WO2006096207A2 - Methods for preparing multivalent constructs for therapeutic and diagnostic applications - Google Patents

Methods for preparing multivalent constructs for therapeutic and diagnostic applications Download PDF

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WO2006096207A2
WO2006096207A2 PCT/US2005/028127 US2005028127W WO2006096207A2 WO 2006096207 A2 WO2006096207 A2 WO 2006096207A2 US 2005028127 W US2005028127 W US 2005028127W WO 2006096207 A2 WO2006096207 A2 WO 2006096207A2
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peptide
group
acid
eddwyyc
agptwc
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PCT/US2005/028127
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French (fr)
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WO2006096207A3 (en
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Hong Fan
Edmund Marinelli
Palaniappa Nanjappan
Radhakrishna Pillai
Rolf E. Swenson
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Bracco International B.V.
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Priority to JP2007525706A priority Critical patent/JP2008512356A/ja
Priority to EP05857561A priority patent/EP1778299A4/en
Priority to CA002575730A priority patent/CA2575730A1/en
Priority to AU2005328644A priority patent/AU2005328644A1/en
Publication of WO2006096207A2 publication Critical patent/WO2006096207A2/en
Publication of WO2006096207A3 publication Critical patent/WO2006096207A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links

Definitions

  • the invention relates to the use of multivalent constructs for therapeutic and diagnostic applications, and methods of preparing such constructs.
  • Chemical ligation techniques employ specialized chemistries that allow construction of multimers of a single peptide or provide a means to couple peptide fragments to prepare larger peptides or proteins.
  • the linking functions are nonpeptidic, but more recently developed methods can provide products with amide linkages.
  • the building blocks are typically single-chain Fv fragments (scFv) which are monovalent.
  • scFv single-chain Fv fragments
  • miniantibodies have properties similar to those of diabodies, but rather than a short 5-20 amino acid linker they have a relatively more flexible linker that allows freer orientation of the binding sites relative to each other, similar to the case in a natural antibody. Like diabodies, miniantibodies do not have the high molecular weight, immunologically active Fc dimer fragment. They can also be made by bacterial systems. Although they have desired advantages over natural antibodies, miniantibodies still suffer from having a relatively large size, which affects their pharmacokinetics, and must be made using biological methods. The smallest miniantibody is about 120 kDa in size.
  • bispecific antibodies e.g., antibodies that bind to two separate targets
  • pretargeting This approach uses a two-step protocol.
  • a bispecific antibody with at least one arm that recognizes a tumor-associated antigen and at least one other arm that recognizes an epitope on a diagnostic or therapy agent is given as a first injection. After the unbound antibody has substantially cleared non-target tissues and has reached a maximum level in the tumor, the smaller, bispecific antibody-recognizable diagnostic or therapeutic agent is given.
  • An alternative to this approach attempts to use a mixed antibody avidin/biotin system in a two-step procedure. For example, a targeting antibody is conjugated with either avidin or biotin and then is injected, whereupon it localizes in the tumor of interest. Thereafter, either biotin or avidin (depending on which was coupled to the targeting antibody), bearing an imaging or radiotherapeutic radionuclide, is injected and becomes localized at the site of the primary antibody by binding to avidin or biotin respectively.
  • Another approach to the use of antibodies as targeting moieties for radiopharmaceuticals or other diagnostic imagining agents has attempted to use a bivalent hapten to increase the avidity for the cell bound bispecif ⁇ c antibody over that of the circulating antibody.
  • This approach relies on bidentate binding occurring with the cell bound antibodies, because the surface density on the cells is sufficiently high, but not occurring with the circulating antibodies, because the concentration is too low. In effect, the system makes use of the increase in avidity caused by the closer presentation of the antibodies/antigen on the cells.
  • Peptides have also been used as targeting moieties.
  • two or more peptide based targeting agents selective for different targets.
  • a hybrid peptide having ligands to two targets selected from the somatostatin-, GRP-, CCK-, Substance P-, or VIP receptor and ⁇ v ⁇ 3 integrin was reportedly made and tested for the ability to bind to tumor cells.
  • the initial evaluation showed no improved tumor uptake for the multiple ligand systems investigated.
