96 
                 CELL 
                 (II) 
               
               
                   
               
               
                 PPLCYFVGTQEWHHCNPF 
                 97 
                 CELL 
                 (II) 
               
               
                   
               
               
                 WWECKREEYRNTTWCAWA 
                 98 
                 CELL 
                 II 
               
               
                   
               
               
                 DSYCMMNEKGWWNCYLY 
                 99 
                 CELL 
                 NA 
               
               
                   
               
               
                 PAQCWESNYQGIFFCDNP 
                 100 
                 CELL 
                 II? 
               
               
                   
               
               
                 GSWCEMRQDVGKWNCFSD 
                 101 
                 CELL 
                 II 
               
               
                   
               
               
                 GWACAKWPWGGEICQPS 
                 102 
                 CELL 
                 (II) 
               
               
                   
               
               
                 ASTCVFHDHPYFPMCQDN 
                 103 
                 CELL 
                 I/III 
               
               
                   
               
               
                 PDTCTMWGDSGRWYCFPA 
                 104 
                 CELL 
                 (II) 
               
               
                   
               
               
                 NWKCEYTQGYDYTECVYL 
                 105 
                 O/N 
                 II 
               
               
                   
               
               
                 NWECGWSNMFQKEFCARP 
                 106 
                 1 HR 
                 (III) 
               
               
                   
               
               
                 SGYCEFESDTGRWFCSSW 
                 107 
                 O/N 
                 II 
               
               
                   
               
               
                 GGWCQLVDHSWWWCGDS 
                 108 
                 O/N 
                 II 
               
               
                   
               
               
                 DNWCEIVVEKGQWFCYGS 
                 109 
                 O/N 
                 II 
               
               
                   
               
               
                 YPGCYETSLSGVWFCADG 
                 110 
                 CELL 
                 II 
               
               
                   
               
               
                 GWCQMDAQGIWSCWAD 
                 111 
                 1 HR 
                 II 
               
               
                   
               
               
                 DRWCMLDQEKGWWLCGPP 
                 112 
                 CELL 
                 II 
               
               
                   
               
               
                 NSECGCPNMLHKEFCARH 
                 113 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 PFWCKFQQSKAMFPCSWF 
                 114 
                 1 HR 
                 II 
               
               
                   
               
               
                 YPWCHEHSDSVTRFCVPW 
                 115 
                 1 HR 
                 III 
               
               
                   
               
               
                 SDLCYNQSGWWELCYFD 
                 116 
                 O/N 
                 I/II? 
               
               
                   
               
               
                 LGYCMYDYENRGWTCYPP 
                 117 
                 O/N 
                 II 
               
               
                   
               
               
                 YYQCQRYWDGKTWWCEYN 
                 118 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 DSWCELEHQSGIWRCDFW 
                 119 
                 CELL 
                 II 
               
               
                   
               
               
                 DWACDEYWSAYSVLCKHP 
                 120 
                 CELL 
                 II 
               
               
                   
               
               
                 LSLCYNDMHGWWEHCQWY 
                 121 
                 CELL 
                 II 
               
               
                   
               
               
                 YSHCIETSMENIWFCDFD 
                 122 
                 CELL 
                 II 
               
               
                   
               
               
                 PPFCIYQEPSGQWWCYDH 
                 123 
                 CELL 
                 II 
               
               
                   
               
               
                 PGWCDFSPQLGQWMCDWF 
                 124 
                 CELL 
                 II 
               
               
                   
               
               
                 LDNCIWNVWKGVQDCEYS 
                 125 
                 O/N 
                 II 
               
               
                   
               
               
                 AGWCEYVAPQGAWRCFHN 
                 126 
                 CELL 
                 II 
               
               
                   
               
               
                 WDDCIWHMWLKKKDCNSG 
                 127 
                 O/N 
                 II 
               
               
                   
               
               
                 PGHCEYIWIDEQPWCVRL 
                 128 
                 CELL 
                 III 
               
               
                   
               
               
                 YSDCLFQLWKGSVCPPS 
                 129 
                 CELL 
                 II 
               
               
                   
               
               
                 YFFCSFADVAYESCHPL 
                 130 
                 CELL 
                 NA 
               
               
                   
               
               
                 NYMCESEDHTYMFPCWWY 
                 131 
                 CELL 
                 NA 
               
               
                   
               
               
                 DAVCYNPWFKYWETCEYN 
                 132 
                 CELL 
                 NA 
               
               
                   
               
               
                 NYMCEYEDHTYMLTCECN 
                 133 
                 CELL 
                 NA 
               
               
                   
               
               
                 WDDCIYSMWMVHTVCDR 
                 134 
                 CELL 
                 NA 
               
               
                   
               
               
                 NWKCDAHQEGRIHICWGY 
                 135 
                 CELL 
                 NA 
               
               
                   
               
               
                 NGSCWYDFGWETEICFHN 
                 136 
                 CELL 
                 II 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Lin20 Library Isolates* 
               
            
           
           
               
               
               
               
            
               
                 Sequence 
                 SEQ ID NO: 
                 Elution 
                 Class 
               
               
                   
               
               
                 QVQYQFFLGTPRYEQWDLDK 
                 137 
                 CELL 
                 II 
               
               
                   
               
               
                 EPEGYAYWEVITLYHEEDGD 
                 138 
                 CELL 
                 (II) 
               
               
                   
               
               
                 WYYDWFHNQRKPPSDWIDNL 
                 139 
                 1 HR 
                 III 
               
               
                   
               
               
                 AFPRFGGDDYWIQQYLRYTD 
                 140 
                 1 HR 
                 (III) 
               
               
                   
               
               
                 GDYVYWEIIELTGATDHTPP 
                 141 
                 O/N 
                 (III) 
               
               
                   
               
               
                 RGDYQEQYWHQQLVEQLKLL 
                 142 
                 1 HR 
                 (III) 
               
               
                   
               
               
                 RSWYLGPPYYEEWDPIPN 
                 143 
                 CELL 
                 II 
               
               
                   
               
               
                 PSNSWAAVWEDDMQRLMRQH 
                 144 
                 CELL 
                 II 
               
               
                   
               
               
                 PRLGDDFEEAPPLEWWWAHF 
                 145 
                 CELL 
                 II 
               
               
                   
               
               
                 MPPGFSYWEQVVLHDDAQVL 
                 146 
                 CELL 
                 II 
               
               
                   
               
               
                 KKEDAQQWYWTDYVPSYLYR 
                 147 
                 1 HR 
                 III? 
               
               
                   
               
               
                 WVTKQQFIDTYGRKEWTILF 
                 148 
                 CELL 
                 II 
               
               
                   
               
               
                 WLYDYWDRQQKSEEFKFWSQ 
                 149 
                 1 HR 
                 III 
               
               
                   
               
               
                 PVTDWTPHHPKAPDVWLFYT 
                 150 
                 1 HR 
                 III? 
               
               
                   
               
               
                 EWYWTEHVGMKHGFFV 
                 151 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 DALEAPKRDWYYDWFLNHSP 
                 152 
                 1 HR 
                 III 
               
               
                   
               
               
                 PDNWKEFYESGWKYPSLYKPL 
                 153 
                 1 HR 
                 NA 
               
               
                   
               
               
                 EWDAQYWHDLRQQYMLDYIQ 
                 154 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 AFEIEYWDSVRNKIWQHFPD 
                 155 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 AFPRFGGDDYWIQQYLRYTF 
                 156 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 AHMPPWRPVAVDALFDWVE 
                 157 
                 CELL 
                 NA 
               
               
                   
               
               
                 AHMPPWWPLAVDAQEDWFE 
                 158 
                 CELL 
                 NA 
               
               
                   
               
               
                 AQMPPWWPLAVDALFDWFE 
                 159 
                 CELL 
                 II 
               
               
                   
               
               
                 ARMGDDWEEAPPHEWGWADG 
                 160 
                 CELL 
                 II 
               
               
                   
               
               
                 DWYWQRERDKLREHYDDAFW 
                 161 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 DWYWREWMPMHAQFLADDW 
                 162 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 DWYYDEILSMADQLRHAFLS 
                 163 
                 1 HR 
                 III 
               
               
                   
               
               
                 EEQQALYPGCEPAEHWVYAG 
                 164 
                 1 HR 
                 III 
               
               
                   
               
               
                 FDVVNWGDGIWYAYPS 
                 165 
                 CELL 
                 II 
               
               
                   
               
               
                 FPSQMWQQKVSHHFFQHKGY 
                 166 
                 CELL 
                 II 
               
               
                   
               
               
                 GSDHVRVDNYWWNGMAWEIF 
                 167 
                 1 HR 
                 II 
               
               
                   
               
               
                 ISPWREMSGWGMPWITAVPH 
                 168 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 LEEVFEDFQDFWYTEHIIVDR 
                 169 
                 1 HR 
                 II 
               
               
                   
               
               
                 MPPGFSYWEQAALHDDAQDL 
                 170 
                 CELL 
                 II 
               
               
                   
               
               
                 PEDSEAWYWLNYRPTMFHQL 
                 171 
                 1 HR 
                 I/III? 
               
               
                   
               
               
                 QIEYVNDKWYWTGGYWNVPF 
                 172 
                 1 HR 
                 II 
               
               
                   
               
               
                 QVQYQFILGTPRYEQWDPDK 
                 173 
                 CELL 
                 II 
               
               
                   
               
               
                 RDEWGWTGVPYEGEMGYQIS 
                 174 
                 1 HR 
                 II 
               
               
                   
               
               
                 STNGDSFVYWEEVELVDHPY 
                 175 
                 O/N 
                 II 
               
               
                   
               
               
                 SYEQWLPQYWAQYKSNYFL 
                 176 
                 1 HR 
                 I/III? 
               
               
                   
               
               
                 TKWGPNPEHWQYWYSHYASS 
                 177 
                 1 HR 
                 I/III? 
               
               
                   
               
               
                 VSKGSIDVGEGISYWEIIEL 
                 178 
                 1 HR 
                 III 
               
               
                   
               
               
                 WESDYWDQMRQQLKTAYMKV 
                 179 
                 1 HR 
                 I/III 
               
               
                   
               
               
                 WYHDGLHNERKPPSHWIDNV 
                 180 
                 1 HR 
                 III 
               
               
                   
               
               
                 APAWTFGTNWRSIQRVDSLT 
                 181 
                 CELL 
                 NA 
               
               
                   
               
               
                 EGWFRNPQEIMGFGDSWDKP 
                 182 
                 CELL 
                 NA 
               
               
                   
               
               
                 GWDLSVNRDKRWFWPWSSRE 
                 183 
                 CELL 
                 NA 
               
               
                   
               
               
                 KSGVDAVGWHIPVWLKKYWF 
                 184 
                 CELL 
                 NA 
               
               
                   
               
               
                 GMDLYQYWASDDYWGRHQEL 
                 185 
                 CELL 
                 NA 
               
               
                   
               
               
                 GVDIWHYWKSSTRYFHQ 
                 186 
                 CELL 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 TN7/IV Library Isolates 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Sequence 
                 SEQ ID NO: 
                 Elution 
                 Class 
               
               
                   
               
               
                   
                 GVECNHMGLCVSW 
                 187 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 GITCDELGRCVHW 
                 188 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 WIQCNHQGQCFHG 
                 189 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 WIECNKDGKCWHY 
                 190 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 WVECNHKGLCREY 
                 191 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 WYWCEFYGVCSEE 
                 192 
                 1 HR 
                 I/III 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 TN9/IV Library Isolates 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                   
               
               
                   
                 Sequence 
                 ID NO: 
                 Elution 
                 Class 
               
               
                   
               
               
                   
                 IDFCKGMAPWLCADM 
                 193 
                 1 HR 
                 (III) 
               
               
                   
               
               
                   
                 PWTCWLEDHLACAML 
                 194 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 DWGCSLGNWYWCSTE 
                 195 
                 CELL 
                 NA 
               
               
                   
               
               
                   
                 MPWCSEVTWGWCKLN 
                 196 
                 CELL 
                 II 
               
               
                   
               
               
                   
                 RGPCSGQPWHLCYYQ 
                 197 
                 O/N 
                 II 
               
               
                   
               
               
                   
                 PWGCDHFGWAWCKGM 
                 198 
                 O/N 
                 NA 
               
               
                   
               
               
                   
                 MPWCVEKDHWDCWWW 
                 199 
                 CELL 
                 NA 
               
               
                   
               
               
                   
                 PGPCKGYMPHQCWYM 
                 200 
                 CELL 
                 NA 
               
               
                   
               
               
                   
                 YGPCAEMSPWLCWYP 
                 201 
                 CELL 
                 NA 
               
               
                   
               
               
                   
                 YGPCKNMPPWMCWHE 
                 202 
                 CELL 
                 NA 
               
               
                   
               
               
                   
                 GHPCKGMLPHTCWYE 
                 203 
                 CELL 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 TN10/IX Library Isolates 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ 
                   
                   
               
               
                   
                 Sequence 
                 ID NO: 
                 Elution 
                 Class 
               
               
                   
               
               
                   
                 NNSCWLSTTLGSCFFD 
                 204 
                 O/N 
                 NA 
               
               
                   
               
               
                   
                 DHHCYLHNGQWICYPF 
                 205 
                 CELL 
                 (III) 
               
               
                   
               
               
                   
                 NSHCYIWDGMWLCFPD 
                 206 
                 CELL 
                 (II) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 MTN13/I Library Isolates 
               
            
           
           
               
               
               
               
            
               
                 Sequence 
                 SEQ ID NO: 
                 Elution 
                 Class 
               
               
                   
               
               
                 SNKCDHYQSGPHGKICVNY 
                 207 
                 CELL 
                 NA 
               
               
                   
               
               
                 SNKCDHYQSGPYGEVCFNY 
                 208 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPYGKVCVSY 
                 209 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPDTSCGSQ 
                 210 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPHGKICVRY 
                 211 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPHGKICVNY 
                 212 
                 CELL 
                 NA 
               
               
                   
               
               
                 RVDCDKVISGPHGKICVNY 
                 213 
                 CELL 
                 NA 
               
               
                   
               
               
                 RTTCHHQISGPHGKICVNY 
                 214 
                 CELL 
                 NA 
               
               
                   
               
               
                 EFHCHHIMSGPHGKICVNY 
                 215 
                 CELL 
                 NA 
               
               
                   
               
               
                 HNRCDFKMSGPHGKICVNY 
                 216 
                 CELL 
                 NA 
               
               
                   
               
               
                 WQECTKVLSGPGTFECSYE 
                 217 
                 CELL 
                 NA 
               
               
                   
               
               
                 WQECTKVLSGPGQFSCVYG 
                 218 
                 CELL 
                 NA 
               
               
                   
               
               
                 WQECTKVLSGPGQFECEYM 
                 219 
                 CELL 
                 NA 
               
               
                   
               
               
                 WQECTKVLSGPNSFECKYD 
                 220 
                 CELL 
                 NA 
               
               
                   
               
               
                 WDRCERQISGPGQFSCVYG 
                 221 
                 CELL 
                 NA 
               
               
                   
               
               
                 WQECTKVLSGPGQFLCSYG 
                 222 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDMVFSGPHGKICVNY 
                 223 
                 CELL 
                 NA 
               
               
                   
               
               
                 KRCDTTHSGPHGIVCVVY 
                 224 
                 CELL 
                 NA 
               
               
                   
               
               
                 SNKCDHYQSGPYGAVCLHY 
                 225 
                 CELL 
                 NA 
               
               
                   
               
               
                 SPHCQYKISGPFGPVCVNY 
                 226 
                 CELL 
                 NA 
               
               
                   
               
               
                 AHQCHHWTSGPYGEVCFNY 
                 227 
                 CELL 
                 NA 
               
               
                   
               
               
                 YDKCSSRFSGPFGEICVNY 
                 228 
                 CELL 
                 NA 
               
               
                   
               
               
                 MGGCDFSFSGPFGQICGRY 
                 229 
                 CELL 
                 NA 
               
               
                   
               
               
                 RTTCHHQISGPFGDVCVSY 
                 230 
                 CELL 
                 NA 
               
               
                   
               
               
                 WYRCDFNMSGPDFTECLYP 
                 231 
                 CELL 
                 NA 
               
               
                   
               
               
                 WMQCNMSASGPKDMYCEYD 
                 232 
                 CELL 
                 NA 
               
               
                   
               
               
                 GISCKWIWSGPDRWKCHHF 
                 233 
                 CELL 
                 NA 
               
               
                   
               
               
                 WQVCKPYVSGPAAFSCKYE 
                 234 
                 CELL 
                 NA 
               
               
                   
               
               
                 GWWCYRNDSGPKPFHCRIK 
                 235 
                 CELL 
                 NA 
               
               
                   
               
               
                 EGWCWFIDSGPWKTWCEKQ 
                 236 
                 CELL 
                 NA 
               
               
                   
               
               
                 FPKCKFDFSGPPWYQCNTK 
                 237 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPYGRVCVKY 
                 238 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPYGNVCVNY 
                 239 
                 CELL 
                 NA 
               
               
                   
               
               
                 RLDCDKVFSGPSMGTCKLQ 
                 240 
                 CELL 
                 NA 
               
               
                   
               
               
                 RTTCHHHISGPHGKICVNY 
                 241 
                 CELL 
                 NA 
               
               
                   
               
               
                 QFGCEHIMSGPHGKICVNY 
                 242 
                 CELL 
                 NA 
               
               
                   
               
               
                 PVHCSHTISGPHGKICVNY 
                 243 
                 CELL 
                 NA 
               
               
                   
               
               
                 SVTCHFQMSGPHGKICVNY 
                 244 
                 CELL 
                 NA 
               
               
                   
               
               
                 PRGCQHMISGPHGKICVNY 
                 245 
                 CELL 
                 NA 
               
               
                   
               
               
                 RTTCHHQISGPHGQICVNY 
                 246 
                 CELL 
                 NA 
               
               
                   
               
               
                 WTICHMELSGPHGKICVNY 
                 247 
                 CELL 
                 NA 
               
               
                   
               
               
                 FITCALWLSGPHGKICVNY 
                 248 
                 CELL 
                 NA 
               
               
                   
               
               
                 MGGCDFSFSGPHGKICVNY 
                 249 
                 CELL 
                 NA 
               
               
                   
               
               
                 KDWCHTTFSGPHGKICVNY 
                 250 
                 CELL 
                 NA 
               
               
                   
               
               
                 AWGCDNMMSGPHGKICVNY 
                 251 
                 CELL 
                 NA 
               
               
                   
               
               
                 SNKCDHIMSGPHGKICVNY 
                 252 
                 CELL 
                 NA 
               
               
                   
               
               
                 SNKCDHYQSGPFGDICVMY 
                 253 
                 CELL 
                 NA 
               
               
                   
               
               
                 SNKCDHYQSGPFGDVCVSY 
                 254 
                 CELL 
                 NA 
               
               
                   
               
               
                 SNKCDHYQSGPFGDICVSY 
                 255 
                 CELL 
                 NA 
               
               
                   
               
               
                 RTTCHHQISGPFGPVCVNY 
                 256 
                 CELL 
                 NA 
               
               
                   
               
               
                 RTTCHHQISGPYGDICVKY 
                 257 
                 CELL 
                 NA 
               
               
                   
               
               
                 PHGKICVNYGSESADPSYIE 
                 258 
                 CELL 
                 NA 
               
               
                   
               
               
                 RYKCPRDLSGPPYGPCSPQ 
                 259 
                 CELL 
                 NA 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 TN11.1 Library Isolates 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 # of 
               
               
                 Sequence 
                 SEQ ID NO: 
                 Elution 
                 isolates 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 GSNMVCMDDSYGGTTCYSMAP 
                 505 
                 D6 
                 107 
               
               
                   
               
               
                 GSYNQCYGDYWGGETCYLIAP 
                 506 
                 Bead 
                 93 
               
               
                   
               
               
                 GSRVNCGAEDGLSFLCMMDAP 
                 507 
                 Bead 
                 40 
               
               
                   
               
               
                 GSIWDCQISEYGGEDCYLVAP 
                 508 
                 D6 
                 29 
               
               
                   
               
               
                 GSYWHCMDDFFGGETCFATAP 
                 509 
                 D6 
                 28 
               
               
                   
               
               
                 GSGEYCFPSIYGGETCYAHAP 
                 510 
                 D6 
                 24 
               
               
                   
               
               
                 GSEQLCFEYQYGGVECFGPAP 
                 511 
                 D6 
                 21 
               
               
                   
               
               
                 GSTGVCSPAPYGGEVCYHFAP 
                 512 
                 D6 
                 20 
               
               
                   
               
               
                 GSHDECWEDIYGGFTCMLMAP 
                 513 
                 D6 
                 19 
               
               
                   
               
               
                 GSQHTCFSDPYGGEVCYADAP 
                 514 
                 D6 
                 18 
               
               
                   
               
               
                 GSWEVCENSNYGGQICYWFAP 
                 515 
                 D6 
                 18 
               
               
                   
               
               
                 GSHEMCWSDVWGGLTCMTMAP 
                 516 
                 D6 
                 15 
               
               
                   
               
               
                 GSLSLCKFFGDGSYYCEPPAP 
                 517 
                 D6 
                 14 
               
               
                   
               
               
                 GSTRFCEPYQWGGEVCYWKAP 
                 518 
                 D6 
                 14 
               
               
                   
               
               
                 GSFSTCATFPWTTKFCSNMAP 
                 519 
                 VEGF 
                 12 
               
               
                   
               
               
                 GSHELCFEGTYGGEVCFSMAP 
                 520 
                 D6 
                 12 
               
               
                   
               
               
                 GSLWHCFNDVYGGENCIPFAP 
                 521 
                 VEGF 
                 12 
               
               
                   
               
               
                 GSQQYCIPAEYGGMECYPFAP 
                 522 
                 Bead 
                 11 
               
               
                   
               
               
                 GSIQNCWKYEFGGIVCMDMAP 
                 523 
                 D6 
                 9 
               
               
                   
               
               
                 GSVSGCKEFWNSSGRCFTHAP 
                 524 
                 D6 
                 9 
               
               
                   
               
               
                 GSLWECRGDFYGGEVCFNYAP 
                 525 
                 D6 
                 8 
               
               
                   
               
               
                 GSNLICYDYYYGGQDCYHDAP 
                 526 
                 D6 
                 8 
               
               
                   
               
               
                 GSEGTCEEYQYGGIVCWWGAP 
                 527 
                 D6 
                 7 
               
               
                   
               
               
                 PGSGDCDWYYEWLFDCPLNAP 
                 528 
                 VEGF 
                 7 
               
               
                   
               
               
                 GSDQMCFNESFGGQICFYSAP 
                 529 
                 VEGF 
                 6 
               
               
                   
               
               
                 GSGMACMSDPYGGQVCYAIAP 
                 530 
                 D6 
                 5 
               
               
                   
               
               
                 GSELTCWDSAYGGNECFFFAP 
                 531 
                 VEGF 
                 4 
               
               
                   
               
               
                 GSHFLCVKEMEGGETCYYSAP 
                 532 
                 VEGF 
                 4 
               
               
                   
               
               
                 GSWEICFAGPYGGSWCIPEAP 
                 533 
                 Bead 
                 4 
               
               
                   
               
               
                 GSAQYCMESYYGGFTCVTLAP 
                 534 
                 Bead 
                 3 
               
               
                   
               
               
                 GSFNACGFEEGLEWMCYRQAP 
                 535 
                 D6 
                 3 
               
               
                   
               
               
                 GSKLLCQYWEHEWWPCMNEAP 
                 536 
                 VEGF 
                 3 
               
               
                   
               
               
                 GSNMNCGAEQGLESLCGWRAP 
                 537 
                 VEGF 
                 3 
               
               
                   
               
               
                 GSNWVCLSEGYGGMTCYPSAP 
                 538 
                 VEGF 
                 3 
               
               
                   
               
               
                 GSPSTCIYSSGLIVDCGLLAP 
                 539 
                 VEGF 
                 3 
               
               
                   
               
               
                 GSTQHCWPSEYGGMTCVPAAP 
                 540 
                 D6/VEGF 
                 3 
               
               
                   
               
               
                 GSTWACEEISAHHTKCTYQAP 
                 541 
                 VEGF/Bead 
                 3 
               
               
                   
               
               
                 GSYTECWEEDYGGVTCFNVAP 
                 542 
                 Bead 
                 3 
               
               
                   
               
               
                 GSDKFCFKDPWGGVTCYHLAP 
                 543 
                 D6 
                 2 
               
               
                   
               
               
                 GSDLDCWTDPYGGEVCYWHAP 
                 544 
                 D6 
                 2 
               
               
                   
               
               
                 GSDYECYNAWFGYFDCPGDAP 
                 545 
                 VEGF/Bead 
                 2 
               
               
                   
               
               
                 GSLSTCWKQAYGGVWCVDHAP 
                 546 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSMQLCRQWAYGGQTCYWYAP 
                 547 
                 D6 
                 2 
               
               
                   
               
               
                 GSNQLCITAQFGGQDCYPIAP 
                 548 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSPMWCAPWPWGGEHCVGSAP 
                 549 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSQLLCGSEPELAWMCEQGAP 
                 550 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSQRQCWDDYFGGIICYVIDA 
                 551 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSREVCWQDFFGGMVCVRDAP 
                 552 
                 Bead 
                 2 
               
               
                   
               
               
                 GSSQWCQRDFWGGDICINLAP 
                 553 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSTDICWPGSYGGEICIPRAP 
                 554 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSTEYCWPEPHGGQACILLAP 
                 555 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSTHFCIDYIWGGKHCIADAP 
                 556 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSTMMCWPAHYGGDECFALAP 
                 557 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSTQMCFPHQYGGQSCYSFAP 
                 558 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSVEGCWVEDQTSPFCWIDAP 
                 559 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSWYTCWDEASGGQVCYQLAP 
                 560 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSYNLCYPEIYGGQVCYRMAP 
                 561 
                 D6 
                 2 
               
               
                   
               
               
                 GSYSQCFPDPFGGTTCFVSAP 
                 562 
                 D6 
                 2 
               
               
                   
               
               
                 GSSMQCFNRVSQLVDCETAAP 
                 563 
                 VEGF 
                 2 
               
               
                   
               
               
                 GSAKTCRSYWAQSGYCYEYAP 
                 564 
                 D6 
                 1 
               
               
                   
               
               
                 GSAQTCWDYVYGGFFCLNTAP 
                 565 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSAWDCFQQDTYSTHCHWRAP 
                 566 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSAWNCEMLDPWSTQCSWDAP 
                 567 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSAWVCHPEQEGGTTCYWVAP 
                 568 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSDELCWPQEFGGWVCIQGAP 
                 569 
                 Bead 
                 1 
               
               
                   
               
               
                 GSDFQCFNWEGYPTNCYSNAP 
                 570 
                 D6 
                 1 
               
               
                   
               
               
                 GSDKKCWPSPYGGQICWAVAP 
                 571 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSDQLCFDQRWGGQVCVFGAP 
                 572 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSDSGCKEFWNSSDRCYTHAP 
                 573 
                 D6 
                 1 
               
               
                   
               
               
                 GSEWICWSSFFGGETCTPKAP 
                 574 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSEWNCLNNTPYQTTCSWRAP 
                 575 
                 Bead 
                 1 
               
               
                   
               
               
                 GSEWRCWPDVFGGQMCFNMAP 
                 576 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSEYECYPDWYGGEVCVQKAP 
                 577 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSFEACWEEAYGGLTCWHDAP 
                 578 
                 D6 
                 1 
               
               
                   
               
               
                 GSFEECMPYRYGGQTCFMIAP 
                 579 
                 D6 
                 1 
               
               
                   
               
               
                 GSFWTCVDTNWHTTECFHSAP 
                 580 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSGQMCWHGQYGGTICVAMAP 
                 581 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSGWVCKQQGPHKTECLFMAP 
                 582 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSHDECWEDIYGGFTCMPYGS 
                 583 
                 D6 
                 1 
               
               
                   
               
               
                 GSHVVCWDDPYGGESCYNTAP 
                 584 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSIDICTDSYWGGITCYKFAP 
                 585 
                 D6 
                 1 
               
               
                   
               
               
                 GSKWICVDVKWGGSACYDIAP 
                 586 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSLWECRIDYYGGEVCFIDAP 
                 587 
                 D6 
                 1 
               
               
                   
               
               
                 GSLWTCVLSVYGGEDCYNLAP 
                 588 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSMTMCGAEPDLWYMCYGIAP 
                 589 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSNQYCMPYDWGGEMCFEVAP 
                 590 
                 D6 
                 1 
               
               
                   
               
               
                 GSNVFCSEGPFGGEICYGIAP 
                 591 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSNWACFIEAMGGWTCAPRPT 
                 592 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSNWTCFIDSFQGETCYPFAP 
                 593 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSNWWCHSEAFGGHTCYNAAP 
                 594 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSPCACNNSYGHSDDCDHLAP 
                 595 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSPGNCKDFWAWSLQCFSFAP 
                 596 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSPRWCYFSSGIMKDCDILAP 
                 597 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSPTYCQFHSGVVTLCSMFAP 
                 598 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSQEICFNSQYGGQVCFDSAP 
                 599 
                 D6 
                 1 
               
               
                   
               
               
                 GSQMICYPHVFGGQDCFPGAP 
                 600 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSQWTCTELSDVMTHCSYTAP 
                 601 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSRVNCGAEDDLSFLCMTEAP 
                 602 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSSGDCIEMYNDWYYCTILAP 
                 603 
                 Bead 
                 1 
               
               
                   
               
               
                 GSSWECGEFGDTTIQCNWVAP 
                 604 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSSWQCFSEAPSGATCVPIAP 
                 605 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSSWQCVQVDDFHTECSFMAP 
                 606 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSSWTCVFYPYGGEVCIPDAP 
                 607 
                 D6 
                 1 
               
               
                   
               
               
                 GSTELCVPYQWGGEVCVAQAP 
                 608 
                 D6 
                 1 
               
               
                   
               
               
                 GSTVYCHNEYFGGQVCFTIAP 
                 609 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSTYGCEYYMPFQHKCSVEAP 
                 610 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSWWGCFPYSWGGEICTSIAP 
                 611 
                 D6 
                 1 
               
               
                   
               
               
                 GSWWNCVDTSFHTTQCKYAAP 
                 612 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSYFMCQDGFWGGQDCFYIAP 
                 613 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSYMWCTESKFGGSTCFNLAP 
                 614 
                 VEGF 
                 1 
               
               
                   
               
               
                 GSGAYSHLLEYHAVCKNVAP 
                 615 
                 VEGF 
                 1 
               
               
                   
               
               
                 PGSWTCQNYEPWATTCVYDAP 
                 616 
                 VEGF 
                 1 
               
               
                   
               
            
           
         
       
         
         
           
             During the course of DNA synthesis, there is always a small percentage of incomplete couplings at each cycle. Since the libraries used for these experiments were constructed using TRIM technology to couple trinucleotides (codons) instead of nucleotides, the library template DNA often has a small percentage of deleted codons. In the case of the TN12 library, for instance, it has been observed that approximately 5.3% of the total library is phage expressing a cyclic 11-mer, rather than a 12-mer, and indeed some phage expressing 11-mers were isolated in the selections described above (see Table 2). 
           
         
       
    
     In the foregoing tables, Class I peptides only bind KDR in the absence of heparin, and therefore presumably target the heparin binding domain of KDR; Class II peptides bind in the presence or absence of heparin or VEGF, and therefore presumably bind at a non-involved site on KDR; Class III 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. In the elution column, 1 HR, 0/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). 
     Example 2 
     Peptide Synthesis and Fluorescein Labeling 
     Selected KDR or VEGF/KDR complex binding peptides corresponding to positive phage isolates were synthesized on solid phase using 9-fluorenylmethoxycarbonyl protocols and purified by reverse phase chromatography. Peptide masses were confirmed by electrospray mass spectrometry, and peptides were quantified by absorbance at 280 nm. For synthesis, two N-terminal and two C-terminal amino acids from the phage vector sequence from which the peptide was excised were retained, and a -Gly-Gly-Gly-Lys-NH 2  linker (SEQ ID NO:262) was added to the C-terminus of each peptide. Each peptide was N-terminally acetylated. For peptides with selected lysine residues, these were protected with 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), which allows selective coupling to the C-terminal lysine, is not removed during peptide cleavage, and can be removed after coupling with 2% hydrazine in DMF or 0.5 M hydroxylamine, pH 8, in water. 
     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). 
     Example 3 
     Fluorescence Anisotropy Measurements and BiaCore Assays 
     Fluorescence anisotropy measurements were performed in 384-well microplates in a volume of 10 μl 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 μg/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. The observed change in anisotropy was fit to the equation below via nonlinear regression to obtain the apparent K D . This equation (1) assumes that the synthetic peptide and KDR form a reversible complex in solution with 1:1 stoichiometry. 
                       r   obs     =       r   free     +       (       r   bound     -     r   free       )     ⁢         (       K   D     +   KDR   +   P     )     -           (       K   D     +   KDR   +   P     )     2     -     4   ·   KDR   ·   P             2   ·   P             ,           (   1   )               
where r obs  is the observed anisotropy, r free  is the anisotropy of the free peptide, r bound  is the anisotropy of the bound peptide, K D  is the apparent dissociation constant, KDR is the total KDR concentration, and P is the total fluorescein-labeled peptide concentration. K D  was calculated in a direct binding assay (K D,B ) (see Table 8), and therefore these values represent KDR binding to the fluorescein labeled peptide.
 
     For BiaCore determinations of K D , KDR-Fc(or other protein targets) was cross-linked to the dextran surface of a CM5 sensor chip by the standard amine coupling procedure (0.5 mg/mL solutions diluted 1:20 with 50 mM acetate, pH 6.0, R L  KDR-Fc=12859). Experiments were performed in HBS-P buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 0.005% polysorbate 20 (v/v)). Peptide solutions quantitated by extinction coefficient were diluted to 400 nM in HBS-P. Serial dilutions were performed to produce 200, 100, 50, and 25 nM solutions. For association, peptides were injected at 20 μl/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 1M NaCl for 25 sec. at 50 μl/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 3.1. Apparent K D  by this method is set forth as BiaK D  in Table 8. Unlike the fluorescence anisotropy experiments above, the unlabeled peptide was used for all testing using this assay and therefore, these values represent KDR binding to the unlabeled peptide. 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 in bold. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Binding Affinities for Synthesized Peptides 
               
            
           
           
               
               
               
               
            
               
                   
                 K D,B   
                 BiaK D   
                 SEQ 
               
               
                 Sequence 
                 (μM) 
                 (μM) 
                 ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 TN8 
                   
                   
                   
               
               
                 AGDSW CSTEYTYC EMIGTGGGK 
                 &gt;2 
                   
                 263 
               
               
                   
               
               
                 AGPKW CEEDWYYC MITGTGGGK 
                 0.28 
                 0.027 
                 264 
               
               
                   
               
               
                 AGVWE CAKTFPFC HWFGTGGGK 
                 2.60 
                   
                 265 
               
               
                   
               
               
                 AGWVE CWWKSGQC YEFGTGGGK 
                 1.3 
                   
                 266 
               
               
                   
               
               
                 AGWLE CYAEFGHC YNFGTGGGK 
                 &gt;10 
                   
                 267 
               
               
                   
               
               
                 AGWIQ CNSITGHC TSGGTGGGK 
                 0.24 
                   
                 268 
               
               
                   
               
               
                 AGWIE CYHPDGIC YHFGTGGGK 
                 0.32 
                 0.32 
                 269 
               
               
                   
               
               
                 AGSDW CRVDWYYC WLMGTGGGK 
                 0.064 
                   
                 270 
               
               
                   
               
               
                 AGANW CEEDWYYC FITGTGGGK 
                 0.310 
                   
                 271 
               
               
                   
               
               
                 AGANW CEEDWYYC WITGTGGGK 
                 0.097 
                   
                 272 
               
               
                   
               
               
                 AGPDW CEEDWYYC WITGTGGGK 
                 0.075 
                   
                 273 
               
               
                   
               
               
                 AGSNW CEEDWYYC YITGTGGGK 
                 0.046 
                   
                 274 
               
               
                   
               
               
                 AGPDW CAADWYYC YITGTGGGK 
                 0.057 
                   
                 275 
               
               
                   
               
               
                 AGPEW CEVDWYYC WLLGTGGGK 
                 0.075 
                   
                 276 
               
               
                   
               
               
                 AGPTW CEDDWYYC WLFGTGGGK 
                 0.0032 
                 0.079 
                 277 
               
               
                   
               
               
                 AGSKW CEQDWYYC WLLGTGGGK 
                 0.400 
                   
                 278 
               
               
                   
               
               
                 AGRNW CEEDWYYC FITGTGGGK 
                 0.190 
                   
                 279 
               
               
                   
               
               
                 AGVNW CEEDWYYC WITGTGGGK 
                 0.260 
                   
                 280 
               
               
                   
               
               
                 AGANW CEEDWYYC YITGTGGGK 
                 0.180 
                   
                 281 
               
               
                   
               
               
                 AGQAWVE CYAETGYC WPRSWGTGGGK 
                 0.71 
                   
                 282 
               
               
                   
               
               
                 AGQAWIE CYAEDGYC WPRSWGTGGGK 
                 1.40 
                   
                 283 
               
               
                   
               
               
                 AGVGWVE CYQSTGFC YHSRDGTGGGK 
                 1.30 
                   
                 284 
               
               
                   
               
               
                 AGFTWVE CHqATGRC VEWTTGTGGGK 
                 2.00 
                   
                 285 
               
               
                   
               
               
                 AGDWWVE CRVGTGLC YRYDTGTGGGK 
                 0.93 
                   
                 286 
               
               
                   
               
               
                 AGDSWVE CDAQTGFC YSFLYGTGGGK 
                 2.30 
                   
                 287 
               
               
                   
               
               
                 AGGGWVE CYWATGRC IEFAGGTGGGK 
                 NB 
                   
                 288 
               
               
                   
               
               
                 AGERWVE CRAETGFC YTWVSGTGGGK 
                 2.10 
                   
                 289 
               
               
                   
               
               
                 AGGGWVE CRAETGHC QEYRLGTGGGK 
                 1.60 
                   
                 290 
               
               
                   
               
               
                 AGVAWVE CYQTTGKC YTFRGGTGGGK 
                 ~2 
                   
                 291 
               
               
                   
               
               
                 AGEGWVE CFANTGAC FTYPRGTGGGK 
                 2.10 
                   
                 292 
               
               
                   
               
               
                 TN12 
                   
                   
                   
               
               
                 GDYPW CHELSDSVTRFC VPWDPGGGK 
                 0.98 
                 0.18 
                 293 
               
               
                   
               
               
                 GDSRV CWEDSWGGEVCF RYDPGGGK 
                 0.069 
                 0.12 
                 294 
               
               
                   
               
               
                 GDDHM CRSPDYQDHVFC MYWDPGGGK 
                 0.48 
                 0.14 
                 295 
               
               
                   
               
               
                 GDPPL CYFVGTQEWHHC NPFDPGGGK 
                 0.60 
                   
                 296 
               
               
                   
               
               
                 GDDSY CMMNEKGWWNCY LYDPGGGK 
                 1.3 
                   
                 297 
               
               
                   
               
               
                 GDPAQ CWESNYQGIFFC DNPDPGGGK 
                 2.3 
                   
                 298 
               
               
                   
               
               
                 GDGSW CEMRQDVGKWNC FSDDPGGGK 
                 0.62 
                 0.18 
                 299 
               
               
                   
               
               
                 GDGWA CAKWPWGGEICQ PSDPGGGK 
                 1.0 
                 1.5 
                 300 
               
               
                   
               
               
                 GDPDT CTMWGDSGRWYC FPADPGGGK 
                 0.49 
                 0.26 
                 301 
               
               
                   
               
               
                 GDNWK CEYTQGYDYTEC VYLDPGGGK 
                 0.82 
                   
                 302 
               
               
                   
               
               
                 GDNWE CGWSNMFQKEFC ARPDPGGGK 
                 0.21 
                 0.99 
                 303 
               
               
                   
               
               
                 GDWWE CKREEYRNTTWC AWADPGGGK 
                   
                   
                 486 
               
               
                   
               
               
                 GDSSV CFEYSWGGEVC FRYDPGGGK 
                 0.058 
                   
                 487 
               
               
                   
               
               
                 GDSRV CWEYSWGGQIC LGYDPGGGK 
                 0.32 
                   
                 488 
               
               
                   
               
               
                 Lin20 
                   
                   
                   
               
               
                 AQQVQYQFFLGTPRYEQWDLDKGGK 
                 1.7 
                   
                 304 
               
               
                   
               
               
                 AQEPEGYAYWEVITLYHEEDGDGGK 
                 0.27 
                 0.73 
                 305 
               
               
                   
               
               
                 AQAFPRFGGDDYWIQQYLRYTDGGK 
                 0.53 
                 0.25 
                 306 
               
               
                   
               
               
                 AQGDYVYWEIIELTGATDHTPPGGK 
                 0.18 
                   
                 307 
               
               
                   
               
               
                 AQRGDYQEQYWHQQLVEQLKLLGGK 
                 0.31 
                 5.3 
                 308 
               
               
                   
               
               
                 AQRSWYLGPPYYEEWDPIPNGGK 
                 1.8 
                   
                 309 
               
               
                   
               
               
                 AQDWYYDEILSMADQLRHAFLSGGGK 
                   
                 0.05 
                 310 
               
               
                   
               
               
                 TN9 
                   
                   
                   
               
               
                 AGIDF CKGMAPWLC ADMGTGGGK 
                 0.73 
                 0.18 
                 311 
               
               
                   
               
               
                 AGPWT CWLEDHLAC AMLGTGGGK 
                 3.9 
                   
                 312 
               
               
                   
               
               
                 AGDWG CSLGNWYWC STEGTGGGK 
                 2.0 
                   
                 313 
               
               
                   
               
               
                 TN10 
                   
                   
                   
               
               
                 GSDHH CYLHNGQWIC YPFAPGGGK 
                 0.26 
                 0.15 
                 314 
               
               
                   
               
               
                 GSNSH CYIWDGMWLC FPDAPGGGK 
                 0.74 
                   
                 315 
               
               
                   
               
               
                 MTN13 
                   
                   
                   
               
               
                 SGRLD CDKVFSGPYGKVC VSYGSGGGK 
                 1.05 
                   
                 316 
               
               
                   
               
               
                 SGRLD CDKVFSGPHGKIC VNYGSGGGK 
                 ~2 
                   
                 317 
               
               
                   
               
               
                 SGRTT CHHQISGPHGKIC VNYGSGGGK 
                 0.65 
                   
                 318 
               
               
                   
               
               
                 SGAHQ CHHWTSGPYGEVC FNYGSGGGK 
                 ~2 
                   
                 319 
               
               
                   
               
            
           
         
       
     
     For the analysis of those peptides that bind specifically to KDR/VEGF complex, each peptide was tested for binding to the complex in both assays (fluorescence anisotropy/Biacore) as above. In the anisotropy assay, 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. As a control, each peptide was also tested for binding to KDR and VEGF alone to assess their specificity for complex. Since none of the peptides bound VEGF to any extent, the presence of excess VEGF in the assay should not affect the K D  determination. As shown in Table 9, below, all of the peptides showed a dramatic binding preference, binding for KDR/VEGF complex over VEGF. Some of them, however, did show some residual binding to free KDR. To confirm the anisotropy results, the unlabeled peptides were tested in Biacore as before, except the chip was saturated with VEGF to form KDR/VEGF complex prior to the injection of the peptides. In the peptides tested, the BiaK D  was within at least 2-fold of the anisotropy measurement. 
                     TABLE 9                  KDR/VEGF Complex Specific Peptides                                     SEQ               K D , B   BiaK D         ID       K D , B   K D , B   (KDR/   (KDR/       NO:   Sequence   (KDR)   (VEGF)   VEGF)   VEGF)                                             320   AGMPW CVEKDHWDC WWWG   NB   10   0.14               TGGGK                               321   AGPGP CKGYMPHQC WYMGT   0.4   NB   0.06   0.08           GGGK                               322   AGYGP CAEMSPWLC WYPGT   3.7   NB   0.13               GGGK                               323   AGYGP CKNMPPWMC WHEGT   1.8   NB   0.18   0.42           GGGK                               324   AGGHP CKGMLPHTC WYEGT   &gt;10   NB   3.3               GGGK                               325   AQAPAWTFGTNWRSIQRVDSL   NB   NB   0.84               TGGGGGK                               326   AQEGWFRNPQEIMGFGDSWD   NB   NB   1.4               KPGGGGGK                    
The putative disulfide-constrained cyclic peptide moiety is underscored.
 
     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. 
     The ability of the 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 (K D  10.00+/−2.36 nM, four experiment average), SEQ ID NO:301 (K D ) 4.03+/−0.86 nM, three experiment average), SEQ ID NO:337 (K D  6.94+/−1.94 nM, one experiment), and SEQ ID NO:338 (K D  3.02+/−0.75 nM, one experiment). 
                     TABLE 10                  KDR, VEGF/KDR Complex Binding Polypeptides                             SEQ ID NO:   Structure (or) Sequence   Mol. Wt.   MS                                     294   Ac-GDSRVC WEDSWGGEVC FRYDPGGGK-NH 2     2801.98   1399.6 [M − H] −                 329   Ac-AGMPW CVEKDHWDC WWGTGGGK-NH 2     2730.14   —               311   Ac-AGIDF CKGMAPWLC ADMGTGGGK-NH 2     2324.02   —               264   Ac-AGPKW CEEDWYYC MITGTGGGK-NH 2     2361   —               266   Ac-AGWVE CWWKSGQC YEFGTGGGK-NH 2     2474.06   —               330   Ac-AQEGWFRNPQEIMGFGDSWDKPGGGK-NH 2     2934.35   —               299   Ac- GDGSWCEMRQDVGK(iv-Dde)WNCFSDDP -   3075.29   1537.5 [M 2− ]           GGGK-NH 2                         299   Ac-GDGSW CEMRQDVGKWNC FSDDPGGGK-NH 2     2869.16   —               303   Ac-GDNWE CGWSNMFQK(iv-Dde)EFC ARPDP-   3160.36   1579.6 [M 2− ]           GGGK-NH 2                         303   Ac-GDNWE CGWSNMFQKEFC ARPDPGGGK-NH 2     2954.23   —               294   Ac-GDSRV CWEDSWGGEVC FRYDPGGGK(Biotin)-   3030.29   1512.4 [M 2− ]           NH 2                         331   Ac-AQRGDYQEQYWHQQLVEQLK(iv-Dde)LLGGGK-NH 2     3318.71   1659.1 [M 2− ]               331   Ac-AQRGDYQEQYWHQQLVEQLKLLGGGK-NH 2     3112.58   —               332   Ac-AGWYW CDYYGIGC K(iv-Dde)WTGGGK-NH 2     2673.18                   333   Ac-AGWYW CDYYGIGC KWTGTGGGK-NH 2     2467.05                   334   Ac-AQWYYDWFHNQRKPPSDWIDNLGGGK-NH 2     3218.51   —               323   Ac-AGYGP CKNMPPWMC WHEGTGGGK-NH 2     2502.05   —               335   Ac-AGPKW CEEDWYYC MITGTGGGK(N,N-Dimethyl-   2836.204   2833.4 [M − H] −             Gly-Ser-Cys(Acm)-Gly)-NH 2                         264   Ac-AGPK(iv-Dde)W CEEDWYYC MITGTGGGK-NH 2     2698.11   2695.7 [M − H] − ;                   1347.8 [M − 2H] 2− /2               336   Ac-WQP CPWESWTFC WDPGGGK(AcSCH 2 C(═O)—)—NH 2     2422.71   2420.7 [M − H] − ,                   1209.9 [M − 2H]/2               264   Ac-AGPKW CEEDWYYC MITGTGGGK(Biotin)-NH 2     2718.13   2833.4 (M − H) −                 264   Ac-AGPKW CEEDWYYC MITGTGGGK(Biotin-JJ-)-   3008.44   1502.6.4 (M − 2H) 2− /2           NH 2                         264   Ac-AGPKW CEEDWYYC MITGTGGGK   2608.96   1304, [M − 2H] 2− /2           (AcSCH 2 C(═O)—)—NH 2                         294   Ac-GDSRV CWEDSWGGEVC FRYDPGGGK(Biotin-   3316.4   1657.8, [M − 2H] 2− /2           JJ-)-NH 2                         294   Ac-GDSRV CWEDSWGGEVC FRYDPGGGK   2917.15   1457.4, [M − 2H] 2− /2           (AcSCH 2 C(═O)—)—NH 2                         294   Biotin-JJGDSRV CWEDSWGGEVC FRYDPGGGK-   3272.34   1636.7, [M − 2H] 2− /2           NH 2                         264   Ac-AGPKW CEEDWYYC MITGT-   2899.28   1449.2, [M − 2H] 2− /2           GGGK(AcSCH 2 C(═O)-JJ-)-NH 2                         277   Ac-AGPTW CEDDWYYC WLFGTGGGK(Biotin-JJ-)-   3066.27   1532.8, [M − 2H] 2− /2           NH 2                         337   Ac-V CWEDSWGGEVC FRYDPGGGK(Biotin-JJ-)-NH 2     2903.24   1449.3, (M − 2H) 2− /2;                    965.8, (M − 3H) 3− /3               338   Ac-AGPTW CEDDWYYC WLFGTJK(Biotin-JJ-)-NH 2     3042.44   1519.7, (M − 2H) 2− /2−;                   1012.8 (M − 3H) 3− /3               294   Ac-GDSRV CWEDSWGGEVC FRYDPGGGK   3208.48   1602.6, [M − 2H] 2− /2           (AcSCH 2 C(═O)-JJ-)-NH 2                         339   Ac-AGPTW CEDDWYYC WLFGTGGGK(N,N-   3242.33   1621.5, [M − 2H] 2− /2           Dimethyl-Gly-Ser-Cys(Acm)-Gly-JJ-)-NH 2                         277   Ac-AGPTW CEDDWYYC WLFGTGGGK   2907.29   1453.1, [M − 2H] 2− /2           (AcSCH 2 C(═O)-JJ-)-NH 2                         340   Ac-AQAHMPPWRPVAVDALFDWVEGG-   3404.64   1701.6, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         341   Ac-AQAHMPPWWPLAVDAQEDWFEGG-   3493.59   1746.2, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         342   Ac-AQAQMPPWWPLAVDALFDWFEGG-   3487.64   1743.2, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         343   Ac-AQDWYWREWMPMHAQFLADDWGG-   3751.64   1874.3, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         344   Ac-AQK(ivDde)K(iv-Dde)EDAQQWYWTDYVPSY-   4220.06   2108.9, [M − 2H] 2− /2           LYRGGGGGK(Biotin-JJ-)-NH 2                         345   Ac-AQPVTDWTPHHPK(iv-Dde)APDVWLFYT-   3781.86   1890.4, [M − 2H] 2− /2           GGGGGK(Biotin-JJ-)-NH 2                         346   Ac-AQDALEAPK(iv-Dde)RDWYYDWFLNHSP-   3897.85   1948.0, [M − 2H] 2− /2           GGGGGK(Biotin-JJ-)-NH 2                         347   Ac-KW CEEDWYYC MITGTGGGK(Biotin-JJ-)-NH 2     2781.2   1390.0, [M − 2H] 2− /2               348   Ac-AGPKW CEEDWYYC MIGGGK(Biotin-JJ-)-NH 2     2747.15   1373.5, [M − 2H] 2− /2               349   Ac-KW CEEDWYYC MIGGGK(Biotin-JJ-)-NH 2     2522.04   1260.8, [M − 2H] 2− /2               350   Ac-AQPDNWK(iv-Dde)EFYESGWK(iv-Dde)-   4377.2   2188.4, [M − 2H] 2− /2           YPSLYK(iv-Dde)PLGGGGGK(Biotin-JJ-)-NH 2                         351   Ac-AQMPPGFSYWEQVVLHDDAQVLGG-   3499.7   1749.2, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         352   Ac-AQARMGDDWEEAPPHEWGWADGG-   3480.5   1740.2, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         353   Ac-AQPEDSEAWYWLNYRPTMFHQLGG-   3751.7   1875.8, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         354   Ac-AQSTNGDSFVYWEEVELVDHPGG-   3554.6   1776.4, [M − 2H] 2− /2           GGGK(Biotin-JJ-)-NH 2                         355   Ac-AQWESDYWDQMRQQLK(iv-Dde)TAYMK(iv-   4187.02   2093.0, [M − 2H] 2− /2           Dde)VGGGGGK(Biotin-JJ-)-NH 2                         356   Ac-   3641.69   1820.9, [M − 2H] 2− /2           AQDWYYDEILSMADQLRHAFLSGGGGGK(Biotin-JJ-)-                   NH 2                 The putative disulfide constrained cyclic peptide is indicated in bold.            
The putative disulfide constrained cyclic peptide is indicated in bold.
 
     Example 5 
     Binding of KDR Binding Peptides/Avidin HRP Complex to KDR Transfected 293H Cells 
     To determine the binding of peptides identified by phage display to KDR expressed in transiently-transfected 293H cells, a novel assay that measures the binding of biotinylated peptides complexed with neutravidin HRP to KDR on the surface of the transfected cells was developed. This assay was used to screen the biotinylated peptides set forth in Example 4. Neutravidin HRP was used instead of streptavidin or avidin because it has lower non-specific binding to molecules other than biotin due to the absence of lectin binding carbohydrate moieties and also due to the absence of the cell adhesion receptor-binding RYD domain in neutravidin. 
     In the experiments described herein, 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 transiently-transfected with KDR. All four tetrameric complexes of KDR-binding peptides were biotinylated and contained the JJ spacer, and bound to the KDR-expressing cells; however, SEQ ID NO:356 exhibited the best K D  (1.81 nM). 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. 
     In 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. 
     In Experiment D, it was shown that this assay is useful in evaluating distinct combinations of KDR and VEGF/KDR complex binding polypeptides for use in multimeric targeting constructs that contain more than one KDR and VEGF/KDR complex binding polypeptide. Moreover, Experiments D and E establish that tetrameric constructs including two or more KDR binding peptides that bind to different epitopes exhibited superior binding to “pure” tetrameric constructs of the targeting peptides alone. 
     Experiment A 
     Preparation of m-RNA &amp; 5′ RACE Ready cDNA Library 
     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. # K1520-02). 12 μg 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 μg of mRNA, oligo dT primer (5′-(T) 25 GC-3′) and/or smart II oligo (5′AAGCAGTGGTAACAACGCAGAGTACGCGGG-3′) (SEQ ID NO:357) using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase. The reaction was performed in a total volume of 20 μl and the reaction mix contained 2 μl of RNA, 1 μsmart II oligo, 1 μl of oligo dT primer, 4 μl of 5× first-strand buffer (250 mM Tris HCl pH 8.3, 375 mM KCl, 30 mM MgCl 2 ) 1 μDTT (20 mM, also supplied with reverse transcriptase), 1 μl dNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP in ddH 2 O, Stratagene, cat. #200415), 9 μl ddH 2 O and 1 μl MMLV reverse transcriptase (Clonetech, cat #8460-1). The reverse transcription reaction was performed for 90 minutes at 42° C., and the reaction was stopped by adding 250 μl of tricine-EDTA buffer (10 mM tricine, 1.0 mM EDTA). The reverse transcription product, a 5′ RACE ready cDNA library, can be stored for 3 months at −20° C. Note that all water used for DNA and RNA application was DNAse and RNAse free from USB (cat. #70783). 
     Cloning of s-KDR into TOPOII Vector 
     In order to clone s-KDR, a 5′ oligo (G ATG GAG AGC AAG GTG CTG CTG G) (SEQ ID NO:358) and a 3′ oligo (C CAA GTT CGT CTT TTC CTG GGC A) (SEQ ID NO:359) were used. These were designed to amplify the complete extracellular domain of KDR (˜2.2 kbps) from the 5′ RACE ready cDNA library (prepared above) using polymerase chain reaction (PCR) with pfu polymerase (Stratagene, cat. #600135). The PCR reaction was done in total volume of 50 μl and the reaction mix contained 2 μl 5′ RACE ready cDNA library, 1 μl 5′ oligo (10 μM), 1 μl 3′ oligo (10 μM), 5 μl 10×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 μl dNTP mix (10 mM) and 40 μl ddH 2 O. The PCR reaction was performed by using a program set for 40 cycles of 1 minute at 94 C, 1 minute at 68 C and 4 minutes at 72 C. 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 μl of ddH 2 O, the 2 μl of 10× Taq polymerase buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl 2 , 0.01% gelatin) and 1 μl of Taq polymerase (Stratagene, cat. #600131) was added to generate an A overhang to each end of the product. After incubating for 1 hour at 72 C the modified product was cloned directly into a TOPOII vector (InVitrogen, Carlsbad, Calif.) following the manufacturer&#39;s protocol to give TOPO-sKDR. The TOPO vector allows easy cloning of PCR products because of the A-overhang in Taq (PCR enzyme)-treated PCR products. 
     Cloning the Transmembrane and Cytoplasmic Domains of KDR into TOPO II Vector 
     In order to clone the transmembrane and cytoplasmic domains of KDR, a 5′ oligo (TCC CCC GGG ATC ATT ATT CTA GTA GGC ACG GCG GTG) (SEQ ID NO:360) and a 3′ oligo (C AGG AGG AGA GCT CAG TGT GGT C) (SEQ ID NO:361) were used. These were designed to amplify the complete transmembrane and cytoplasmic domains of KDR (˜1.8 kbps) from the 5′ RACE ready cDNA library (described above) using polymerase chain reaction (PCR) with pfu polymerase. PCR reaction conditions and the program were exactly the same as described above for s-KDR. Just as with the s-KDR sequence, the PCR product was purified using phenol chloroform extraction, treated with Taq polymerase and cloned into TOPOII vector from Invitrogen to give TOPO-CYTO. 
     Cloning of Full-Length KDR into pcDNA6 Vector 
     To create the full-length receptor, the extra-cellular domain and the cytoplasmic domain (with trans-membrane 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 Not1 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. Similarly, the 5′ oligo (ATC ATT ATT CTA GTA GGC ACG GCG GTG) (SEQ ID NO:364) and the 3′ oligo, with a Not1 site (A TAA GAA TGC GGC CGC AAC AGG AGG AGA GCT CAG TGT GGT C) (SEQ ID NO:365), were used to amplify by PCR the cytoplasmic domain of KDR (with transmembrane domain) from TOPO-CYTO. Both PCR products were digested with Not1 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-His C vector. Purification of DNA and ligation was done as described earlier for psKDR. The ligation reaction was used to transform a culture of DH5a 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. 
     Cell Culture 
     293H cells were obtained from Invitrogen (cat. #11631) and grown as monolayer culture in their recommended media plus 1 mL/L pen/strep (Invitrogen, cat. #15140-148). All the cells were grown in presence of antibiotic for everyday culture but were split into antibiotic free media for 16-20 hours prior to transfection. 
     Preparation of DNA for Transfection 
       E. coli  bacteria DH5α containing pf-KDR was streaked onto LB with 50 μg/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 μl of bacterial culture from the 3 mL tube was transferred 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 shaker. The next day, the bacterial culture was processed using maxi-prep kit (QIAGEN, cat. #12163). Generally, about 1 mg of plasmid DNA (as quantitated by absorbance at 260 nm) was obtained from 250 mL of bacterial culture. 
     Transfection of 293H Cells in 96 Well Plate 
     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 0.1 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. 
     Preparation of M199 Media 
     In order to prepare M199 media for the assay, one M199 medium packet (GIBCO, cat. #31100-035), 20 mL of 1 mM HEPES (GIBCO, cat. #15630-080) and 2 μm of DIFCO Gelatin (DIFCO, cat. #0143-15-1) were added to 950 mL of ddH 2 O and the pH of the solution was adjusted to 7.4 by adding approximately 4 mL of 1N 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 μm filters (Corning, cat. #43109), and stored at 4° C. to be used later in the assay. 
     Preparation of SoftLink Soft Release Avidin-Sepharose 
     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 2 O. A fresh 50% slurry of avidin-sepharose was prepared for each experiment. 
     Preparation of Peptide/Neutravidin HRP Solution 
     Biotinylated peptides SEQ ID NOS:294, 264, 277, 356, and the non-binding biotinylated control peptide were used to prepare 250 μM stock solutions in 50% DMSO and a 33 μM stock solution of neutravidin-HRP was prepared by dissolving 2 mg of neutravidin-HRP (Pierce, cat. #31001) in 1 mL of ddH 2 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. To prepare peptide/neutravidin-HRP complexes, 10 μl of 250 μM biotinylated peptide stock solution and 10 μl of 33 μM 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 μl of soft release avidin-sepharose (50% slurry in ddH 2 O) to remove excess peptides and another incubation for 30 minutes on a rotator at 4° C. Finally, the soft release avidin-sepharose was pelleted by centrifuging at 12,000 rpm for 5 minutes at room temperature, and the resulting supernatant was used for the assays. Fresh peptide/neutravidin-HRP complexes were prepared for each experiment. 
     Preparation of Peptide/Neutravidin HRP Dilutions for the Assay 
     For saturation binding experiments, 120 μl, 60 μl, 20 μl, 10 μl, 8 μl, 6 μl, 4 μl, and 1 μl of peptide/neutravidin HRP complex were added to 1.2 mL aliquots of M199 medium to create dilutions with final concentrations of 33.33 nM, 16.65 nM, 5.55 nM, 2.78 nM, 1.67 nM, 1.11 nM and 0.28 nM complex, respectively. 
     Preparation of Blocking Solution for Transfected 293H Cells 
     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. 
     Assay to Detect the Binding of Peptide/Neutravidin-HRP 
     24 hours after transfection, each well of the 293H cells was washed once with 100 μl of M199 medium and incubated with 80 μl of blocking solution at 37° C. After one hour, cells were washed twice with 100 μl of M199 media and incubated with 70 μl 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). After incubation at room temperature, plates were transferred to 4° C. for another half-hour incubation. Subsequently, cells were washed 5 times with ice-cold M199 media and once with ice-cold PBS (in that order). After the final wash, 100 μl of ice cold TMB solution (KPL, cat. #50-76-00) was added to each well and each plate was incubated for 30 minutes at 37° C. in an air incubator. Finally, the HRP enzyme reaction was stopped by adding 50 μl of 1N phosphoric acid to each well, and binding was quantitated by measuring absorbance at 450 nm using a microplate reader (BioRad Model 3550). 
     Binding of Peptide/Neutravidin HRP to KDR-Transfected Cells 
     In this assay, complexes of control peptide, SEQ ID NO:264, SEQ ID NO:294, SEQ ID NO:277, and SEQ ID NO:356 peptides, each biotinylated with the JJ spacer and conjugated with neutravidin-HRP, were prepared as described above and tested for their ability to bind 293H cells that were transiently-transfected with KDR. During the peptide/neutravidin complex preparation, a 7.5-fold excess of biotinylated peptides over neutravidin-HRP was used to make sure that all four biotin binding sites on neutravidin were occupied. After complex formation, the excess of free biotinylated peptides was removed using soft release avidin-sepharose to avoid any competition between free biotinylated peptides and neutravidin HRP-complexed biotinylated peptides. The experiment was performed at several different concentrations of peptide/neutravidin-HRP, from 0.28 nM to 33.33 nM, to generate saturation binding curves for SEQ ID NO:264 and SEQ ID NO:294 ( FIG. 1A ) and 0.28 to 5.55 nM to generate saturation binding curve for SEQ ID NO:277 and SEQ ID NO:356 ( FIG. 1B ). In order to draw the saturation binding curve, the background binding to mock-transfected cells was subtracted from the binding to KDR-transfected cells for each distinct peptide/neutravidin HRP complex at each concentration tested. Therefore, absorbance on the Y-axis of  FIG. 1  is differential absorbance (KDR minus mock) and not the absolute absorbance. Analysis of the saturation binding data in  FIG. 1  using Graph Pad Prism software (version 3.0) yielded a K D  of 10.00 nM (+/−2.36) for the tetrameric SEQ ID NO:294, 14.87 nM (+/−5.066) for the tetrameric SEQ ID NO:264, 4.031 nM (+/−0.86) for the tetrameric SEQ ID NO:277, and 1.814 nM (+/−0.27) for the tetrameric SEQ ID NO:356 peptide complexes. These 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). As expected, no saturation of binding for the control (non-binder) peptide/neutravidin HRP-complex was observed. 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, 294 and 277) from a non-binding peptide. 
     Experiment B 
     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. In this experiment, 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:294 without the spacer), and a non-KDR binding, biotinylated control peptide (with and without spacer, prepared as set forth above) was used as a control. The peptide structure of all the KDR-binding sequences tested in this experiment is shown in  FIG. 3 . 
     This experiment was performed as set forth in Experiment A described above, except that it was only done at a single concentration of 2.78 nM. 
     Results: 
     It is evident from the results shown in the  FIG. 4  that the spacer enhances binding of SEQ ID NO:294 and SEQ ID NO:264. The spacer between the binding sequence and biotin can be helpful in enhancing binding to target molecule by multiple mechanisms. First, it could help reduce the steric hindrance between four biotinylated peptides after their binding to a single avidin molecule. Second, it could provide extra length necessary to reach multiple binding sites available on a single cell. 
     Experiment C 
     Experiment C was designed to look at the serum effect on the binding of SEQ ID NOS: 294, 264, 277 and 356. In this procedure, 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: 
     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 (c) 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. 97 ). 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 
     Experiment D was designed to evaluate the binding of tetrameric complexes of KDR and VEGF/KDR complex binding polypeptides SEQ ID 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 experiment. 
     Preparation of Peptide/Neutravidin Solutions: 
     250 μM stock solutions of biotinylated peptides SEQ ID NOs:264, 294 and control peptide were prepared in 50% DMSO and a 33 μM stock solution of Neutravidin HRP was prepared by dissolving 2 mg of Neutravidin HRP (Pierce, cat. #31001) in 1 mL of ddH 2 O. Peptide stock solutions were stored at −20 C, whereas the Neutravidin HRP stock solution was stored at −80 C. The sequences of the biotinylated peptides are shown above. To prepare peptide/neutravidin HRP complexes, a total 5.36 μL of 250 μM biotinylated peptide stock solution (or a mixture of peptide solutions, to give peptide molecules four times the number of avidin HRP molecules) and 10 μL of 33 μM 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 μL of soft release avidin-sepharose (50% slurry in ddH 2 O) to remove excess peptides and another incubation for 30 minutes on a rotator at 4 C. Finally, the soft release avidin-sepharose was pelleted by centrifuging at 12,000 rpm for 5 minutes at room temperature, and the resulting supernatant was used for the assays. Fresh peptide/neutravidin HRP complexes were prepared for each experiment. 
     Assay to Detect the Binding of Peptide/Neutravidin HRP: 
     24 hours after transfection, each well of the 293H cells was washed once with 100 μL of M199 medium and incubated with 80 μL of blocking solution at 37 C. After one hour, cells were washed twice with 100 μL of M199 media and incubated with 70 μL of 3.33 nM peptide (or peptide mix)/neutravidin HRP solutions (prepared by adding 10 μL of stock prepared earlier to 1 mL of M199 media) 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. After incubation at room temperature, plates were transferred to 4 C for another half-hour incubation. Subsequently, cells were washed five times with ice-cold M199 media and once with ice-cold PBS (in that order). After the final wash, 100 μL of ice cold TMB solution (KPL, Gaithersburg, Md.) was added to each well and each plate was incubated for 30 minutes at 37 C in an air incubator. Finally, the HRP enzyme reaction was stopped by adding 50 μL of 1N phosphoric acid to each well, and binding was quantitated by measuring absorbance at 450 nm using a microplate reader (BioRad Model 3550). 
     Results: 
     This experiment establishes that SEQ ID NO:294 and SEQ ID NO:264 bind to KDR in multimeric fashion, and cooperate with each other for binding to KDR in 293H transfected cells. A biotinylated control peptide that does not bind to KDR was used. As expected, a tetrameric complex of the control peptide with avidin-HRP did not show enhanced binding to KDR-transfected cells. Tetrameric complexes of SEQ ID NO:294 and SEQ ID NO:264 bound to KDR-transfected cells significantly better than to mock-transfected cells (see  FIG. 6 ). SEQ ID NO:294 tetramers, however, bound much better than SEQ ID NO:264 tetramers. If the control peptide was added to the peptide mixture used to form the tetrameric complexes, the binding to the KDR-transfected cells decreased. The ratio of specific binding of tetramer to monomer, dimer and trimer was calculated by dividing the specific binding (obtained by subtracting the binding to mock transfected cells from KDR transfected cells) of tetramer, trimer and dimer with that of monomer. Results indicate that there is co-operative effect of multimerization of SEQ ID NOS:264, 294 and 356 on the binding to KDR-transfected cells. 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 Tetramer 
                 Trimer 
                 Dimer 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ ID NO: 264 
                 45.4 
                 5 
                 4.3 
               
               
                   
                 SEQ ID NO: 294* 
                 38.6 
                 7.1 
                 2.7 
               
               
                   
                 SEQ ID NO: 277 
                 1 
                 1.1 
                 1.1 
               
               
                   
                 SEQ ID NO: 356 
                 16 
                 5.7 
                 2.3 
               
               
                   
                   
               
               
                   
                 *monomeric peptide binding at 2.22 nM was zero, therefore ratios were calculated using binding at 5.55 nM. 
               
            
           
         
       
     
     A mixture of 25% non-binding control peptide with 75% SEQ ID NO:264 did not bind significantly over background to KDR-transfected cells, indicating that multivalent binding is critical for the SEQ ID NO:264/avidn-HRP complex to remain bound to KDR throughout the assay. This phenomenon also held true for SEQ ID NO:294, where substituting 50% of the peptide with control peptide in the tetrameric complex abolished almost all binding to KDR on the transfected cells. 
     Surprisingly, 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 ID 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. This may be because multimeric binding to a single target requires that the multimeric binding entity span two or more separate target molecules that are close enough together for it to bind them simultaneously, whereas a multimeric binder that 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 
     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 μM) of biotinylated control peptide with spacer, SEQ ID NO:264 and SEQ ID NO:294, none of which were complexed with avidin. 
     Results: 
     It is evident from  FIG. 7  that SEQ ID NO:264 does compete with SEQ ID NO:264/avidin HRP solution for binding to recombinant KDR-Fc fusion protein whereas control peptide and SEQ ID NO:294 do not compete with SEQ ID NO:264/avidin HRP solution for binding to recombinant KDR-Fc fusion protein. Thus, SEQ ID NO:264 and SEQ ID NO:294 bind to distinct and complementary sites on KDR. 
     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. 
     Example 7 
     Bead-Binding Assay to Confirm Ability of Peptides Identified by Phage Display to Bind KDR-Expressing Cells 
     The following procedures were performed to assess the ability of KDR-binding peptides to bind to KDR-expressing cells. In this procedure, 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. 
     Protocol 
     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&#39;s instructions. 
     Preparation of Peptide-Conjugated Fluorescent Beads: 
     0.1 mL of a 0.2 mM stock solution of each biotinylated peptide (prepared as set forth above, in 50% DMSO) was incubated with 0.1 mL of Neutravidin-coated red fluorescent microspheres (2 micron diameter, custom-ordered from Molecular Probes) and 0.2 mL of 50 mM 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, 0.12 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 1×MEM Non-Essential Amino Acids Solution (NEAA) (GIBCO 11140-050) and 40% FBS (Hyclone SH30070.02) 0.03 mL of the sonicated bead preparations was added. 96-well plates seeded with 293H cells that have been mock-transfected in columns 1 to 6, and KDR-transfected in columns 7 to 12 (as in Example 5), were drained and washed once with DMEM, high glucose with 1×NEAA and 40% FBS. To each well 0.1 mL of bead solution was added, six wells per bead preparation. After incubating at room temperature for 30 minutes, the wells were drained by inverting the plates and washed four times with 0.1 mL PBS with Ca ++ Mg ++  (GIBCO #14040-117) with shaking at room temperature for 5 minutes each wash. After draining, 0.1 mL of PBS was added per well. The plates were then read on a Packard FluoroCount fluorometer at excitation 550 nm/emission 620 nm. Unconjugated neutravidin beads were used as a negative control while beads conjugated with a biotinylated anti-KDR antibody were used as the positive control for the assay. 
     To calculate the number of beads bound per well, a standard curve with increasing numbers of the same fluorescent beads was included in each assay plate. The standard curve was used to calculate the number of beads bound per well based on the fluorescence intensity of each well. 
     Results: 
     The positive control beads with anti-KDR attached clearly bound preferentially to the KDR-expressing cells while avidin beads with nothing attached did not bind to either cell type ( FIG. 9 ). Biotinylated SEQ ID NO:264 beads did not bind to the KDR-transfected cells significantly more than to mock-transfected cells, but adding a hydrophilic spacer between the peptide moiety and the biotin group (biotinylated SEQ ID NO:264 with a JJ spacer beads) enhanced binding to KDR cells without increasing the binding to mock-transfected cells. 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 ID NO:294 with the JJ spacer) significantly improved the specific binding to KDR in the transfected cells. Thus, 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  125 I Labeled VEGF for Binding to KDR-Transfected 293H Cells 
     KDR-binding polypeptides were next assessed for their ability to compete with 125I-labeled VEGF for binding to KDR expressed by transfected 293H cells. The results indicate that KDR-binding polypeptide SEQ ID NO:263 (Ac-AGDSWCSTEYTYCEMIGTGGGK-NH 2 ) did not compete significantly with  125 I-labeled VEGF, and SEQ ID NOS:294, 264, and SEQ ID NO: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. 
     Transfection of 293H 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. 
     Preparation of M199 Media: 
     M199 medium was prepared as described in Example 5. 
     Preparation of Peptide Solutions: 
     3 mM stock solutions of peptides SEQ ID NO:294, SEQ ID NO:263, SEQ ID NO:264 and SEQ ID NO:277 were prepared as described above in 50% DMSO. 
     Preparation of  125 I-Labeled VEGF Solution for the Assay: 
     25 μCi of lyophilized  125 I-labeled VEGF (Amersham, cat. # IM274) was reconstituted with 250 μl of ddH 2 O to create a stock solution, which was stored at −80 C for later use. For each assay, a 300 μM 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 μM and 0.3 μM Peptide Solution in 300 μM  125 I-Labeled VEGF: 
     For each 96 well plate, 10 mL of 300 μM  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 μl of M199 media with 300 μM  125 I-labeled VEGF to prepare 30 μM and 0.3 μM peptide solutions containing 300 μM of  125 I-labeled VEGF. Once prepared, the solutions were kept on ice until ready to use. The dilution of peptides in M199 media containing 300 μM  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 M199 medium was removed from the plate and replaced with 75 μl of 300 μM  125 I-labeled VEGF in M199 medium (prepared as above) with the polypeptides. Each dilution was added to three 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. 100 μl of solubilizing solution (2% Triton X-100, 10% Glycerol, 0.1% BSA) was added to each well and the plates were incubated at room temperature for 30 minutes. The solubilizing solution in each well was mixed by pipeting up and down, and transferred to 1.2 mL tubes. Each well was washed twice with 100 μl of solubilizing solution and the washes were added to the corresponding 1.2 mL tube. Each 1.2 mL tube was then transferred to a 15.7×100 cm tube to be counted in an LKB Gamma Counter using program 54 ( 125 I window for 1 minute). 
     Competition of Peptides with  125 I Labeled VEGF in 293H Cells: 
     The ability of KDR-binding peptides SEQ ID NO:294, SEQ ID NO:263, SEQ ID NO:264 and SEQ ID NO:277 to specifically block  125 I-labeled VEGF binding to KDR was assessed in mock-transfected and KDR-transfected cells. SEQ ID NO:263 was used in the assay as a negative control, as it exhibited poor binding to KDR in the FP assays described herein and would therefore not be expected to displace or compete with VEGF. To calculate the specific binding to KDR, the binding of  125 I-labeled VEGF to mock-transfected cells was subtracted from KDR-transfected cells. Therefore, the binding of  125 I-labeled VEGF to sites other than KDR (which may or may not be present in 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], where Y1 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. 
     As shown in  FIG. 10 , in 293 cells, SEQ ID NO:263, which due to its relatively high K d  (&gt;2 μM) was used as a negative control, did not compete significantly with  125 I-labeled VEGF, 12.69±7.18% at 30 μM and −5.45±9.37% at 0.3 μM ( FIG. 10 ). At the same time, 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 μM and 52.27±3.78% and 80.96±3.8% at 0.3 μM ( FIG. 10 ) respectively. The percentage inhibition with SEQ ID NO:264 was 47.95±5.09% of  125 I-labeled VEGF binding at 30 μM and 24.41±8.43% at 0.3 μM ( FIG. 10 ). Thus, 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 
     The ability of KDR-binding peptides identified by phage display to inhibit 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. 
     Protocol 
     Human umbilical vein endothelial cells (HUVECs) (Biowhittaker Catalog #CC-2519) were obtained frozen on dry ice and stored in liquid nitrogen until thawing. These cells were thawed, passaged, and maintained as described by the manufacturer in EGM-MV medium (Biowhittaker Catalog #CC-3125). Cells seeded into 100 mm dishes were allowed to become confluent, then cultured overnight in basal EBM medium lacking serum (Biowhittaker Catalog #CC-3121). The next morning, the medium in the dishes was replaced with 10 mL fresh EBM medium at 37 C 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). In some cases, a neutralizing anti-KDR antibody (Catalog #AF357, R&amp;D Systems) was used as a positive control inhibitor of activation. In 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. in a 37° C. tissue culture incubator they were washed three times with ice-cold D-PBS containing calcium and magnesium and placed on ice without removing the last 10 mL of Delbecco&#39;s phosphate buffered saline (D-PBS). 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 (phenylmethylsulfonylfluoride), 1 mM sodium orthovanadate, 100 mM NaF, 50 mM sodium pyrophosphate, 10 μg/mL leupeptin, 10 μg/mL aprotinin). The cells were quickly scraped into the lysis buffer using a cell scraper (Falcon, Cat No. #353087), dispersed by pipeting up and down briefly, and the resulting lysate was transferred to the second drained dish of the pair. Another 0.5 mL of lysis buffer was used to rinse out the first dish then transferred to the second dish, which was then also scraped and dispersed. The pooled lysate from the two dishes was transferred to a 1.5 mL Eppindorf tube. The above procedure was repeated for each of the controls and test samples (KDR-binding peptides), one at a time. The lysates were stored on ice until all the samples had been processed. At this point samples were either stored at −70° C. or processed to the end of the assay without interruption. 
     The lysates, freshly prepared or frozen and thawed, were precleared by adding 20 μl 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% 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×g and the supernatants transferred to new 1.5 mL tubes. Twenty μg of anti-Flk-1 antibody (Santa Cruz Biotechnology, Catalog #sc-504) was added to each tube, and the tubes were incubated overnight (16-18 hr.) at 4 C on a rotator to immunoprecipitate KDR. The next day 40 μl of protein A-sepharose beads were added to the tubes that were then incubated 4 C for 1 hr. on a rotator. The beads in each tube were subsequently washed three times by centrifuging for 2 min. in a Picofuge, discarding the supernatant, and dispersing the beads in 1 mL freshly added TBST buffer (20 mM Tris base pH 7.5, 137 mM NaCl, and 0.1% Tween 20). After centrifuging and removing the liquid from the last wash, 40 μl of Laemmli SDS-PAGE sample buffer (Bio-Rad, Catalog #161-0737) was added to each tube and the tubes were capped and boiled for 5 min. After cooling, the beads in each tube were pelleted by centrifuging and the supernatants containing the immunoprecipitated KDR were transferred to new tubes and used immediately or frozen and stored at −70 C for later analysis. 
     Detection of phosphorylated KDR as well as total KDR in the immunoprecipitates was carried out by immunoblot analysis. Half (20 μL) 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)). 
     Using a Bio-Rad mini-Protean 3 apparatus (Catalog #165-3302), the resolved proteins in each gel were electroblotted to a PVDF membrane (Bio-Rad, Cat. No. 162-0174) in a Bio-Rad mini Trans-Blot cell (Catalog #170-3930) in CAPS buffer (10 mM CAPS, Sigma Catalog #C-6070, 1% ACS grade methanol, pH 11.0) for 2 hr. at 140 mA according to the method of Matsudaira ( J. Biol. Chem.,  262:10035-10038 (1987)). 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. Subsequently, 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. 
     To confirm that similar amounts of KDR were compared in the assay, 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-Flk-1 polyclonal antibody (Catalog #sc-315 from Santa Cruz Biotech), 1:200 in 5% Blotto-TBST with 1% normal 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. Subsequently, 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. 
     Results: 
     Immunoblots of KDR immunoprecipitates prepared from HUVECs with and without prior VEGF stimulation demonstrated that activated (phosphorylated) KDR could be detected when the HUVECs were stimulated with VEGF. An anti-phosphotyrosine antibody (PY-20) detected no phosphorylated proteins close to the migration position of KDR from unstimulated HUVECs on the blots, but after five minutes of VEGF stimulation, an intense band was consistently observed at the expected location ( FIG. 11 , upper panel). When the blots were stripped of bound antibodies by incubation in acidic solution then reprobed with an anti-KDR antibody (sc-315), the identity of the phosphorylated protein band was confirmed to be KDR. Moreover, it was observed that immunoprecipitates from unstimulated HUVECs contained about as much total KDR as immunoprecipitates from VEGF-stimulation HUVECs ( FIG. 11 , lower panel). 
     The foregoing results indicate that the phosphorylated KDR detected was formed from pre-existing KDR through autophosphorylation of KDR dimers resulting from VEGF binding, as five minutes is not enough time to synthesize and process a large glycosylated cell-surface receptor such as KDR. 
     The ability of 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&amp;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. This result was expected since the ability of the antibody to block VEGF-induced DNA synthesis is part of the manufacturer&#39;s quality control testing of the antibody. Re-probing the blot with an anti-KDR antibody ( FIG. 12 , lower panel) indicated that slightly less total KDR was present in the VEGF+ antibody-treated lane (+V+α-KDR) relative to the VEGF-only-treated lane (+V), but the difference was not great enough to account for the much lower abundance of phosphorylated KDR in the antibody-treated lane. 
     To assess the potency of a linear KDR-binding peptide (AFPRFGGDDYWIQQYLRYTD, SEQ ID NO:140) 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. Re-probing the blot for total KDR showed that there is even more total KDR in the VEGF+SEQ ID NO:306-treated cells (+V+SEQ ID NO:306) than in the VEGF only-treated cells (+V) ( FIG. 13 , lower panel). Thus, it is clear that the decreased phosphorylation of KDR in the presence of SEQ ID NO:306 is not due to differential sample loading, but rather the ability of the polypeptide to inhibit VEGF-activation of KDR. 
     Repeating the foregoing assay, the following polypeptides demonstrated at least a 50% inhibition of VEGF-induced KDR phosphorylation at 10 μM: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 269) 
               
            
           
           
               
               
            
               
                   
                 Ac-AGWIE CYHPDGIC YHFGTGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 267) 
               
            
           
           
               
               
            
               
                   
                 Ac-AGWLE CYAEFGHC YNFGTGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 294) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDSRV CWEDSWGGEVC FRYDPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 366 having a blocked K) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDWWE CK(ivDde)REEYRNTTWC AWADPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 301) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDPDT CTMWGDSGRWYC FPADPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 305) 
               
            
           
           
               
               
            
               
                   
                 Ac-AQEPEGYAYWEVITLYHEEDGDGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 306) 
               
            
           
           
               
               
            
               
                   
                 Ac-AQAFPRFGGDDYWIQQYLRYTDGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 307) 
               
            
           
           
               
               
            
               
                   
                 Ac-AQGDYVYWEIIELTGATDHTPPGGK-NH 2 . 
               