  • the investigators assumed that steric impairment leads to a reduction of the receptor affinities of the dimeric structures.
  • a variation on these approaches uses a bispecif ⁇ c diabody targeted to two different epitopes on the same antigen.
  • This approach attempts to increase the avidity of the construct for the target, because, although the binding is monovalent for each epitope, the construct as a whole is bivalent to its target, as each of the binding epitopes is located within the same target molecule.
  • scFv fragments have been found to have insufficient affinity and an increase in avidity was required.
  • Two rationales underlie the approaches described above. The first rationale uses two different targeting moieties to overcome some of the pharmacokinetic problems associated with the delivery of antibodies to solid tumors.
  • the second rationale uses two different targeting moieties to increase the avidity of the construct for a given target, such as a single molecule or a whole tumor.
  • a given target such as a single molecule or a whole tumor.
  • all of the approaches described suffer from various drawbacks.
  • diagnostic and therapeutic agents with increased affinity and/or avidity for a target of interest.
  • diagnostic and therapeutic agents that, when administered in vivo to a mammal, have acceptable pharmacokinetic properties.
  • Angiogenesis the formation of new blood vessels, occurs not only during embryonic development and normal tissue growth and repair, but is also associated with the female reproductive cycle, establishment and maintenance of pregnancy, and repair of wounds and fractures.
  • 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 rumor from hyperplastic to neoplastic growth, that inhibition of angiogenesis has become an active cancer therapy research field.
  • VEGF vascular endothelial growth factor
  • VEGF also referred to as VEGF-A
  • VEGF-A is synthesized as five different splice isoforms of 121, 145, 165, 189, and 206 amino acids.
  • VEGF 121 and VEGF 165 are the main forms produced, particularly in tumors ⁇ see Cohen et al. 1999, supra).
  • VEGF 121 lacks a basic domain encoded by exons 6 and 7 of the VEGF gene and does not bind to heparin or extracellular matrix, unlike VEGF 165 .
  • VEGF family members act primarily by binding to receptor tyrosine kinases.
  • receptor tyrosine kinases are glycoproteins having an extracellular domain capable of binding one or more specific growth factors, a transmembrane domain (usually an alpha helix), a juxtamembrane domain (where the receptor may be regulated by, e.g., 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.
  • a transmembrane domain usually an alpha helix
  • juxtamembrane domain where the receptor may be regulated by, e.g., phosphorylation
  • a tyrosine kinase domain the catalytic component of the receptor
  • carboxy-terminal tail which in many receptors is involved in recognition and binding of the substrates for the tyrosine kinase.
  • FIt-I VEGFR-I
  • KDR FIk-I
  • VEGFR-3 VEGFR-3
  • FIt-I and KDR have been identified as the primary high affinity VEGF receptors. While FIt-I 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).
  • KDR KDR-induced angiogenic vessel
  • angiogenic vessel angiogenic vessels
  • 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, 5 th Edition (B.C. Decker Inc.; Ontario, Canada, 2000) pp. 132-152).
  • KDR kinase domain region
  • 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.
  • 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. Diagnostic applications, such as detecting malignant tumor growth, and therapeutic applications, such as targeting tumoricidal agents or angiogenesis inhibitors to the tumor site, would be particularly beneficial.
  • Hepatocyte growth factor also known as scatter factor
  • HGF is a multi-functional growth factor involved in various physiological processes such as embryogenesis, wound healing and angiogenesis. It has become apparent that HGF, through interactions with its high affinity receptor (cMet), is involved in tumor growth, invasion and metastasis.
  • cMet high affinity receptor
  • dysregulated cMet expression for example, the overexpression of cMet in neoplastic epithelium of colorectal adenomas and in other carcinomas as compared to normal mucosa
  • cMet receptor for example, the overexpression of cMet in neoplastic epithelium of colorectal adenomas and in other carcinomas as compared to normal mucosa
  • hyperactivity of the cMet receptor through an autocrine stimulatory loop with HGF 3 has been demonstrated in a variety of tumor tissues and induces oncogenic transformation of specific cell lines.
  • HGF is produced by the stromal cells, which form part of many epithelial tumors; however, it is believed that the production of HGF by tumor cells themselves comprises the main pathway leading to the hyperproliferation of specific tumors.