            
           
         
       
     
     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 μM. 
     The following peptides were tested in the assay and did not produce significant inhibition of KDR activation at 10 μM: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 264) 
               
            
           
           
               
               
            
               
                   
                 Ac-AGPK(ivDde)W CEEDWYYC MITGTGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 314) 
               
            
           
           
               
               
            
               
                   
                 Ac-GSDHH CYLHNGQWIC YPFAPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 293) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDYPW CHELSDSVTRFC VPWDPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 295) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDDHM CRSPDYQDHVFC MYWDPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 296) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDPPL CYFVGTQEWHHC NPFDPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 299) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDGSW CEMRQDVGK(ivDde)WNC FSDDPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 331) 
               
            
           
           
               
               
            
               
                   
                 Ac-AQRGDYQEQYWHQQLVEQLK(ivDde)LLGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 303) 
               
            
           
           
               
               
            
               
                   
                 Ac-GDNWE CGWSNMFQK(ivDde)EFC ARPDPGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 367) 
               
            
           
           
               
               
            
               
                   
                 Ac-AGPGP CK(ivDde)GYMPHQC WYMGTGGGK-NH 2   
               
               
                   
               
            
           
           
               
            
               
                 (SEQ ID NO: 322) 
               
            
           
           
               
               
            
               
                   
                 Ac-AGYGP CAEMSPWLC WYPGTGGGK-NH 2 . 
               
            
           
         
       
     
     In addition, tetrameric complexes of biotinylated derivatives of SEQ ID 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 KDR-transfected 293H cells. Tc-labeled SEQ ID NO:277 (i.e., Ac-AGPTWCEDDWYYCWLFGT-GGGK(N,N-dimethyl-Gly-Ser-Cys-Gly-di(aminodioxaocta-))-NH 2 ) 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. 
     Preparation of Peptidic Chelate for Binding to Tc by SPPS ( FIG. 35 ) 
     To a 250 mL of SPPS reaction vessel was added 6.64 mmol of H-Gly-2-Cl-trityl resin (0.84 mmol/g, Novabiochem). It was swelled in 80 mL of DMF for 1 h. For each coupling cycle the resin was added 26.6 mmol of DIEA, 26.6 mmol of a Fmoc-amino acid in DMF (EM Science), 26.6 mmol of HOBT (Novabiochem) in DMF, and 26.6 mmol of DIC. The total volume of DMF was 80 mL. The reaction mixture was shaken for 4 h. The resin then was filtered and washed with DMF (3×80 mL). A solution of 20% piperidine in DMF (80 mL) was added to the resin and it was shaken for 10 min. The resin was filtered and this piperidine treatment was repeated. The resin finally was washed with DMF (3×80 mL) and ready for next coupling cycle. At the last coupling cycle, N,N-dimethyl glycine (Aldrich) was coupled using HATU/DIEA activation. Thus, to a suspension of N,N-dimethyl glycine (26.6 mmol) in DMF was added a solution of 26.6 mmol of HATU (Perseptive Biosystems) in DMF and 53.1 mmol of DIEA. The clear solution was added to the resin and shaken for 16 h. Following the synthesis, the resin was filtered and washed with DMF (3×80 mL), CH 2 Cl 2  (3×80 mL) and dried. The resin was mixed with 80 mL of AcOH/CF 3 CH 2 OH/DCM (1/1/8, v/v/v) and shaken for 45 min. The resin was filtered and the filtrate was evaporated to a paste. Purification of the crude material by silica gel chromatography using 25% MeOH/DCM afforded 2.0 g of the final product. 
     Coupling of the Peptidic Chelate to the Peptide (Fragment Coupling) 
     Diisopropylcarbodiimide (0.0055 mmol) was added to a mixture of purified Me 2 N-Gly-Cys-(Trt)-Ser(tBu)-Gly-OH and hydroxybenzotriazole (0.0055 mmol) in DMF (0.25 mL), and the mixture was stirred at RT for 6 h. The peptide (0.005 mmol) in DMF (0.25 mL) was then added to the reaction mixture, and stirring was continued for an additional 6 h. DMF was removed under vacuum and the residue was treated with reagent B and stirred for 3 h. TFA was removed under reduced pressure and the residue was purified by preparative HPLC using acetonitrile-water containing 0.1% TFA. Fractions containing the pure product were collected and freeze dried to yield the peptide. The peptide was characterized by ES-MS and the purity was determined by RP-HPLC (acetonitrile-water/0.1% TFA) gradient. 
     Synthesis of  99m Tc-Labeled SEQ ID NO:339 
     A stannous gluconate solution was prepared by adding 2 mL of a 20 μg/mL SnCl 2 .2H 2 O solution in nitrogen-purged 1N 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 μl (20-40 μg) of SEQ ID NO:339 ligand dissolved in 50/50 ethanol/H 2 O, 6-12 mCi of  99m TcO 4   −  in saline and 100 μl of stannous glucoheptonate solution. The mixture was heated at 100° C. for 22 min. The resulting radiochemical purity (RCP) was 10-47% when analyzed using a Vydac C18 Peptide and Protein column that was eluted at a flow rate of 1 mL/min. with 66% H 2 O (0.1% TFA)/34% ACN(0.085% TFA). The reaction mixture was purified by HPLC on a Vydac C18 column (4.6 mm×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 μl of a stabilizing buffer containing 5 mg/mL ascorbic acid and 16 mg/mL hydroxypropyl-α-cyclodextrin in 50 mM phosphate buffer. The mixture was concentrated using a speed vacuum apparatus to remove acetonitrile, and 200 μl of 0.1% HSA in 50 mM pH 5 citrate buffer was added. The resulting product had an RCP of 100%. Prior to injection into animals, the compound was diluted to the desired radioconcentration with normal saline. 
     Transfection of 293H Cells 
     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. 
     Preparation of Opti-MEMI Media with 0.1% HSA 
     Opti-MEMI was obtained from Invitrogen (cat. #11058-021) and human serum albumin (HSA) was obtained from Sigma (cat. # A-3782). To prepare opti-MEMI media with 0.1% HSA, 0.1% w/v HSA was added to opti-MEMI, stirred at room temperature for 20 min. and then filter sterilized using 0.2 μm filter. 
     Preparation of Tc-Labeled SEQ ID NO:339 Dilutions for the Assay 
     Stock solution of Tc-labeled SEQ ID NO:339 (117 μCi/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 μCi/mL of Tc-labeled SEQ ID NO:339. 
     Assay to Detect the Binding of Tc-Labeled SEQ ID NO:339 
     Cells were used 24 hours after transfection, and to prepare the cells for the assay, they were washed once with 100 μl of room temperature opti-MEMI with 0.1% HSA. After washing, the opti-MEMI with 0.1% HSA was removed from the plate and replaced with 70 μl of 1.17, 2.34, 4.68 and 11.7 μCi/mL of Tc-labeled SEQ ID NO:339 (prepared as above). Each dilution was added to three separate wells of mock- and KDR-transfected cells. After incubating at room temperature for 1 hour, the plates were transferred to 4° C. for 15 minutes and washed 5 times with 100 μl of cold binding buffer (opti-MEMI with 0.1% HSA), gently blotted dry and checked under a microscope for cell loss. 100 μl of solubilizing solution (2% Triton X-100, 10% Glycerol, 0.1% BSA) was added to each well and the plates were incubated at 37° C. for 10 minutes. The solubilizing solution in each well was mixed by pipeting up and down, and transferred to 1.2 mL tubes. Each well was washed once with 100 μl of solubilizing solution and the washes were added to the corresponding 1.2 mL tube. Each 1.2 mL tube was then transferred to a 15.7×100 cm tube to be counted in an LKB Gamma Counter using program 12 (Tc-window for 20 sec). 
     Binding of Tc-Labeled SEQ ID NO:339 to KDR Transfected Cells 
     The ability of Tc-labeled SEQ ID NO:339 to specifically bind to KDR was assessed using transiently transfected 293H cells. 
     As shown in  FIG. 14 , Tc-labeled SEQ ID NO:339 bound significantly better to KDR transfected 293H cells as compared to mock transfected 293H cells. To calculate specific binding to KDR, 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. 96 ). Linear binding was not surprising because concentration of Tc-labeled SEQ ID NO:339 was only ˜100 μM (even at the highest concentration, 11.7 μCi/mL, tested in the assay), which is far below the K D  value of ˜3-4 nM of SEQ ID NO:277 (as calculated using avidin HRP assay), so no saturation of binding would be expected. 
     Example 11 
     Preparation of Peptides and Dimeric Peptide Construction 
     The following methods were used for the preparation of individual peptides and dimeric peptide constructs described in the following Examples (11-15). 
     Automated Peptide Synthesis 
     Peptide synthesis was carried out on a ABI-433A Synthesizer (Applied Biosystems Inc., Foster City, Calif.) on a 0.25 mmol scale using the FastMoc protocol. In each cycle of this protocol preactivation was accomplished by dissolution of 1.0 mmol of the requisite dry N α -Fmoc side-chain protected amino acid in a cartridge with a solution of 0.9 mmol of HBTU, 2 mmol of DIEA, and 0.9 mmol of HOBt in a DMF-NMP mixture. The peptides were assembled on NovaSyn TGR (Rink amide) resin (substitution level 0.2 mmol/g). Coupling was conducted for 21 min. Fmoc deprotection was carried out with 20% piperidine in NMP. At the end of the last cycle, the N-terminal Fmoc group was removed and the fully protected resin-bound peptide was acetylated using acetic anhydride/DIEA/HOBt/NMP. 
     Cleavage, Side-Chain Deprotection and Isolation of Crude Peptides 
     Cleavage of the peptides from the resin and side-chain deprotection was accomplished using Reagent B for 4.5 h at ambient temperature. The cleavage solutions were collected and the resins were washed with an additional aliquot of Reagant B. The combined solutions were concentrated to dryness. Diethyl ether was added to the residue with swirling or stirring to precipitate the peptides. The liquid phase was decanted, and solid was collected. This procedure was repeated 2-3 times to remove impurities and residual cleavage cocktail components. 
     Cyclization of Di-Cysteine Peptides 
     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 stirred 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. 
     Preparation of Peptides Containing Linkers 
     In a typical experiment, 400 mg of the resin-bound peptide bearing an ivDde-protected lysine) was treated with 10% hydrazine in DMF (2×20 mL). The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). 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) and 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 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 stirred 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 stirred for 16 h to effect the disulfide cyclization and the reaction was monitored by analytical HPLC. After completion of the cyclization, 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 product were combined and lyophilized to provide the required peptide. 
     Preparation of Biotinylated Peptides Containing Linkers 
     In a typical experiment, 400 mg of the resin-bound peptide bearing an ivDde-protected lysine, was treated with 10% hydrazine in DMF (2×20 mL). The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). 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) and 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 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 stirred 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 stirred for 4-6 h to effect the disulfide cyclization, which was monitored by HPLC. Upon completion of the cyclization, 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 product were collected and lyophilized to provide the required biotinylated peptide. 
     Preparation of DOTA-Conjugated Peptides for Labeling with Selected Gadolinium or Indium Isotopes 
     In a typical experiment, 400 mg of the resin-bound peptide bearing an N ε -ivDde-protected lysine moiety was treated with 10% hydrazine in DMF (2×20 mL). The resin was washed with DMF (2×20 mL) and DCM (1×20 mL). 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,-1,4,7-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. Upon completion of the reaction, the resin was washed with DMF (2×10 mL) and with DCM (1×10 mL) and treated with Reagent B (20 mL) for 4 h. The mixture was filtered and the filtrate concentrated to dryness. The residue was stirred 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 stirred 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. 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 product were combined and lyophilized to provide the required biotinylated peptide. 
     The following monomeric peptides of Table 11 were prepared by the above methods, “PnAO6”, as used herein, refers to 3-(2-amino-3-(2-hydroxyimino-1,1-dimethyl-propylamino)-propylamino)-3-methyl-butan-2-one oxime. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Sequence or Structure of Monomeric Peptides and Peptide Derivatives 
               
            
           
           
               
               
            
               
                 Structure or Sequence 
                 SEQ. ID NO: or dimer 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK(BiotinJJ- K )-NH 2   
                 277 
               
               
                 (Ac-AGPTWCEDDWYYCWLFGTGGGK K (BiotinJJ-)-NH 2)   
                 373 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTJK(DOTAJJ-K)-NH 2   
                 493 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTJK(JJ)-NH 2   
                 493 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(ivDde)]-NH 2   
                 373 
               
               
                   
               
               
                 Ac-VCWEDSWGGEVCFRYDPGGGK(Biotin-JJ K )-NH 2   
                 337 
               
               
                   
               
               
                 (Ac-VCWEDSWGGEVCFRYDPGGGK K (Biotin-JJ)-NH 2 ) 
                 494 
               
               
                   
               
               
                 Ac-VCWEDSWGGEVCFRYDPGGGK(JJ)-NH 2   
                 337 
               
               
                   
               
               
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK(J)-NH 2   
                 356 
               
               
                 Seq 12 derivative 
                   
               
               
                   
               
               
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGKK(ivDde) 
                 495 
               
               
                 Application seq 12 derivative 
                   
               
               
                   
               
               
                 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH 2   
                 294 
               
               
                 Seq 5 derivative 
                   
               
               
                   
               
               
                 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH 2  Seq 5 deriv 
                 294/D10 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[(PnAO6- 
                 277/D10 
               
               
                 C(═O)(CH 2 ) 3 C(═O)—K]—NH 2  A Seq 11 derivative 
                   
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[(DOTA-JJK(iV-Dde)]- 
                 277/D11 
               
               
                 NH 2  A Seq 11 derivative 
                   
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[(PnAO6- 
                 476/D12 
               
               
                 C(═O)(CH 2 ) 3 C(═O))K]—NH 2  A Seq 11 derivative 
                   
               
               
                   
               
               
                 Ac-VCWEDSWGGEVCFRYDPGGGK-NH 2  A Seq 5 derivative 
                 337/D12 
               
               
                 specifically: Seq 5 residues 5-25 
                   
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(BOA)]-NH 2  Seq 11 
                 277/D13 
               
               
                 derivative 
                   
               
               
                   
               
               
                 Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK[PnAO6- 
                 356/D14 
               
               
                 C(═O)(CH 2 ) 3 C(═O)—K(iV-Dde)]-NH 2  Application seq 12 
                   
               
               
                 derivative 
                   
               
               
                   
               
               
                 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH 2  Seq 5 deriv 
                 294/D15 
               
               
                 linker = Glut 
                   
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK-[PnAO6- 
                 277/D16 
               
               
                 C(═O)(CH 2 ) 3 C(═O)—K]—NH 2  A Seq 11 derivative, new sequence 
                   
               
               
                   
               
               
                 Ac-AQDWYYEILJGRGGRGGRGGK[K(ivDde)]-NH 2   
                 496/D17 
               
               
                 A Seq 12 (1-9) derivative 
                   
               
               
                   
               
               
                 Ac-APGTWCDYDWEYCWLGTFGGGK[(6PnAO- 
                 497/D18 
               
               
                 C(═O)(CH 2 ) 3 C(═O)—K]—NH 2  A scrambled Seq 11 derivative used 
                   
               
               
                 as a control. 
                   
               
               
                   
               
               
                 Ac-GVDFRCEWSDWGEVGCRSPDYGGGK(JJ)-NH 2  A 
                 489/D18 
               
               
                 scrambled Seq 5 derivative. 
                   
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK(Biotin-K)—NH 2 , A Seq 
                 294/D19 
               
               
                 11 derivative 
                   
               
               
                   
               
               
                 JJAGPTWCEDDWYYCWLFGTGGGK(iV-Dde)-NH 2  (SEQ ID 
                 277/D20 
               
               
                 NO: 277) 
                   
               
               
                   
               
               
                 JJVCWEDSWGGEVCFRYDPGGG-NH 2   
                 370/D20 
               
               
                   
               
               
                 JJAGPTWCEDDWYYCWLFGTGGGK(iV-Dde)-NH 2   
                 277/D21 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[K(SATA)]-NH 2   
                 373/D22 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK[SATA-JJ-K]-NH 2   
                 339/D23 
               
               
                   
               
               
                 Ac-GDSRVCWEDSWGGEVCFRYDPGGGK(JJ)-NH 2   
                 294/D24 
               
               
                   
               
               
                 H 2 N-AGPTWCEDDWYYCWLFGTGGGK[K(iV-Dde)]-NH 2   
                 373/D25 
               
               
                   
               
               
                 Ac-AGPTWCEDDWYYCWLFGTGGGK{Biotin-JJK[NH 2 - 
                 339/D26 
               
               
                 Ser(GalNAc(Ac) 3 -alpha-D)-Gly-Ser(GalNAc(Ac) 3 -alpha-D]}-NH 2   
                   
               
               
                   
               
               
                 Ac-VCWEDSWGGEVCFRYDPGGGK(NH 2 -Ser(GalNAc(Ac) 3 - 
                 337/D26 
               
               
                 alpha-D)-Gly-Ser(GalNAc(Ac) 3 -alpha-D)-NH 2   
                   
               
               
                   
               
               
                 Ac-GSPEMCMMFPFLYPCNHHAPGGGK[(PnAO6)- 
                 482/D27 
               
               
                 C(═O)(CH 2 ) 3 C(═O)—K]}-NH 2  A modified cMet Binding Sequence 
               
               
                   
               
            
           
         
       
     
     Example 12 
     Preparation of Homodimeric and Heterodimeric Constructs 
     The purified peptide monomers mentioned above in Example 8 were used in the preparation of various homodimeric and heterodimeric constructs. 
     Preparation of Homodimer-Containing Constructs 
     To prepare homodimeric compounds, half of the peptide needed to prepare the dimer was dissolved in DMF and treated with 10 equivalents of glutaric acid bis-N-hydroxysuccinimidyl 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 the 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. The reaction was allowed to proceed for 24 h. This mixture was applied directly to a YMC reverse phase HPLC column and purified by elution with a linear gradient of acetonitrile into water (both containing 0.1% TFA). 
     Preparation of Heterodimer-Containing Constructs 
     In the case of heterodimers, one of the monomers (“A”) was reacted with the bis-NHS ester of glutaric acid and after washing off the excess of bis-NHS ester (as described for the homodimeric compounds), the second monomer (“B”) was added in the presence of DIEA. After the reaction the mixture was purified by preparative HPLC. Typically, to a solution of glutaric acid bis N-hydroxysuccinimidyl ester (0.02 mmol, 10 equivalents) in DMF (0.3 mL) was added a solution of peptide “A” and DIEA (2 equiv) in DMF (0.5 mL) and the mixture was stirred for 2 h. 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 (3×1.0 mL) to remove the unreacted bis-NHS ester. The residue was dried, re-dissolved in anhydrous DMF (0.5 mL) and treated with a solution of peptide “B” and DIEA (2 equiv) in DMF (0.5 mL) for 24 h. The mixture was diluted with water (1:1,v/v) and applied directly to a YMC C-18 reverse phase HPLC column and purified by elution with a linear gradient of acetonitrile into water (both containing 0.1% TFA). Fractions were analyzed by analytical HPLC and those containing the pure product were combined and lyophilized to obtain the required dimer. The dimers depicted in  FIGS. 36-63  were prepared by this method (structure, name, compound reference number as described in the “Brief Description of the Drawings”). 
     For the preparation of the dimer D5, after the coupling reaction of the individual peptides, 50 μL of hydrazine was added to the reaction mixture (to expose the lysine N ε -amino group) and the solution was stirred 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. 
     The HPLC analysis data and mass spectral data for the dimeric peptides are given in Table 12 below. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Analytical Data for Homodimeric and Heterodimeric Peptide Constructs HPLC Analysis System 
               
            
           
           
               
               
               
            
               
                   
                 Retention 
                   
               
               
                   
                 Time 
               
               
                   
                 (System) 
                 Mass Spectral data (API-ES, - ion) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                 D1 
                 8.98 
                 min. (A) 
                 1987.7 (M − 3H)/3, 1490.6 (M − 4H)/4, 1192.3 (M − 5H)/5 
               
               
                 D2 
                 16.17 
                 min (B) 
                 2035.3 (M − 3H)/3, 1526.1 (M − 4H)/4, 1220.7 (M − 5H)/5 
               
               
                 D3 
                 8.74 
                 min (C) 
                 1933.6 (M − 3H)/3, 1449.9 (M − 4H)/4, 1159.4 (M − 5H)/5 
               
               
                 D4 
                 10.96 
                 min (D) 
                 2032.8 (M − 3H)/3 
               
               
                 D5 
                 6.57 
                 min (E) 
                 1816.2 (M − 3H)/3, 1361.8 (M − 4H)/4, 1089.4 (M − 5H)/5, 907.7 (M − 6H)/6 
               
               
                 D6 
               
               
                 D7 
               
               
                 D8 
                 4.96 
                 min; (F) 
                 2379.3 [M − 3H]/3 
               
               
                 D9 
                 5.49 
                 min; (G) 
                 2146.4 [M − 3H]/3 
               
               
                 D10 
                 5.44 
                 min; (H) 
                 2082.7 [M − 3H]/3, 1561.7 [M − 4H]/4, 1249.1 [M − 5H]/5, 1040.7 [M − 6H]/6 
               
               
                 D11 
                 7.23 
                 min; (E) 
                 2041.8 [M − 3H]/3, 1531.1 [M − 4H]/4, 1224.6 [M − 5H]/5 
               
               
                 D12 
                 5.84 
                 min; (H) 
                 1877.1 [M − 3H]/3, 1407.6 [M − 4H]/4, 1125.9 [M − 5H]/5, 938.1 [M − 6H]/6. 
               
               
                 D13 
                 5.367 
                 min; (E) 
                 1965.3 [M − 3H]/3, 1473.8 [M − 4H]/4, 1178.8 [M − 5H]/5, 982.2 [M − 6H]/6 
               
               
                 D14 
                 4.78 
                 min; (I) 
                 2275.0 [M − 3H]/3, 1362.8 [M − 5H]/5 
               
               
                 D15 
                 5.41 
                 min; (H) 
                 1561.3 [M − 4H]/4, 1249.1 [M − 5H]/5, 1040.8 [M − 6H]/6, 891.8 [M − 7H]/7. 
               
               
                 D16 
                 5.44 
                 min; (J) 
                 2150.8 [M − 3H]/3, 1613.1 [M − 4H]/4, 1289.9 [M − 5H]/5, 1074.8 [M − 6H]/6, 
               
               
                   
                   
                   
                 920.9 [M − 7H]/7. 
               
               
                 D17 
                 4.78 
                 min; (K) 
                 1789.4 [M − 3H]/3, 1347.7 [M − 4H]/4. 
               
               
                 D18 
                 4.74 
                 min; (L) 
                 2083.1 [M − 3H]/3, 1562.7 [M − 4H]/4, 1249.5 [M − 5H]/5. 
               
               
                 D19 
                 7.13 
                 min; (O) 
                 1891.9 [M − 3H]/3, 1418.4 [M − 4H]/4, 1134.8 [M − 5H]/5, 945.5 [M − 6H]/6. 
               
               
                 D20 
                 9.7 
                 min; (P) 
                 2700.4 [M − 2H]/2, 1799.3[M − 3H]/3 
               
               
                 D21 
                 6.1 
                 min; (P) 
                 2891.3 [M − 2H]/2, 1927.2[M − 3H]/3, 1445.1 [M − 4H]/4, 1155.8 [M − 5H]/5. 
               
               
                 D22 
                 6.23 
                 min; (Q) 
                 1994.4 [M − 3H]/3, 1495.7 [M − 4H]/4, 1196.3 [M − 5H]/5 
               
               
                 D23 
                 7.58 
                 min; (J) 
                 1854.4 [M − 3H]/3, 1390.8 [M − 4H]/4, 1112.7 [M − 5H]/5, 927 [M − 6H]/6 
               
               
                 D24 
                 8.913 
                 min; (R) 
                 1952.1 [M − 3H]/3, 1463.4 [M − 4H]/4, 1171.1 [M − 5H]/5, 975.3 [M − 6H]/6 
               
               
                 D25 
                 5.95 
                 min; (E) 
                 1954.9 [M − 3H]/3, 1466.1 [M − 4H]/4, 1172.4 [M − 5H]/5, 976.8 [M − 6H]/6. 
               
               
                 D26 
                 6.957 
                 min; (S) 
                 1759.1 [M − 3H]/3, 1319.6 [M − 4H]/4, 1055.1 [M − 5H]/5 
               
               
                 D27 
                 5.5 
                 min; (M) 
                 2317.6 [M − 3H]/3, 1737.2[M − 4H]/4, 1389.3[M − 5H]/5, 1157.7 [M − 6H]/6 
               
               
                 D30 
                 4.29 
                 min (T) 
                 [M + H]: 5782.3, [M + 4H]/4: 1146.6, [M + 5H]/5: 1157.4, [M + 6H]/6: 964.7 
               
               
                 D31 
                 6.6 
                 min (U) 
                 [M − 3H]/3: 2045.3. 
               
               
                 Monomer 
                 6.0 
                 min (U) 
                 [M − 2H]/2: 1307.4 
               
               
                 Compound 2 
               
               
                 Monomer 
                 5.3 
                 min (U) 
                 [M − 2H]/2: 1307.4 
               
               
                 Compound 4 
               
               
                 (SEQ ID NO: 
               
               
                 374-related 
               
               
                 sequence) 
               
               
                   
               
            
           
         
       
     
                     TABLE 13                  Dimer sequences and linkers                     Dimer #   Sequence               D1   Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277)[(Biotin-JJK-       (FIG. 36)   (O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-           VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337))-NH)CONH 2 ]—NH 2 )               D2   Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277) [(Biotin-JJK-       (FIG. 37)   (O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-           AGPTWCEDDWYYCWLFGTJK(SEQ ID NO: 493))-NH)CONH 2 ]—NH 2                 D3   Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)[(Biotin-JJK-       (FIG. 38)   (O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-           VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337))-NH)CONH 2 ]—NH 2                 D4   Ac-AGPTWCEDDWYYCWLFGTJK(SEQ ID NO: 338)[DOTA-JJK-       (FIG. 39)   (O═)C(CH 2 ) 3 C(═O)-JJ-NH(CH 2 ) 4 —(S)—CH((Ac-           VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337))-NH)CONH 2 ]—NH 2                 D5   Ac-VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)       (FIG. 40)   (JJ-C(═O)(CH 2 ) 3 C(═O)—K—NH(CH 2 ) 4 —(S)—CH((Ac-           AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277))-           NH)CONH 2 )—NH 2                 D6   GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 294)-       (FIG. 63)   AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO: 277) (see FIG. 63 for linkage)               D7   GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 294)-       (FIG. 64)   AGPKWCEEDWYYCMITGTGGGK (SEQ ID NO: 264) (see FIG. 64 for linkage)               D8   Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK{Ac-       (FIG. 41)   AQDWYYDEILSMADQLRHAFLSGGGGGK(J-Glut-)-NH 2 }K(Biotin-JJ)-NH 2                 D9   Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK{[Ac-       (FIG. 42)   GDSRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut-)]-NH 2 }K—NH 2                 D10   Ac-AGPTWCEDDWYYCWLFGTGGGK{[Ac-       (FIG. 43)   GDSRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut-NH(CH 2 ) 4 —(S)—CH(PnAO6-Glut-           NH)(C═O-)]-NH 2 }—NH 2                 D11   Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-       (FIG. 44)   VCWEDSWEDSWGGEVCFRYDPGGGK[JJ-Glut-NH(CH 2 ) 4 —(S)—CH(DOTA-JJ-NH—)(C═O)—]—NH 2 }—NH 2                 D12   Ac-AGPTWCEDDYCWLFGTGGGK{[PnAO6-Glut-K(Ac-       (FIG. 45)   VCWEDSWGGEVCFRYDPGGGK(-C(═O)CH 2 (OCH 2 CH 2 ) 2 OCH 2 C(═O)—)—NH 2 ]}—NH 2                 D13   Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[JJ-       (FIG. 46)   Glut-K(BOA)]-NH 2 }—NH 2 : Dimer 13 (D13)               D14   Ac-AQDWYYDEILSMADQLRHAFLSGGGGGK{PnAO6-Glut-K[Ac-       (FIG. 47)   GSDRVCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH 2 ]}—NH 2                 D15   Ac-AGPTWCEDDWYYCWLFGTGGGK{[[Ac-       (FIG. 48)   GDSRVCWEDSWGGEVCFRYDPGGGKJJ-Glut]-NH 2 ]—K(PnAO6-Glut)}-NH 2                 D16   Ac-AGPTWCEDDWYYCWLFGTGGGGK{PnAO6-Glut-K[Ac-       (FIG. 49)   GDSRVCWEDSWGGEVCFRYDPGGGK[-           C(═O)CH 2 O(CH 2 CH 2 O) 2 CH 2 C(═O)NH(CH 2 ) 3 O(CH 2 CH 2 O) 2 (CH 2 ) 3 NHC(═O)CH 2 O(CH 2 CH 2 O) 2 CH 2 C(═O)—]—NH 2 ]}—NH 2                 D17   Ac-AQDWYYDEILJGRGGRGGRGGK{K[Ac-       (FIG. 50)   VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH 2 ]}—NH 2                 D18   Ac-AGPTWCDYDWEYCWLGTFGGGK{PnAO6-Glut-K[Ac-       (FIG. 51)   GVDFRCEWSDWGEVGCRSPDYGGGK(JJ-Glut)-NH 2 ]}—NH 2                 D19   Ac-AGPTWCEDDWYYCWLFGTGGGK{Biotin-K[Ac-       (FIG. 52)   VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH 2 ]}—NH 2                 D20   (-JJAGPTWCEDDWYYCWLFGTGGGGK-NH 2 )-Glut-       (FIG. 53)   VCWEDSWGGEVCFRYDPGGG-NH 2                 D21   [-JJAGPTWCEDDWYYCWLFGTGGGGK(PnAO6-Glut)-NH 2 ]-Glut-       (FIG. 54)   VCWEDSWGGEVCFRYDPGGG-NH 2                 D22   Ac-GDSRVCWEDSWGGEVCFRYDPGGGK{JJ-Glut-JJ-       (FIG. 55)   AGPTWCEDDWYYCWLFTGGGK-NH 2 }—NH 2                 D23   Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-VCWEDSWGGEVCFRYDPGGGK[JJ-       (FIG. 56)   Glut-K(SATA)]-NH 2 }—NH 2                 D24   Ac-AGPTWCEDDWYYCWLFGTGGGK{SATA-JJK[Ac-       (FIG. 57)   VCWEDSWGGEVCFRYDPGGGK(JJ-Glut)-NH 2 ]}—NH 2                 D25   Ac-AGPTWCEDDWYYCWLFGTGGGK{Ac-       (FIG. 58)   GDSRVCWEDSWGGEVCFRYDPGGGK[JJ-Glut-NH(CH 2 )4—(S)—CH(NH 2 )C(═O)—]—NH 2 }—NH 2                 D26   AGPTWCEDDWYYCWLFGTGGGGK{(-Glut-JJ-VCWEDSWGGEVCFRYDPGGG-       (FIG. 59)   NH 2 )—K}—NH 2                 D27   Ac-AGPTWCEDDWYYCWLFGTGGGGK{Ac-       (FIG. 60)   VCWEDSWGGEVCFRYDPGGGK[S(GalNAc(Ac) 3 -alpha-D)-G-S(GalNAc(Ac) 3 -           alpha-D)-Glut-S(GalNAc(Ac) 3 -alpha-D)-G-S(GalNAc(Ac) 3 -alpha-D)-NH(CH 2 ) 4 —(S)—CH(Biotin-           JJNH-)C(═O)—]—NH 2 }—NH 2                 D28   AQEPEGYAYWEVITLYHEEDGDGGK (SEQ ID NO: 305)-       (FIG. 61)   AQAFPRFGGDDYWIQQYLRYTDGGK (SEQ ID NO: 306) (see FIG. 61 for linkage)               D29   AGPTWCEDDWYYCWLFGTGGGK (SEQ ID NO: 277)-       (FIG. 62)   VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 337) (see FIG. 62 for linkage)               D30   Ac-VCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 337){[PnAO 6 -Glut-K(-Glut-JJ-       (FIG.   NH(CH 2 ) 4 —(S)—CH(Ac-AQDWYYDEILJGRGGRGGRGG(SEQ ID NO: 478)-       87C)   NH)C(═O)NH 2 ]—NH2}—NH 2  (see FIG. 87C)               D31   Ac-AGPTWCEDDWYYCWLFGTGGGK(SEQ ID NO: 277)[Ac-       (FIG.   VCWEDSWGGEVCFRYDPGGGK(SEQ ID NO: 337)[SGS-Glut-SGS—(S)—NH(CH 2 ) 4 —CH(Biotin-       88D)   JJ-NH)—C(═O)]—NH 2 ]—NH 2  (see FIG. 88D)                    
HPLC Analysis Systems
 
     System A: Column: YMC C-4 (4.6×250 mm); Eluents: A: Water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 25% B, linear gradient 25-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm. 
     System B: Column: YMC C-4 (4.6×250 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: UV @ 220 nm. 
     System C: Column: YMC C-4 (4.6×250 mm); Eluents: A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 30% B, linear gradient 30-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm. 
     System D: Column: YMC C-4 (4.6×250 mm); Eluents: A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient 20-60% B in 10 min; flow rate: 2.0 mL/min; Detection: UV @ 220 nm. 
     System E: Column: Waters XTerra, 4.6×50 mm; Eluents:A:water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, linear gradient 10-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System F: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: Acetonitrile (0.1% TFA); Elution: Initial condition, 30% B, Linear Gradient 30-70% B in 10 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm. 
     System G: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (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 XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient 20-52% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System I: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, linear gradient 10-65% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System J: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient 20-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System K: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 5% B, linear gradient 5-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System L: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); 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, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); 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 XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, linear gradient 20-80% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System O: Column: YMC-C18, 4.6×250 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 30% B, linear gradient 30-60% B in 10 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm. 
     System P: Column: YMC-C18, 4.6×250 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient 20-80% B in 20 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm. 
     System Q: Column: YMC-C18, 4.6×250 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 20% B, linear gradient 20-60% B in 6 min; flow rate: 2.0 mL/min; detection: UV @ 220 nm. 
     System R: Column: YMC-C18, 4.6×250 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); 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, 4.6×100 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 10% B, linear gradient 10-60% B in 10 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System T: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 5% B, linear gradient 5-65% B in 8 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     System U: Column: Waters XTerra, 4.6×50 mm; Eluents:A: water (0.1% TFA), B: ACN (0.1% TFA); Elution: initial condition, 15% B, linear gradient 15-50% B in 8 min; flow rate: 3.0 mL/min; detection: UV @ 220 nm. 
     Example 13 
     Competition with  125 I-VEGF for Binding to KDR on HUVECs and KDR-Transfected Cells 
     The following experiment assessed the ability of KDR-binding peptides to compete with  125 I-labeled VEGF for binding to KDR expressed by transfected 293H cells. 
     Protocol 
     293H cells were transfected with the KDR cDNA or mock-transfected by standard techniques. The cells were incubated with  125 I-VEGF in the presence or absence of competing compounds (at 10 μM, 0.3 μM, and 0.03 μM). After washing the cells, the bound radioactivity was quantitated on a gamma counter. The percentage inhibition of VEGF binding was calculated using the formula [(Y1−Y2)×100/Y1], where Y1 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. 
     Results 
     As shown in  FIG. 15 , all of the KDR-binding peptides assayed were able to compete with  125 I-VEGF for binding to KDR-transfected cells. The heterodimer (D1) was clearly the most effective at competing with  125 I-VEGF, even over the two homodimers (D2 and D3), confirming the superior binding of D1. 
     Example 14 
     Receptor Activation Assay 
     The ability of KDR-binding peptides to inhibit VEGF induced activation (phosphorylation) of KDR was assessed using the following assay. 
     Protocol 
     Dishes of nearly confluent HUVECs were placed in basal medium lacking serum or growth factors overnight. The dishes in group (c), below were then pretreated for 15 min in basal medium with a KDR-binding peptide, and then the cells in the dishes in groups (a), (b), and (c) were placed in fresh basal medium containing: 
     (a) no additives (negative control), 
     (b) 5 ng/mL VEGF (positive control), or 
     (c) 5 ng/mL VEGF plus the putative competing/inhibiting peptide. 
     After 5 min of treatment, lysates were prepared from each set of dishes. KDR was immunoprecipitated from the lysates was analyzed sequentially by immunoblotting for phosphorylation with an anti-phosphotyrosine antibody, and for total KDR with an anti-KDR antibody (to control for sample loading).
 
Results
 
     As shown in  FIG. 16 , D1 was able to completely block the VEGF-induced phosphorylation of KDR in HUVECs at 10 nM. More than half of the phosphorylation was inhibited by the compound at 1 nM. Homodimers D2 and D3, made up of the two individual binding moieties that are contained in D1, had no effect on phosphorylation at up to 100 nM, demonstrating the benefit of heterodimer constructs in blocking a receptor-ligand interaction. In multiple experiments, the IC 50  for D1 in this assay varied between 0.5 and 1 nM. A different heterodimer containing unrelated binding sequences, D28, a tail-to-tail heterodimer comprising the polypeptides of SEQ ID NO:305 and SEQ ID NO:306 ( FIG. 61 ), had no effect on phosphorylation at 100 nM in spite of it&#39;s high binding affinity (11 nM for KDR by SPR), suggesting that the choice of KDR-binding moieties is important when constructing a multimer to compete with VEGF for binding to KDR. One of ordinary skill in the art would be able to construct suitable heteromultimers using the binding polypeptides provided herein and routine screening assays. 
     Even though the affinity of D1 for KDR is 10-fold higher than that of D2 (by SPR analysis), the IC 50  of D1 in the activation assay is at least 100-fold lower. This suggests that targeting two distinct epitopes on KDR with a single binding molecule can generate greater steric hindrance than a molecule with similar affinity that only binds to a single epitope on KDR and, therefore, improve the ability to inhibit VEGF induced KDR activity. Similarly, it should be pointed out that the two KDR-binding moieties within D1, when tested as monomeric free peptides (SEQ ID NO:277 and SEQ ID NO:337 in the receptor activation assay, had IC 50 s of 0.1 and 1 micromolar, respectively. The IC 50  for the monomeric free peptides were 100 to 1000-fold higher than the IC 50  for D1 in the assay and 14 to 30-fold higher than the K D s for the fluoresceinated derivatives of the monomeric peptides. Thus, creating a dimer containing two peptides with weak VEGF-blocking activity has resulted in a molecule with very potent VEGF-blocking activity that goes well beyond the increased binding affinity of D1. 
     Example 15 
     Migration Assay 
     The following experiment assessed the ability of D1 to block the VEGF-induced migration of HUVECs in culture. 
     Protocol 
     Serum-starved HUVECs were placed, 100,000 cells per well, into the upper chambers of BD Matrigel-coated FluoroBlok 24-well insert plates (#354141). Basal medium, containing either nothing or different attractants such as VEGF (10 ng/mL) or serum (5% FBS) in the presence or absence of potential VEGF-blocking/inhibiting compounds, was added to the lower chamber of the wells. After 22 hours, quantitation of cell migration/invasion was achieved by post-labeling cells in the insert plates with a fluorescent dye and measuring the fluorescence of the invading/migrating cells in a fluorescent plate reader. The VEGF-induced migration was calculated by subtracting the migration that occurred when only basal medium was placed in the lower chamber of the wells. 
     Results 
     VEGF induced a large increase in endothelial cell migration in the assay, which was potently blocked by D1. At 5 nM D1, the VEGF-stimulated endothelial cell migration was 84% blocked (see  FIG. 17 ). At 25 nM D1, this migration was almost completely blocked. In other experiments, a known KDR inhibitor, SU-1498 (Strawn, L. et al., 1996 , Cancer Res.,  56:3540-3545) was tested in the assay. SU-1498 at 3 micromolar did not block the VEGF-induced migration as well as D1 (47% blocked at 3 micromolar). D6 (structure shown below in Example 18), at 50 nM, also produced essentially complete inhibition of the migration stimulated by VEGF. Serum was a very powerful attractant in the assay when used in place of VEGF, but its effect was not significantly diminished by D1, indicating that D1 specifically inhibits endothelial migration induced by VEGF. 
     Example 16 
     Preparation of Labeled Compounds 
     The following experiments describe methods used to prepare Tc, In, and I-labeled compounds. 
     Preparation of  99m Tc-378 (Ac-AGPTWC*EDDWYYC*WLFGTGGGK(PnAO6-NH—(O═)C(CH 2 ) 3 C(═O)-JJ)-NH 2 ; SEQ ID NO:378). 
     SnCl 2 .2H 2 O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of this solution was added to 1 mL of a DTPA solution that was prepared by dissolving 10 mg of Ca Na 2 DTPA 2.5 H 2 O (Fluka) in 1 mL of water. The pH of the stannous DTPA solution was adjusted to pH 6-8 using 1N NaOH. SEQ ID NO:378 (50 μg in 50 μL of 10% DMF) was mixed with 20 μL of  99m TcO 4   −  (2.4 to 4 mCi, Syncor), followed by 100 μL of the stannous Sn-DTPA solution. After 30 minutes at RT, the radiochemical purity (RCP) was 93%. The product was purified on a Supelco Discovery C16 amide column (4×250 mm, 5 um pore size) eluted at a flow rate of 0.5 mL/min using an aqueous/organic gradient of 1 g/L ammonium acetate in water (A) and acetonitrile (B). The following gradient was used: 30.5% B to 35% B in 30 minutes, ramp up to 70% B in 10 min. The compound, which eluted at a retention time of 21.2 minutes was collected into 500 μL of 50 mM citrate buffer (pH 5.2) containing 1% ascorbic acid and 0.1% HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After purification, the compound had an RCP of &gt;98%. 
     Preparation of  111 In-Ac-AGPTWCEDDWYYCWLFGTJK(JJ-DOTA)-NH 2  (SEQ ID NO:338). 
     SEQ ID NO:338 (50 μg in 50 μL of 10% DMF) was mixed with  111 InCl 3  (50 μL, 400 μCi, Mallinckrodt) and 100 μL of 0.2M ammonium acetate or citrate buffer at a pH of 5.3. After being heated at 85° C. for 45 minutes, the radiochemical purity (RCP) ranged from 44% to 52.2% as determined using HPLC. The  111 In-labeled compound was separated from unlabeled ligand using a Vydac C18 column (4.6×25 cm, 5 micron pore size) under following conditions: aqueous phase, 1 g/L ammonium acetate (pH 6.8); organic phase, acetonitrile. Gradient: 23% org. to 25% org. in 30 minutes, up to 30% org. in 2 minutes, hold for 10 minutes. The compound, which eluted at a retention time of 20.8 min, was collected into 200 μL of 50 mM citrate buffer (pH 5.2) containing 1% ascorbic acid and 0.1% HSA, and the acetonitrile was removed using a Speed Vacuum (Savant). After purification the compound had an RCP of &gt;93%. 
     Preparation of  111 In-D4 
     A histidine buffer was prepared by adjusting a 0.1M solution of histidine (Sigma) to pH 6.25 with concentrated ammonium hydroxide. Ammonium acetate buffer was prepared by adjusting a 0.2 M solution of ammonium acetate (99.99%, Aldrich) to pH 5.5 using concentrated HCl (J. T. Baker, Ultra Pure). High purity  111 InCl 3  (100 μL, 1.2 mCi, Malinckrodt, Hazelwood, Mo.) was added to D4 (200 μg in 200 of 50% DMF, 10% DMSO, 20% acetonitrile and 20% water), followed by addition of 300 μL of histidine buffer. The final pH was 5.5. After incubation of the reaction mixture at 85° C. for 45 minutes, the RCP was 20%. 
     Alternatively,  111 InCl 3  provided with a commercially available OctreoScan™ Kit (134 μL, 0.6 mCi, Mallinkrodt) was added to D4 (135 μg) in 162 μL of 0.2M ammonium acetate buffer. The final pH was 5.5. After incubation of the reaction mixture at 85° C. for 45 min. the RCP was 20%. 
     Preparation of  125 I-D5 
     D5 (200 μg), in 30 μL of DMF that had been previously adjusted to pH 8.5-9.0 using diisopropyl amine, was added to 1 mCi of mono-iodinated  125 I Bolton-Hunter Reagent (NEX-120, Perkin-Elmer) that had been evaporated to dryness. The vial was shaken and then incubated on ice for 30 minutes with occasional shaking. After this time, the RCP was 23%.  125 I-D5 was purified by HPLC at a flow rate of 1 mL/min using a Vydac C18 column (4.6×250 mm, 5 micron pore size) under the following conditions. Aqueous phase: 0.1% TFA in water; organic phase: 0.085% TFA in acetonitrile. Gradient: 30% org. to 36% org. in 30 minutes, up to 60% org. in 5 minutes, hold for 5 minutes. The compound was collected into 200 μL of 50 mM citrate buffer (pH 5.2) containing 1% ascorbic acid and 0.1% HSA. Acetonitrile was removed using Speed Vacuum (Savant). The resulting compound had an RCP of 97% (see  FIG. 65 ). 
     Preparation of  177 Lu-D11 
     D11 (5 μL of a ˜1 μg/μL solution in 0.05N NH 4 OH/10% EtOH) was added to a glass insert microvial containing 80 μL of 0.2M NaOAc buffer, pH 5.6. Enough  177 Lu was added to bring the ligand:Lu ratio to ≦2:1 (1-5 mCi). The vial was crimp-sealed and heated at 100° C. for 15-20 minutes, cooled for 5 minutes, and treated with 3 μL of 1% Na 2 EDTA.2H 2 O in H 2 O. The entire reaction mixture was injected onto a Supelco Discovery RP Amide C16 column (4 mm×250 mm×5 μm). The following HPLC conditions were used: Column temperature=50° C., Solvent A=H 2 O w/0.1% TFA, Solvent B=ACN w/0.085% TFA, gradient 0.6/0.25 mL/min A/B at t=0 minutes to 0.5/0.4 mL/min A/B at t=60 minutes. The retention time for D11 was ˜40 minutes; that of  177 Lu-D11 was ˜42 minutes. The radioactive peak was collected into 0.7 mL of 0.05M citrate buffer, pH 5.3 containing 0.1% Human Serum Albumin Fraction V and 1.0% Ascorbic Acid, and the mixture was spun down in a Savant Speed Vac to remove organic solvents. Radiochemical purities of greater than 80% were obtained. 
     Preparation of  177 Lu-D13 
     D13 (306 μg) was added to a 2-mL autosampler vial with a ˜450 μL conical insert and dissolved in 0.01N NH 4 OH (50 μL). To this was added 300 μL of 0.5M Ammonium Acetate containing Sodium Ascorbate, Sodium Gentisate, L-Methionine and L-Tryptophan each at 10 mg/mL, plus Human Serum Albumin Fraction V at 2 mg/mL, final pH=7.6 adjusted with NaOH. A 6.8 μL aliquot of  177 LuCl 3  in 0.05N HCl (39.3 mCi) was added, the vial was crimp-sealed, warmed for 15 min at 37 C, cooled for ˜5 minutes, and 10 μL of 1% Na 2 EDTA 2H 2 O in H 2 O was added. A 350 μL aliquot of the reaction mixture was injected onto a Supelco Discovery RP Amide C16 column (4 mm×250 mm×5 μm). The following HPLC conditions were used: column temperature=37 C, Solvent A=H 2 O containing 2 g/L NH 4 OAc buffer, pH 7.0, Solvent B=80% ACN/20% H 2 O, gradient 0.56/0.24 mL/min A/B at t=0 minutes to 0.47/0.33 mL/min A/B at t=30 minutes. The retention time for D13 was ˜28 minutes; the retention time for  177 Lu-BRU 1339 was ˜29 minutes. The radioactive peak was collected into 1 mL of a buffer containing Sodium Ascorbate, Sodium Gentisate, L-Methionine and L-Tryptophan each at 10 mg/mL, plus Human Serum Albumin Fraction V at 2 mg/mL, final pH=7.6 adjusted with NaOH). It was then spun down ˜40 minutes using a Speed Vacuum (Savant) to remove ACN. The RCP of the isolated product was 86%. 
     Preparation of  99m Tc-D10 
     SnCl 2  2H 2 O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of this solution was added to 1 mL of a DTPA solution that was prepared by dissolving 10 mg of Ca Na 2 DTPA 2.5 H 2 O (Fluka) in 1 mL of water. D10 (100 μg in 100 μL of 50% DMF) was mixed with 75 μL of 0.1 M, pH 9 phosphate buffer and 50 μL of  99m TcO 4   −  (2.4 to 5 mCi, Syncor), followed by 100 μL of the stannous Sn-DTPA solution. After 15 min at RT, the radiochemical purity (RCP) was 72%. The product was purified on a Supelco Discovery C16 amide column (4×250 mm, 5 um pore size) eluted at a flow rate of 0.7 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA in acetonitrile (B; “ACN”). The following gradient was used: 30% B to 42% B in 36 min, ramp up to 70% B in 10 min. The compound, which eluted at a retention time of 32 min., was collected into 500 μL of 50 mM citrate buffer (pH 5.2) containing 0.2% HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After purification, the compound had an RCP of &gt;90%. 
     Preparation of  99m Tc-D12 
     SnCl 2 .2H 2 O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of this solution was added to 1 mL of a DTPA solution that was prepared by dissolving 10 mg of Ca Na 2 DTPA 2.5 H 2 O (Fluka) in 1 mL of water. D12 (100 μg in 100 μL of 50% DMF) was mixed with 75 μL of 0.1 M, pH 9 phosphate buffer and 60 μL of  99m TcO 4   −  (2.4 to 4 mCi, Syncor), followed by 100 μL of the stannous Sn-DTPA solution. After 10 min at 40° C., the radiochemical purity (RCP) was 16%. The product was purified on a Supelco Discovery C16 amide column (4×250 mm, 5 um pore size) eluted at a flow rate of 0.7 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA in acetonitrile (B). The following gradient was used: 30% B to 42% B in 36 min, ramp up to 70% B in 10 min. The compound, which eluted at a retention time of 37.1 min. was collected into 500 μL of 50 mM citrate buffer (pH 5.2) containing 0.2% HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After purification, the compound had an RCP of &gt;90%. 
     Preparation of  99m Tc-D14 
     SnCl 2 .2H 2 O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of this solution was added to 1 mL of a DTPA solution that was prepared by dissolving 10 mg of Ca Na 2 DTPA 2.5 H 2 O (Fluka) in 1 mL of water. D14 (100 μg in 100 μL of 50% DMF) was mixed with 50 μL of  99m TcO 4   −  (6 mCi, Syncor) and 125 μL of 0.1M phosphate buffer, pH 9 followed by 100 μL of the stannous Sn-DTPA solution. After 15 min at 40° C., the radiochemical purity (RCP) was 21%. The product was purified on a Vydac peptide C18 column (4.6×250 mm) eluted at a flow rate of 1 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA in acetonitrile (B). The following gradient was used: 30% B to 45% B in 40 min. The compound, which eluted at a retention time of 34.9 min., was collected into 500 μL of 50 mM citrate buffer (pH 5.3) containing 0.2% HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After purification, the compound had an RCP of 92.5%. 
     Preparation of  99m Tc-D18 
     SnCl 2  2H2O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of this solution was added to 1 mL of a DTPA solution that was prepared by dissolving 10 mg of Ca Na e  DTPA 2.5 H 2 O (Fluka) in 1 mL of water. D18 (100 μg in 100 μL of 50% DMF) was mixed with 50 μL of 0.1 M, pH 9 phosphate buffer and 90 μL of  99m TcO 4   −  (14 mCi, Syncor), followed by 100 μL of the stannous Sn-DTPA solution. The reaction was warmed for 20 minutes at 37 C. The entire reaction was injected on a Vydac 218TP54 C18 column (4.6×250 mm, 5 um silica) and eluted at a flow rate of 1.5 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA in ACN (B). The following gradient was used: 32% to 39% B in 30 minutes, ramp up to 80% B in 2 min. The free ligand eluted at a retention time of 19 minutes. The complex, which eluted at 24 minutes, was collected into 500 μL of 50 mM citrate buffer (pH 5.3) containing 0.1% HSA and 1% Ascorbic Acid. ACN and excess TFA were removed using a Speed Vacuum (Savant) for 40 minutes. After purification, the compound had an RCP of 93%. 
     Preparation of  99m Tc-D30 
     SnCl 2 .2H 2 O (20 mg) was dissolved in 1 mL of 1 N HCl, and 10 μL of this solution was added to 1 mL of a DTPA solution that was prepared by dissolving 10 mg of Ca Na 2 DTPA 2.5 H 2 O (Fluka) in 1 mL of water. D30 (100 μg in 100 μL of DMF) was mixed with 150 μL of 0.1 M pH 8 phosphate buffer and 50 μL of  99m TcO 4   −  (5.2 mCi, Syncor), followed by 100 μL of the stannous Sn-DTPA solution. After 15 min at 100° C., the radiochemical purity (RCP) was 13%. The product was purified on a Vydac C18 peptide column (4.6×250 mm, 5 um pore size) eluted at a flow rate of 1 mL/min using an aqueous/organic gradient of 0.1% TFA in water (A) and 0.085% TFA in acetonitrile (B). The following gradient was used: 10% B to 50% B in 30 min, hold 50% B for 5 min, back to 70% B in 5 min. The compound, which eluted at a retention time of 33.2 min., was collected into 3 mL of 50 mM citrate buffer (pH 5.5) containing 0.2% HSA, and acetonitrile was removed using a Speed Vacuum (Savant). After purification, the compound had an RCP of 92.4%. 
     Example 17 
     Binding to KDR-Transfected Cells 
     An experiment was performed to test the ability of  125 I-labeled D5 to bind to KDR-transfected 293H cells. In this experiment, different amounts of  125 I-labeled D5 (1-4 μCi/mL, labeled with  125 I-Bolton-Hunter reagent and HPLC-purified) were incubated with mock and KDR-transfected 293H cells in 96-well plates for 1 hr at room temperature. Binding was performed with and without 40% mouse serum to evaluate the serum effect on binding to KDR-transfected cells. After washing away the unbound compound, the cells in each well were lysed with 0.5 N NaOH and the lysates were counted with a gamma counter. 
     The results of this experiment are summarized in  FIG. 18  and  FIG. 19 .  125 I-labeled D5 is able to specifically bind to KDR-transfected cells, and its binding is not affected by the presence of 40% mouse serum. Somewhat more binding to KDR-transfected cells was observed in the absence of serum as compared to binding in the presence of 40% mouse serum. However, the binding of  125 I-D5 to mock-transfected cells was also increased by about the same extent when serum was omitted during the assay, indicating that the increased binding in the absence of serum was non-specific ( FIG. 18 ). Specific binding to KDR-transfected cells (after subtracting binding to mock-transfected cells) looked almost identical with or without mouse serum (as shown in  FIG. 19 ). In this experiment, 10-14% of the total CPM added were specifically bound to KDR-transfected cells (data not shown). 
     Example 18 
     Biacore Analysis of Heterodimer Binding to KDR-Fc and Determination of Affinity Constant 
     A peptide heterodimer ( FIG. 63 ) composed of SEQ ID NO:277 and SEQ ID NO:294 was prepared as previously described in Example 12 using glutaric acid bis N-hydroxysuccinimidyl ester. The heterodimer was tested for binding to KDR-Fc using Biacore, and an affinity constant was determined as follows. 
     Three densities of KDR-Fc were cross-linked to the dextran surface of a CM5 sensor chip by the standard amine coupling procedure (0.5 mg/mL solution diluted 1:100 or 1:50 with 50 mM acetate, pH 6.0). Flow cell 1 was activated and then blocked to serve as a reference subtraction. Final immobilization levels achieved: 
     R L  Fc 2 KDR-Fc=1607 
     R L  Fc 3 KDR-Fc=3001 
     R L  Fc 4 KDR-Fc=6319 
     Experiments were performed in PBS (5.5 mM phosphate, pH 7.65, 0.15 M NaCl)+0.005% P-20 (v/v)). D6 was diluted to 250 nM in PBS and serial dilutions were performed to produce 125, 62.5, 31.3 15.6, 7.8, and 3.9 nM solutions. All samples were injected in duplicate. For association, peptides were injected at 20 μL/min for 12.5 minutes using the kinject program. Following a 10 minute dissociation, any remaining peptide was stripped from the KDR surface with a quickinject of 50 mM NaOH+1 M NaCl for 12 s at 75 μL/min. Sensorgrams were analyzed using BIAevaluation software 3.1 and a hyperbolic double rectangular regression equation in SigmaPlot 6.0. Heterodimer steady state binding affinities (K DAV ) were determined at all three KDR immobilization densities (Table 14). 
                     TABLE 14                  Summary of Parameters                                         K D1  (nM)   R max1     K DAV  (nM)   R maxAV     R 2 *                                                     D6   Vs.   46   13.1   1.5   12.6   0.995           1600RU           Vs.   25.5   21.2   0.665   22.7   0.991           3000RU           Vs.   17   61.3   0.662   62.2   0.993           6000RU                    
From these data, it appears that at the higher immobilization densities, the heterodimer binds KDR with a sub-nanomolar affinity (˜0.6 nM).
 
     To assess the in vivo clearance of this peptide heterodimer, a small amount of material was iodinated using iodogen and Na 125 I according to standard protocols (Pierce). One tube coated with the iodogen reagent was pre-wet with 1 mL of 25 mM Tris, 0.4M NaCl, pH 7.5. This was discarded and 100 μL of the same buffer added. Using a Hamilton syringe 11 μL of the  125 I—NaI was transferred to the reaction tube. Based on original estimates of the Na 125 I concentration of 143.555 mCi/mL, the 11 μL should contain about 1.5 mCi. After addition, the sample was swirled and set in a lead pig to incubate for 6 min with a swirl every 30 sec. After 6 min, the entire sample was transferred to the protein that was in an Eppendorf tube. The sample was swirled and set to incubate for 8 min, with a swirl every 30 sec. After 8 min the reaction was quenched (terminated) with tyrosine (10 mg/mL, a saturated solution), allowed to sit for 5 min, and then 2 μL was removed for a standard. 
     For purification a 10 mL column of the D-salt polyacrylamide 1800 was used to separate the labeled peptide from labeled tyrosine. The column was first washed with 10 mL saline, then 5 mL of 25 mM Tris, 0.4M NaCl, pH 7.5 containing 2.5% HSA to block non-specific sites. After the HSA buffer wash, the column was eluted with 60 mL of the 25 mM Tris, 0.4 M NaCl buffer, and the column was stored overnight at 4° C. The labeled sample contained 1.355 mCi, as determined by the dose calibrator. The 2 μL sample that was removed as a standard contained 8.8 μCi. The peptide sample was applied to the D-salt 1800 column and eluted with the Tris/NaCl buffer, pH 7.5. The flow was controlled by applying single 0.5 mL aliquots for each fraction, #1-14, and then 1.0 mL for fractions 25-43.  FIG. 21  shows the elution profile of activity versus fraction number. The peak of activity in fractions #9, 10, and 11, was assumed to be the peptide. The radioactivity in 24 through ˜40 is likely the labeled tyrosine. From this purification, fractions #9-12 were pooled together and used for the subsequent clearance study (concentration of  125 I-D6 in pool is 7.023 μg/mL; 100 μL=0.702m with 8.6 μCi). 
     A total of 15 mice were injected with 100 μL  125 I-D6 and mice (in sets of 3) were sacrificed at the following time points: 0, 7, 15, 30, 90 minutes. After injection more than 2 μCi was found remaining in the syringe, so actual activity injected was about 6 μCi. With 6 μCi injected, the corresponding protein administered was ˜0.5 μg per animal. Once sacrificed, the counts were determined in a 50 μL plasma sample from each animal. For each set of three animals at each time point, the counts were averaged, converted to % injected dose/ml plasma (ID %/mL), and then plotted to assess the rate of clearance ( FIG. 20 ). This data was fit to either a 4 or 5 parameter equation to determine the biphasic half life of this molecule. The 4 parameter fit resulted in a T 1/2α  of 2.55 minutes and a T 1/2β  of 64.66 minutes. The 5 parameter fit resulted in a T 1/2α  of 2.13 minutes and a T 1/2β  of 23.26 minutes. 
     Larger volumes of plasma were also taken from mice sacrificed at the 0, 30, and 90 minute time points. These samples were injected onto a Superdex peptide column (Pharmacia) coupled to a radioactivity detector to assess the association of the peptide with serum proteins ( FIG. 21 ). As shown, the labeled peptide does associate with higher MW proteins, which could explain its biphasic half life clearance behavior. 
     To help assess the potency of the peptide as an anti-angiogenesis inhibitor, D6 was tested in an endothelial cell proliferation assay using HUVECs and BrdU detection. Briefly, freshly isolated HUVECs (between p3-6) were cultured in RPMI+10% FCS+1% antibiotics+1% L-glutamine+0.4% BBE (bovine brain extract) and seeded per well, 5000-10000/well in 100 μL. The cells were allowed to recover for 24 hrs prior to use. Then the cells were washed with PBS twice and treated for 48 hrs with anti-VEGF antibody (positive control) or peptides A, B and C (0.1 and 10 ug/mL) in RPMI+0.1% BSA+1% L-glutamine. The following 6 variables were tested in 2 series (n=4): 
     Series I: w/o VEGF 
     Series II: w/VEGF (30 ng/mL) 
     
         
         1. Standard medium: RPMI+10% FCS+1% antibiotics+1% L-glutamine+0.4% BBE 
         2. Negative control 1: RPMI (true starvation) 
         3. Negative control 2: RPMI+0.1% BSA+1% L-glutamine 
         4. Positive control: anti-VEGF 10 μg/mL in RPMI+0.1% BSA+1% L-glutamine 
         5. 0.1 μg/mL KDR peptides in RPMI+0.1% BSA+1% L-glutamine 
         6. 10 μg/mL KDR peptides in RPMI+0.1% BSA+1% L-glutamine
 
Protocol:
 
         1) cells are incubated for 48 hours under various conditions 
         2) 10 μL BrdU dilution (1:100 in EBM) is added to each well at 24 hours 
         3) incubate for another 24 hours (total 48 hrs) 
         4) aspirate the culture medium 
         5) add 100 μL FixDenat (Roche Applied Science, Indianapolis, Ind.) to each well, incubate at room temperature for 30 min. 
         6) Discard FixDenat solution 
         7) 100 μL antibody-solution (PBS 1% BSA and anti-BrdU PO) added to each well. 
         8) incubate at RT for 90 minutes. 
         9) wash 3 times with PBS, 200 μL/well, 5 min. 
         10) add substrate solution (TMB), incubate for 10-30 minutes 
         11) transfer all to a flexible plate 
         12) stop the reaction by adding 2 M H 2 SO 4 , 25 μL/well 
         13) read absorbance at 450 nm within 5 minutes after stopping the reaction. 
       
    
     Background binding was determined by omitting the anti-BrdU antibody in 4 wells with control cells (cultured in complete medium; EBM+BulletKit (Clonetics, BioWhittaker, Inc., MD) and by complete labeling of cells that was not exposed to BrdU. 
     Of the two KDR binding peptide tested (D6 and SEQ ID NO:277) as shown in  FIG. 22 , D6 (A) completely inhibits HUVEC proliferation at 10 μg/mL in the presence of VEGF, similar to an anti-VEGF antibody (positive control). On the other hand, SEQ ID NO:277 (B, one of the peptides that make up the heterodimer) did not inhibit proliferation in this assay at the highest concentration tested (10 μg/mL). As a result, the heterodimer shows an enhanced ability to compete with VEGF in comparison with SEQ ID NO:277 alone. 
     Example 19 
     BIAcore Analysis—murine KDR-Fc Binding of Peptide Dimers D1 and D7 
     Using BIAcore, the binding constants of peptide dimers D1 (a heterodimer of SEQ ID NO:277 and SEQ ID NO:294 and D7 (a heterodimer of SEQ ID NO:264 and SEQ ID NO 294; see  FIG. 67 ) for murine KDR-Fc were determined. 
     Procedure 
     Three densities of recombinant murine KDR-Fc were cross-linked to the dextran surface of a CM5 sensor chip by the standard amine coupling procedure (0.5 mg/mL solution diluted 1:100 or 1:40 with 50 mM acetate, pH 6.0). Flow cell 1 was activated and then blocked to serve as a reference subtraction. Final immobilization levels achieved: 
     R L  Fc 2 KDR-Fc=2770 
     R L  Fc 3 KDR-Fc=5085 
     R L  Fc 4 KDR-Fc=9265 
     Experiments were performed in PBS buffer (5.5 mM phosphate, pH 7.65, 0.15 M NaCl)+0.005% P-20 (v/v)). SEQ ID NO:277, run as a control, was diluted to 125 nM in PBS. Serial dilutions were performed to produce 62.5, 31.3, 15.6, 7.8, and 3.9 nM solutions. D1 and D6 were diluted to 50 nM in PBS and serial dilutions were performed to produce 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 nM solutions. All samples were injected in duplicate. For association, peptides were injected at 30 μL/min for 3 minutes using the kinject program. Following a 10 minute dissociation, any remaining peptide was stripped from the rmKDR-Fc surface with a quickinject of 50 mM NaOH+1 M NaCl for 12 s at 75 μL/min. 
     Sensorgrams were analyzed using the simultaneous k a /k d  fitting program in the BIAevaluation software 3.1. The Results are shown in Table 15 and  FIGS. 23-25 . The fact that about the same K D 2 constant was achieved for both heterodimers even when the density of receptor on the sensor chip was reduced by half is consistent with multimeric binding of the heterodimers to individual receptors rather than cross-link-type binding between receptors. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Summary of Kinetic Parameters 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 ka1 (1/Ms) 
                 Kd1 (1/s) 
                 ka2 (1/RUs) 
                 kd2 (1/s) 
                 KD1 #  (nM) 
                 KD2 ‡  (nM) 
                 Chi 2 * 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 D1 
                 vs. 2700RU 
                 7.94E+05 
                 0.0139 
                 3.31E−04 
                 5.96E−04 
                 17.5 
                 0.751 
                 0.077 
               
               
                   
                 vs. 5000RU 
                 5.54E+05 
                 8.88E−03 
                 1.17E−04 
                 4.57E−04 
                 16.0 
                 0.825 
                 0.323 
               
               
                 D7 
                 vs. 2700RU 
                 7.59E+05 
                 0.011 
                 3.36E−04 
                 6.44E−04 
                 14.5 
                 0.848 
                 0.082 
               
               
                   
                 vs. 5000RU 
                 5.21E+05 
                 7.39E−03 
                 1.17E−04 
                 4.68E−04 
                 14.2 
                 0.898 
                 0.278 
               
               
                 Fluorescein 
                 vs. 2700RU 
                 1.02E+06 
                 0.037 
                 — 
                 — 
                 36.4 
                 — 
                 0.073 
               
               
                 SEQ ID NO: 277 
                 vs. 5000RU 
                 5.18E+05 
                 0.0174 
                 — 
                 — 
                 33.6 
                 — 
                 0.167 
               
               
                   
               
               
                   # K D1  is a calculated K D  based on kd 1 /ka 1   
               
               
                   ‡ K D2  is a calculated K D  based on kd 2 /ka 1  (i.e., avidity factor) 
               
               
                 *The chi2 value is a standard statistical measure of the closeness of the fit. For good fitting to ideal data, chi2 is of the same order of magnitude as the instrument noise in RU (typically &lt;2). 
               
            
           
         
       
     
     Example 20 
     In Vivo Inhibition of Tumor Growth 
     Conditions are described providing methods for determining efficacy of three (3) concentrations for Test Article (binding peptide, D6) suspected of having anti-angiogenic activity on SW-480 human colon carcinoma cells using an in vivo xenograft tumor model. 
     Athymic nude mice are acceptable hosts for the growth of allogenic and heterogenic cells. Nude mice are required in  Points to Consider in the Characterization of Cell Lines used to Produce Biologicals  ( Points to Consider in the Characterization of Cell Lines used to Produce Biologicals , FDA 1993). 
     D6 is a synthetic heterodimeric peptide suspected of having anti-angiogenic activity. This peptide binds to the human VEGF receptor 2 (KDR) with high affinity and competes with VEGF binding. 
     SW-480 Human Carcinoma Cells 
     Colon carcinoma, SW-480, cells (ATCC) were cultured in Dulbecco&#39;s Modified Eagles Medium (DMEM) supplemented with 4 mM L-glutamine, 0.1 mM non-essential amino acids, 50 mg/mL Gentamicin, 250 mg/mL Fungizone and 10% heat inactivated fetal bovine serum at 37° C. in 95% air and 5% CO 2 . 
     Exponentially growing cells were harvested, washed twice in phosphate buffered saline (PBS) to remove any traces of trypsin or serum. Cells were suspended in Hanks Balanced Salt Solution (HBSS) for injections. 
     Sterile phosphate buffered saline (BioWhittaker) was manufactured in accordance with cGMP regulations and was cell culture tested to assure compatibility; having a pH of 7.3-7.7 and an osmolarity of 271-287 mOsm/kg. PBS was the vehicle used to reconstitute Test Articles and for vehicle control injections. 
     Cisplatin (American Pharmaceutical Partners, Inc.; Los Angeles, Calif.) was prepared according to manufacture&#39;s specifications. Cisplatin was prepared in an aseptic fashion using a BL2 BioChem guard hood. 
     Test System 
     
         
         A. Species/Strain:  Mus musculus , Crl:NU/NU-nuBR mice (nude mice) 
         B. Sex: Female 
         C. Age: 6-8 weeks at initiation of treatment 
         D. Weight Range: No weight requirement 
         E. Source: Animals were received from the Gnottobiotic Department at Charles River Laboratories, Wilmington, Mass. 
         F. Number: A total of 115 animals were received and injected for this study, with 90 mice used on study. 
         G. Method of Identification: 
       
    
     Mice were uniquely numbered using an ear tag system. Additionally, cages were marked with cage cards minimally identifying group number, animal number, study number and IACUC protocol number.
     H. Randomization:   

     Animals were randomly assigned to treatment groups using Microsoft® Excel 97 SR-1 program.
     I. Humane Care of Animals:   

     Treatment and care of the animals were in accordance with the standard operating procedures of Charles River Laboratories, which adheres to the regulations outlined in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and the  Guide for the Care and Use of Laboratory Animals.    
     This study protocol was covered under the Charles River Laboratories Institutional Animal Care and Use Committee (IACUC Protocol Number: P071820011). 
     Animal Care 
     A. Diet and Drinking Water: 
     Mice were fed gamma-irradiated rodent chow ad libitum. Tap water was sterilized and supplied via bottle and sipper tube ad libitum. 
     B. Animal Environment: 
     Animals were housed by groups in semi-rigid isolators. Mice were housed in flat bottom caging containing five to ten animals. Cages contained gamma-irradiated contact bedding. The number of mice in each cage may have been altered due to the behavior of the mice, changes were noted in the isolator inventory. The housing conforms to the recommendations set forth in the  Guide for the Care and Use of Laboratory Animals , National Academy Press, Washington, D.C., 1996 and all subsequent revisions. 
     Environmental controls were set to maintain a temperature of 16-26° C. (70±8° F.) with a relative humidity of 30-70. A 12:12 hour light: dark cycle was maintained. 
     C. Acclimation: 
     Once animals were received, they were allowed to acclimate to the laboratory environment for 24-hours prior to the study start. Mice were observed for signs of disease, unusual food and/or water consumption or other general signs of poor condition. At the time of animal receipt, animals were clinically observed and appeared to be healthy. 
     Experimental Design 
     A. General Description: 
     Female athymic nude mice (Crl:NU/NU-nuBR) at 6-8 weeks of age were used in this study. A total of 115 mice were injected subcutaneously into the right lateral thorax with 5×10 6 SW-480, human colon carcinoma cells. When tumors reached a target window size of approximately 150±75 mg, 90 tumor-bearing mice were randomly selected and distributed into one of nine groups. Test Articles and vehicle were administered intraperitoneally (IP), Cisplatin was administered intravenously (IV). Tumor measurements were recorded twice weekly using hand-held calipers. Mice were monitored daily for signs of toxicity and morbidity. At study termination, animals were euthanized by carbon dioxide overdose and necropsied for tissue collection. 
     B. Group Assignments: 
     A total of nine (9) groups were used in this study. Each group contained ten (10) tumor-bearing mice. Groups 1 and 2 contained untreated and vehicle treated negative control mice, respectively. Groups 3, 4, and 5 contained mice that received one of three different concentrations of the D6 anti-angiogenic peptide. Group 9 contained mice that received cisplatin, a standard chemotherapeutic compound as a positive control. 
     C. Dosing Levels and Regiment: 
     Dose levels for each group are provided in Table 16. Dosing began the same day that animals were randomly sorted into groups (Study Day 7). Each dose was removed from the dose vial using aseptic technique for each animal and the injection site was wiped with an alcohol swab prior to dose administration. Doses were administered with a 1.0 mL syringe and a 27-gauge×½″ needle for each mouse 
     The Test Article- and vehicle-treated mice received daily intraperitoneal (IP) injections for 15 days. Cisplatin was administered every other workday for a total of five (5) doses via an intravenous route. 
                     TABLE 16                  Study Treatment Groups                                             Concentration   Number of           Group   Test Article   mg/kg   Animals                                                 1   Untreated   —   10           2   Vehicle   0   10           3   D6   0.05   10           4   D6   0.5   10           5   D6   5.0   10           9   Cisplatin   6.0   10                        
D. Clinical Observations of Animals:
 
     Clinical Observations of each animal were performed and recorded at least once daily for toxicity, morbidity and mortality. Morbidity included signs of illness such as, but not limited to, emaciation, dehydration, lethargy, hunched posture, unkempt appearance, dyspnea and urine or fecal staining. 
     E. Tumor Measurements: 
     In accordance with the protocol tumor measurements were taken twice weekly throughout the study by measuring the length and width of tumors with calibrated calipers. Measurements occurred a minimum of 3-4 days apart, except when animals were euthanized and measurements were taken; this sometimes resulted in an interval of less than 3 days. Tumor weights were calculated using the following formula: mg=(L×W 2 )/2. Animals were euthanized either when mean tumor weight was ≧1000 mg per group over two (2) consecutive measurements, or if tumors became ulcerated, impaired the animal&#39;s ability to ambulate or obtain food and water. 
     F. Unscheduled Euthanasia and Unexpected Deaths: 
     1. Unscheduled Euthanasia: 
     None of the animals required unscheduled euthanasia while on study. 
     2. Unexpected Deaths: 
     None of the animals died while on study. 
     G. Necropsy: 
     1. Euthanasia and Necropsy Order: 
     All mice in groups 1, 2, 3, 4, and 5 (50 total) were submitted for necropsy when tumors reached a group mean target size of ≧1000 mg over two (2) consecutive measurements within a group. Animals were submitted for necropsy to the Charles River Laboratories Health Monitoring Laboratory (HM), Wilmington, Mass. All animals were euthanized on Study Day 22, short of received the full 28 day treatment regiment with Test Articles because mean tumor size was ≧1000 mg in Test Article Treated Groups 3-8. 
     All animals were humanely euthanized by carbon dioxide (CO 2 ) inhalation. 
     2. Tissue Collection: 
     Tumors were dissected free of surrounding tissue and overlying skin. Additionally the kidneys were collected. Any abnormalities noted on the renal surfaces were noted. 
     Frozen blocks were made of tumors and kidneys for each animal. A representative section of the tissue (tumor, kidneys) was taken. Kidney sections included the cortex and medulla. Tissue sections were placed in the bottom of a labeled plastic-freezing mold. Tissue was embedded with OCT medium. Blocks were submerged into isopentane chilled with dry ice until frozen. Blocks were briefly examined for quality, and stored on dry ice. 
     Blocks were labeled with the animal number and a letter code corresponding to tissue (A=left kidney; B=right kidney; C=mass). Blocks from one animal were placed into a labeled bag. 
     Results 
     A. In-Life Measurements and Observations: 
     1. Clinical Observations, Morbidity and Mortality Summary Statement: 
     All animals appeared healthy and were within normal limits throughout the study. D6 showed no signs of toxicity at the doses used in this study. 
     Animals were euthanized on Study Day 22. All mice, except Group 9 mice, were euthanized prior to completing Test Article administration, because mean tumor size was &gt;1000 mg in Groups 1-8. Group 9, Cisplatin-treated animals were euthanized on Study Day 22 when mean tumor weight was 995 mg. No animals died while on study. 
     2. Mass Palpation Summary Statement: 
     Throughout the study palpable masses were detected in all mice, with tumors progressively growing for the duration of the study. As expected tumors in untreated and vehicle treated negative control mice (Groups 1 and 2) grew the fastest, reaching a mean tumor size of 1000 mg on or before Study Day 20. In addition, animals treated with Cisplatin (Group 9) developed tumors that grew the slowest reaching a mean tumor size of 995 mg at study termination (Day 22). 
     In general, except for Group 3 mice, all animals treated with Test Article resulted in slower tumor growth ( FIG. 65 ). Animals in Group 3, which were treated with the low dose of D6 (0.05 mg/kg) had tumors that grew at approximately the same rate as the tumors in untreated and vehicle treated animals in Groups 1 and 2. Animals treated with either higher doses of D6 (Groups 4 and 5) had tumors that grew slower; reaching a mean tumor size of 1000 mg on Study Day 21. When compared to control Groups 1 and 2 mice, Test Article treatment resulted in a delay of tumor growth of approximately 1 day. 
     B. Conclusions: 
     Data from this study validate the model used because tumor-bearing mice in negative control Groups 1 and 2 and positive control Group 9 responded as expected. 
     Throughout the study palpable masses were observed in all groups. In addition, all animals were healthy and within normal limits throughout the study. Furthermore, D6 did not adversely affect the animals. Therefore, these data would suggest that animals treated with D6 Test Article had tumors that grew slowly (approximately 1 day slower over the 22 day test period than controls). Also, since the Test Article did not show any significant toxic effects, higher concentrations of Test Article could also be used with potentially better tumor regression. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 17 
               
             
            
               
                   
                   
               
               
                   
                 Test Article 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Untreated 
                 Vehicle 
                 D6 
                 Cisplatnin 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Control 
                 Control 
                 0.005 mg/kg 
                 0.05 mg/kg 
                 0.5 mg/kg 
                 6 mg/kg 
                   
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Days 
                 4 
                 48 
                 49 
                 43 
                 51 
                 50 
                 34 
                 Tumor 
               
               
                 After 
                 7 
                 164 
                 156 
                 157 
                 163 
                 154 
                 160 
                 Weights 
               
               
                 Cell 
                 8 
                 180 
                 164 
                 156 
                 133 
                 168 
                 173 
                 (mg) 
               
               
                 Injection 
                 11 
                 340 
                 388 
                 333 
                 298 
                 310 
                 407 
               
               
                   
                 14 
                 684 
                 648 
                 726 
                 596 
                 577 
                 675 
               
               
                   
                 20 
                 1064 
                 986 
                 973 
                 857 
                 978 
                 635 
               
               
                   