  • HGF/cMet autocrine stimulatory loops have been detected in gliomas, osteosarcomas, and mammary, prostate, breast, lung and other carcinomas.
  • HGF Interrupting the HGF interaction with the cMet receptor slows tumor progression in animal models.
  • HGF In addition to stimulating proliferation of certain cancer cells through activation of cMet, HGF also protects against DNA-damaging agent-induced cytotoxicity in a variety of cell lines susceptible to hyperproliferative phenotypes (e.g., breast cancer). Therefore, preventing HGF from binding to cMet could predispose certain cancer cells to the cytotoxicity of certain drugs.
  • cMet In addition to hyperproliferative disorders, cMet also has been linked to angiogenesis. For example, stimulation of cMet leads to the production of vascular endothelial growth factor (VEGF), which, in turn, stimulates angiogenesis. Additionally, stimulation of cMet also has been implicated in promoting wound healing.
  • VEGF vascular endothelial growth factor
  • stimulation of cMet also has been implicated in promoting wound healing.
  • the large discrepancy between expression levels of neoplastic and corresponding normal tissues indicates that cMet is an attractive target for imaging applications directed to hyperproliferative disorders.
  • the present invention is directed, in one embodiment, to novel methods for preparing multivalent constructs for therapeutic and diagnostic applications using a novel linker D comprising a derivative of a dicarboxylic acid. More specifically, in one preferred embodiment, the present invention is directed to a method for preparing a multivalent compound comprising the formula A-B-C-D-E-B' -F comprising the steps of attaching in any order any of said A, B, C, D, E, B' or F to any of its respective adjacent component or components in the formula A, B, C, D, E, B' and F until the compound A-B-C-D-E-B'-F is formed, wherein D is a linker comprising a derivative of a dicarboxylic acid.
  • the present invention is directed to novel methods for preparing multivalent constructs for therapeutic and diagnostic applications using a novel linker D comprising a dicarboxylic acid derivative.
  • a glutaric acid bis-NHS ester or a derivative therof is a preferred dicarboxylic acid derivative.
  • the linker D holds or connects two or more peptides of the construct together.
  • the present invention is directed to a method for preparing a multivalent compound comprising the formula A-B-C-D-E-B'-F comprising the steps of attaching in any order any of said A, B, C, D, E, B' or F to any of its respective adjacent component or components in the formula A, B, C, D, E, B' and F until the compound A-B-C-D-E-B' -F is formed, wherein D is a linker comprising a glutaric acid bis N-hydroxysuccinimidyl ester or a derivative therof.
  • the present invention is directed to novel methods for preparing multivalent constructs for therapeutic and diagnostic applications using a novel linker D comprising a diamine or a derivative therof. More specifically, in another preferred embodiment, the present invention is directed to a method for preparing a multivalent compound comprising the formula A-B-C-D-E-B' -F comprising the steps of attaching in any order any of said A, B, C, D, E, B' or F to any of its respective adjacent component or components in the formula A, B, C, D, E, B' and F until the compound A-B-C-D-E-B' -F is formed, wherein D is a linker comprising a diamine or a derivative thereof.
  • A is a first peptide
  • B is a first branching group
  • C is an optional first spacer
  • E is an optional second spacer which may be the same as or different from said first spacer C
  • B' is an optional second branching group which may be the same as or different from said first branching group B
  • F is a second peptide which may be the same as or different from said first peptide A.
  • peptides A and F include any of the monomers described herein, and specifically include those in Table 1 in Section 19.
  • At least one of peptide A and peptide F is a compound or compounds comprising two or more amino acids.
  • the peptides A and F may be different peptides, optionally targeting peptides, and may target different eptiopes of the same target or may target different epitopes of the same receptor.
  • the receptors may include KDR or cMET.
  • One or both of the first peptide A and second peptide F may be disulfide peptides.
  • Branching groups B and B' provide a point of attachment for an optional reporter-sp group to the compound.
  • One or both of said first branching group B and said second branching group B' may be, but are not limited to, lysine, ornithine, aspartic acid or glutamic acid.
  • Spacers C and E attach the linker D to the other components A, B, B' or F of the compound while keeping those other components at a distance or "space" from the linker D.