                 21 
                 1412 
                 1571 
                 1468 
                 983 
                 1056 
                 839 
               
               
                   
                 22 
                 1967 
                 1863 
                 2026 
                 1474 
                 1526 
                 995 
               
               
                   
               
            
           
         
       
     
     Example 21 
     In Vitro Cell Proliferation Assay 
     Microvascular endothelial cells (MVECs, Cascade Biologics, Portland, Oreg.) were used to assess the in vitro efficacy of D6 and related analogues for their ability to inhibit VEGF-stimulated proliferation. MVECs (passage 2) were grown to 90% confluency, trypsinized and plated in gelatin-coated 96-well microtiter plates at a density of 4-8×10 3  cells/well. Sixteen to 24 hours after plating, the cells were washed one time (200 μL/well) with media devoid of fetal bovine serum but containing 0.1% bovine serum albumin (BSA). Fresh BSA-containing media was added to each well and the cells were incubated for an additional 24 hours. After this 24 hour period of starvation, fresh BSA-containing media (containing 25 ng/mL VEGF) with or without D6 was added and the cells were incubated for an additional 48 hours at 37 C. To assess the dose response in this assay, multiple D6 concentrations were tested in duplicate wells. The media was removed and fresh BSA-containing media was added with or without BrdU and the cells were incubated for an additional 24 hours prior to determining the level of incorporation exactly as described by the manufacturer. Results are shown in  FIG. 84 . 
     Example 22 
     The following experiment assessed the ability of D25 and D27 to block the VEGF-induced migration of HUVECs in culture and demonstrated that the added glycosylation and/or distinct spacer structure used in D27 enhanced its potency. 
     Protocol: 
     Serum-starved HUVECs were placed, 100,000 cells per well, into the upper chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal medium, with or without VEGF (10 ng/mL) in the presence or absence of D25 or D27, was added to the lower chamber of the wells. After 22 hours, quantitation of cell migration/invasion was achieved by post-labeling cells in the insert plates with a fluorescent dye and measuring the fluorescence of the invading/migrating cells in a fluorescent plate reader. The VEGF-induced migration was calculated for each experimental condition by subtracting the amount of migration that occurred when only basal medium was added to the lower chamber of the wells. 
     Results: 
     VEGF induced a large increase in endothelial cell migration in the assay, which was potently blocked by both D25 and D27 ( FIG. 66 ). D27 was ten-fold more potent than D25 (IC 50  0.5 nM and 5 nM respectively), indicating that the glycosylation of D27 and/or its distinct spacer properties has enhanced its ability to bind KDR and block the effects of VEGF. 
     Example 23 
     The following experiment assessed the ability of “Adjunct A” multimeric construct of TKPPR peptide (SEQ ID NO:503; binds to NP-1, a VEGF receptor that enhances the effects of VEGF mediated by KDR), to enhance the inhibition of the VEGF-induced migration of HUVECs in culture produced by D6. Adjunct A=5 CF-Gly-N{[CH 2 CH 2 C(═O)-Gly-N(CH 2 CH 2 C(═O)-Adoa-Thr-Lys-Pro-Pro-Arg-OH] 2 } 2  where Adoa=3,6-dioxa-8-aminooctanoyl, 5 CF=5-carboxyfluoresceinyl. See  FIG. 67B . 
     Protocol: 
     Serum-starved HUVECs were placed, 100,000 cells per well, into the upper chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal medium, containing with or without VEGF (10 ng/mL) in the presence or absence of varying concentrations of D6, or varying concentrations of D6 in combination with a constant 100 nM Adjunct A (synthesized as described in WO 01/91805 A2), was added to the lower chamber of the wells. After 22 hours, quantitation of cell migration/invasion was achieved by post-labeling cells in the insert plates with a fluorescent dye and measuring the fluorescence of the invading/migrating cells in a fluorescent plate reader. VEGF-induced migration was calculated for each experimental condition by subtracting the amount of migration observed in the absence of VEGF. 
     Results: 
     VEGF induced a large increase in endothelial cell migration in the assay, which was potently blocked by D6 (IC 50  about 12.5 nM), but not by 100 nM Adjunct A alone ( FIG. 67A ). Surprisingly however, Adjunct Awas able to enhance the potency of D6 by about ten-fold when used in the assay simultaneously with D6 (IC 50  about 2.5 nM). This indicates that compounds containing the TKPPR sequence (or similar) found in Adjunct A can be used to enhance the potency of certain compounds such as D6, which compete with VEGF for binding to KDR. In addition, a heteromultimer containing the peptide sequences found in D6 or similar) as well as the TKPPR sequence (or similar), in one or more repetitions, would likely possess enhanced activity in this assay. See U.S. patent application Ser. No. 09/871,974, incorporated by reference in its entirety, for details regarding the preparation of TKPPR constructs. 
     Example 24 
     Synthesis of D27 
     Synthesis of 1 and 3 (see  FIGS. 68 and 69 ) 
     Synthesis of the monomers were carried out as described in Method 5 on a 0.25 mmol scale employing as the starting resin Fmoc-GGGK(iV-Dde)NH-PAL-PEG-PS resin. The peptide resin was washed and dried before cleavage or further derivatization by automated or manual methods. 
     Procedure Synthesis of Peptide 2 and Peptide 4 (see  FIGS. 68 and 69 ) 
     Appendage of Biotin-JJ, Lysyl, Glycyl and Serinyl (GalNAc(Ac) 3 -α-D moieties onto 1 and 3 was done by manual SPPS such as described in Method 6 and Method 8. The coupling of amino acids was carried out in DMF using HOBt/DIC activation (except for Ser(GalNAc(Ac) 3 -α-D). Fmoc removal was carried out with 20% piperidine in DMF. All couplings were 5-16 hours duration. After each coupling, the completion was confirmed by the Kaiser test. In the case of Ser(GalNAc(Ac) 3 -α-D, the coupling was performed in DMF employing HATU/DIEA as the coupling agent. In cases where the Kaiser test indicated unreacted amino groups the coupling was repeated. Removal of the N-terminal Fmoc group and cleavage from resin was performed. The crude peptide was precipitated in ether and washed twice by ether and dried under vacuum. The linear crude peptide was directly cyclized by dissolving the peptide in DMSO (40 mg/mL). The pH of the solution was adjusted to 8 by addition of aqueous N-methylglucamine and the solution was stirred in air for 48 h at room temperature. The peptides were then purified employing gradient HPLC as described in Method 1 employing a Waters-YMC C-18 ODS preparative column (250 mm×4.6 mm i.d.). The pure product-containing fractions were combined and lyophilized to provide the needed peptides. 
     Procedure: Synthesis of D27—Compound 6 (see  FIG. 70 ) 
     To a solution of glutaric acid bis-NHS ester (0.122 mmol, Pierce Scientific Co.) in anhydrous DMF was added dropwise a solution of 4 in DMF (40 mg, 0.0122 mmol, DIEA was added to neutralize the trifluoroacetic acid bound to the peptide and N-hydroxysuccinimide formed during the reaction). This 0.7 mL solution was stirred for 4 h. The reaction was monitored by HPLC and mass spectroscopy. DMF was removed under vacuum. The excess diester was removed by addition of ethyl acetate, which precipitated the peptide-monoester 5 while dissolving glutaric acid bis-NHS ester. The mixture was centrifuged and the liquid portion decanted. This was repeated twice. The residue was kept under vacuum for 10 min. The residue was dissolved in DMF and mixed with a solution of 2 (37 mg, 0.009 mmol) in DMF (pH 7). It was stirred at ambient temperature for 16 h. The volatiles were removed under high vacuum and the acetate functions were removed by treatment of the residue with 1 mL of hydrazine/MeOH (15/85, v/v) solution with stirring for 2.5 h at ambient temperature. Acetone was added to quench the excess of hydrazine and the volatiles were removed under vacuum. The resulting residue was dissolved in DMSO and purified by preparative HPLC as described above to provide 9 mg of the pure material. 
     Sequence and Analytical Data for Peptides 2, 4 and 6 
                                                 HPLC           Compound       Ret. time   Mass Spectrum       identifier   Sequence   (System)   (ESI, neg. ion)                  Peptide 2:   Ac-    7.4 min   2041.3 [M − 2H]/2       New Seq, a   AGPTWCEDDWYYCWLFGTGGGK{Biotin-   (T)           Seq 11   JJK[NH 2 -Ser(GalNAc(Ac) 3 -α-D)-Gly-               derivative   Ser(GalNAc(Ac) 3 -α-D]}-NH 2                         Peptide 4:   Ac-VCWEDSWGGEVCFRYDPGGGK(NH 2     8.0 min   1636.3 [M − 2H]/2       New Seq, a   Ser(GalNAc(Ac) 3 -α-D)-Gly-   (T)           Seq 5   Ser(GalNAc(Ac) 3 -α-D)-NH 2                 derivative                           D27   Ac-AGPTWCEDDWYYCWLFGTGGGGK{Ac-   5.50 min   1737.2 (M − 4H)/           VCWEDSWGGEVCFRYDPGGGK[S(GalNAc-α-D)-   (M)   4;           G-S(GalNAc-α-D)-Glut-S(GalNAc-α-D)-G-S(GalNAc-       1389.3 (M − 5H)/5;           α-D)-NH(CH 2 ) 4 -(S)—CH(Biotin-JJNH—)C(═O)—]—NH 2 }—NH 2         1157.7 [M − 6H]/6                    
System T: Column: Waters XTerra, 4.6×50 mm; Eluents:A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA): Elution: Initial condition, 15% B, Linear Gradient 15-50% B in 8 min; Flow rate: 3.0 mL/min; Detection: UV @ 220 nm.
 
     Example 25 
     Demonstration of the Distinction Between Binding Affinity and Biological Potency Through In Vitro Assays 
     The following experiments showed that heteromultimeric peptides can display much greater biological potency than a monomeric peptide with similar binding affinity to the same target. 
     Protocol Experiment 1: 
     293H cells were transfected with the KDR cDNA or mock-transfected by standard techniques described in Example 5. The cells were incubated with  125 I-VEGF in the presence or absence of SEQ ID NO:504 or D1 (at 300, 30, 3, and 0.3 nM). After washing the cells, the bound radioactivity was quantitated on a gamma counter. The percentage inhibition of VEGF binding was calculated using the formula [(Y1−Y2)×100/Y1], where Y1 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. 
     Protocol Experiment 2: 
     Serum-starved HUVECs were placed, 100,000 cells per well, into the upper chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal medium, with or without VEGF (10 ng/mL) in the presence or absence of increasing concentrations of SEQ ID NO:504 or D1, was added to the lower chamber of the wells. After 22 hours, quantitation of cell migration/invasion was achieved by post-labeling cells in the insert plates with a fluorescent dye and measuring the fluorescence of the invading/migrating cells in a fluorescent plate reader. VEGF-stimulated migration was derived by subtracting the basal migration measured in the absence of VEGF. 
     Results Experiment 1: 
     As shown in  FIG. 71 , SEQ ID NO:504 AND D1 competed about equally well with  125 I-VEGF for binding to KDR-transfected cells, indicating that they possess comparable binding affinities as well as a comparable ability to inhibit VEGF from binding to KDR. 
     Results Experiment 2: 
     Despite the fact that both SEQ ID NO:504 and D1 potently block  125 I-VEGF binding to KDR-expressing cells to the same degree ( FIG. 72 ), the heterodimeric D1 was much more potent in blocking the biological effects of VEGF as demonstrated in an endothelial cell migration assay ( FIG. 72 ) than the monomeric SEQ ID NO:504. At up to 62.5 nM, a peptide comprising SEQ ID NO:504 had no effect on VEGF-stimulated migration whereas D1 completely blocked VEGF-stimulated migration at 50 nM. These data suggest that heteromultimeric binding can more effectively block the biological activity of a ligand than a monomer, even when the monomer has a comparable ability to inhibit ligand binding to its receptor. 
     Example 26 
     Identification of Fragments of SEQ ID NO:356 with KDR Binding Activity 
     The following experiment showed that fragments of SEQ ID NO:356 can maintain significant KDR binding activity. 
     Protocol: 
     293H cells were transfected with the KDR cDNA or mock-transfected by standard techniques described in Example 6. Binding of the neutravidin-HRP complexes to the cells was carried out as in Example 6 with a complex concentration of 5.5 nM in the presence of 0 to 250 nM or 0 to 1000 nM of the following competing peptides: SEQ ID NOS:356, 462, 463, and 465. After determining the specific binding under each experimental condition, the IC 50  for each peptide was determined (where possible). 
     Results: 
     As shown in Table 18, SEQ ID NO:462, composed of just the Asp-Trp-Tyr-Tyr (SEQ ID NO:490) binding motif that is also shared with SEQ ID NO:286 along with the non-targeted Gly-Gly-Gly-Lys (SEQ ID NO:262) sequence that was added to most monomeric peptides synthesized based on phage display data, was the smallest fragment able to block peptide/neutravidin-HRP complex binding with an IC 50  below one micromolar. Surprisingly, a larger fragment comprising SEQ ID NO:356, failed to significantly inhibit complex binding at one micromolar. However, when a solubilising motif, (Gly-Arg-Gly) 3  was added to the latter peptide to make SEQ ID NO:465, it was able to compete with the complex for binding with an IC 50  of 175 nM, confirming that certain fragments of SEQ ID NO:356 containing the Asp-Trp-Tyr-Tyr (SEQ ID NO:490) motif retain KDR-binding activity. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Fragments of SEQ ID NO: 356 in a displacement assay competing 
               
               
                 with a complex composed of binding peptide and neutravidin- 
               
               
                 HRP for binding to KDR-expressing cells. 
               
            
           
           
               
               
               
            
               
                   
                 Fragment (SEQ ID NO) 
                 IC 50 , nM 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 356 
                 93 
               
               
                   
                 462 
                 850 
               
               
                   
                 463 
                 &gt;1000 
               
               
                   
                 465 
                 175 
               
               
                   
                   
               
            
           
         
       
     
     Example 27 
     Cell Based Assay for Binding of KDR/VEGF Complex Binders 
     The ability of a KDR/VEGF complex-binding peptide to selectively bind to the KDR/VEGF complex was demonstrated. 
     Reagent Preparation 
     The reagents for this assay were prepared as described in Example 5 except where noted. 
     Preparation of Peptide- 125 I-Neutravidin Solution 
     Biotinylated peptides SEQ ID NOS:321, 320 and 323, and a biotinylated non-binding control peptide were used to prepare 1.25 μM stock solutions in 50% DMSO. A 33.33 nM stock solution of  125 I-neutravidin was purchased from Amersham (Buckinghamshire, UK). A stock solution of 13.33 nM  125 I-neutravidin/100 nM VEGF was prepared by mixing 850 mL of  125 I-neutravidin with 22 μL of 10 μM VEGF and 1275 μL of M199 media. Another stock solution was prepared in the same manner, but lacking VEGF. To prepare 13.33 nM peptide- 125 I-neutravidin complex solutions ±VEGF, 500 μL, of the  125 I-neutravidin (with and without VEGF) stock solutions (prepared in last step) were mixed with 24 μL of 1.25 μM peptide solution of SEQ ID NOS:321, 320 and 323, or control peptide. The mixtures were incubated on a rotator at 4 C for 60 minutes, followed by addition of 50 μL of soft release avidin-sepharose (50% slurry in ddH 2 O) to remove excess peptides and another incubation for 30 minutes on a rotator at 4 C. Finally, the soft release avidin-sepharose was pelleted by centrifuging at 12,000 rpm for 5 minutes at room temperature, and the resulting supernatants were used for the assays. 
     Binding of Peptide/Neutravidin HRP to KDR-Transfected Cells 
     Complexes of control peptide and the test peptides (SEQ ID NOS:321, 320 and 323) with  125 I-neutravidin in the presence or absence of VEGF (prepared as above) were tested for their ability to bind 293H cells that were transiently-transfected with KDR. The complex of SEQ ID NO:321 with  125 I-neutravidin specifically bound to KDR-transfected 293H cells as compared to mock transfected cells in the presence of VEGF ( FIG. 73 ), but not where VEGF was omitted ( FIG. 74 ). SEQ ID NO:321, was also the best KDR/VEGF complex binder among the peptides tested using fluorescence polarization and SPR (BIAcore) assays (Table 9). This example shows that peptide (SEQ ID NO:321) can specifically bind to the KDR/VEGF complex present on the cell surface. This establishes a utility for the assay as useful for targeting the KDR/VEGF complex in vitro and in vivo for diagnostic or therapeutic purposes. Since the KDR/VEGF binding peptide only detects the functional and active KDR receptor and not all the KDR present on cell surface, it will be useful in detecting and/or treating active angiogenesis in tumors, metastasis, diabetic retinopathy, psoriasis, and arthropathies. 
     Example 28 
     This example provides more evidence that heterodimeric peptides targeting two epitopes on KDR are superior to a homodimeric peptide that binds one of the two epitopes on the target molecule. The following experiment provides further evidence that heterodimeric constructs are superior to homodimeric peptides in their ability to block the biological effects of VEGF. 
     Protocol: 
     Serum-starved HUVECs were placed, 100,000 cells per well, into the upper chambers of BD fibronectin-coated FluoroBlok 24-well insert plates. Basal medium, containing either nothing or VEGF in the presence or absence of increasing concentrations of homodimeric D8 or heterodimeric D17, was added to the lower chamber of the wells. After 22 hours, quantitation of cell migration/invasion was achieved by post-labeling cells in the insert plates with a fluorescent dye and measuring the fluorescence of the invading/migrating cells in a fluorescent plate reader. 
     Results: 
     As shown in  FIG. 75 , VEGF induced a large increase in endothelial cell migration in the assay, which was potently blocked by D17 but not D8. D17 blocked VEGF-induced migration with an IC 50  of about 250 nM while D8 had no significant effect on migration even at 800 nM. This is in spite of the fact that D8 used the full targeting sequence found in SEQ ID NO:356 while D17 contained a truncated version of the SEQ ID NO:356 sequence (as seen in SEQ ID NO:465) with a lower affinity for KDR (as demonstrated in Example 26). Thus a heterodimer with the capability of binding two separate epitopes on KDR is more effective at blocking ligand binding to KDR than a homodimer containing the same or even more potent targeting sequences. 
     Example 29 
     Preparation of KDR-Binding Peptides in which the Disulfide Bond has been Replaced 
     Disulfide bond substitution analogs of SEQ ID NO:301, where the Cys residues at position 6 and 13 are replaced by a pair of amino acids, one with a carboxy-bearing side-chain (either Glu or Asp) and the other with an amino-bearing side chain [(Lys or Dpr (2,3-diaminopropanoic acid)] were prepared. The cycle, encompassing the same sequence positions as those included in SEQ ID NO:301 (made by formation of the disulfide bond) was made by condensation of the side-chain amino and side-chain acid moieties, resulting in a lactam ring that bridges the residues 6-13 as does the disulfide bond of SEQ ID NO:301. 
     Table 19 below displays some examples of the substitutions made for Cys 6  and Cys 13  of SEQ ID NO:301 in lactam analogs. 
                     TABLE 19                  Lactam Analogs of SEQ ID NO: 277                                         Difference in                   Ring Size vs       Sequence   Position 6   Position 13   SEQ ID NO: 277               SEQ ID NO: 277   Cys   Cys   —       (parent seq)       453   Glu   Lys   4       454   Lys   Glu   4       455   Dpr   Asp   0       456   Asp   Dpr   0       457   Asp   Lys   3                    
Synthesis of Resin Bound SEQ ID NO:453
 
     Synthesis of 1 was carried out using Method 5 on a 0.25 mmol scale. The peptide resin 1 was washed and dried for further derivatization manually (see  FIG. 76 ). 
     Synthesis of 4 (SEQ ID NO: 453) 
     To 1 (240 mg, 0.06 mmol) was added NMM (N-methyl morpholine)/HOAc/DMF 1/2/10 (v/v/v) (65 mL). Palladium tris-triphenylphosphine [Pd(PPh 3 ) 4 , 554.4 mg, 0.48 mmol] was added and the resin was shaken for 20 h shielded from light. The resin was filtered and washed with a solution of sodium diethyldithiocarbamate (0.5 g)/DIEA (0.5 mL)/DMF (100 mL), and finally with DMF (3×70 mL). This treatment served to expose only the carboxy and amino groups of Glu6 and Lys13 that are required for the lactam forming reaction. The on-resin cyclization of 2 was carried out using HATU (114 mg, 0.3 mmol), NMM (66 μL, 0.6 mmol) and DMF (10 mL) for 3 h. The completion of the cyclization was monitored by Kaiser test. The peptide was cleaved from the peptide resin 3 using reagent B for 4 h. The resin was filtered and the filtrate was evaporated to a paste. The crude peptide was precipitated in ether and washed twice with ether. The cyclic peptide was purified by preparative reverse phase linear gradient HPLC using a Waters-YMC C-18 column (250 mm×30 mm i.d.) with CH 3 CN into H 2 O (both with 0.1% TFA) as the eluent. Lyophilization of the product-containing fractions afforded 8 mg of (SEQ ID NO:453). SEQ ID NOS:454, 455, 456 and 457 were prepared similarly. 
     Example 30 
     Replacement of a Disulfide Bridge while Retaining KDR-Binding Activity 
     The following experiment demonstrated that the lactam SEQ ID NO:454 replaced a chemically reactive disulfide bridge to maintain significant KDR binding activity. 
     Protocol: 
     293H cells were transfected with the KDR cDNA or mock-transfected by standard techniques described in Example 5. Neutravidin-HRP complexes were prepared as in Example 5. Binding of the neutravidin-HRP complexes to the cells was carried out as in Example 5 with a complex concentration of 5.5 nM in the presence of 0 to 250 nM SEQ ID NO:277 or SEQ ID NO:454. After determining the specific binding under each experimental condition, the IC 50  for each peptide was determined. 
     Results: 
     As shown in Table 20, SEQ ID NO:454, containing a lactam disulfide bridge replacement, was still able to compete with peptide-neutravidin-HRP complexes for binding to KDR although some affinity was lost (IC 50  108 nM versus 13 nM). 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 SEQ ID NO: 277 and SEQ ID NO: 454 (disulfide bridge 
               
               
                 replacement analog) in a displacement assay competing 
               
               
                 with a neutravidin-HRP/binding peptide complex 
               
               
                 for binding to KDR-expressing cells. 
               
            
           
           
               
               
               
            
               
                   
                 Fragment (SEQ ID NO) 
                 IC 50 , nM 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 277 
                 13 
               
               
                   
                 454 
                 108 
               
               
                   
                   
               
            
           
         
       
     
     Example 31 
     Use of the Neutravidin/Avidin HRP Assay with Biotinylated Peptides Identified by Phage Display Allows Identification of Peptides Capable of Binding to the Target Even where the Affinity of the Peptides is Too Low for Other Assays 
     This example confirms that the neutravidin/HRP screening assay described herein is an effective technique for screening peptides whose affinity as monomers is too low for use in conventional screening assays, such as, for example, an ELISA. 
     Three different derivatives of SEQ ID NO:482, which was identified by phage display as a peptide that binds to cMet, were prepared as described in U.S. Patent Application No. 60/451,588 (incorporated herein by reference in its entirety), filed on the same date as U.S. patent application Ser. No. 10/382,082, of which the present application is a continuation-in-part. 
     These three peptides and a control peptide that does not bind to cMet, were tested as tetrameric complexes with neutravidin HRP for their ability to bind cMet-expressing MB-231 cells. All three tetrameric complexes of cMet-binding peptides bound to the MB231 cells as compared to control peptide. 
     Cell Culture: 
     MDA-MB231 cells were obtained from ATCC and grown as monolayer culture in their recommended media plus 1 mL/L pen/strep (InVitrogen, Carlsbad, Calif.). Cells were split the day before the assay, 35000 cells were added to each well of a 96 well plate. The rest of the experiment was conducted as in Example 6, except as noted below. 
     Binding of Peptide/Neutravidin HRP to MDA-MB-231 Cells: 
     Complexes of control peptide, and SEQ ID NO:482 derivatives with 0, 1 or 2 J spacers with neutravidin-HRP were prepared as described above and tested for their ability to bind MDA-MB-231 cells. During the peptide/neutravidin-HRP complex preparation, a 7.5-fold excess of biotinylated peptides over neutravidin-HRP was used to make sure that all four biotin binding sites on neutravidin were occupied. After complex formation, the excess of free biotinylated peptides was removed using soft release avidin-sepharose to avoid any competition between free biotinylated peptides and neutravidin HRP-complexed biotinylated peptides. The experiment was performed at several different concentrations of peptide/neutravidin-HRP, from 0.28 nM to 33.33 nM, to generate saturation binding curves for derivatives with no or one spacer ( FIG. 77 ) and 0.28 to 16.65 nM to generate a saturation binding curve for the derivative with two spacers ( FIG. 77 ). In order to draw the saturation binding curve, the background binding of the control peptide/neutravidin HRP complex was subtracted from the binding of the binding derivative peptide/neutravidin-HRP complexes for each concentration tested. Therefore, absorbance on the Y-axis of  FIG. 77  is differential absorbance (cMet-binding peptide minus control peptide) and not the absolute absorbance. Analysis of the saturation binding data in  FIG. 77  using Graph Pad Prism software (version 3.0) yielded a K D  of 12.62 nM (+/−3.16) for the tetrameric derivative with two spacers, 155.4 nM (+/−86.56) for the tetrameric derivative with one spacer and 123.8 nM (+/−37.71) for the tetrameric derivative without a spacer. These binding constants are, as expected, lower than that measured by FP for the related monodentate peptide SEQ ID NO:482 (880 nM). 
     Results: 
     As was the case where the binding target was KDR, the neutravidin-HRP assay with biotinylated peptides identified with phage display was useful for identifying peptides capable of binding to an immobilized target even when the affinity of the monomeric binding sequence is too low for an ELISA-type assay (with washing steps after binding) to work well (see  FIG. 77 ). 
     Example 32 
     Binding of Tc-Labeled Heterodimeric Polypeptides to KDR-Transfected 293H Cells 
     The ability of Tc-labeled D10 to bind KDR was assessed using KDR-transfected 293H cells. The results show that Tc-labeled D10 binds significantly better to KDR transfected 293H cells than to mock transfected 293H cells, and good binding was maintained in the presence of 40% mouse serum. In addition, a derivative of Tc-labeled D10 with its amino acid sequence scrambled, D18, was shown to possess no affinity for KDR-expressing cells, confirming the specificity of the D10 binding to those cells. 
     Transfection of 293H 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 cells in one half of the plate (48 wells) were mock-transfected (without DNA) and the cells in the other half of the plate 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 (the assay was aborted if these conditions were not satisfied). 
     Preparation of Opti-MEMI Media with 0.1% HSA 
     Opti-MEMI was obtained from InVitrogen (Carlsbad, Calif.) and human serum albumin (HSA) was obtained from Sigma (St. Louis, Mo.). Opti-MEMI media was prepared by adding 0.1% HSA, 0.1% w/v HSA to opti-MEMI, followed by stirring at room temperature for 20 minutes. The media was filter sterilized using 0.2 μM filter. 
     Preparation of Tc-Labeled Peptide Dilutions for the Assay 
     D10 and D18 were diluted in opti-MEMI with 0.1% HSA to provide solutions with final concentrations of 1.25, 2.5, 5.0, and 10 μCi/mL of each Tc-labeled heterodimer. A second set of dilutions was also prepared using a mixture of 40% mouse serum/60% opti-MEMI with 0.1% HSA as the diluent. 
     Assay to Detect the Binding of the Tc-Labeled Heterodimers 
     Cells were used 24 h after transfection, and to prepare the cells for the assay, they were washed once with 100 μL of room temperature opti-MEMI with 0.1% HSA. After washing, the opti-MEMI with 0.1% HSA was removed from the plate and replaced with 70 μL of 1.25, 2.5, 5.0, and 10 μCi/mL of Tc-labeled D10 or D18 (prepared as above with both diluent solutions). Each dilution was added to three separate wells of mock- and KDR-transfected cells. After incubating at room temperature for 1 h, the plates were washed 5 times with 100 μL of cold binding buffer (opti-MEMI with 0.1% HSA). 100 μL of solubilizing solution (0.5 N NaOH) was added to each well and the plates were incubated at 37 C for 10 minutes. The solubilizing solution in each well was mixed by pipeting up and down, and transferred to 1.2 mL tubes. Each well was washed once with 100 μL of solubilizing solution and the washes were added to the corresponding 1.2 mL tube. Each 1.2 mL tube was then transferred to a 15.7 mm×100 cm tube to be counted in an LKB Gamma Counter. 
     Binding of Tc-Labeled Peptide to KDR Transfected Cells 
     The ability of Tc-labeled D10 and D18 to bind specifically to KDR was demonstrated using transiently transfected 293H cells. As shown in  FIG. 78 , Tc-labeled D10 bound better to KDR transfected 293H cells, as compared to mock-transfected (with a scrambled peptide) 293H cells in both the presence and absence of 40% mouse serum, although there was some inhibition in the presence of serum. The total specific binding of this Tc-labeled heterodimer to KDR-expressing cells was greater than that observed previously with a Tc-labeled monomeric peptide (Example 10). Tc-labeled D18, the scrambled peptide, displayed no affinity for either mock-transfected or KDR-transfected 293H cells (not shown), confirming the specificity of D10 binding. 
     Example 33 
     Binding of a Lu-Labeled Heterodimeric Polypeptide to KDR-Transfected 293H Cells 
     The ability of Lu-labeled D13 to bind KDR was assessed using KDR-transfected 293H cells. The results show that Lu-labeled D13 binds better to KDR transfected 293H cells than to mock transfected 293H cells, and significant binding was maintained in the presence of 40% mouse serum. 
     Transfection of 293H Cells 
     293H cells were transfected using the protocol described in Example 5. Transfection was performed in black/clear 96-well plates (Becton Dickinson, San Jose, Calif.). The cells in one half of the plate (48 wells) were mock-transfected (without DNA) and the cells in the other half of the plate 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 assay (the assay was aborted if these conditions were not satisfied). 
     Preparation of Opti-MEMI Media with 0.1% HSA 
     Opti-MEMI was prepared as in Example 32. 
     Preparation of Lu-Labeled Peptide Dilutions for the Assay 
     A stock solutions of Lu-labeled D13 was diluted in opti-MEMI with 0.1% HSA to provide solutions with final concentrations of 1.25, 2.5, 5.0, and 10 μCi/mL of labeled heterodimer. A second set of dilutions was also prepared using a mixture of 40% mouse serum/60% opti-MEMI with 0.1% HSA as the diluent. 
     Assay to Detect the Binding of the Lu-Labeled Heterodimers 
     Detection of binding was measured as detailed in Example 32 except that Lu-labeled D13 was used in place of the Tc-labeled heterodimers. 
     Binding of Lu-Labeled Peptide to KDR Transfected Cells 
     The ability of Lu-labeled D13 to bind specifically to KDR was demonstrated using transiently-transfected 293H cells. As shown in  FIG. 95 , Lu-labeled D13 bound significantly better to KDR transfected 293H cells, as compared to mock-transfected 293H cells in both the presence and absence of 40% mouse serum, although there was some binding inhibition in the presence of serum. 
     Example 34 
     Radiotherapy with a Lu-Labeled Heterodimeric Peptide in Tumor-Bearing Mice 
     In this example, the ability of Lu-labeled D13 to inhibit the growth of PC3 cell tumors implanted in nude mice is demonstrated. 
     Animal Model 
     PC3 cells from ATCC, grown as recommended by the supplier, were injected subcutaneously between the shoulder blades of nude mice. When their tumors reached 100-400 mm 3 , twelve mice were injected i.v. with 500 microcuries of Lu-labeled D13 and their growth monitored for an additional 18 days. Mice were sacrificed if they lost 20% or more of their body weight or their tumors exceeded 2000 mm 3 . Tumor growth in the treated mice was compared with the average tumor growth in 37 untreated nude mice implanted with PC3 tumors. 
     Results 
     In 6 of the 12 treated mice in the study, the tumors experienced a significant or complete growth delay ( FIG. 80 ) relative to untreated tumor mice, indicating that D13 was effective in slowing PC3 tumor growth under the conditions employed. 
     Example 35 
     Preparation of Ultrasound Contrast Agents Conjugated to KDR-Binding Peptides 
     Ultrasound contrast agents comprising phospholipid-stabilized microbubbles conjugated to KDR-binding polypeptides of the invention were prepared as described below. 
     200 mg of DSPC (distearoylphosphatidylcholine), 275 mg of DPPG.Na (distearoylphosphatidylglycerol sodium salt), 25 mg of N-MPB-PE were solubilized at 60 C in 50 mL of Hexan/isopropanol (42/8). The solvent was evaporated under vacuum, and then PEG-4000 (35.046 g) was added to the lipids and the mixture was solubilized in 106.92 g of t-butyl alcohol at 60 C, in a water bath. The solution was filled in vials with 1.5 mL of solution. The samples were rapidly frozen at −45 C and lyophilized. The air in the headspace was replaced with a mixture of C 4 F 10 /Air (50/50) and vials capped and crimped. The lyophilized samples were reconstituted with 10 mL saline solution (0.9%-NaCl) per vial. 
     Peptide Conjugation 
     Peptides, e.g., SEQ ID NO:356, SEQ ID NO:294 and SEQ ID NO:480, were conjugated to a preparation of microbubbles as above described, according to the following methodology. 
     The thioacetylated peptide (200 μg, SEQ ID NO:356) was dissolved in 20 μL DMSO and then diluted in 1 mL of Phosphate Buffer Saline (PBS). This solution was mixed to the N-MPB-functionalized microbubbles dispersed in 18 mL of PBS-EDTA 10 mM, pH 7.5 and 2 mL of deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) was added. The headspace was filled with C 4 F 10 /Air (35/65) and the mixture was incubated for 2.5 hours at room temperature under gentle agitation (rotating wheel), in the dark. Conjugated bubbles were washed by centrifugation. 
     Example 36 
     Preparation of Ultrasound Contrast Agents Conjugated to KDR Binding Peptides 
     Ultrasound contrast agents comprising phospholipid-stabilized microbubbles conjugated to KDR-binding polypeptides of the invention were prepared as described below. 
     Distilled water (30 mL) containing 6 mg of dipalmitoylphosphatidylserine (DPPS, Genzyme), 24 mg of distearoylphosphatidylcholine (DSPC, Genzyme) and 3 g of mannitol was heated to 65 C in 15 minutes then cooled to room temperature. N-MPB-DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] Na salt—Avanti Polar Lipids) was added (5% molar—1.9 mg). This derivatized phospholipid was dispersed in the aqueous phase using an ultrasonic bath (Branson 1210—3 minutes). 
     Perfluoroheptane (2.4 mL from Fluka) was emulsified in this aqueous phase using a high speed homogenizer (Polytron®, 10000 rpm, 1 minute). 
     The emulsion was washed once by centrifugation (200 g/10 min) then resuspended in 30 mL of a 10% solution of mannitol in distilled water. The washed emulsion was frozen (−45 C, 5 minutes) then freeze dried (under 0.2 mBar, for 24 hours). 
     Atmospheric pressure was restored by introducing a mixture of C 4 F 10  and air. The lyophilizate was dissolved in distilled water (30 mL). Microbubbles were washed once by centrifugation and redispersed in 10 mL of Phosphate Buffer Saline. 
     Peptide Conjugation 
     Thioacetylated peptide (200 μg, SEQ ID NO:356) was dissolved in 20 μL DMSO and then diluted in 1 mL of Phosphate Buffer Saline (PBS). This solution was mixed to 5 mL of the N-MPB-functionalized microbubbles. 0.6 mL of deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) was added and the suspensions were stirred by inversion for 2 h30. 
     Microbubbles were washed twice with a solution of maltose 5% and Pluronic F68 0.05% in distilled water, by centrifugation (200 g/10 minutes). The final volume was fixed to 5 mL. 
     Example 37 
     Preparation of Ultrasound Contrast Agents Conjugated to KDR Binding Peptides 
     Ultrasound contrast agents comprising microballoons conjugated to KDR-binding polypeptides of the invention were prepared as described below. 
     Distilled water (30 mL) containing 40 mg of distearoylphosphatidylglycerol (DSPG, Genzyme) was heated to 65 C during 15 minutes then cooled to 40 C. 
     DPPE-PEG2000-Maleimide (3.5 mg—Avanti Polar Lipids) and tripalmitine (60 mg—Fluka) were dissolved in cyclohexane (0.6 mL) at 40 C in a ultrasound bath for 2 min. 
     This organic phase was emulsified in the aqueous phase using a high speed homogenizer (Polytron®, 10000 rpm, 1 minute). 
     Polyvinylalcohol (200 mg) dissolved in distilled water (5 mL) was added to the emulsion. The mixture was cooled to 5 C, then frozen (−45 C, 10 minutes) and finally freeze dried (under 0.2 mBar, for 24 hours). 
     The lyophilisate was dispersed in distilled water (15 mL). The mixture was stirred for 30 min to obtain a homogenous suspension of microballoons. 
     Peptide Conjugation 
     The thioacetylated peptide (200 μg) was dissolved in 20 μL DMSO then diluted with PBS (1 mL). 
     7.5 mL of the suspension of microballoons obtained as above described were centrifuged (500 rpm for 5 min). The infranatant was discarded and microballoons were redispersed in Phosphate Buffer Saline (2 mL). 
     The microcapsule suspension was mixed with the solution of peptide. Three hundred microliters of a hydroxylamine solution (10.4 mg in PBS 50 mM, pH: 7.5) was added to the suspension to deprotect the thiol. The suspension was stirred by inversion for two and a half hours. 
     The microballoons were washed twice by centrifugation (500 g/5 min) with distilled water containing 5% maltose and 0.05% Pluronic F68 and finally redispersed in 3 mL of this solution. 
     Example 38 
     Ultrasound Contrast Agents Conjugated to KDR Binding Polypeptides Bind to KDR-Expressing Cells In Vitro and In Vivo 
     The ability of ultrasound contrast agents conjugated to peptides of the invention to bind to KDR-expressing cells in vitro was assessed using 293H cells transfected to expresses KDR. Additionally, the ability of ultrasound contrast agents conjugated to KDR binding polypeptides of the invention to bind to KDR-expressing tissue in vivo was assessed using two known models of angiogenesis, the rat matrigel model and the rat MatB III tumor model. 
     Transfection of 293H Cells on Thermanox® Coverslips 
     293H cells were transfected with KDR DNA as set forth in Example 5. The transfected cells were incubated with a suspension of peptide-conjugated ultrasound contrast agents or with a control peptide (a scrambled version of the conjugated peptide having no affinity for KDR). 
     For the incubation with the transfected cells a small plastic cap is filled with a suspension containing 1 to 3×10 8  peptide-conjugated microbubbles and the cap covered with an inverted Thermanox® coverslip as to put the transfected cells in contact with the conjugated microbubbles. After about 20 min at RT, the coverslip is lifted with tweezers, rinsed three times in PBS and examined under a microscope to assess binding of the conjugated microbubbles. 
       FIG. 85  indicates that microballoons conjugated to peptides of the invention bind specifically to KDR-expressing cells. Indeed, microballoons conjugated to KDR-binding peptide bound to KDR-expressing cells while they did not bind appreciably to mock transfected cells and microballoons bearing a scrambled control peptide showed no appreciable binding. 
     Determination of the % of Surface Covered by Microvesicles 
     Images were acquired with a digital camera DC300F (Leica) and the percent of surface covered by bound microbubbles or microballoons in the imaged area was determined using the software QWin (Leica Microsystem AG, Basel, Switzerland). 
     The following table shows the results of the binding affinity (expressed as coverage % of the imaged surface) of targeted microvesicles of the invention to KDR transfected cells, as compared to the binding of the same targeted microvesicles towards Mock-transfected cells or (only in the case of the peptide) to the binding of microvesicles targeted with a scrambled peptide to the same KDR transfected cells. 
     As shown in Table 21, targeted microvesicles show increased binding affinity for KDR. 
                                 TABLE 21                          Coverage %                                         SEQ ID           Scrambled           NO   KDR   Mock   peptide                                                     Example 35   356   14.2%   1.4%   2.1%               277   3.5%   0.9%   n.a.               480   16.8%   1.0%   n.a.           Example 36   356   18.3%   0.4%   2.2%           Example 37   356   6.7%   0.2%   0.1%                        
In Vivo Animal Models
 
     Known models of angiogenic tissue (rat matrigel model and rat Mat B III model) were used to examine the ability of the peptide conjugated ultrasound conjugates to localize to and provide an image of angiogenic tissue. 
     Animals: 
     Female Fisher 344 rat (Charles River Laboratories, France) weighing 120 to 160 g were used for the MATBIII tumor implantation. Male OFA rats (Charles River Laboratories, France) weighing 100 to 150 g were used for Matrigel injection. 
     Anesthesia: 
     Rats were anesthetized with an intramuscular injection (1 mL/kg) of Ketaminol/xylazine (Veterinaria AG/Sigma) (50/10 mg/mL) mixture before implantation of Matrigel or MatBIII cells. For imaging experiments, animals were anesthetized with the same mixture, plus subcutaneous injection of 50% urethane (1 g/kg). 
     Rat MATBIII Tumor Model: 
     A rat mammary adenocarcinoma, designated 13762 Mat B III, was obtained from ATCC(CRL-1666) and grown in McCoy&#39;s 5a medium+10% FCS. 1% glutamine and 1% pen/strep (Invitrogen cat#15290-018). Cells in suspension were collected and washed in growth medium, counted, centrifuged and resuspended in PBS or growth medium at 1.10 7  cells per mL. For tumor induction: 1×10 6  cells in 0.1 mL were injected into the mammary fat pad of anesthetized female Fisher 344 rat. Tumors usually grow to a diameter of 5-8 mm within 8 days. 
     Rat Matrigel Model: 
     Matrigel (400 μL) (ECM, Sigma, St Louis, Mo.) containing human bFGF (600 ng/mL) (Chemicon: ref: GF003) was subcutaneously injected in the dorsal flank of each rat. 
     Matrigel solution was kept liquid at 4 C until injection. Immediately after matrigel injection, the injection site was maintained closed for a few seconds with the hand in order to avoid leaking of the matrigel. At the body temperature, matrigel becomes gelatinous. Ten days post-injection, neoangiogenesis was observed in matrigel plug of rat and imaging experiment were performed. 
     In Vivo Ultrasound Imaging: 
     Mat B III tumor or matrigel imaging was performed using an ultrasound imaging system ATL HDI 5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversion at low acoustic power (MI=0.05) was used to follow accumulation of peptide conjugated-microbubbles on the KDR receptor expressed on the endothelium of neovessels. For the control experiments, an intravenous bolus of unconjugated microbubbles or microbubbles conjugated to non-specific peptide was injected. The linear probe was fixed on the skin directly on line with the implanted tumors or matrigel plug and accumulation of targeted bubbles was followed during thirty minutes. 
     In both models, a perfusion of SonoVue® was administrated before injecting the test bubble suspension. This allows for the evaluation of the vascularization status; the video intensity obtained after SonoVue® injection is taken as an internal reference. 
     A baseline frame was recorded and then insonation was stopped during the bubble injection. At various time points after injection (1, 2, 5, 10, 15, 20, 25, 30 minutes) insonation was reactivated and 2 frames of one second were recorded on a videotape. 
     Video frames from matrigel or Mat B III tumor imaging experiments were captured and analysed with the video-capture and Image-Pro Plus 2.0 software respectively. The same rectangular Area of Interest (AOI) including the whole sectional area of the tumor or matrigel was selected on images at different time points (1, 2, 5, 10, 15, 20, 25, 30 minutes). At each time point, the sum of the video pixel inside the AOI was calculated after the subtraction of the AOI baseline. Results are expressed as the percentage of the signal obtained with SonoVue, which is taken as 100%. Similarly, a second AOI situated outside from matrigel or tumor, and representing the freely circulating contrast agent, is also analysed. 
     Results 
     The results indicate that ultrasound contrast agents bearing KDR binding moieties of the invention localize to angiogenic (and thus KDR expressing) tissue in animal models. Specifically,  FIG. 81  shows uptake and retention of bubble contrast in the tumor up to 30 minutes post injection for suspensions of phospholipids stabilized microbubbles conjugated to KDR peptides of the invention prepared according to Example 35. In contrast, the same bubbles showed only transient (no more than 10 minutes) visualization/bubble contrast in the AOI situated outside the tumor site. Similarly,  FIG. 82  and  FIG. 83  show uptake and retention of bubble contrast in the matrigel at up to 30 minutes post injection for suspensions of phospholipids stabilized microbubbles conjugated to KDR peptides of the invention (e.g., SEQ ID NOS:374 and 294, respectively) prepared according to Example 35. In contrast, the same bubbles showed only transient (no more than 10 minutes) visualization/bubble contrast in the AOI situated outside the matrigel site. 
     Example 39 
     Enhancing the Serum Residence of KDR-Binding Peptides 
     Compounds that contain maleimide and other groups that can react with thiols react with thiols on serum proteins, especially serum albumin, when the compounds are injected. The adducts have serum life times similar to serum albumin, more than 14 days in humans for example. 
     Conjugation to Maleimide 
     Methods are available that allow for the direct synthesis of maleimide-labeled linear peptides encompassed by the present invention (Holmes, D. et al., 2000 . Bioconjug. Chem.,  11:439-444). 
     Peptides that include disulfides can be derivatized with maleimide in one of several ways. For example, a third cysteine can be added at the carboxy terminus. The added cysteine is protected with protecting group that is orthogonal to the type of groups used for the cysteines that are to form the disulfide. The disulfide is formed by selectively deprotecting the intended cysteines and oxidizing the peptide. The final cysteine is then deprotected and the peptide reacted with a large molar excess of a bismaleimide. The resulting compound has one of the maleimides free to react with serum albumin or other thiol-containing serum proteins. 
     Alternatively, a cyclic peptide of the present invention is synthesized with a lysine-containing C-terminal extention, such as -GGGK (SEQ ID NO:262). Lysines of the KDR-binding motif are protected with ivDde and the C-terminal lysine is deprotected. This lysine is reacted with a maleimide-containing compound, such as N-[e-maleimidocaproyloxy]succinimide ester (Pierce Biotechnology, Rockford, Ill.) or N-(a-Maleimidoacetoxy)succinimide ester (Pierce Biotechnology). 
     Conjugation to a Moiety that Binds Serum Albumin Non-Covelently 
     Polypeptides having a molecular weight less than 50-60 kDa are rapidly excreted. Many small molecules, such as fatty acids, bind to serum albumin. Fatty acids containing 10 to 20 carbon atoms have substantial affinity for serum albumin. Linear and branched fatty acids can be used. This binding in serum can reduce the rate of excretion. Using methods known in the art, serum-albumin-binding moieties can be conjugated to any one of the peptides herein disclosed. The serum-albumin-binding moiety can be joined to the KDR-binding peptide through a linker. The linker can be peptidic or otherwise, such as PEG. Linkers of zero to about thirty atoms are preferred. It is preferred that the linker be hydrophilic. The serum-albumin-binding moiety can be conjugated to the KDR-binding peptide at either end or though a side group of an appended amino acid. Suitable side groups include lysine and cysteine. Such compounds can also comprise chelators for radionuclides, as discussed herein. A KDR-binding peptide joined to a serum-albumin-binding moiety will bind KDR. 
     Conjugation to PEG 
     Attachment of poly(ethyleneglycol) (PEG) to proteins and peptides enhances the serum residence of these molecules. Attachment of PEG (linear or branched) to a KDR-binding peptide is expected give substantial enhancement of serum residence time. The molecular weight of the PEG should be at least 10 kDA, more preferably at least 20 kDa, and most preferably 30 kDa or more. The PEG could be attached at the N- or C-terminus. Methods of attaching PEG to peptides are well known in the art (Roberts M. et al., 2002 . Adv. Drug. Deliv. Rev.,  54:459-476). PEG can be attached to reactive side groups such as lysine or cysteine. 
     Fusion to Serum Protein 
     Proteins comprising serum albumin (SA) and other proteins have enhanced serum residence times. The amino-acid sequence of human SA (hSA) is shown in Table 22. Table 23 shows a fusion protein comprising: AGDWWVE CRVGTGLC YRYDTGTGGGK (SEQ ID NO:286):: PGGSGGEGGSGGEGGRPGGSEGGTGG::mature hSA:: GGSGGEGGSGGEGGSGPGEGGEGSGGRP::GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO:294). The KDR-binding peptides are separated from mature hSA by linkers that are rich in glycine to allow flexible spacing. One need not use all of hSA to obtain an injectable protein that will have an enhanced serum residence time. Chemical groups, such as maleimide and alpha bromo carboxylates, react with the unpaired cysteine (residue 34) to form stable adducts. Thus, one can attach a single chelator to hSA fusion proteins so that the adduct will bind a radionuclide. One can prepare a chelator with a maleimide group and couple that to hSA or an hSA derivative. Alternatively, hSA or an hSA derivative can be reacted with a bismaleimide and a chelator carrying a reactive thiol could be reacted with the bismaleimide-derivatized hSA. 
     Construction of genes that encode a given amino-acid sequence are known in the art. Expression of HSA fusions in  Saccharomyces cerevisiae  is known in the art (Sleep, D et al., 1991 . Biotechnology  ( NY ), 9:183-187). 
     Pretargeting Radioactivity or Toxins to KDR Expressing Tumors 
     Conventional radioimmune cancer therapy is plagued by two problems. The generally attainable targeting ratio (ratio of administered dose localizing to tumor versus administered dose circulating in blood or ratio of administered dose localizing to tumor versus administered dose migrating to bone marrow) is low. Also, the absolute dose of radiation or therapeutic agent delivered to the tumor is insufficient in many cases to elicit a significant tumor response. Improvement in targeting ratio or absolute dose to tumor would be of great importance for cancer therapy. 
     The present invention provides methods of increasing active agent localization at a target cell site of a mammalian recipient. The methods include, for example, a) administering to a recipient a fusion protein comprising a targeting moiety and a member of a ligand-anti-ligand binding pair; b) thereafter administering to the recipient a clearing agent capable of directing the clearance of circulating fusion protein via hepatocyte receptors of the recipient, wherein the clearing agent incorporates a member of the ligand-anti-ligand binding pair; and c) subsequently administering to the recipient an active agent comprising a ligand/anti-ligand binding pair member. 
     Hexoses, particularly the hexoses galactose, glucose, mannose, mannose-6-phosphate, N-acetylglucosamine, pentamannosyl phosphate, N-acetylgalactosamine, thioglycosides of galactose, and mixtures thereof are effective in causing hepatic clearance. Binding of sugars to hepatic receptors is not, however, the only means of directing a molecule to the liver.) 
     Clearance of carcinoembryonic antigen (CEA) from the circulation is by binding to Kupffer cells in the liver. We have shown that CEA binding to Kupffer cells occurs via a peptide sequence YPELPK representing amino acids 107-112 of the CEA sequence. This peptide sequence is located in the region between the N-terminal and the first immunoglobulin like loop domain. Using native CEA and peptides containing this sequence complexed with a heterobifunctional crosslinking agent and ligand blotting with biotinylated CEA and NCA we have shown binding to an 80 kD protein on the Kupffer cell surface. This binding protein may be important in the development of hepatic metastases. (Thomas, P. et al., 1992 . Biochem. Biophys. Res. Commun.,  188:671-677 
     To use YPELPK (SEQ ID NO:498) as a clearance agent, one fuses this sequence via a linker to a moiety that binds the fusion protein (Ab). For example, if the Ab has affinity for DOTA/Re, one would make a derivative having YPELPK attached to DOTA/Re; for example, rvYPELPKpsGGG-DOTA. ‘rvYPELPKps’ is a fragment of CEA that includes the YPELPK sequence identified by Thomas et al. Any convenient point on DOTA can be use for attachment. RVYPELPKPSGGG-DOTA/cold Re (SEQ ID NO:499) would then be used as a clearing agent. The Fab corresponding to the fusion Ab would have affinity for the clearing agent of Kd&lt;100 nM, preferably Kd&lt;10 nM, and most preferably Kd&lt;1 nM. 
     The therapeutic agent would contain DOTA/ 185 Re. In a preferred embodiment, the therapeutic agent would contain two or more DOTA moieties so that the Ab immobilized on the tumor would bind the bis-DOTA compound with high avidity. The two DOTA moieties would preferably be connected with a hydrophilic linker of ten to thirty units of PEG. PEG is a preferred linker because it is not degraded, promotes solubility. Ten to thirty units of PEG is not sufficient to give the bis DOTA compound a very long serum residence time. A half-life of 30 minutes to 10 hours is acceptable. The serum half life should be longer than the radioactive half life of the radionuclide used so that most of the radiation is delivered to the tumor or to the external environment. 
     In one embodiment, a “fusion protein” of the present invention comprises at least one KDR-binding peptide fused to the amino terminus or the carboxy terminus of either the light chain (LC) or the heavy chain (HC) of a human antibody. Optionally and preferably, two or more KDR-binding peptides are fused to the antibody. The antibody is picked to have high affinity for a small molecule that can be made radioactive or have a toxin attached. Preferably, the affinity of the Fab corresponding to the Ab has affinity for the small molecule with Kd less than 100 nM, more preferably less than 10 nM, and most preferably less than 1 nM. The small molecule could be a chelator capable of binding a useful radioactive atom, many of which are listed herein. The small molecule could be a peptide having one or more tyrosines to which radioactive iodine can be attached without greatly affecting the binding property of the peptide. 
     Any KDR-binding peptide (KDR-BP) of the present invention can be fused to either end of either chain of an antibody that is capable of binding a small radioactive compound. Useful embodiments include: 
     1) KDR-BP#1::link::LC/HC, 
     2) LC::link::KDR-BP#1/HC, 
     3) LC/KDR-BP#1::link::HC, 
     4) LC/HC::link::KDR-BP#1, 
     5) KDR-BP#1::link1::LC::link2::KDR-BP#2/HC, 
     6) LC/KDR-BP#1::link1::HC::link2::KDR-BP#2, 
     7) KDR-BP#1::link1::LC/KDR-BP#2::link2::HC, 
     8) KDR-BP#1::link1::LC/HC::link2:: KDR-BP#2, 
     9) LC::link1::KDR-BP#1/KDR-BP#2::link2::HC, 
     10) LC::link1::KDR-BP#1/HC::link2:: KDR-BP#2, 
     11) KDR-BP#1::link1::LC::link2::KDR-BP#2/KDR-BP#3::link3::HC, 
     12) KDR-BP#1::link1::LC::link2::KDR-BP#2/HC::link3::KDR-BP#3, 
     13) KDR-BP#3::link3::LC/KDR-BP#1::link1::HC::link2::KDR-BP#2, 
     14) LC::link3::KDR-BP#3/KDR-BP#1::link1::HC::link2::KDR-BP#2, and 
     15) KDR-BP#1::link1::LC::link2::KDR-BP#2/KDR-BP#3::link3::HC::link4::KDR-BP#4. 
     In cases (5)-(15), the linkers (shown as “link1”, “link2”, “link3”, and “link4”) can be the same or different or be absent. These linkers, if present, are preferably hydrophilic, protease resistant, non-toxic, non-immunogenic, and flexible. Preferably, the linkers do not contain glycosylation sites or sequences known to cause hepatic clearance. A length of zero to fifteen amino acids is preferred. The KDR-binding peptides (KDR-BP#1, #2, #3, and #4) could be the same or different. If the encoded amino-acid sequences are the same, it is preferred that the DNA encoding these sequences is different. 
     Since antibodies are dimeric, each fusion protein will present two copies of each of the fused peptides. In case (15), there will be eight KDR-BPs present and binding to KDR-displaying cells should be highly avid. It is possible that tumor penetration will be aided by moderate KDR affinity in each of the KDR-BPs rather than maximal affinity. 
     One group of preferred embodiments have SEQ ID NO:294 as one of the KDR-BPs and SEQ ID NO:286 as the other. For example, in case (7) (KDR-BP#1::link1::LC/KDR-BP#2::link2::HC), KDR-BP#1 is SEQ ID NO:294 and KDR-BP#2 is SEQ ID NO:286 and link1 is between 10 and 20 amino acids and link2 is also between ten and twenty amino acids. A suitable sequence for link1 is GGSGGEGRPGEGGSG (SEQ ID NO:491) and a suitable sequence for link2 is GSESGGRPEGGSGEGG (SEQ ID NO:492). Other sequences rich in Gly, Ser, Glu, Asp, Thr, Gln, Arg, and Lys are suitable. To reduce the risk of proteolysis, it is preferred to follow Arg or Lys with Pro. To avoid difficulties in production and poor solubility, it is preferred to avoid long stretches (more than twelve) of uncharged residues. Since the peptides are displayed at the amino termini of LC and HC, the combined linker length will allow them to bind to KDR simultaneously. Additionally, in case (15) (KDR-BP#1::link1::LC::link2::KDR-BP#2/KDR-BP#3::link3::HC::link4::KDR-BP#4), KDR-BP#1 and KDR-BP#2 are SEQ ID NO:294 and KDR-BP#3 and KDR-BP#4 are SEQ ID NO:29. Link1 and link3 are 10 to 20 amino acids and link2 and link4 are each 15 to 30 amino acids. Link2 and link4 are longer because they need to allow a peptide on the carboxy terminus of LC to reach a peptide on the carboxy terminus of HC. 
     The fusion protein is produced in eukaryotic cells so that the constant parts of the HC will be glycosylated. Preferably, the cells are mammalian cells, such as CHO cells. 
     The fusion proteins are injected into a patient, and time is allowed for the fusion protein to accumulate at the tumor. A clearing agent is injected so that fusion protein that has not become immobilized at the tumor will be cleared. In previous pretargeting methods, the antibody combining site has been used to target to the tumor and biotin/avidin or biotin/streptavidin has been used to attach the radioactive or toxic agent to the immobilized antibody. The biotin/avidin or streptavidin binding is essentially irreversible. Here we fuse a target-binding peptide to the antibody that is picked to bind a radioactive or toxic agent. Because the fusion protein contains 2, 4, 6, or 8 KDR-BPs, binding of the fusion protein to the tumor is very avid. A clearing agent that will cause fusion protein not immobilized at the tumor to clear can be administered between 2 and 48 hours of the injection of the fusion protein. Because the clearance agent is monomeric in the moiety that binds the antibody, complexes of clearance agent and immobilized fusion protein will not have very long life times. Within 4 to 48 hours of injecting clearance agent, the immobilized antibody will have lost any clearance agent that binds there. The active agent is, preferably, dimeric in the moiety that binds the fusion protein. The active agent is injected between 2 and ˜48 hours of injection of clearance agent. 
     
       
         
           
               
             
               
                 TABLE 22 
               
               
                   
               
               
                 Amino-acid sequence of Mature HSA from 
               
               
                 GenBank entry AAN17825 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV 
               
               
                   
               
               
                   
                 KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL 
               
               
                   
               
               
                   
                 RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 
               
               
                   
               
               
                   
                 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR 
               
               
                   
               
               
                   
                 YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC 
               
               
                   
               
               
                   
                 ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 
               
               
                   
               
               
                   
                 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE 
               
               
                   
               
               
                   
                 KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA 
               
               
                   
               
               
                   
                 EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYKTTLEKC 
               
               
                   
               
               
                   
                 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE 
               
               
                   
               
               
                   
                 YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH 
               
               
                   
               
               
                   
                 PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 
               
               
                   
               
               
                   
                 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE 
               
               
                   
               
               
                   
                 RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK 
               
               
                   
               
               
                   
                 ADDKETCFAE EGKKLVAASR AALGL (SEQ ID NO: 500) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
               
                   
               
               
                 SEQ ID NO: 286::linker1::HSA::linker2::SEQ 
               
               
                 ID NO: 294 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 AGDWWVE CRVGTGLC YRYDTGTGGGK 
               
               
                   
               
               
                   
                 PGGSGGEGGSGGEGGRPGGSEGGTGG 
               
               
                   
               
               
                   
                 DAHKSEVAHR FKDLGEENFK ALVLIAFAQY LQQCPFEDHV 
               
               
                   
               
               
                   
                 KLVNEVTEFA KTCVADESAE NCDKSLHTLF GDKLCTVATL 
               
               
                   
               
               
                   
                 RETYGEMADC CAKQEPERNE CFLQHKDDNP NLPRLVRPEV 
               
               
                   
               
               
                   
                 DVMCTAFHDN EETFLKKYLY EIARRHPYFY APELLFFAKR 
               
               
                   
               
               
                   
                 YKAAFTECCQ AADKAACLLP KLDELRDEGK ASSAKQRLKC 
               
               
                   
               
               
                   
                 ASLQKFGERA FKAWAVARLS QRFPKAEFAE VSKLVTDLTK 
               
               
                   
               
               
                   
                 VHTECCHGDL LECADDRADL AKYICENQDS ISSKLKECCE 
               
               
                   
               
               
                   
                 KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA 
               
               
                   
               
               
                   
                 EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYKTTLEKC 
               
               
                   
               
               
                   
                 CAAADPHECY AKVFDEFKPL VEEPQNLIKQ NCELFEQLGE 
               
               
                   
               
               
                   
                 YKFQNALLVR YTKKVPQVST PTLVEVSRNL GKVGSKCCKH 
               
               
                   
               
               
                   
                 PEAKRMPCAE DYLSVVLNQL CVLHEKTPVS DRVTKCCTES 
               
               
                   
               
               
                   
                 LVNRRPCFSA LEVDETYVPK EFNAETFTFH ADICTLSEKE 
               
               
                   
               
               
                   
                 RQIKKQTALV ELVKHKPKAT KEQLKAVMDD FAAFVEKCCK 
               
               
                   
               
               
                   
                 ADDKETCFAE EGKKLVAASR AALGL 
               
               
                   
               
               
                   
                 GGSGGEGGSGGEGGSGPGEGGEGSGGRP 
               
               
                   
               
               
                   
                 GDSRVCWEDSWGGEVCFRYDPGGGK (SEQ ID NO: 501) 
               
               
                   
               
            
           
         
       
     
     Example 40 
     Synthesis of Dimers D30 and D31 
     Preparation of Ac-VCWEDSWGGEVCFRYDPGGGK{[PnA06-Glut-K(-Glut-JJ-NH(CH 2 ) 4 —(S)—CH(Ac-AQDWYYDEILJGRGGRGGRGG-NH)C(═O)NH 2 ]—NH 2 }—NH 2 : Dimer D30 
     Preparation of Ac-VC WEDS WGGEVCFRYDPGGGK[PnAO6-Glut-K]—NH 2  (Compound 3;  FIG. 87A ) 
     Ac-VCWEDSWGGEVCFRYDPGGGK[K(iV-Dde)]-NH 2  [(1), comprising SEQ ID NO:494, is a SEQ ID NO:374 derivative; specifically Acetyl-(SEQ ID NO:374, 5-21)-GGGK[K(iV-Dde), 48 mg] was prepared by the procedures of Method 5. The compound was dissolved in DMF (0.85 mL) and treated with compound B and DIEA (7 μL) was added to maintain the basicity of the reaction mixture. The progress of the reaction was monitored by HPLC and mass spectroscopy. At the completion of the reaction (20 h), the volatiles were removed in vacuo. The residue, which consists of a compound 2 (SEQ ID NO:374, 5-21) derivative, specifically Acetyl-(SEQ ID NO:374, 5-21)-GGGK[(PnAO6-Glut-)K(iV-Dde)]-NH2), was treated with 10% hydrazine in DMF (5 μL) for 10 min. HPLC analysis and mass spectroscopy indicated the completion of the reaction. The mixture then was applied directly to a Waters Associates XTerra MSC18 preparative HPLC column (50 mm×19 mm i.d.) and purified by elution with a linear gradient of acetonitrile into water (both containing 0.1% TFA) to provide 11 mg of pure Compound 3. 
     Preparation of the Dimer D30 from Compound 3 and Ac-AQDWYYDEIL-Adoa-GRGGRGGRGGGK(Adoa-Adoa)-NH 2  (Compound 4 (Comprising SEQ ID NO:617 with Modified Lysine Side Chains; Based on the Peptide Binding Moiety of SEQ ID NO:376)). 
     Disuccinimidyl glutarate (12 mg) was dissolved in DMF (500 μL), and DIEA was added (1 μL). Compound 3 in DMF was added into the DMF solution of disuccinimidyl glutarate/DIEA. The mixture was stirred for 2.5 h. HPLC and mass spectroscopy indicated the completion of the reaction. The volatiles were removed in vacuo and the residue was washed with ether (3×) to remove the unreacted bis-NHS ester. The residue was dried, re-dissolved in anhydrous DMF and treated with the Compound 4, Ac-AQDWYYDEIL-Adoa-GRGGRGGRGGGK(Adoa-Adoa)-NH 2 , which was prepared by Method 5 and Method 8, in the presence of 2 equivalents of DIEA. The reaction was allowed to proceed for 20 h. The mixture then was applied directly to a Waters Associates MSC18 reverse phase preparative (50 mm×19 mm i.d.) HPLC column and purified by elution with a linear gradient of acetonitrile into water (both containing 0.1% TFA) to provide 2 mg of D30 (For purification and structure of D30, see below and also  FIGS. 87B  and C, respectively). 
     Synthesis of Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK[SGS-Glut-SGS-(S)—NH(CH 2 ) 4 —CH(Biotin-JJ-NH)—C(═O)]—NH 2 ]—NH 2 : D31 
     Preparation of Monomer Compound 2 and Monomer Compound 4 
     See  FIG. 88B . 
     Synthesis of Monomer Peptide 1 and Monomer Peptide 3 
     Monomer Peptide 1 comprises SEQ ID NO:378 with the following modification: it is an Ne22-iV-Dde-SEQ ID NO:378 peptide. 
     Monomer peptide 3 comprises SEQ ID NO:370, and is a derivative of SEQ ID NO:337. It is an Ne25-iV-Dde-SEQ ID NO:370 peptide. 
     Synthesis of the monomers 1 and 3 were carried out using the procedures of Method 5 for the ABI 433A synthesizer. 
     Synthesis of Monomer Peptide 2 and Monomer Peptide 4 
     See  FIGS. 88A and 88B . 
     Appendage of Biotin-JJ, Lys, Gly and Ser onto Compounds 1 and 3 was done by SPPS manually using the appropriate Fmoc amino acids, Biotin-JJ and Fmoc-J (J=8-amino-3,6-dioxaoctanoic acid) according to the procedures of Methods 6, 7, 8, 9 and 10. Cleavage of the peptides from the resin, processing of the crude peptides was carried out as described in Method 1 for the synthesis of peptides. Cyclization of the cysteine moieties to form the cyclic disulfide peptides was performed by the procedures of Method 9. 
     Purification of the peptides was carried out using a Shimadzu LC-10A HPLC system and a YMC C-18 ODS preparative HPLC column employing a linear gradient elution of acetonitrile (0.1% TFA) into 0.1% aqueous TFA. Pure fractions were combined and lyophilized. 
     The dimer D31 was prepared using monomer compound 4 to generate, in situ, the activated monomer compound 5, which was then reacted with monomer compound 2 using the procedures described in Method 13, entitled: ‘Preparation of Heterodimer Containing Constructs’. The crude compound D31 was purified by preparative reverse phase HPLC using a Waters-YMC C-18 ODS column to provide 10 mg of the dimer D31. 
     Example 41 
     In Vitro Competition Experiments on KDR-Transfected Cells 
     The following experiment assessed the specificity of the binding of peptide-conjugated microbubbles to KDR-expressing cells. 
     Protocol: 
     293H cells were transfected with KDR cDNA. The transfected cells were incubated with a suspension of peptide-conjugated microbubbles in presence or absence of the corresponding free peptide (at 100, 30, 10, 3, 1, 0.3, 0.1 μM). Microbubbles were conjugated to a SATA-modified peptide comprising SEQ ID NO:480, a SATA-modified peptide comprising SEQ ID NO:356, or a SATA-modified peptide comprising SEQ ID NO:356 and a JJ linker. Competition was also performed using the corresponding non-binding or control free peptide as competing compound. At the end of the incubation, the transfected cells were rinsed three times in PBS and examined under a microscope. Binding of the conjugated bubbles was quantified and expressed as percent of surface covered by the targeted microbubbles. 
     Results: 
     All the KDR-conjugated microbubbles were competed off by the corresponding free KDR-specific peptide whereas the presence of control peptide had no effect. Example of curves obtained by plotting the fraction of residual binding as a function of the competitor concentration are shown in  FIG. 89 . 
     Example 42 
     In Vitro Competition Experiments on KDR-Transfected Cells 
     The following experiment assessed the specificity of the binding of peptide-conjugated microbubbles to KDR-expressing cells. 
     Protocol: 
     293H cells were transfected with KDR cDNA. The transfected cells were incubated with a suspension of peptide-conjugated microbubbles in presence or absence of the corresponding free peptide (between 100 μM to 3 nM). Competition was also performed using a non-binding peptide as competing compound. At the end of the incubation, the transfected cells were rinsed three times in PBS and examined under a microscope. Binding of the conjugated bubbles was quantified and expressed as percent of surface covered by the targeted microbubbles. 
     Results: 
     Microbubbles conjugated to KDR-specific dimer (D23) or monomer (SEQ ID NO:338) molecules were competed off by the corresponding free KDR-specific peptide whereas the presence of control peptide had no effect. Example of curves obtained by plotting the fraction of residual binding as a function of the competitor concentration are shown in  FIG. 90 . 
     In Vitro Competition Experiments on KDR-Transfected Cells 
     The following experiment compares the binding efficiency of monomers and dimers conjugated to microbubbles on KDR-transfected cells. 
     Protocol: 
     293H cells were transfected with KDR cDNA. The transfected cells were incubated with a suspension of microbubbles conjugated to different peptides (monomers or dimers) in presence or absence of increasing concentrations of free dimer (at 1000, 300, 100, 30, 10, 3, 1 nM). At the end of the incubation, the transfected cells were rinsed three times in PBS and examined under a microscope. Binding of the conjugated bubbles was quantified and expressed as percent of surface covered by the targeted microbubbles. 
     Results: 
     Microbubbles conjugated to D23 were more resistant to competition and less easily displaced by the corresponding free dimeric peptide than KDR-specific monomer-conjugated microbubbles conjugated to SEQ ID NO:338 or SEQ ID NO:376. Representative curves obtained by plotting the fraction of residual binding as a function of the competitor concentration are shown in  FIG. 91 . 
     Example 43 
     In Vitro Binding of Heteromultimers and Dimers Compared to Multimeric Monomers 
     The following experiment aims at comparing the binding efficiency of mixed monomers, dimers and monomers conjugated to microbubbles in the KDR-transfected cells assay. 
     Protocol: 
     Microbubbles were conjugated to either a dimer (D23) or two different peptides monomers (SEQ ID NO:294 or SEQ ID NO:480). A fourth conjugation reaction was performed using equal quantities of each monomer (and the same total peptide load). 293H cells were transfected with KDR cDNA. The transfected cells were incubated with the same number of targeted microbubble and in presence of 50% human serum. At the end of the incubation, the transfected cells were rinsed three times in PBS and examined under a microscope. Binding of the conjugated bubbles was quantified and expressed as percent of surface covered by the targeted microbubbles. 
     Results: 
     As shown in  FIG. 92 , microbubbles conjugated with SEQ ID NO:294 bound poorly compared with microbubbles conjugated with SEQ ID NO:480 or dimer D23. Surprisingly, microbubbles conjugated to D23 bound equivalently to those conjugated to SEQ ID NO:480 although D23 has half the load. Moreover, the “mixed monomer” conjugated microbubbles, which also have half the SEQ ID NO:480 load, bound as well as microbubbles conjugated with SEQ ID NO:480 or D23. These results show the increased binding capacity of heteromultimers. 
     Example 44 
     Blocking VEGF-Enhanced Peritoneal Vascular Permeability with a Heterodimeric Peptide 
     In this example, the ability of heterodimer D10 to inhibit the enhanced vascular permeability caused by VEGF injected into the peritoneum of nude mice is demonstrated. 
     Protocol 
     Male balb/c nu/nu mice were injected intraperitoneally with 2 mL vehicle (1% bovine serum albumin in 95% saline/5% DMSO), vehicle+1.2 nM VEGF 165 , or vehicle+1.2 nM VEGF 165 +20 μM D10. Immediately after, the mice were injected with Evan&#39;s Blue Dye (0.5% in saline, 4 mL/kg) i.v. via their tail veins. After 60 min, mice were sacrificed by CO 2  asphyxiation and the peritoneal fluid was retrieved. After centrifuging the samples briefly, the absorbance at 590 nm was measured for each. 
     Results 
     As shown in  FIG. 93 , VEGF, when added to the fluid injected intraperitoneally, significantly increased the dye leakage into the peritoneum, and this increase was substantially blocked by including D10 with the VEGF. 
     Example 45 
     Mouse Xenograft Tumor Model of Human Colon Cancer 
     This example assesses the effects of dimer D6 that has been processed into biodegradable sustained release pellets. Since D6 has a half-life on the order of 1 hour, a way of improving the residence time in sera was sought. The compound is formulated into a sustained release format so that greater therapeutic benefit to animal models is observed. 
     The effect of D6 on the tumor model is determined, for example, by measuring tumor size with and without treatment. Additionally, the effect of D6, engineered to have a longer residence time in sera, is compared to the effect of unmodified D6 (see Example 39). 
     Briefly, 140 nude mice are injected subcutaneously with the cell line, SW-480. Tumors are measured, and when tumors reach 100-200 mg, 100 animals are selected and randomized into 10 study groups of 10 animals each. The overall study is summarized in Table 24 below. The dosing schedule follows the chart shown in Table 25. Tumor measurements are taken on each animal twice a week during the normal workweek. Measurements are made by hand-held vernier caliper. Body weights and tumor measurements are recorded twice a week. This study is based on a typical four week study from beginning of dosing and includes removal of 30 tumors. 
     
       
         
           
               
             
               
                 TABLE 24 
               
               
                   
               
               
                 D6 Mouse Tumor Study 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Cell line 
                 SW-480, human colon carcinoma 
               
               
                   
                   
                 5 × 10 6 , subcutaneous 
               
               
                   
                 Test Animal 
                 nude mouse (CRL: NU/NU = nuBR) 
               
               
                   
                   
                 female 
               
               
                   
                   
                 n = 10/test group 
               
               
                   
                 Study Initiation 
                 &gt;6 weeks age 
               
               
                   
                   
                 Tumor ~100 −/+ 50 mg 
               
               
                   
                 Control 
                 1. untreated 
               
               
                   
                   
                 2. Vehicle 
               
               
                   
                   
                 3. Placebo pellet 
               
               
                   
                   
                 4. Cisplatin 
               
               
                   
                 Test Article 
                 D6 
               
               
                   
                   
                 0.5 mg/kg/d × 21 d 
               
               
                   
                   
                 2.0 mg/kg/d × 21 d 
               
               
                   
                   
                 2.0 mg/kg/d × 21 d pellet 
               
               
                   
                 Test Article Form 
                 1. solution for injection (PBS, IP) 
               
               
                   
                   
                 2. sustained release pellet (nominal 
               
               
                   
                   
                 21 day, subcutaneous) 
               
               
                   
                 Primary endpoints 
                 1. tumor growth 
               
               
                   
                   
                 2. histopathology (necropsy) 
               
               
                   
                 Supplementary 
                 1. angiogenesis (CD-31+) 
               
               
                   
                 measures 
                 2. cell proliferation (PCNA) 
               
               
                   
                 (representative 
                 3. circulating D6 
               
               
                   
                 samples) 
               
               
                   
                   
                 4. 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 25 
               
               
                   
               
               
                   
                   
                 Vehicle 
                 D6 Dose 
                 Cisplatin Dose 
               
               
                 Treatment 
                   
                 Administra- 
                 Administra- 
                 Administra- 
               
               
                 Group 
                 n 
                 tion 
                 tion 
                 tion 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 10 
                 — 
                 — 
                 — 
               
               
                 2 
                 10 
                 PBS 
                 — 
                 — 
               
               
                   
                   
                 1 IP inj/ 
               
               
                   
                   
                 d, 21 d 
               
               
                 3 
                 10 
                 — 
                 0.5 mg/kg/day 
                 — 
               
               
                   
                   
                   
                 1 IP inj/ 
               
               
                   
                   
                   
                 d, 21 d 
               
               
                 4 
                 10 
                 — 
                 2.0 mg/kg/day 
                 — 
               
               
                   
                   
                   
                 1 IP inj/ 
               
               
                   
                   
                   
                 d, 21 d 
               
               
                 5 
                 10 
                 Vehicle 
                 — 
                 — 
               
               
                   
                   
                 pellet 
               
               
                   
                   
                 (1), sc 
               
               
                 6 
                 10 
                 — 
                 2.0 mg/kg/day 
                 — 
               
               
                   
                   
                   
                 pellet (1), sc 
               
               
                 7 
                 10 
                 — 
                 — 
                 [6 mg/kg] 
               
               
                   
                   
                   
                   
                 1 IV inj/2 days, 
               
               
                   
                   
                   
                   
                 to 5 Ttl 
               
               
                 8 
                 10 
                 — 
                 2.0 mg/kg/day 
                 [6 mg/kg] 
               
               
                   
                   
                   
                 pellet (1), sc 
                 1 IV inj/2 days 
               
               
                   
                   
                   
                   
                 to 5 Ttl 
               
               
                 9 
                 10 
                 — 
                 2.0 mg/kg/day 
                 [3 mg/kg] 
               
               
                   
                   
                   
                 pellet (1), sc 
                 1 IV/2 days, 
               
               
                   
                   
                   
                   
                 to 5 Ttl 
               
               
                 10 
                 10 
                 — 
                 2.0 mg/kg/day 
                 [1 mg/kg] 
               
               
                   
                   
                   
                 pellet (1), sc 
                 1 IV/2 days, 
               
               
                   
                   
                   
                   
                 to 5 Ttl 
               
               
                   
               
            
           
         
       
     
     Example 46 
     The following example describes the preparation of an ultrasound contrast agent conjugated to a KDR-binding heterodimer of the invention and the ability of the heterdimer conjugated contrast agent to localize to KDR-expressing cells in vitro and angiogenic tissue in vivo. 
     Preparation of Derivatized Microbubbles for Peptide Conjugation 
     200 mg of DSPC (distearoylphosphatidylcholine), 275 mg of DPPG.Na (distearoylphosphatidylglycerol sodium salt) and 25 mg of N-MPB-PE were solubilized at 60° C. in 50 mL of Hexan/isopropanol (42/8). The solvent was evaporated under vacuum, and then PEG-4000 (35.046 g) was added to the lipids and the mixture was solubilized in 106.92 g of t-butyl alcohol at 60° C., in a water bath. The solution was filled in vials with 1.5 mL of solution. The samples were rapidly frozen at −45° C. and lyophilized. The air in the headspace was replaced with a mixture of C 4 F 10 /Air (50/50) and vials capped and crimped. The lyophilized samples were reconstituted with 10 mL saline solution (0.9%-NaCl) per vial, yielding a suspension of phospholipids stabilized microbubbles. 
     Peptide Conjugation 
     D23 was conjugated with a preparation of microbubbles as above described, according to the following methodology. The thioacetylated peptide (200 μg) was dissolved in 20 μL DMSO and then diluted in 1 ml of Phosphate Buffer Saline (PBS). This solution was mixed to the N-MPB-functionalized microbubbles dispersed in 18 mL of PBS-EDTA 10 mM, pH 7.5, and 2 mL of deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCl, pH 7.5) was added. The headspace was filled with C 4 F 10 /Air (50/50) and the mixture was incubated for 2.5 hours at room temperature under gentle agitation (rotating wheel), in the dark. Conjugated bubbles were washed by centrifugation. Similarly, the monomer peptides making up D23 were separately conjugated to two different microbubble preparations according to the methodology described above. 
     In Vitro Assay on Transfected Cells 
     The ability of phospholipid stabilized microbubbles conjugated to peptides and heteromultimeric peptide constructs of the invention to bind to KDR-expressing cells was assessed using 293H cells transfected to expresses KDR. 
     Transfection of 293H Cells on Thermanox® Coverslips 
     293H cells were transfected with KDR DNA as set forth in Example 5. The transfected cells were incubated with a suspension of peptide-conjugated microbubbles prepared as described above. For the incubation with the transfected cells a small plastic cap is filled with a suspension containing 1 to 3×10 8  peptide-conjugated microbubbles and the cap covered with an inverted Thermanox® coverslip is placed so that the transfected cells are in contact with the conjugated microbubbles. After about 20 min at room temperature, the coverslip is lifted with tweezers, rinsed three times in PBS and examined under a microscope to assess binding of the conjugated microbubbles. 
     Determination of the Percent of Surface Covered by Microvesicles 
     Images were acquired with a digital camera DC300F (Leica) and the percent of surface covered by bound microbubbles in the imaged area was determined using the software QWin (Leica Microsystem AG, Basel, Switzerland). Table 26 shows the results of the binding affinity (expressed as coverage % of the imaged surface) of targeted microvesicles of the invention to KDR transfected cells, as compared to the binding of the same targeted microvesicles to Mock-transfected cells. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 26 
               
             
            
               
                   
                   
               
               
                   
                 Conjugated microbubbles 
                   
                 % of covered 
                   
               
               
                   
                 prepared as described above 
                   
                 surface 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Peptide code 
                 Batch Id 
                 KDR 
                 Mock 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ ID NO: 294 
                 BG1979T02 
                 3.5% 
                 0.9% 
               
               
                   
                 Derivative 
               
               
                   
                 SEQ ID NO: 480 
                 BG1980T02 
                 16.8% 
                 1.0% 
               
               
                   
                 Derivative 
               
               
                   
                 D23 (dimer) 
                 BG2002T02 
                 22.9% 
                 3.3% 
               
               
                   
                 SEQ ID NO. 
                 BG1958T02 
                 12.9% 
                 0.8% 
               
               
                   
                 294/SEQ ID 
               
               
                   
                 NO: 480 Deriv. 
               