  • the spacers C or E are optional, and may comprise one or more of the following: (a) GIy, (b) Ser-Gly-Ser; (c) Ser(S)-Gly-Ser(S) wherein the group S comprises a hydrophilic function such as a sugar moiety which may be selected from N-acetylgalactoseamine, D-(+)- allose, D-(+)-altrose, D-(+)-glucose, D-(+)-mannose, D-(-)-gulose, D-(-)-idose, D-(+)- galactose, D-(-)-talose, D-(-)-ribose, D-(-)-arabinose, D-(+)-xylose or D-(-)-
  • the linker D comprises a substituted aromatic or heterocyclic diamine of the form NH 2 -Q-Z-Q '-NH 2 , where
  • the linker D comprises a derivative of a dicarboxylic acid, which may be derived from a D or L amino acid, or a D or L amino acid derived from lysine, ornithind or 2,3-diaminopropionic acid.
  • the linker D may be selected from among the following: 4-aminomethylbenzylamine, 4-aminomethyl-4'-aminomethylbiphenyl, N- methylpyrrole-2,5-dicarboxylic acid bis 2-aminoethylamide, pyridine-2,6-dicarboxylic acid bis-2-aminoethylamide, N-methylpyrrole-2,3 -dicarboxylic acid bis-2-aminoethylamide, N- methylpyrrole-2,4-dicarboxylic acid bis-2-aminoethylamide, pyridine 2,3 -dicarboxylic acid bis-2-aminoethylamide, pyridine-3, 5 -dicarboxylic acid bis-2-aminoethylamide and pyridine 3,4-dicarboxylic acid bis-2-aminoethylamide.
  • the linker D comprises a glutaric acid bis N- hydroxysuccinimid
  • said first peptide A and said second peptide F may be connected in a number of arrangements, including, e.g.,: (a) from C- terminus to C-terminus; (b) from N-terminus to C-terminus; (c) from C-terminus to N- terminus; or (d) from N-terminus to N-terminus.
  • the preferred method includes the step of attaching a first reporter-sp group to one of said branching groups B or B'. Additionally, where both branching groups B and B' are present, a preferred method includes the step of attaching a second reporter-sp group to the other of said branching group B or B' to which the first reporter-sp group was not attached.
  • Reporter-sp groups or molecules serve as reporters or special purpose molecules, or may serve both functions.
  • One or both of the first reporter-sp group and second reporter-sp group may comprise a phospholipid or lipid moiety.
  • One or both of the first reporter-sp group and second reporter-sp group may comprise, e.g., a diamagnetic or magnetic metal chelate, a metal chelator system, a radiometal chelating system, a radioisotope-labeled molecule, a dye molecule, a fluorescent molecule, a phosphorescent molecule, a molecule absorbing in the UV spectrum, a molecule capable of absorption of near or far infrared radiation; or a molecule or supramolecular construct that absorbs and/or emits x-rays, gamma rays, beta particles, alpha particles, ultrasounds, optical or magnetic signals.
  • first reporter-sp group and second reporter-sp group may possess the ability to act as a therapeutic, and/or the capability to act as a toxin employed for selective killing and/or inhibiting the growth of cancer cells or normal tissue.
  • the final multivalent compound may comprise one, two or even three or more reporter-sp groups.
  • the present methods allow a high degree of control over the incorporation of reporter-sp groups into one or more of the constituent monomers. This is also controllable with respect to the stage of synthesis at which the incorporation of the reporter-sp groups are conducted.
  • novel method of using peptides which target different epitopes of the same targetor or receptor imparts unexpectedly superior binding characteristics to the multivalent constructs of the present invention.
  • FIG. 1 shows the binding of fluorescent beads to KDR-transfected and mock- transfected cells.
  • Neutravidin-coated beads with the indicated biotinylated ligands attached were tested for binding to KDR-expressing and non-expressing 293H cells. Specific binding to KDR was detected for both P5 (with hydrophilic spacer) and P6. Further details are provided in Example 2.
  • FIG. 2 shows the percentage inhibition of 125 I-labeled VEGF binding by peptides [P6, P4, P5-X-B and P12-X-B) at two different concentrations (30 ⁇ M and 0.3 ⁇ M) to KDR-transfected 293H cells, as described in Example 3.
  • the results for P6, P4 and P5-X- B are the average of three experiments ⁇ SD, whereas the result for P12-X-B is based on one experiment.
  • FIG. 3 depicts immunoblots of KDR immunoprecipitates from unstimulated (-
  • FIG. 4 depicts immunoblots demonstrating inhibition of KDR phosphorylation (activation) with a neutralizing anti-KDR antibody, as described in Example 4.
  • Immunoprecipitates from unstimulated (-V), VEGF-stimulated (+V), and simultaneously VEGF/anti-KDR (l ⁇ g/mL) (+V+ ⁇ -KDR)-treated HUVECs were resolved by SDS-PAGE, blotted, and sequentially probed with anti-phosphotyrosine (“Phospho KDR”) and anti-KDR ("Total KDR”) antibodies.
  • Phospho KDR anti-phosphotyrosine
  • Total KDR total KDR
  • FIG. 5 depicts immunoblots demonstrating inhibition of KDR phosphorylation (activation) with a KDR-binding peptide (repeat experiment).
  • Immunoprecipitates from unstimulated (-V), VEGF-stimulated (+V), and a KDR-binding peptide (10 ⁇ M) (+V+P10)-treated HUVECs were resolved by SDS-PAGE, blotted, and sequentially probed with anti-phosphotyrosine (“Phospho KDR") and anti-KDR ("Total KDR").
  • Phospho KDR anti-phosphotyrosine
  • Total KDR Total KDR
  • FIG. 6 depicts binding of Tc-labeled P 12-C to mock and KDR-transfected
  • FIG. 7 depicts specific binding of Tc-labeled P12-C to KDR-transfected 293H cells, as described in Example 5.
  • FIG. 8 depicts saturation binding of peptide/Neutravidin HRP complexes, as described in Example 6.
  • FIG. 8A shows the results obtained using P6-XB and P5-XB.
  • FIG. 8B shows the results obtained using P12-XB and P13-XB. Calculated
  • K D values were: 10.00 nM (P6-XB), 14.87 nM (P5-XB), 4.03 nM (P12-XB) and 1.81 nM (P13-XB).
  • FIG. 9 depicts binding of peptide/neutravidin HRP complexes (P 1 -X-B, P5-
  • FIG. 10 depicts binding of peptide/neutravidin HRP complexes (Pl-XB, Pl-
  • FIG. 11 depicts specific binding (binding to KDR-transfected cells minus binding to mock-transfected cells) of peptide/neutravidin HRP complexes (P6-XB, P5-XB, P 12-XB and Pl 3-XB) with and without 40% rat serum, as described in Experiment C of Example 6.
  • concentration of peptide/avidin HRP solution was 6.66 nM for P6-XB and P5-XB, 3.33 nM for P12-XB and 2.22 nM for P13-XB.
  • FIG. 12 shows the binding of peptide/avidin HRP with mock- and KDR- transfected cells, plotted as absorbance at 450 run. The proportions of control and KDR- binding peptides used to form each tetrameric complex are indicated for each tested multimer, in the legend.
  • FIG. 13 depicts specific binding of a P5-XB/avidin-HRP complex to KDR- transfected cells (background binding to mock-transfected cells subtracted), plotted as absorbance at 450 nm. Increasing concentrations (as indicated by the X axis) of uncomplexed peptides were added to the assay as indicated in the legend. Only free P5-XB was able to decrease the binding of the P5-XB/avidin complex to KDR-transfected cells. [0059] FIG.
  • FIG. 14 is a graph showing the percentage inhibition of 125 I-labeled VEGF binding by peptides (P12-XB, D2, D1, D3, and P13-D) at three different concentrations (10 ⁇ M, 0.3 ⁇ M, and 0.03 ⁇ M) to KDR-transfected 293H cells. The results are from one experiment carried out in tripicate +/- S. D.
  • FIG. 15 is a photograph showing the ability of D1 to completely block the
  • FIG. 16 shows 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. 17 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. 18 is a graph showing the specific binding (KDR-MOCK) of 125 I-labeled
  • FIG. 19 is a graph of plasma clearance as percent injected dose per mL versus time.