               
                   
                   
               
            
           
         
       
     
     Where the SEQ ID NO:294-derived sequence and the SEQ ID NO:480-derived sequence are separately attached to phospholipid stabilized microbubbles as monomers the resulting preparations achieve binding of the bubbles to KDR transfected cells in vitro to a different extent (3.5% and 16.8%). When a preparation of phospholipid stabilized microbubbles resulting from the addition of equal quantities of each of these peptide monomers (but the same total peptide load) is tested in the same system, 12.9% binding is achieved. Binding is a little more than the average of the two but as it is achieved with two sequences that bind to different sites on the target will be more resistant to competition at one or other of the sites on the target. However, for D23, the dimer, binding is increased to 22.9% (with the same peptide load). These results indicate that hetromultimers of the invention permit increased binding and increased resistance to competition. 
     In Vivo Animal Models 
     A known model of angiogenic tissue (the rat Mat B III model) was used to examine the ability of phospholipids-stabilized microbubbles conjugated to a heteromultimer of the invention to localize to and provide images of angiogenic tissue. 
     Female Fisher 344 rat (Charles River Laboratories, France) weighing 120 to 160 g were used for the MATBIII tumor implantation. Male OFA rats (Charles River Laboratories, France) weighing 100 to 150 g were used for Matrigel injection. 
     Anesthesia 
     Rats were anesthetized with an intramuscular injection (1 mL/kg) of Ketaminol®/xylazine (Veterinaria AG/Sigma) (50/10 mg/mL) mixture before implantation of Matrigel or MatBIII cells. For imaging experiments, animals were anesthetized with the same mixture, plus subcutaneous injection of 50% urethane (1 g/kg). 
     Rat MATBIII Tumor Model 
     A rat mammary adenocarcinoma, designated 13762 Mat B III, was obtained from ATCC(CRL-1666) and grown in McCoy&#39;s 5a medium+10% FCS. 1% glutamine and 1% pen/strep (InVitrogen cat#15290-018). Cells in suspension were collected and washed in growth medium, counted, centrifuged and resuspended in PBS or growth medium at 1×10 7  cells per mL. For tumor induction: 1×10 6  cells in 0.1 mL were injected into the mammary fat pad of anesthetized female Fisher 344 rat. Tumors usually grow to a diameter of 5-8 mm within 8 days. 
     In Vivo Ultrasound Imaging 
     Tumor imaging was performed using an ultrasound imaging system ATL HDI 5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversion at low acoustic power (MI=0.05) was used to follow accumulation of peptide conjugated-microbubbles on the KDR receptor expressed on the endothelium of neovessels. For the control experiments, an intravenous bolus of unconjugated microbubbles or microbubbles conjugated to non-specific peptide was injected. The linear probe was fixed on the skin directly on line with the implanted tumors and accumulation of targeted bubbles was followed during thirty minutes. 
     A perfusion of SonoVue® was administrated before injecting the test bubble suspension. This allows for the evaluation of the vascularization status and the video intensity obtained after SonoVue® injection is taken as an internal reference. 
     A baseline frame was recorded and then insonation was stopped during the injection of the microbubbles. At various time points after injection (1, 2, 5, 10, 15, 20, 25, 30 minutes) insonation was reactivated and 2 frames of one second were recorded on a videotape. 
     Video frames from tumor imaging experiments were captured and analysed with the video-capture and Image-Pro Plus 2.0 software respectively. The same rectangular Area of Interest (AOI) including the whole sectional area of the tumor was selected on images at different time points (1, 2, 5, 10, 15, 20, 25, 30 minutes). At each time point, the sum of the video pixel inside the AOI was calculated after the subtraction of the AOI baseline. Results are expressed as the percentage of the signal obtained with SonoVue®, which is taken as 100%. Similarly, a second AOI situated outside the tumor, and representing the freely circulating contrast agent, is also analyzed. 
       FIG. 94  shows uptake and retention of bubble contrast in the tumor up to 30 minutes post injection for suspensions of phospholipid stabilized microbubbles conjugated to a heteromultimeric construct of the invention prepared as described above (D23). In contrast, the same bubbles showed only transient (no more than 10 minutes) visualization/bubble contrast in the AOI situated outside the tumor site. 
     Example 47 
     The study described below was performed to assess the ability of contrast agents containing representative KDR-binding peptides described herein to detect, monitor and assess the anti-angiogenic therapeutic effect of therapeutic agents such as Sunitinib (a potent tyrosine kinase inhibitor, which inhibits KDR). 
     Material and Methods 
     Four (4) female OFA rats (Charles River, Lyon, France) were investigated. At the specific age of 42 days, animals received intraperitoneal injection of N-methyl-N-nitrosourea 50 mg/kg (NMU, Sigma-Aldrich, ref D3254) prepared in 0.9% NaCl, in order to develop a KDR-expressing tumor in the animals (see latency period infra). Weight of the animals was 241.8±6.3 g when entering the protocol. Animals received food and water (tap water) ad libitum. 
     Study Treatment 
     Sunitinib malate salt (LC Laboratories, ref S-8803) was dissolved in NaCl 0.9% containing 10% polyethylene glycol 300 (PEG 300, Sigma-Aldrich, ref 90878), 0.5% polysorbate 80 (Tween 80, Sigma-Aldrich, ref) pH 3.75. Stock solution was made weekly and stored at 4° C. Rats received daily oral gavage of 53.6 mg/kg of sunitinib malate salt, which corresponds to 40 mg/kg of sunitinib. 
     Animal Preparation 
     Gaseous anesthesia of the rat was achieved by Isoflurane inhalation 5% in 1 L/min oxygen enriched (95%) air. At the level of the tumor site, skin was gently shaved and an ultrasound coupling gel (Aquasonic 100, Parker, USA) was applied between the clinical ultrasound probe and the skin. 
     Imaging Examination 
     Ultrasound imaging was performed using a clinical ultrasound device (Philips iU22, vision 2008, Philips Medical System, Eindhoven, The Netherlands), operating in a contrast-specific imaging mode. The linear probe L12-5 was used. 
     Experimental Procedure 
     Dosing of Animals 
     Vials of a contrast agent containing D5 were reconstituted in 2 ml glucose 5%. D5 is described in more detail in Examples 50 and 51. As described in Example 51, D5 includes the peptide monomers (12) (SEQ ID NO. 277) and (13) (SEQ ID NO. 337) which form dimer (16) (D5). Lipopeptides comprising the dimer D5 were prepared as described in Example 52. The lyophilized precursor of the contrast agent was obtained according to the procedure illustrated in Example 59a, by replacing DSPE-PEG2000 with DPPE-PEG5000 (2.3 mg) and admixing it with 3.3. mg of the dimeric phospholipid conjugate (11) of  FIG. 5  in 60 mL of a distilled water, containing 10% PEG4000. Stearate was further replaced by palmitic acid (1.0 mg) and admixed with DSPC (59 mg) in cyclooctane (4.8 ml). Emulsification was effected at 12′000 rpm (five minutes). The lyophilizate was reconsistituted as described above. The contrast agent was quickly injected through the catheter placed in the caudal vein at the dose of 2 μl gas per kg. The injection volume ranged from 49 to 61 μl followed by a flush of 100 μsaline. Injections were performed at a constant rate of 4 ml/min. 
     Echographic Evaluations 
     Tumor size was assessed in fundamental-B mode using calipers. 
     For each injection, tumor imaging was performed in contrast mode starting 10 sec before contrast injection until 10 to 14 minutes after injection 
     Quantification of Tumor Contrast Enhancement and Targeting 
     Contrast enhancement was quantified from Dicom files using an in-house quantification and providing, among other parameters, mean echo-signal values in areas of interest as a function of time. The amplitude of the echo signal was expressed as linearized echo amplitude values. Individual frames were analyzed, time-intensity curves were generated and Maximum intensity (Imax) was determined as an index of tumor vascularization and late phase opacification (LPO) was determined at 10 min after injection in the median plane of the tumor. 
     Results 
     Latency for tumor appearance ranged between 44 days and 93 days after NMU-treatment for the four tested rats. When the lesion was detected, tumor size, assessed by fundamental-B mode was around 0.3 cm 2  (largest cross section). Performing in vivo imaging with D5-containing microbubbles showed that Imax, an index of tumor vascularization, was quite variable throughout the follow up of tumor growth whereas LPO, which correlates with the expression of KDR receptor, was more stable over the duration of the protocol. 
     In rats no. 1, no. 2 and no. 3, treatment with sunitinib (a potent tyrosine kinase inhibitor, which inhibits KDR) was initiated on day 106 after NMU treatment, at a dose of 40 mg/kg/day. Tumor size was determined in fundamental B-mode. Values were multiplied by 10 to fit with Y abscissa scale. Imax and LPO were measured using contrast mode and after injection of the D5-containing contrast agent. 
     The results for rat no. 1 are shown in  FIG. 98  and in Table 28 below. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 28 
               
               
                   
                   
               
               
                   
                 day of imaging 
                 Imax 
                 LPO 
                 Area *10 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 79 
                 436.0 
                 12 
                 5.60 
               
               
                   
                 85 
                 1294.0 
                 20 
                 13.90 
               
               
                   
                 91 
                 1220.0 
                 24 
                 18.10 
               
               
                   
                 105 
                 1090.0 
                 20 
                 21.80 
               
               
                   
                 108 
                 966.0 
                 14 
                 20.80 
               
               
                   
                 115 
                 1095.0 
                 10 
                 11.00 
               
               
                   
                 116 
                 931.5 
                 8 
                 10.10 
               
               
                   
                   
               
            
           
         
       
     
     Sunitinib treatment period is indicated by the red box in  FIG. 98 . Treatment started on day 106. 
     The results for rat no. 2 are shown in  FIG. 99  and in Table 29 below. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 29 
               
               
                   
                   
               
               
                   
                 day of imaging 
                 Imax 
                 LPO 
                 Area *10 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 79 
                 930 
                 25 
                 3.40 
               
               
                   
                 85 
                 1629 
                 31 
                 7.20 
               
               
                   
                 93 
                 2660 
                 54 
                 16.80 
               
               
                   
                 105 
                 1523 
                 36 
                 26.90 
               
               
                   
                 108 
                 1882 
                 10 
                 26.70 
               
               
                   
                 109 
                 406 
                 5 
                 20.20 
               
               
                   
                   
               
            
           
         
       
     
     Sunitinib treatment period is indicated by the red box in  FIG. 99 . Treatment started on day 106. On day 109, tumor was harvested and processed for immunohistochemistry.  FIG. 103  provides immunohistochemistry images for this sample. 
     The results for rat no. 3 are shown in  FIG. 100  and in Table 30 below. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 30 
               
               
                   
                   
               
               
                   
                 day of imaging 
                 Imax 
                 LPO 
                 Area *10 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 93 
                 2600 
                 30 
                 9.1 
               
               
                   
                 101 
                 3456 
                 24 
                 9.8 
               
               
                   
                 105 
                 2900 
                 30 
                 10.8 
               
               
                   
                 108 
                 3200 
                 10 
                 7.5 
               
               
                   
                 114 
                 1327 
                 7 
                 2.9 
               
               
                   
                 116 
                 1524 
                 5 
                 3.0 
               
               
                   
                   
               
            
           
         
       
     
     Sunitinib treatment period is indicated by the red box in  FIG. 100 . Treatment started on day 106. The results for rat no. 4 are shown in  FIG. 101  and in Table 31 below. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 31 
               
               
                   
                   
               
               
                   
                 day of imaging 
                 Imax 
                 LPO 
                 Area *10 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 44 
                 127 
                 5 
                 3.370 
               
               
                   
                 50 
                 441 
                 2 
                 4.570 
               
               
                   
                 56 
                 439 
                 9 
                 5.000 
               
               
                   
                 63 
                 912 
                 18 
                 8.590 
               
               
                   
                 78 
                 825 
                 20 
                 14.500 
               
               
                   
                   
               
            
           
         
       
     
     On day 78, tumor was harvested and processed for immunohistochemistry staining  FIG. 104  provides immunohistochemistry images for this sample. 
     48 hours after treatment (day 108) tumor size remained unchanged in rats no. 1 and no. 4 (respectively −4.6% and −0.7% when expressed as a percentage of the tumor prior sunitinib treatment). In rat no. 3 there was a reduction of tumor size by 30%. Imax was slightly decreased in rat no. 1 (−11%) and increased in both rats no. 2 and no. 3 (+23% and +11% respectively). Finally, the measurement of LPO, i.e. targeted microbubbles bound to KDR receptor, revealed a strong decrease in all 3 rats 24 hours after sunitinib treatment (−30%, −70% and −66% in rats no. 1, no. 2 and no. 3 respectively). Sunitinib treatment was maintained up to day 109 for rat no. 2 and up to day 116 for rats no. 1 and no. 3. Treatment with sunitinib for longer periods (up to 3 days for rat no. 2 and 10 days for rats no. 1 and no. 3) resulted in a decrease in all measured parameters, tumor size, Imax and LPO. 
     Interestingly in rat no. 2, immunohistochemistry performed 3 days after initiation of sunitinib treatment, showed large area of necrosis in the tumor, in agreement with the reduction of all echographic parameters (as shown in  FIG. 103 ). 
     Finally, in rat no. 4, which did not receive sunitinib, immunohistochemistry was performed on day 78 after NMU treatment. Tumor size was around 1.5 cm 2 . Staining for CD31, a marker of endothelial cells, revealed a strong staining of vessels (mostly straight) in stroma tissue surrounding tumor nodules, whereas the staining was weak inside the tumor nodules. Conversely KDR staining in tumor vessels was very strong and showed tortuous vessels in tumor nodules, whereas only a few vessels were stained for KDR in the stroma (as shown in  FIG. 104 ). 
     Conclusion 
     These data indicate that use of targeted microbubbles comprising KDR binding peptides such as D5, allows for the early evaluation of the efficacy of anti-angiogenic treatment therapies. Thus, according to certain embodiments, the present invention allows for adjustments to therapeutic regimens to be made as needed and in a timely manner. 
     Materials and Analytical Methods for Examples 48-64 
     Solvents for reactions, chromatographic purification and HPLC analyses were E. Merck Omni grade solvents from VWR Corporation (West Chester, Pa.). N-Methylpyrrolidinone (NMP) and N,N-dimethylformamide (DMF) were obtained from Pharmco Products Inc. (Brookfield, Conn.), and were peptide synthesis grade or low water/amine-free Biotech grade quality. Piperidine (sequencing grade, redistilled 99+%) and trifluoroacetic acid (TFA) (spectrophotometric grade or sequencing grade) were obtained from Sigma-Aldrich Corporation (Milwaukee, Wis.) or from the Fluka Chemical Division of Sigma-Alrich Corporation. N,N′-Diisopropylcarbodiimide (DIC), phenol (99%), N,N-diisopropylethylamine (DIEA) and triisopropylsilane (TIS) were purchased from Sigma-Aldrich Corporation. Fmoc-protected amino acids, pseudoproline dipeptides, Fmoc-Asp(O-tBu)-Ser(ψMe,Mepro)-OH and Fmoc-Gly-Thr(ψMe,Mepro)-OH and N-hydroxybenzotriazole (HOBt) were obtained from Novabiochem (San Diego, Calif.). Fmoc-8-amino-3,6-dioxaoctanoic acid (Adoa) was obtained from NeoMPS Corp (San Diego, Calif.) or Suven Life Sciences (Hyderabad, India). Disuccinimidyl glutarate (DSG) and 1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethyleneglycol)2000] ammonium salt, [DSPE-PEG2000-NH2] were obtained from Pierce Chemical Co. (Rockford, Ill.) and Avanti® Polar Lipids (Alabaster, Ala.), respectively. Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH were prepared in-house from the corresponding triglycine or diglycine by the reaction with Fmoc-OSu. An AG MP-50 ion-exchange resin was obtained from Bio-Rad (Hercules, Calif.). 
     Analytical HPLC data were generally obtained using a Shimadzu LC-10AT VP dual pump gradient system employing a Waters XTerra MS-C18 4.6×50 mm column, (particle size: 5 μA; 120 Å pore size) and gradient or isocratic elution systems using water (0.1% TFA) as eluent A and CH3 CN (0.1% TFA) or CH3 CN—CH 3 OH (1:1, v/v) (0.1% TFA) as eluent B. Detection of compounds was accomplished using UV at 220 and 254 nm. The purity of the phospholipid-PEG-peptide derivatives was determined on a YMC C-4 (5 μM, 300 Å, 4.6×250 mm) column or on a Zorbax 300 SB-C3 (3.5 μM; 300 Å, 3×150 mm) column using a SEDEX 55 Light Scattering Detector (LSD) and with a UV detector. 
     Preparative HPLC was conducted on a Shimadzu LC-8A dual pump gradient system equipped with a SPD-10AV UV detector fitted with a preparative flow cell. Generally the solution containing the crude peptide was loaded onto a reversed phase C18, C4 or C3 column, depending on the compound characteristics, using a third pump attached to the preparative Shimadzu LC-8A dual pump gradient system. After the solution of the crude product mixture was applied to the preparative HPLC column the reaction solvents and solvents employed as diluents, such as DMF or DMSO, were eluted from the column at low organic phase composition. Then the desired product was eluted using a gradient elution of eluent B into eluent A. Product-containing fractions were combined based on their purity as determined by analytical HPLC and mass spectral analysis. The combined fractions were freeze-dried to provide the desired product. 
     Amino acid composition analyses were performed at the Keck Biotechnology Resource Laboratory at Yale University, New Haven, Conn. Mass spectral data were obtained from MScan Inc. (606 Brandywine Parkway, West Chester Pa. 19380) or obtained in-house on an Agilent LC-MSD 1100 Mass Spectrometer. For the purposes of fraction selection and characterization of the products mass spectral values were usually obtained using API-ES in negative ion mode. Generally the molecular weight of the target peptides was ˜3000; the mass spectra usually exhibited doubly or triply negatively charged ion mass values rather than [M-H]-. These were generally employed for selection of fractions for collection and combination to obtain the pure peptide during HPLC purification. In some cases fractions exhibited dominant peaks attributable to [M-2H]/2+57 or [M-2H]/2+114 in the mass spectrum. These peaks are due to the formation of adducts of one or two molecules of trifluoroacetic acid per molecule of the peptide. After careful collection of fractions by comparing MS results and HPLC purities and freeze-drying process, a small amount of the isolated fluffy solid was dissolved in water (0.5 mg/mL) and treated with a drop of aqueous N-methyl-D-glucamine (˜0.5 M). This solution was analyzed by HPLC and MS for final purity results of the purified peptide. Peptide solutions in the presence of N-methyl-D-glucamine did not exhibit [M-2H]/2+57 or [M-2H]/2+114 mass value peaks in the mass spectrum, instead the expected [M-2H]/2 or [M-3H]/3 peaks were observed. 
     Examples 48-49 below refer to the monomeric peptide phospholipid conjugate shown in  FIG. 106 . A process for synthesizing this compound is shown in  FIG. 105 . Although these Examples refer more specifically to the process for synthesizing the compound shown in  FIG. 106 , similar processes may used to prepare the monomeric peptide phospholipid conjugate shown in  FIG. 114  and the linear peptide monomer (32) shown in  FIG. 113  as well as other monomer peptide-phospholipid conjugates including, for example, those formed using peptide monomers Ac-RAQDWYYDEILSMADQLRHAFLSGAGSGK-NH 2  (SEQ. ID NO. 6) and Ac-RAQDWYYDEILSMADQLRHAFLSGSAGSK-NH 2  (SEQ. ID NO. 7), as well as analogs and derivatives of any of the above. Additionally, co-pending U.S. application Ser. No. 10/661,156, filed Sep. 11, 2003, sets forth methods for the preparation of the peptide monomers and is incorporated by reference herein in its entirety. 
     Example 48 
     Solid Phase Synthesis (SPPS) and Purification of Linear Peptide Monomer (2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH2, (SEQ ID NO. 2) Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L- phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide 
     The linear peptide monomer (2) was synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DIC-mediated HOBt ester activation in DMF. The peptide sequence was synthesized in stepwise fashion by SPPS methods on the Fmoc-Pal-Peg-PS resin, typically on a 10 mmol scale. The amino acid couplings were carried out with a 4-fold excess each of amino acid and the DIC-HOBt reagent pair in DMF. 
     In a typical coupling of an amino acid, 5 mL of dry DMF per gram of resin was used. The total volume of DMF, calculated on the basis of resin used, was allocated among amino acid, HOBt and DIC for solution preparation. For example, for the synthesis involving 50 g (10 mmol scale) of resin, the calculated volume of 250 mL of DMF was distributed among amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vessel on the Sonata Pilot Peptide Synthesizer was charged with the solid dry amino acid (4-fold excess with respect to the resin). At inception of the coupling step, the software of the instrument was employed to deliver successively the chosen volume of DMF (for dilution of the amino acid) and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF and mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to pre-activate the amino acid and to insure complete dissolution of all components of the mixture. After activation, the software mediated the transfer of the solution of the activated Fmoc-amino acid to the reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g, 6×) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2×10 min). The resin was thoroughly washed with DMF (5 mL/g, 6×) to assure complete removal of piperidine from the resin in preparation for the ensuing amino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was not conducted in order to minimize the formation of diketopiperazine during the activation time as discussed in the text. Therefore, in these two cases, the solutions of amino acid, HOBt and DIC were added to the reaction vessel sequentially and the coupling process was conducted with ‘in situ’ activation. 
     After chain elongation was completed, the Fmoc group of the N-terminal amino acid was removed in the standard manner followed by the standard wash with DMF (vide supra). The N-terminal amino acid was then capped by treatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF/6 mL/g of resin), 2×20 min. After completion of the peptide synthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’ (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h. The volatiles were removed and the paste thus obtained was triturated with ether to provide a solid which was washed with ether (3×) with intervening centrifugation (to compact the suspended solids in order to allow decantation of the supernatant) and then dried under vacuum to provide the required peptide as an off-white solid. A 10 mmol scale synthesis of the linear peptide monomer (2) gave 33.82 g (103% of theory) of the crude peptide. The greater than theoretical yield was most likely due to moisture and residual solvents. 
     Purification of the Linear Peptide Monomer (2) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK-NH 2  (SEQ ID NO. 2); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-NH2; N-acetyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L-aspartyl-L-glutamyl-L-isoleucyl-L-leucyl-L-seryl-L-methionyl-L-alanyl-L-aspartyl-L-glutamyl-L-leucyl-L-arginyl-L-histidyl-L-alanyl-L- phenylalanyl-L-leucyl-L-seryl-glycyl-glycyl-glycyl-glycyl-glycyl-L-lysinamide 
     A ˜0.5 g portion of the crude linear peptide monomer (2) was dissolved in a minimum amount of CH3 CN (˜20 mL). The volume of the solution was adjusted to ˜100 mL with water and employing a third pump the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min) which had been pre-equilibrated with 10% CH3 CN in water (0.1% TFA). The column was not eluted with the equilibrating eluent during application of the sample solution. After the sample solution was applied to the column, the composition of the eluent was ramped to 20% CH3 CN-water (0.1% TFA) over 1 min, and a linear gradient at a rate of 0.6%/min of CH3 CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The collected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (5 particle, 120 Å pore) and product-containing fractions of &gt;95% purity were pooled and freeze-dried to afford the corresponding pure linear peptide monomer (2). Typically the purification of 0.5 g of crude (2) afforded 0.12 g (24% yield) of the desired product (&gt;95% purity). 
     Example 49 
     Preparation of Monomeric Peptide Phospholipid Conjugate (1) Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH2 (SEQ ID NO. 1); Ac-Arg-Ala-Gln-Asp-Trp-Tyr-Tyr-Asp-Glu-Ile-Leu-Ser-Met-Ala-Asp-Gln-Leu-Arg-His-Ala-Phe-Leu-Ser-Gly-Gly-Gly-Gly-Gly-Lys-(DSPE-PEG2000-NH-Glut)-NH2; N-acetyl-L-arginyl-L-alanyl-L-glutaminyl-L-aspartyl-L-tryptophyl-L-tryptophyl-L-aspartyl-L-isoleucyl-L-glutamyl-L-leucyl-L-serinyl-L-methionyl-L-alanyl-L-aspartyl-L-glutaminyl-L-leucyl-L- arginyl-L-histidyl-L-alanyl-L-phenylalanyl-L-leucyl-L-serinyl-glycyl-glycyl-glycl-glycyl-glycyl-L-lysinamide 
     The monomeric peptide phospholipid conjugate (1), Ac-RAQDWYYDEILSMADQLRHAFLSGGGGGK(DSPE-PEG2000-NH-Glut)-NH 2  (SEQ ID NO. 1), was prepared by conjugation of (3), the glutaric acid monoamide mono-NHS ester of peptide monomer (2), with DSPE-PEG2000-NH2 phospholipid ammonium salt (4). 
     A round-bottomed flask equipped with magnetic stir bar and septum cap was charged sequentially with anhydrous dimethylformamide (7.5 mL), disuccinimidyl glutarate (DSG, 0.25 g, 0.75 mmol) and diisopropylethylamine (0.10 g, 0.78 mmol) with stirring. Solid linear peptide monomer (2) (0.5 g, 0.152 mmol) was added portionwise to the above solution over a period of 2 min; then the solution was stirred for 30 min at ambient temperature. The reaction mixture was diluted to ˜50 mL with anhydrous ethyl acetate; this resulted in precipitation of the intermediate mono-NHS ester (3), the glutaric acid monoamide mono-NHS ester of peptide monomer (2). The solution was centrifuged to bring down mono-NHS ester (3) as a colorless solid. The supernatant containing excess DSG was decanted from the compacted solid mono-NHS ester (3) which was again dispersed in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG. The solid intermediate mono-NHS ester (3) thus obtained was dissolved in anhydrous DMF (10.0 mL); diisopropylethylamine (0.10 g, 0.78 mmol) was added; and the mixture was stirred. 
     Meanwhile, DSPE-PEG2000-NH 2  phospholipid ammonium salt (4) (0.38 g, 0.14 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask and trifluoroacetic acid (2 drops) was added to protonate the phosphodiester oxygen facilitating solubilization of phospholipid ammonium salt in dichloromethane. The clear solution was then evaporated on a rotary evaporator to remove the volatiles and dried further under vacuum. 
     The solid phospholipid ammonium salt (4) was dissolved in DMF (5 mL) and transferred to the stirred solution of mono-NHS ester (3) and the resulting mixture was stirred for 24 h at ambient temperature. The reaction mixture was diluted to 100 mL with a 1:1 mixture of CH 3 OH and CH 3 CN-water (1:1, v/v) and the insolubles were filtered. Half of the filtered solution was loaded onto a reversed phase C2 preparative column (Kromasil® Prep C2, 10∥, 300 Å, 50×250 mm) which had been pre-equilibrated with 3:1 (v/v) mixture of water (0.1% TFA) and CH 3 OH—CH 3 CN (1:1, v/v, 0.1% TFA) at a flow rate of 100 mL/min. Note that the column was not eluted with the equilibrating eluent during loading of the sample. After the sample solution was loaded the column was washed with the equilibration eluent until the plug of DMF was eluted. The composition of the eluent was ramped to 70% CH 3 OH—CH 3 CN (1:1, 0.1% TFA) over 9 min and a linear gradient of 0.75%/min of CH 3 OH—CH 3 CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated and run for 40 min. Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution. Fractions were checked for purity on an analytical HPLC system (column: YMC C-4, 5μ, 300 Å, 4.6×250 mm) using UV at 220 nm and an evaporative light scattering detector (ELSD). The latter detector (ELSD) was employed to detect DSPE-PEG2000-NH2 phospholipid ammonium salt (4) which has very little UV absorbance at 220 nm. Product-containing fractions of &gt;98% purity, and devoid of DSPE-PEG2000-NH2 phospholipid ammonium salt (4) were combined and concentrated on a rotary evaporator to reduce the content of CH3OH. The concentrated solution was then diluted with 10% CH3 CN in water until a faint flocculent precipitate formed. The resulting solution was freeze-dried to provide monomeric peptide phospholipid conjugate (1) as a colorless solid. The second portion of crude monomeric peptide phospholipid conjugate (1) was purified as described above. The combined yield of the target monomeric peptide phospholipid conjugate (1) was 0.40 g (47% yield). 
     Examples 50-52 below refer to the dimeric peptide phospholipid conjugate shown in  FIG. 109 . Representative methods of synthesizing the dimeric conjugate are shown in  FIGS. 107, 108, 110, 111 and 112 . 
     Example 50 
     Solid Phase Synthesis (SPPS), Cyclization and Purification of Monomer Peptides (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]—NH2 and (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 
     The linear peptides were synthesized by an established automated protocol on a SONATA®/Pilot Peptide Synthesizer using Fmoc-Pal-Peg-PS resin (0.2 mmol/g), Fmoc-protected amino acids and DCI-mediated HOBt ester activation in DMF. The peptide sequence on the Fmoc-Pal-Peg-PS resin was synthesized in stepwise fashion by SPPS methods typically on a 10 mmol scale. The amino acid coupling was carried out with a 4-fold excess each of amino acid and DIC-HOBt reagent in DMF. 
     In a typical coupling of an amino acid in the sequence, 5 mL of dry DMF per gram of resin was used. The total volume of DMF, calculated on the basis of resin used, was allocated among amino acid, HOBt and DIC for solution preparation. For example, for the synthesis involving 50 g of resin, the calculated volume of 250 mL of DMF was distributed among amino acid (150 mL), HOBt (50 mL) and DIC (50 mL). The amino acid vessel on the Sonata® Pilot Peptide Synthesizer was charged with the solid dry amino acid (4-fold excess with respect to the resin). At inception of the coupling step, the chosen volume of DMF and HOBt (4 eq.) in DMF and DIC (4 eq.) in DMF were delivered successively and after each delivery mixing by nitrogen bubbling was conducted. After the last reagent was delivered mixing by nitrogen bubbling was initiated and conducted for 4 min. This served to preactivate the amino acid and to insure complete dissolution of all components of the mixture. 
     After activation, the solution of the activated Fmoc-amino acid was transferred to the reaction vessel containing the resin. After transfer was complete the vessel was agitated for 3 h with recurrent nitrogen bubbling. After the 3 h coupling time, the resin was washed thoroughly with DMF (5 mL/g, 6×) and the cleavage of the Fmoc-group was performed with 25% piperidine in DMF (5 mL/g) containing HOBt (0.1M) (2×10 min). The resin was thoroughly washed with DMF (5 mL/g, 6×) to assure complete removal of piperidine from the resin in preparation for the ensuing amino acid coupling. In the case of Fmoc-Gly-Gly-Gly-OH and Fmoc-Gly-Gly-OH, the pre-activation in the amino acid bottle was not conducted in order to minimize the formation of diketopiperazine during the activation time as discussed in the text. Therefore, in these two cases, the solution of the amino acid, HOBt and DIC were added to the reaction vessel sequentially and the coupling process was conducted with ‘in situ’ activation. After chain elongation was completed, the fmoc group of the N-terminal amino acid was removed in the standard manner followed by the standard wash with DMF (vide supra). The N-terminal amino acid was then capped by treatment with freshly prepared acetylation mixture (0.5M acetic anhydride, 0.125M DIEA and 0.015M HOBt in DMF—6 mL/g of resin), 2×20 min. 
     Functionalization of the ε-amino group of C-terminal Lysine moieties of the monomer peptides (with Fmoc-Adoa or with Fmoc-Lys(ivDde) as required) was accomplished by first removing the ivDde group of the ε-amino group with freshly prepared 10% hydrazine in DMF (5 mL/g of resin −2×10 min). For appending of Fmoc-Adoa or Fmoc-Lys(ivDde) the coupling time was increased to 10 h. After completion of the peptide synthesis, the resin was treated with the cleavage cocktail, ‘Reagent B’ (TFA:water:phenol:triisopropylsilane, 88:5:5:2, v/v/w/v) (10 mL/g of resin) for 4 h. After evaporation of the volatiles under vacuum, the paste was triturated with ether to provide a solid which was collected by filtration washed with diethyl ether and dried. A 10 mmol scale synthesis of (12), Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH 2  gave 30 g (103% of theory) of the crude peptide. In the case of (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 , a 10 mmol scale synthesis gave 28 g (107% of theory) of crude peptide. The greater than theoretical yields are most likely due to moisture and residual solvents. 
     Cyclization of the Linear Di-Cysteine Peptides to Cyclic Disulfide Peptides 
     Cyclic disulfide peptides were prepared from the corresponding linear di-cysteine peptides by DMSO-assisted oxidation using DMSO/water (95/5, v/v). The crude linear peptide was dissolved in the solvent mixture (5 mL/g) in a wide mouth beaker, and the pH of the solution was adjusted to 8.5 by the addition of solid N-methyl-D-glucamine in portions. The resulting mixture was stirred for 36 h at ambient temperature. The solution was then diluted with acetonitrile (50 mL/g) and the mixture was stirred for 2 min. The solid cyclic disulfide peptide was collected by filtration, washed with diethyl ether and dried. 
     Purification of Monomer Peptides 
     Peptide Monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK[K(ivDde)]-NH 2 ; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Lys(ivDde)]-NH 2  cyclic (6-13) Disulfide 
     A ˜0.5 g portion of the crude cyclic disulfide peptide monomer (12) was dissolved in a minimum amount of DMSO (˜3 mL). The volume of the solution was adjusted to ˜100 mL with 20% CH 3 CN-water and employing a third pump, the solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10μ, 300 Å, 50×250 mm, flow rate 100 mL/min), which had been pre-equilibrated with 10% CH 3 CN in water (0.1% TFA). During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was applied to the column, the flow of equilibrating eluent from the gradient HPLC system was reinitiated and the column was eluted with 10% CH 3 CN-water (0.1% TFA) until the DMSO was eluted. Then the eluent composition was ramped to 35% CH 3 CN-water (0.1% TFA) over 1 min after which a linear gradient at a rate of 0.5%/min CH 3 CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The collected fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (5μ particle, 120 Å pore) and product-containing fractions of &gt;95% purity were pooled and freeze-dried to afford the corresponding cyclic disulfide peptide monomer (12). Typically the purification of 0.5 g of crude peptide monomer (12) afforded 0.1 g (20% yield) of the desired product (&gt;95% purity). 
     Peptide Monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 ; Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(Adoa-Adoa)-NH 2  cyclic (2-12) disulfide 
     Following the procedure employed for the HPLC purification of peptide monomer (2), the crude cyclic disulfide peptide monomer (13) Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH2 (0.5 g) dissolved in 20% CH 3 CN-water mixture (100 mL) was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 50×250 mm, 10μ particle, 300 Å pore, flow rate 100 mL/min), which had been pre-equilibrated with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA). During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was applied to the column, the flow of equilibrating eluent from the gradient HPLC system was reinitiated and the column was eluted with 10% CH 3 CN-water (0.1% TFA) for 5 min. Then the eluent composition was ramped to 30% CH 3 CN (0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient elution at a rate of 0.5%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA) was initiated and maintained for 50 min. Fractions (15 mL) were manually collected using UV at 220 nm as an indicator of product elution. The fractions were analyzed on a Waters XTerra analytical reversed phase C-18 column (4.6 mm i.d.×50 mm, 5μ particle, 120 Å pore) and product-containing fractions of &gt;95% purity were pooled and freeze-dried to afford the corresponding cyclic disulfide peptide monomer (13). Typically the purification of 0.5 g of crude peptide monomer (3) afforded 0.12 g (24% yield) of the desired product (&gt;95% purity). 
     Example 51 
     Preparation and Purification of Precursor Dimer Peptide (16) Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[—NH 2  cyclic (2-12) disulfide]-NH2 cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys[Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys(-Adoa-Adoa-Glut-Lys)]-NH2 cyclic (2-12) disulfide]-NH 2  cyclic (6-13) disulfide 
     As shown in  FIG. 107 , disuccinimidyl glutarate (DSG, 0.28 g, 0.86 mmol) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added in one portion. Then solid peptide monomer (12) Ac-AGPTWC*EDDWYYC*WLFGTGGGK-[K(ivDde)]—NH 2  (0.50 g, 0.17 mmol) was added in portions to the stirred solution of DSG over a period of two min. After stirring for 30 min at room temperature, the solution was diluted with anhydrous ethyl acetate to ˜50 mL, (this served to precipitate intermediate mono-NHS ester (14)). The entire mixture was centrifuged and the supernatant was decanted leaving intermediate mono-NHS ester (14) as a colorless solid. The solid was resuspended with ethyl acetate; the solution containing the suspended solid mono-NHS ester (14) was centrifuged to separate the solid and the supernatant was again decanted. This washing process was repeated twice to remove completely the excess DSG. 
     The solid mono-NHS ester (14) was dissolved in stirred anhydrous dimethylformamide (2.0 mL) and diisopropylethylamine (0.11 g, 0.85 mmol) was added. Then solid peptide monomer (13), Ac-VC*WEDSWGGEVC*FRYDPGGGK(Adoa-Adoa)-NH 2 , (0.50 g, 0.19 mmol, 1.12 eq.) was added in portions to the stirred solution over a three min. period and the resulting mixture was stirred for 18 h. The reaction was monitored by mass spectrometry; after the complete consumption of the peptide monomer glutaric acid monoamide mono-NHS ester (14) was confirmed, neat hydrazine (0.1 mL) was added to remove the ivDde protecting group of the ivDde-bearing dimer (15) and the mixture was stirred for 20 min at room temperature. 
     The solution was then acidified by dropwise addition of TFA and the mixture was diluted to 100 mL with 10% CH 3 CN (0.1% TFA) in water (0.1% TFA). The solution was filtered to remove particulates and half of the clarified solution was loaded onto a reversed phase C18 preparative column (Waters, XTerra® Prep MS C18, 10 50×250 mm, flow rate 100 mL/min) pre-equilibrated with 10% CH3 CN in water (0.1% TFA). During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was applied to the column, the flow of equilibrating eluent from the gradient HPLC system was reinitiated and the column was eluted with 10% CH 3 CN-water (0.1% TFA) in order to flush DMF from the column. After elution of the DMF plug was completed the eluent composition was increased to 20% CH 3 CN over one min. and the elution was continued with a linear gradient rate of 0.6%/min of CH 3 CN (0.1% TFA) into water (0.1% TFA). Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution. The fractions were analyzed on a reversed phased C18 column (Waters MS C18, 4.6 mm i.d.×50 mm, 5μ particle, 120 Å pore) and the product-containing fractions of &gt;95% purity were pooled and freeze-dried to provide precursor dimer peptide (16) as a colorless, fluffy solid. The remaining crude precursor dimer peptide (16) was purified in the same manner. From 0.5 g each of monomer peptides (12) and (13), 320 mg (overall yield 33%) of the desired dimer (16) was obtained (&gt;95% purity). 
     Example 52 
     Preparation of KDR-Binding Dimeric Peptide Phospholipid Conjugate (11) Acetyl-L-alanyl-glycyl-L-prolyl-L-threonyl-L-tryptophyl-L-cystinyl-L-glutamyl-L-aspartyl-L-aspartyl-L-tryptophyl-L-tyrosyl-L-tyrosyl-L- cystinyl-L-tryptophyl-1-leucyl-L-phenylalanyl-glycyl-L-threonyl-glycyl-glycyl-glycyl-L-lysyl[Acetyl-L-valyl-L-cystinyl-L-tryptophyl-L-glutamyl-L-aspartyl-L-seryl-L-tryptophyl-glycyl-glycyl-L-glutamyl-L-valyl-L-cystinyl-L-phenylalanyl-L-arginyl-L-tyrosyl-L-aspartyl-L-prolyl-glycyl-glycyl-glycyl-L- lysyl(distearylphosphoethanolaminocarbonoxy-PEG2000-amino-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-glutaryl-L-lysyl) amide cyclic (2-12) disulfide]-amide cyclic (6-13) disulfide; Ac-AGPTWCEDDWYYCWLFGTGGGK {Ac-VCWEDSWGGEVCFRYDPGGGK[-Adoa-Adoa-Glut-K(DSPE-PEG2000-NH-Glut)]-NH 2  cyclic (2-12) disulfide}-NH 2  cyclic (6-13) disulfide; Ac-Ala-Gly-Pro-Thr-Trp-Cys-Glu-Asp-Asp-Trp-Tyr-Tyr-Cys-Trp-Leu-Phe-Gly-Thr-Gly-Gly-Gly-Lys {Ac-Val-Cys-Trp-Glu-Asp-Ser-Trp-Gly-Gly-Glu-Val-Cys-Phe-Arg-Tyr-Asp-Pro-Gly-Gly-Gly-Lys[-Adoa-Adoa-Glut-Lys(DSPE-PEG2000-NH-Glut)-]-NH 2  cyclic (2-12) disulfide}-NH2 cyclic (6-13) disulfide. 
     The KDR-binding dimer (11) may be prepared by conjugation of precursor dimer peptide (16), Ac-AGPTWCEDDWYYCWLFGTGGGK[Ac-VCWEDSWGGEVCFRYDPGGGK(-Adoa-Adoa-Glut-K)[—NH2 cyclic (2-12) disulfide]-NH 2  cyclic (6-13) disulfide, with DSPE-PEG2000-NH 2  phospholipid ammonium salt (18) as shown in  FIG. 108 . 
     Solid precursor dimer peptide (16) (0.5 g, 0.092 mmol) was added portionwise to a solution of disuccinimidyl glutarate (DSG, 0.15 g, 0.46 mmol), and diisopropylethylamine (0.06 g, 0.47 mmol) in anhydrous DMF (3.0 mL) with stirring over a period of 3 min. Then the solution was stirred at ambient temperature for 30 min. The reaction mixture was diluted to 50 mL with anhydrous ethyl acetate; this resulted in precipitation of the dimer glutaric acid monoamide mono-NHS ester (17), the glutaric acid monoamide mono-NHS ester of the precursor dimer peptide (16). The solution was centrifuged to pellet 6 (m/z, neg. ion, 1887.3 (M-3H)/3, 1415.1 (M-4H)/4, 1131.9 (M-5H)/5) as a colorless solid. The supernatant ethyl acetate layer containing excess DSG was decanted from the compacted solid dimer glutaric acid monoamide mono-NHS ester (17) which was again resuspended in ethyl acetate, centrifuged and washed twice more to remove the remaining traces of DSG. The solid intermediate glutaric acid monoamide mono-NHS ester dimer derivative (17) thus obtained was dissolved in anhydrous DMF/CH 2 Cl 2  (8:2, v/v) (3.0 mL); diisopropylethylamine (0.06 g, 0.47 mmol) was added and the solution was stirred. 
     Meanwhile, DSPE-PEG2000-NH 2  phospholipid ammonium salt (18) (0.235 g, 0.084 mmol, 0.9 eq.) was suspended in dry dichloromethane (2 mL) in a separate flask and TFA (2 drops) was added to protonate the phosphodiester oxygen, facilitating solubilization of phospholipid ammonium salt (18) in dichloromethane. The clear solution was concentrated to remove the volatiles and dried further under vacuum. 
     The solid phospholipid ammonium salt (18) was dissolved in DMF (2 mL) and transferred to the stirred solution of glutaric acid monoamide mono-NHS ester dimer derivative (17) and the resulting mixture was stirred for 24 h at ambient temperature. The reaction mixture was diluted with a solution of 50% CH 3 OH, 25% CH 3 CN and 25% water (1:1) to 100 mL and the insolubles were filtered. Half of the filtered solution was loaded onto a reverse phased C4 preparative column (Kromasil® Prep C4, 10μ, 300 Å, 50×250 mm) which had been pre-equilibrated with 1:1 mixture of CH 3 OH and CH 3 CN (1:1, 0.1% TFA) and water (0.1% TFA) at a flow rate of 100 mL/min. During application of the sample solution to the column the flow of the equilibrating eluent from the preparative HPLC system was stopped. After the sample solution was loaded the flow of the equilibrating eluent was reinitiated and the column was washed until the plug of DMF was eluted. 
     The composition of the eluent was then ramped to 70% CH 3 OH—CH 3 CN (1:1, 0.1% TFA)-water (0.1% TFA) over 1 min and a linear gradient of 0.75%/min of CH 3 OH—CH 3 CN (1:1, 0.1% TFA) into water (0.1% TFA) was initiated. The elution was continued after reaching 100% B in order to achieve complete elution of the product from the column. Fractions (15 mL) were collected using UV (220 nm) as an indicator of product elution and after the main product was eluted fraction collection was continued for several minutes in order to insure elution of trace amounts of starting phospholipid ammonium salt (18). Fractions were checked for purity on an analytical HPLC system (column: YMC C4, 5 μM, 300 Å, 4.6×250 mm) using UV at 220 nm and an evaporative light scattering detector (ELSD). The latter detector is employed to detect DSPE-PEG2000-NH 2  which has a weak chromophore at 220 nm. Product-containing fractions of &gt;98% purity, and devoid of DSPE-PEG2000-NH2 phospholipid ammonium salt (8) were combined and concentrated to reduce the content of CH 3 OH. The solution was then diluted with 10% CH 3 CN in water until a faint flocculent precipitate formed. The resulting solution was freeze-dried to afford the dimeric peptide phospholipid conjugate (11) as a colorless solid. The second portion of crude dimeric peptide phospholipid conjugate (11) was purified as described above. The combined yield of the target dimeric peptide phospholipid conjugate (11) was 0.39 g (57% yield). The samples of the dimeric peptide phospholipid conjugate (11) made from different sample purification runs were pooled together, dissolved in tert-butanol-acetonitrile-water mixture and re-lyophilized to provide the dimeric peptide phospholipid conjugate (11) as a colorless, fluffy solid which was further dried under vacuum. 
     Examples 53-55 below refer to the preparation of the dimer peptide-phospholipid conjugate shown in  FIG. 109 , wherein the dimeric conjugate contains very low levels of TFA.  FIGS. 110-112  illustrate the methods described in the Examples below. 
     Example 53 
     Preparation of Dimeric Conjugate Having Low TFA Levels Via the Use of a Glutaryl Linker 
     Preparation of (23), (26) and Dimer Peptide (27) Acetate Salt by Conversion of (22), (25) and Dimer Peptide 27●nTFA Salts to Acetates by AG MP-50 Ion-Exchange resin 
     For compound (23) an AG MP-50 ion-exchange resin (1.5 meq/mL resin bed) was suspended in 20% of CH 3 CN/H 2 O. The suspension was packed in a 3×30 cm glass column and the final volume was 150 mL. The column was connected to a pump and a conductivity meter. It was washed with 20% of CH 3 CN/H 2 O at 17 mL/min flow rate until the conductivity was below 1 μs/cm. Compound (22) (210 mg) was dissolved in 20% of CH 3 CN/H 2 O (80 mL) and the resulting solution was loaded to the column. The column was washed again with the same eluent until its conductivity was below 1 μs/cm. A gradient of NH 4 OAc in 20% of CH 3 CN/H 2 O was applied at 200 mM, 400 mM, 600 mM and 800 mM, 250 mL each. The compound came out at 600 mM NH 4 OAc. The fractions were analyzed by HPLC and the ones containing the compound were combined and lyophilized several times until the weight of the material was constant. 176 mg of the pure material (23) was obtained as a white fluffy solid. The yield was 83.8%. 
     Additional parameters and results were as follows: HPLC: Ret. Time: 5.6 min; Assay &gt;98% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H2O (0.1% TFA), B: CH3 CN (0.1% TFA); Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1441.7 [M-2H]/2, 960.9 [M-3H]/3. CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.3%; acetate 1.1%. 
     For compound (26), following the same procedure for compound (23), 300 mg of the peptide TFA salt (25) in 80 mL of water was loaded at 17 mL/min. to a 150 mL of AG MP-50 column, which was washed with H 2 O to conductivity of 1 μs/cm. The column was then washed with H 2 O again after loading, and the same step gradient of aqueous NH 4 OAc into H 2 O as employed for the ion exchange of compound (23) was applied. Lyophilization of the combined fractions to a constant weight afforded 200 mg of the acetate (26) as a white fluffy solid. The yield was 66.7%. 
     Additional parameters and results were as follows: HPLC: Ret. Time: 5.6 min; Assay 97.0% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H 2 O (0.1% TFA), B: CH 3 CN (0.1% TFA); Elution: Initial condition: 15% B, linear gradient 15-50% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1336.9 [M-2H]/2, 890.8 [M-3H]/3; CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.4%; acetate 4.2%; IC analysis (F %): 0.26. 
     For the dimer peptide (27) acetate salt, similar to the procedure for compound (23), an AG MP-50 column (100 mL wet volume) was washed with 30% CH 3 CN/H 2 O until the conductivity was below 1 μs/cm. Compound (27) as the TFA salt, (120 mg in 80 mL of 30% of CH 3 CN/H 2 O) was loaded onto the column and the column was washed with the same eluent until the conductivity was stable at 1 μs/cm. A step gradient of NH 4 OAc 30% of CH 3 CN/H 2 O into 30% of CH 3 CN/H 2 O was run as for compound (23) and the compound was eluted at ca 600 mM NH 4 OAc. The combined fractions were lyophilized and then relyophilized several times until the material displayed a constant weight to provide 104 mg of the pure material (27) as an acetate salt. The yield was 86.7%. 
     Additional parameters and results were as follows: HPLC: Ret. time: 5.2 min; Assay &gt;99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5μ particle, 120 Å pore; Eluent: A: H 2 O (0.1% TFA), B: CH 3 CN (0.1% TFA); Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; 1816.3 [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion % wt./wt.): TFA estimated to be 0.2%; acetate 0.15%. 
     Preparation and Purification of the Dimer Peptide (27) Acetate Salt from Compound (23) and Compound (26) 
     To a solution of disuccinimidyl glutarate (18 mg, 0.055 mmol) in anhydrous DMF (0.1 mL) was added a solution of compound (23) (61 mg, 0.021 mmol) in 0.2 mL of anhydrous DMF dropwise (pH 8, neutralized by DIEA). The clear solution was stirred at RT for 0.5 h. HPLC and MS showed the completion of the reaction. Solvent was removed in vacuo and EtOAc (8 mL) was added to precipitate the intermediate (24). The mixture was centrifuged and decanted to remove excess glutarate. This EtOAc washing was repeated 3 more times and the resulting solid was dried using a stream of dry nitrogen. It was then dissolved in 0.3 mL of anhydrous DMF. Compound (26), (56 mg, 0.021 mmol) was added and the pH of the solution was adjusted to 8 by addition of DIEA. The solution was stirred for 16 h at room temperature after which by HPLC and MS analysis indicated completion of the reaction. A 30 μL aliquot of NH 2 NH 2  was added and the mixture was stirred for 5 min to cleave the ivDde group. The reaction mixture was analyzed by HPLC and MS, which indicated complete removal of the ivDde group. 
     Before purification of the dimer peptide (27) acetate, caution was taken to carefully wash the whole preparative HPLC system including the column with TFA-free eluents, CH 3 CN/H 2 O/10 mM NH 4 OAc. The crude reaction mixture was then applied to a reverse phase C-18 preparative column (Atlantis C-18, 5 μm particle, 100 Å pore, 30×150 mm, flow rate 30 mL/min), pre-equilibrated with 15% B (A: 10 mM NH 4 OAc in H 2 O; B: 10 mM NH 4 OAc in CH 3 CN/H 2 O, 9/1, v/v). The column was washed with the same eluent until the DMF plug was eluted. The eluent composition was increased to 25% B over 2 min. and then ramped to 65% B over 40 min. The fractions were analyzed on an analytical reverse phase C-18 column (Waters MS C-18, 4.6×50 mm, 5 μm particle, 100 Å pore, flow rate 3 mL/min) and the product-containing fractions of &gt;95% purity were pooled and freeze-dried to afford 25 mg of the dimer peptide (27) as its acetate salt as a fluffy white solid. The yield was 21.8%. 
     Additional parameters and results were as follows: HPLC: Ret. time: 5.2 min; Assay &gt;99% (area %); Column: Waters XTerra MS-C18, 4.6×50 mm, 5 g particle, 120 Å pore; Eluent: A: H 2 O (0.1% TFA), B: CH 3 CN (0.1% TFA); Elution: Initial condition: 20% B, linear gradient 20-60% B over 8 min; Flow rate: 3 mL/min; Detection: UV at 220 nm; Mass Spectrum: API-ES; Mode: Negative ion; [M-3H]/3, 1362.0 [M-4H]/4, 1089.2 [M-5H]/5; CE analysis (counter-ion % wt./wt.): TFA estimated to be less than 0.2%; acetate 1.1%. 
     Example 54 
     FIG.  111   
     Preparation of Dimer Peptide-Phospholipid Conjugates Having Low TFA Levels Via Ion Exchange Resin 
     Preparation and Purification of the Phospholipid Peptide Conjugate (21) as its Acetate Salt from Dimer Peptide (27) Acetate Salt 
     To a solution of disuccinimidyl glutarate-DSG (3.7 mg, 11.3 μmol) in anhydrous DMF (0.1 mL) was added a solution of neutralized dimer peptide (27) acetate salt, (15 mg, 2.75 μmol) in anhydrous DMF (0.2 mL), dropwise. The reaction solution was stirred at RT for 0.5 h. HPLC analysis with a Waters Xterra C-18 column and MS showed the completion of the reaction. The solvent was evaporated and EtOAc (8 mL) was added to precipitate the intermediate (28). The vessel containing the precipitated intermediate (28) was centrifuged and the liquid layer was decanted. This procedure was repeated 3 times to remove the excess of DSG. The solid was dried with a stream of dry nitrogen and then dissolved in 0.3 mL of anhydrous DMF. DSPE-PEG2000-NH 2  ammonium salt (29) (6.5 mg, 2.33 μmol) was added in solid form and the pH of the mixture was adjusted to (28). The reaction mixture was stirred at RT for 16 h. The mixture was analyzed by MS and HPLC with a Zorbax 300 SB-C3 column and this indicated that the reaction was complete. 
     To minimize the potential contamination of the product with TFA, the crude reaction mixture was purified by preparative HPLC equipped using a new Zorbax 300SB-C3 column (21.2×150 mm, 5 g particle) which had never been exposed to TFA. The HPLC system was pre-washed by CH 3 CN/H 2 O/NH 4 OAc extensively to remove traces of TFA. The reaction mixture was loaded onto the column which was pre-equilibrated with 20% B (A: 10 mM NH 4 OAc in H 2 O; B: 10 mM NH 4 OAc in CH 3 CN/H 2 O, 9/1 v/v) at a flow rate of 30 mL/min. The column was eluted at 30 mL/min with the same eluent until the plug of DMF was eluted. The eluent composition was then increased to 40% B over 3 min and then ramped to 90% B over 50 min. The collected fractions were analyzed on an analytical reverse phase C-3 column (Zorbax 300SB-C3, 3×150 mm, 3.5 μm particle, 300 Å pore, flow rate: 0.5 mL/min), where detection was accomplished using UV at 220 nm and an evaporative light scattering detector (ELSD). The fractions containing the pure product were pooled and lyophilized. A 6.5 mg portion of the final product (21) acetate salt was obtained. The yield was 33.0%. 
     Additional parameters and results were as follows: HPLC: Ret. Time: 13.3 min; Assay &gt;99% (area %); Column: Zorbax 300SB-C3, 3×150 mm, 3.5 μm, 300 Å pore; Eluent: A: H 2 O (0.1% TFA), B: CH 3 CN/MeOH 1/1 (0.1% TFA); Elution: Initial condition: 60% B, linear gradient 60-90% B over 3 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm and ELSD; CE analysis (counter-ion % wt./wt.): % wt. TFA: 0.3%; % wt acetate 0.4%. 
     Example 55 
     FIG.  112   
     Preparation of Dimeric Conjugate Having Low TFA Levels Via Sequential Purification Using Zorbax C-3 RP Preparative HPLC and Sephadex G-25 Gel Permeation Chromatography 
     Materials used and conditions for the analytical HPLC system include the following: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle; Eluent A: H 2 O(HPLC Grade with 0.1% TFA by volume); Eluent B: CH 3 CN (0.1% TFA by volume). Elution: Initial condition: 50% B then a linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm. Ret. time: (Compound (21)): 6.77 min, Rt (lyso): 4.06 min. 
     Preparative HPLC Using Preparative Zorbax C-3 Column to Remove the Lyso-Compound from (21) 
     The crude compound was loaded at a concentration of 30% eluent B. Materials used and conditions include: Conditions: Column: Waters Zorbax 300SB C-3; 21.2 mm i.d.×150 mm; 3.5 μm particle; Eluents: Eluent A: H2O(HPLC Grade with 10 mM NH4OAc); Eluent B: CH3 CN/H2O, 9/1 (final NH4OAc concentration:10 mM). 
     The composition of the eluent was then changed to 45% B over 2 min, then the column was eluted with a linear gradient of 45-100% B over 40 min; Flow rate: 30 mL/min; Detection: UV at 220 nm. 
     The crude compound (100 mg) was dissolved in 25 mL of a solution of 30% B. The preparative HPLC system was equilibrated at 30% B. The compound was loaded on to the Zorbax C-3 column. The mobile phase composition was ramped to 45% B over 2 min. A linear gradient from 45-100% B over 40 min was used for the elution of (21). The product eluted between 26.5-33 min. 
     The fractions that contained (21) were combined and lyophilized to give a white fluffy solid. This was dissolved in water-acetonitrile, then lyophilized again. This provided 70 mg product devoid of the lyso-compound. The recovery was about 70%. After chromatography was completed, the system was washed with 95% B for 15 min at a flow rate of 30 mL/min. The column was then washed with CH 3 CN/H 2 O (50/50, without TFA or buffer) for 30 min at a flow rate of 15 mL/min. The column was then stored at room temperature for future use. Analytical HPLC confirmed the absence of the lyso-compound in the isolated material. Further analysis confirmed that no lyso-compound formed after 5 days at room temperature. The material still contained significant amounts (4.2 wt %) of TFA. 
     Removal of TFA from (21) by Gel Permeation Chromatography on Sephadex G-25 
     A Sephadex G-25 column (100 g resin, bead size 20-80 μm, total gel volume ˜500 mL, column height: 27 cm) was equilibrated with 4 L of 50 mM ammonium bicarbonate. Then (21) (70 mg) was dissolved in 30 mL (final volume) of 60 mM ammonium bicarbonate in 10% aqueous acetonitrile. The solution was filtered and then loaded on to the Sephadex G-25 column. The column was eluted with 50 mM ammonium bicarbonate buffer with collection of 10 mL fractions. The collected fractions were monitored by analytical HPLC (UV detection at 220 nm). The results are provided in Table 4-A below. 
     