  • FIG. 20 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 the time point, animal number and volume injected for HPLC analysis.
  • FIG. 21 is a graph showing the results of testing of KDR peptides in HUVEC proliferation assay.
  • A represents D6;
  • B represents P12-G;
  • C represents PNC-I (negative control);
  • F PNC-I (negative control).
  • FIG. 22 shows the kinetic analysis of D1 (heterodimer of a truncated form of
  • FIG. 23 shows the kinetic analysis of D7 (heterodimer of P5-D and P6-D) binding to murine KDR-Fc. All sensograms are fit to the bivalent analyte model.
  • FIG. 24 shows kinetic analysis of fluorescein-labeled P12-G binding to murine KDR-Fc. All sensograms are fit to the 1 : 1 Langmuir model.
  • FIG. 25 is a graph showing the specific binding (binding to KDR-transfected cells minus binding to mock-transfected cells) of 99m Tc-labeled P12C in the presence and absence of 40% rat serum, as described in Experiment C of Example 6. Results are plotted as specific CPM bound +/- s.d.
  • FIG. 26 is a graph depicting % inhibition ⁇ s.d. of specific 125 I-VEGF binding to KDR-transfected cells by PG-I (squares) D1 (diamonds).
  • FIG. 27 is a graph depicting % maximum VEGF-stimulated migration ⁇ s.d. of HUVEC cells in the presence of the indicated concentrations of PG-I (diamonds) D1 (squares).
  • FIG. 28 displays a first graph depicting the binding of Tc-labeled D10 to
  • FIG. 3OA is a graph illustrating the specificity of binding of peptide- conjugated microbubbles to KDR-expressing cells.
  • FIG. 3OB is a graph showing the binding efficiency of monomers and dimers conjugated to microbubbles on KDR-expressing cells.
  • FIG. 3OC is a graph showing the binding efficiency of mixed monomers, dimers and monomers conjugated to microbubbles on KDR-expressing cells.
  • FIG. 31 is a graph summarizing 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 21. [0079] FIG.
  • FIG. 32 is a graph showing the total binding of complexes of control peptide and the test peptides (P30-XB, P31-XB, P32-XB) with 125 I-streptavidin (in the presence of VEGF) to mock-transfected and KDR-transfected cells. Only the complex containing P30- XB showed specific binding (KDR-mock).
  • FIG. 33 is a graph showing that D26 (squares) with its glycosylation and modified spacer is able to block VEGF-stimulated migration even more potently than D24
  • FIG. 34 is a graph showing that TK-1 enhances the potency of D6 in blocking the biological effects of VEGF in a migration assay with cultured HUVECs.
  • FIG. 35 is a graph showing that homodimeric D8 (squares) is less able than heterodimeric D17 (diamonds) to block the effects of VEGF in the migration assay as carried out in Example 25.
  • FIG. 36 is a graph showing cell proliferation data for D6 as described in
  • FIG. 37A is an illustration of examples of (A) a C-terminus to C-terminus linked dimer, (B) an N-terminus to C-terminus linked dimer, and (C) an N-terminus to N- terminus linked dimer.
  • FIG. 37B is an illustration of a general structure for C-terminus and C- terminus linked dimer s.
  • FIG. 37C is an illustration of a general structure for N-terminus to C-terminus linked dimers.
  • FIG. 37D is an illustration of a general structure for N-terminus to N-terminus linked dimers.
  • FIG. 37E is an illustration of an alternate structure for N-terminus to N- terminus linked dimers.
  • FIG. 38 A illustrates examples of branching groups B for peptide 1.
  • FIG. 38B illustrates additional examples of branching groups B for peptide 1.
  • FIG. 39 illustrates examples of synthetic methods for preparing intermediate compounds of the invention.
  • FIG. 40 illustrates examples of cyclic dicarboxylic acids HOOC-Z-COOH employed to prepare linkers D.
  • FIG. 41 illustrates examples of leaving groups employed in active acylating derivatives of linker D compounds.
  • FIG. 42 illustrates an example of a reaction using the 4-mercaptobutanoyl group as a bifunctional linker D.
  • FIG. 43A illustrates examples of branching groups B' for peptide2.
  • FIG. 43B illustrates additional examples of branching groups B' for peptide2.
  • FIG. 44 illustrates a preparation of a peptide 1— branching group B-linker D- leaving group compound.
  • FIG. 45 shows an example of an assembly of the peptide 1 -branching group
  • FIG. 46 shows an example of an attachment of a reporter-sp molecule by reaction of an active acylating species with the amine-containing dimer.
  • FIG. 47 shows an example of a preparation of a reporter-sp molecule-labeled dimer by reaction of an active acylating dimer with an amine-bearing reporter-sp.
  • FIG. 48 illustrates alternative methods for attachment of reporter-sp molecule to the peptide construct.
  • FIG. 49 depicts an example of a preparation of dimeric peptides from active acyl peptide branching group spacers employing diamines as the linker D function.
  • FIG. 50 shows examples of cyclic diamines having the formula NH 2 -Q-Z-
  • FIG. 51 shows examples of cyclic aliphatic diamines having the formula
  • FIG. 52 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. 53 is a bar graph showing that D25 blocks increased peritoneal vascular permeability induced by VEGF injected intraperitoneally.
  • FIGS. 54A-54R are lists of KDR-binding peptides isolated from a TNl 1/1 library.
  • the present invention is based, in part, on the discovery that compounds having two or more binding moieties, wherein at least two of the binding moieties bind to different binding sites on the same target, have unexpected and significantly improved ability to bind the target.
  • the target is a receptor or a receptor/ligand complex.
  • the improved ability of compounds of the invention (variously referred to as "multivalent targeting constructs,” “heterodimers,” “heterodimeric constructs,” “heterotrimers,” “heterotetramers,” “heteromultimers” and/or “heteromultimeric constructs” herein) to bind a target may be demonstrated by comparison to the ability of an individual, constituent, binding moiety to bind the target.
  • the binding strength of a heteromultimer of the invention may be compared to the binding strength of one of the monomers that constitute the structure of the heteromultimer.
  • a heteromultimer of the invention exhibits an increase in affinity
  • the present invention is directed to novel methods for preparing multivalent constructs for therapeutic and diagnostic applications using novel linkers (linker D).
  • the term "recombinant” is used to describe non-naturally altered or manipulated nucleic acids, host cells transfected with exogenous nucleic acids, or polypeptides expressed non-naturally, through manipulation of isolated DNA and transformation of host cells.
  • Recombinant is a term that specifically encompasses DNA molecules which have been constructed in vitro using genetic engineering techniques, and use of the term "recombinant” as an adjective to describe a molecule, construct, vector, transfected cell, polypeptide or polynucleotide specifically excludes naturally occurring such molecules, constructs, vectors, cells, polypeptides or polynucleotides.
  • bacteriophage is defined as a bacterial virus containing a DNA core and a protective shell built up by the aggregation of a number of different protein molecules.
  • the terms “bacteriophage” and “phage” are used herein interchangeably.
  • polypeptide is used to refer to a compound of two or more amino acids joined through the main chain (as opposed to side chain) by a peptide amide bond (— C(:O)NH-).
  • peptide is used interchangeably herein with “polypeptide” but is generally used to refer to polypeptides having fewer than 40, and preferably fewer than 25 amino acids.
  • binding 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, surface plasmon resonance and the monitoring of spectroscopic changes that result from binding.
  • binding polypeptide refers to any polypeptide capable of forming a binding complex with another molecule. Also included within the definition of "binding polypeptides" are polypeptides that are modified or optimized as disclosed herein.
  • modifications include substitution of amino acids for those in the parent polypeptide sequence to optimize properties, to remove or alter the characteristics of 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" 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, to assist in immobilization of binding peptides according to this invention on solid supports
  • binding polypeptides may be linked or conjugated to a radiotherapeutic agent, a cytotoxic agent, a tumorcidal agent or enzyme, a liposome (e.g., loaded with a therapeutic agent, an ultrasound appropriate gas, or both).
  • binding polypeptides of the invention may be bound or linked to a solid support, such as a well, plate, bead, tube, slide, filter, or dish.