       
         
           
               
               
               
             
               
                 TABLE 4-A 
               
               
                   
               
               
                   
                   
                 Compound present 
               
               
                   
                   
                 (by HPLC analysis 
               
               
                 Fraction # 
                 Volume (mL) 
                 of fraction) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 10 
                 No 
               
               
                 3 
                 10 
                 No 
               
               
                 6 
                 10 
                 No 
               
               
                 9 
                 10 
                 No 
               
               
                 12 
                 10 
                 No 
               
               
                 15 
                 10 
                 No 
               
               
                 18 
                 10 
                 No 
               
               
                 19 
                 10 
                 No 
               
               
                 20 
                 10 
                 Yes 
               
               
                 21 
                 10 
                 Yes 
               
               
                 24 
                 10 
                 Yes 
               
               
                 27 
                 10 
                 Yes 
               
               
                 28 
                 10 
                 Yes 
               
               
                 29 
                 10 
                 No 
               
               
                   
               
            
           
         
       
     
     Fractions 20-28 were pooled and lyophilized. The lyophilized material obtained was re-dissolved in a small volume of water and the solution was frozen and lyophilized to remove residual amounts of ammonium bicarbonate. The final weight of the desired material was 58 mg. The recovery was 83%. 
     To ascertain the effective removal of TFA, the sample was subjected to CE analysis for TFA and acetate ions. The TFA is clearly present in the starting material (4.2%) according to the previous assay, while it is hardly detected (0.2%) after the gel permeation procedure. No acetate ion was detected. 
     Analytical Data for (21) Obtained by Serial Zorbax C-3 Preparative HPLC and Sephadex G-25 Gel Permeation Chromatography 
     Materials used and conditions for collecting analytical data include: Fluorine analysis (IC by QTI): 751 ppm (0.15% TFA wt/wt); Mass Spectrum: Method: MALDI-TOF; Mode: Positive Ion; Average molecular weight detected was 8461 the typical PEG2000 mass distribution curve was observed. HPLC: System A: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle; Eluent A: Water (HPLC Grade with 0.1% TFA by volume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution: linear gradient of 50-90% B over 3 min, hold at 90% B for 11 min; Flow rate: 0.5 mL/min; Detection: UV at 220 nm. Ret time: 6.77 min; Area %:99.6%. System B: Column: Zorbax 300SB C-3; 3 mm i.d.×150 mm; 3.5 μm particle; Eluent A: Water (HPLC Grade with 0.1% TFA by volume); Eluent B: Acetonitrile (0.1% TFA by volume). Initial condition: 50% B; Elution: linear gradient of 50-90% B over 3 min then ramp to 100% B over 12 min. Flow rate: 0.5 mL/min; Detection: LSD; Ret: time: 13.98 min. Area %:99.3%. 
     Table 5-A below provides definitions for the abbreviations used and the sources of materials referred to in Examples 56-59. 
     
       
         
           
               
               
             
               
                 TABLE 5-A 
               
               
                   
               
             
            
               
                 DSPA.Na 
                 (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3- 
               
               
                   
                 phosphosphatidic acid, sodium salt 
               
               
                 DPPG.Na 
                 (Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3- 
               
               
                   
                 phosphoglycerol, sodium salt 
               
               
                 DPPE 
                 (Genzyme) IUPAC: 1,2-Dipalmitoyl-sn-glycero-3- 
               
               
                   
                 phosphoethanolamine 
               
               
                 DSPC 
                 Distearoyl-glycero-phosphatidylcholine (Genzyme) 
               
               
                   
                 IUPAC: 1,2-Distearoyl-sn-glycero-3-phosphocholine 
               
               
                 DSPG.Na 
                 (Genzyme) IUPAC: 1,2-Distearoyl-sn-glycero-3- 
               
               
                   
                 phosphoglycerol, sodium salt 
               
               
                 DSPE-PEG1000 
                 Distearoyl-glycero-phosphoethanolamine-N- 
               
               
                   
                 methoxy(polyethylene glycol)1000 (Avanti Polar) 
               
               
                 DSPE-PEG2000 
                 Distearoyl-glycero-phosphoethanolamine-N- 
               
               
                   
                 methoxy(polyethylene glycol)2000 (Avanti Polar) 
               
               
                 Stearate* 
                 Sodium Stearate (Fluka) 
               
               
                 PEG4000 
                 (polyethylene glycol) MW 4000 (Fluka) 
               
               
                 Mannitol 
                 (Fluka) 
               
               
                   
               
               
                 *the acid form, i.e., stearic acid, can also be used in any of the microbubble preparations herein. 
               
            
           
         
       
     
     Example 56 
     Preparation of Targeted Microbubbles with DSPC/DPPG Envelope 
     Example 56A 
     383 mg of a mixture of DSPC/DPPG/ and the dimeric peptide phospholipid conjugate (11) shown in  FIG. 109  (molar ratio 49.75/49.75/0.5, corresponding to 187.1, 176.4 and 19.8 mg of the three components, respectively) and PEG-4000 (22.6 g) were solubilized in 120 g of t-butyl alcohol at 60° C., in a water bath. The solution was filled in vials with 0.8 mL of solution each. The samples were frozen at −45° C. and lyophilized. The air in the headspace was replaced with a mixture of C4F10/Nitrogen (50/50) and vials capped and crimped. The lyophilized samples were reconstituted with 5 mL of H2O per vial. 
     Example 56B 
     Example 56A was repeated using a mixture of DSPC/DPPG/ and the monomeric peptide phospholipid conjugate (31) shown in  FIG. 114  (molar ratio 49.5/49.5/1, corresponding to 182.8, 172.3 and 28.2 mg of the three components, respectively). 
     Example 57 
     Preparation of Targeted Microbubbles with DPPE/DPPG Envelope 
     Example 57A 
     An aqueous suspension of DSPE-PEG1000 (0.43 mg-0.24 μmole) and the monomeric peptide phospholipid conjugate (31) shown in  FIG. 114  (3.0 mg-0.5 μmole) was prepared in 500 μL of distilled water at 60° C. to obtain a micellar suspension. 
     Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) were dispersed in a solution of mannitol 10% in distilled water (20 mL) at 70° C. for 20 minutes. The dispersion was then cooled to room temperature. Perfluoroheptane (1.6 mL) was emulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 10500 rpm to obtain an emulsion. 
     The micellar suspension was added to the emulsion and the resulting mixture was heated at 60° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was divided in 4 mL fractions in 50 mL round bottom flasks. The emulsion was frozen at −45° C. for 5 minutes and freeze-dried at 0.2 mBar for 24 hours (Freeze-Drier Christ Beta 1-8K). 
     Before redispersion, the lyophilisate was exposed to an atmosphere containing C4F10/nitrogen (50/50 by volume). The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking. 
     Example 57B 
     An aqueous suspension of DSPE-PEG1000 (0.5 mg-0.27 μmole) and dimeric peptidephospholipid conjugate (11) shown in  FIG. 109  (5.3 mg-0.63 μmole) was prepared in 500 μL of distilled water at 60° C. to obtain a micellar suspension. 
     Separately, DPPE (15.8 mg-22.8 μmoles) and DPPG (4.2 mg-5.7 μmoles) were dispersed in a solution of PEG4000 10% in distilled water (20 mL) at 70° C. for 20 minutes. The dispersion was then cooled to room temperature. Perfluoroheptane (1.6 mL) was emulsified in the aqueous phase using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 10000 rpm to obtain an emulsion. 
     The micellar suspension was added to the emulsion and the resulting mixture was heated at 80° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (200 g/10 min-Sigma centrifuge 3K10) to eliminate the excess of phospholipid. The separated pellet (containing emulsified microdroplets of solvent) was recovered and re-suspended with the initial volume of a 10% PEG4000 aqueous solution. 
     The obtained emulsion was sampled into DIN8R vials (1 mL/vial). Then vials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. 
     Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product was redispersed in a volume of water twice the initial one by gentle hand shaking. 
     Example 58 
     Preparation of Targeted Microbubbles with DSPC/DSPA Envelope 
     Example 58A 
     An aqueous suspension of DSPE-PEG1000 (2.5 mg-1.4 μmole) and dimeric peptide conjugate (11) shown in  FIG. 109  (7.0 mg-0.84 μmole) was prepared in 1 mL of distilled water at 60° C. to obtain a micellar suspension. 
     DSPC (16.3 mg-20.6 μmoles) and DSPA (3.7 mg-5.15 μmoles) were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase was added to a PEG4000 10% solution in water (20 mL) using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at 8000 rpm, to obtain an emulsion. 
     The micellar suspension was mixed with the emulsion and the resulting mixture was heated at 80° C. for 1 hour under agitation. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of the phospholipid. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution. 
     The obtained suspension was sampled into DIN8R vials (1 mL/vial). Then vials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. Vials were exposed to an atmosphere containing C4F10/Nitrogen (35/65 by volume) and sealed. 
     The lyophilized product was then dispersed in a volume of water twice the initial one by gentle hand shaking. 
     Example 58B 
     Example 58A was repeated, but using 0.7 mg of DSPE-PEG2000 (0.26 moles) and 1.6 mg of monomeric peptide-phospholipid conjugate (1) shown in  FIG. 106  (0.26 μmole) to prepare the micellar suspension. 
     Example 58C 
     DSPC (16.3 mg-20.6 μmoles), DSPA (3.7 mg-5.15 μmoles) and monomeric peptide phospholipid conjugate (1) shown in  FIG. 105  (1.6 mg-0.26 μmole) were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase was emulsified in a PEG4000 10% aqueous phase (20 mL) using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for 1 minute at 8000 rpm to obtain an emulsion. 
     The resulting emulsion was heated at 80° C. for 1 hour under stirring. After cooling to room temperature (1 hour), the obtained emulsion was diluted with 20 ml of a PEG4000 10% aqueous solution. 
     The emulsion was sampled into DIN8R vials (1 mL/vial). Then vials were cooled at −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mBar for 12 hours with a final drying step at 30° C. and 0.1 mBar for 7 hours. 
     Vials were exposed to an atmosphere containing C4F10/nitrogen (35/65 by volume) and sealed. The lyophilized product was redispersed in a volume of water twice the initial one by gentle hand shaking. 
     Example 59 
     Preparation of Targeted Microbubbles with DSPC/Stearate Envelope 
     Example 59A 
     An aqueous suspension of DSPE-PEG2000 (2.5 mg-0.9 μmoles) and the dimeric phospholipid conjugate (11) shown in  FIG. 109  (2.5 mg-0.3 μmoles) was prepared in 660 μL of distilled water at 60° C. to obtain the micellar suspension. 
     Separately, DSPC (18.2 mg-23.1 μmoles) and stearate (1.8 mg-5.8 μmoles) were dissolved in cyclooctane (1.6 mL) at 80° C. This organic phase was added to a PEG4000 10% solution in water (20 mL) using a high speed homogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute at 9000 rpm, to obtain an emulsion. 
     The micellar solution was mixed with the emulsion and the resulting mixture was heated at 80° C. for 1 hour under agitation. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of phospholipids. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended with twice the initial volume of a 10% PEG 4000 aqueous solution. 
     The obtained suspension was sampled into DIN8R vials (1 mL/vial). Then vials were cooled to −50° C. (Christ Epsilon 2-12DS Freeze Dryer) and freeze-dried at −25° C. and 0.2 mbar for 12 hours, with a final drying step at 30° C. and 0.1 mbar for 7 hours. 
     Vials were exposed to an atmosphere containing C4F10/Nitrogen (35/65 by volume) and sealed. 
     The lyophilized product was dispersed in a volume of water twice the initial one by gentle hand shaking. 
     Example 59B 
     Example 59A was repeated by replacing the dimeric peptide phospholipid conjugate (11) shown in  FIG. 109  with the same relative molar amount of the monomeric peptide phospholipid conjugate (1) shown in  FIG. 106 . 
     Example 59C 
     Example 58C was repeated with DSPC (18.2 mg-23.1 μmoles), sodium stearate (1.8 mg-5.8 μmoles) and the dimeric peptide phospholipid conjugate (11) shown in  FIG. 109  (2.2 mg-0.26 μmole). The agitation speed for emulsification was fixed to 9000 rpm. After cooling to room temperature (1 hour), the obtained emulsion was washed once by centrifugation (1500 g/10 min—Sigma centrifuge 3K10) to eliminate the excess of the phospholipid. The separated supernatant (containing emulsified microdroplets of solvent) was recovered and re-suspended in twice the initial volume of a 10% PEG 4000 aqueous solution. 
     Example 60 
     Static Binding Test on KDR-Transfected Cells 
     Plasmid Production and Purification 
     Full-length KDR was cloned into the pcDNA6 vector and the plasmid was amplified in competent DH5a  E. coli . Plasmid amplification and purification was performed using  E. coli  JM 109 and a kit from Quiagen. 
     Transfection of 293H cells on Thermanox® Coverslips 
     Cells were grown on poly-D-lysine-coated Thermanox® circular coverslips in 24-well plate. Transfection was done as recommended in the lipofectamine 2000 protocol (Invitrogen, cat#11668-019) using 1 μg of DNA (pc-DNA6-fKDR)/per coverslip (1.3 cm2) in 0.1 mL. Transfection was done in serum-free media, the transfection reagent mix was removed from cells after 2 hours and replaced with regular serum-containing medium. Some of the cell-coated coverslips were mock-transfected (with no DNA). The next day, expression of the KDR receptor was assessed by immunocytochemistry and the binding assay was performed. 
     Bubble Binding Assay 
     The transfected cells were incubated with KDR-targeted microbubbles resuspended in 50% human plasma in PBS. For the incubation with the transfected cells a small plastic cap was filled with a suspension containing a 1.3×108 bubbles and the cap was covered with an inverted Thermanox® coverslip so as to put the transfected cells in contact with the targeted microbubbles. After 30 min of incubation at RT, the coverslip was lifted with tweezers, rinsed three times in PBS and examined under a microscope to assess binding of the targeted microbubbles. 
     Determination of the % of Surface Covered by Microbubbles 
     Images were acquired with a digital camera DC300F (Leica) and the percent of surface covered by bound microbubbles in the imaged area was determined using the software QWin version 3.1 (Leica Microsystem AG, Basel, Switzerland). Pictures were taken of each Thermanox® coverslip. For each preparation of Examples 56 and 57, the binding assay was repeated a minimum of two times thus obtaining an average value of the surface covered. In the following Tables 6-A and 7-A, the binding activity of the microbubbles prepared according to Examples 56 and 57 above are recorded. 
     As indicated by the Tables, the same peptide may show different binding activities when included (as a lipopeptide) in different phospholipid formulations forming the stabilizing envelope of the microbubble. Microbubbles containing KDR binding lipopeptides of the invention bind specifically to KDR-expressing cells while they did not bind appreciably to mock transfected cells. 
     Example 61 
     Dynamic Binding Test on rh VEGF-R2/Fc Chimeric Protein 
     Preparation of Fc-VEGF-R2-Coated Coverslips 
     Glass coverslips (40 mm in diameter, Bioptechs Inc., Butler, Pa., USA) were coated with recombinant human VEGF-R2/Fc Chimeric protein (R&amp;D Systems) according the following methodology. 
     A surface of dimensions 14×25 mm was delimited on the glass coverslip using a special marker (Dako Pen) and 400 μL of Fc-VEGF-R2 solution at 4 μg/mL in PBS was deposited on this surface. After an overnight incubation at 4° C., the solution was aspirated, replaced by 0.5 mL of a solution of BSA 1% w/v in PBS-0.05% Tween 80, pH 7.4 and incubated for 3 hours at RT. Then the coverslip was washed three times with 5 ml of PBS-0.05% Tween 80. 
     Binding Assay 
     Binding studies of targeted bubbles were carried out using a parallel-plate flow chamber (FCS2, Bioptech Inc., Butler, Pa., USA) with a chamber gasket of 0.25 mm in thickness, with a customized adapter for upside-down chamber inversion. The coated coverslip was inserted as a plate of the flow chamber. Microbubbles (5×106 bubbles/mL in 50% human plasma in PBS) were drawn through the flow chamber using an adjustable infusion pump (Auto Syringe® AS50 Infusion Pump, Baxter, Deerfield, Ill., USA) with a 60 mL syringe (Terumo). The pump flow rate was adjusted to 1 mL/min to obtain the desired shear rate of about 114 s-1. After 10 minutes, the flow was stopped and pictures were taken randomly at different positions on the coverslip (on areas of about 0.025 mm2) using a 40× objective and a CCD monochrome camera (F-View II, Soft Imaging Systems, Germany) connected to an inverted Olympus IX 50 microscope. 
     The number of microbubbles on each picture was determined, averaged with respect to the total number of pictures and the obtained value was then divided by ten (to obtain the “slope”, i.e. the average amount of bound microbubbles per minute). 
     For each preparation of Examples 58 and 59, the binding assay was repeated four times thus obtaining an average value of the slope. 
     The slope represents the bubble binding rate on the target substrate. For instance, a slope value of 8 indicates that an average of eighty (80) microbubbles was bound on the coated coverslip in ten minutes. A higher slope indicates a better capacity of bubbles to bind to the target under flow conditions. 
     In the following tables 8-A and 9-A, the binding activity of the microbubbles prepared according to Examples 58 and 59 above were illustrated. 
     As inferable from the tables, the same peptide may show different binding activities when included (as a peptide-phospholipid conjugate or lipopeptide) in different phospholipid formulations forming the stabilizing envelope of the microbubble. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 6-A 
               
               
                   
                   
               
               
                   
                 Example 
                 KDR 
                 Mock 
                 KDR-Mock 
               
               
                   
                   
               
             
            
               
                   
                 56A 
                 28.6% 
                 0.4% 
                 28.3% 
               
               
                   
                 56B 
                 28.0% 
                 0.3% 
                 27.7% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 7-A 
               
               
                   
                   
               
               
                   
                 Example 
                 KDR 
                 Mock 
                 KDR-Mock 
               
               
                   
                   
               
             
            
               
                   
                 57A 
                 23.6% 
                 0.2% 
                 23.5% 
               
               
                   
                 58B 
                 28.0% 
                 0.0% 
                 28.0% 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 8-A 
               
               
                   
                   
               
               
                   
                 Example 
                 Slope 
               
               
                   
                   
               
             
            
               
                   
                 58A 
                 8.2 
               
               
                   
                 58B 
                 8.1 
               
               
                   
                 58C 
                 5.8 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 9-A 
               
               
                   
                   
               
               
                   
                 Example 
                 Slope 
               
               
                   
                   
               
             
            
               
                   
                 59A 
                 9.0 
               
               
                   
                 59B 
                 8.0 
               
               
                   
                 59C 
                 7.8 
               
               
                   
                   
               
            
           
         
       
     
     Example 62 
     In Vivo Evaluation of Ultrasound Contrast Agents Targeted to KDR 
     The ability of ultrasound contrast agents containing KDR binding lipopeptides of the invention to bind to KDR-expressing tissue in vivo was assessed using a known model of angiogenesis: the rabbit VX2 tumor model. 
     A known model of angiogenic tissue was used to examine the ability of the KDR-targeted ultrasound microbubbles to localize to and provide an image of angiogenic tissue. The VX2 rabbit carcinoma was serially implanted in the dorsal muscle of New Zealand rabbits (Charles River Laboratories, France) weighting 2.5/3 kg. 
     Preparation of Tumor Homogenate 
     Tumor was surgically removed, placed into McCoy&#39;s culture medium containing 10% fetal calf serum, antibiotics, 1.5 mM Glutamax I and cut into small pieces that were rinsed to remove blood and debris. Then tumor pieces (3 to 5 cm3) were placed in a 50 ml Falcon tube containing 5 mL of complete medium. The tumor tissue was ground (Polytron) until no more solid pieces were visible. The murky fluid was centrifuged for 5 minutes at 300 g and the supernatant discarded. Seven mL of fresh medium was added per 5 mL of pellet. 
     Tumor Implantation 
     Rabbits received first 0.3 mL of Vetranquil (Acepromazine, Sanofi, injected intramuscularly) and were then anesthetized with an intramuscular injection of Ketaminol®5/Xylazine (Veterinaria AG/Sigma) mixture (50/10 mg/mL, 0.7 mL/kg). One hundred microliters of VX2 tumor homogenate was injected intramuscularly. Fifteen days after implantation of VX2 tumors, animals were anesthetized again with the same mixture, plus subcutaneous injection of 50% Urethane (2 mL/kg, s.c.) (Sigma) for imaging experiments. 
     In Vivo Ultrasound Imaging 
     VX2 tumor imaging was performed using an ultrasound imaging system ATL HDI 5000 apparatus equipped with a L7-4 linear probe. B-mode pulse inversion at high acoustic power (MI=0.9) was used to evaluate accumulation of targeted microbubbles on the KDR receptor expressed on the endothelium of neovessels. The linear probe was fixed on the skin directly over the implanted tumors. 
     After bubble injection (0.1 μL/kg of gas) using the preparations of either Example 63 or Example 64, insonation was stopped allowing bubbles to accumulate for 25 minutes. Then, insonation was reactivated at high acoustic power (MI 0.9) destroying all the bubbles present in the tumor. The amount of free circulating bubbles was then assessed by recording the signal obtained after 20 sec accumulation without insonation. 
     Video frames from VX2 tumor imaging experiments were captured with video-capture and analysed with Image-Pro Plus 2.0 software. The image representing free circulating bubbles was subtracted from the image obtained at 25 min, to provide an image representing bound bubbles. 
     Referring to  FIG. 115  (which shows the results with the preparation of Example 63) and  FIG. 116  (which shows the results with the preparation of Example 64),  FIGS. 115A and 116A  show an image before bubble injection (baseline);  FIGS. 115B and 116B  show retention of bubble contrast in the tumor 25 minutes post injection; and  FIGS. 115C and 116C  show the result obtained after subtraction of the baseline and free circulating bubbles and represent bound microbubbles containing KDR lipopeptides according to the present invention. Examples 62-64 and  FIGS. 115 and 116  confirm that ultrasound contrast agents bearing such KDR binding moieties localize to KDR expressing (and thus angiogenic) tissue in animal models. 
     Example 63 
     Example 59A was repeated by replacing DSPE-PEG2000 with DSPE-PEG1000 (2.7 mg, 1.54 μmol) and using 2.5 mg (0.31 μmol) of dimeric peptide phospholipid conjugate (11) shown in  FIG. 109 . 
     Example 64 
     Example 63 was repeated by replacing the dimeric peptide phospholipid conjugate with the same molar amount of monomeric phospholipid conjugate (1) shown in  FIG. 106 . 
     While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. The publications, patents and other references cited herein are incorporated by reference herein in their entirety.