  • dimers or multimers of one or more binding polypeptides may be formed. Such constructs may, for example, exhibit increased ability to bind to the target. All such modified polypeptides are also considered "binding polypeptides" so long as they retain the ability to bind the targets.
  • homologous polypeptides may be produced using any of the modification or optimization techniques described herein or known to those skilled in the art. Such homologous polypeptides will be understood to fall within the scope of the present invention and the definition of "binding polypeptides” so long as the substitution, addition, or deletion of amino acids or other such modification does not eliminate its ability to bind to the target.
  • homologous refers to the degree of sequence similarity between two polymers ⁇ i.e., polypeptide molecules or nucleic acid molecules). When the same nucleotide or amino acid residue or one with substantially similar properties (i.e.
  • 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 binding sequences disclosed herein.
  • KDR-binding polypeptide is a binding polypeptide that forms a complex in vitro or in vivo with vascular endothelial growth factor receptor-2 (or KDR, FIk-I).
  • VEGF/KDR complex-binding polypeptide is a binding polypeptide that forms a complex in vitro or in vivo with a binding complex formed between vascular endothelial growth factor (VEGF) and KDR, in particular the complex of homodimeric VEGF and one or two KDR molecules that is believed to form at the surface of endothelial cells during angiogenesis.
  • KDR and VEGF/KDR-binding polypeptides include but are not limited to the peptides presented discussed herein, and in U.S. S.N. 60/360,851 and U.S. S.N. 60/440,441, both of which are incorporated by reference herein in their entirety, and in copending application U.S. S.N. 10/661,156, entitled “KDR and VEGF/KDR-binding peptides and their use in diagnosis and therapy," and include hybrid and chimeric polypeptides incorporating such peptides as well as homologues.
  • cMet binding polypeptide is a binding polypeptide that forms a complex in vitro or in vivo with the HGF receptor, cMet.
  • cMet/HGF complex-binding polypeptide is a binding polypeptide that forms a complex in vifro or in vivo with a binding complex formed between hepatocyte growth factor (HGF) and cMet.
  • HGF hepatocyte growth factor
  • Specific examples of cMet and cMet/HGF binding polypeptides include but are not limited to the peptides presented discussed herein, and in U. S. S. N. copending applications U.S.S.N. 60/451,588 and U.S.S.N.
  • a “labeling group” or “detectable label,” as used herein, is a group or moiety capable of generating a signal for diagnostic imaging, such as, e.g., magnetic resonance imaging, radioimaging, ultrasound imaging, x-ray imaging, light imaging, or carrying a moiety such as a radioactive metal or other entity that may be used in radiotherapy or other forms of therapy.
  • diagnostic imaging such as, e.g., magnetic resonance imaging, radioimaging, ultrasound imaging, x-ray imaging, light imaging, or carrying a moiety such as a radioactive metal or other entity that may be used in radiotherapy or other forms of therapy.
  • the term “specificity” refers to a binding polypeptide having a higher binding affinity for one target over another. Binding specificity may be characterized by a dissociation equilibrium constant (K D ) or an association equilibrium constant (K a ) for the two tested target materials.
  • K D dissociation equilibrium constant
  • K a association equilibrium constant
  • binding polypeptides of the invention have a dissociation constant for a desired target that is lower than about 10 ⁇ M, more preferably lower than about 1 ⁇ M, and most preferably less than about 0.5 ⁇ M or even lower.
  • KDR specificity refers to a KDR-binding moiety having a higher affinity for KDR than an irrelevant target.
  • VEGF/KDR specificity refers to a VEGF/KDR complex-binding moiety having a higher affinity for a VEGF/KDR complex than an irrelevant target.
  • heteromultimers according to the present invention are specific for KDR or the VEGF/KDR complex, and preferably have a dissociation constant that is lower than about 10 ⁇ M, more preferably less than about 1 ⁇ M, most preferably less than about 0.5 ⁇ M or even lower.
  • cMet specificity refers to a cMet binding moiety having a higher affinity for cMet than an irrelevant target.
  • cMet/HGF specificity refers to a cMet/HGF complex-binding moiety having a higher affinity for a cMet/HGF complex than an irrelevant target.
  • binding heteromultimers according to the present invention are specific for cMet or the cMet/HGF complex, and preferably have a dissociation constant that is lower than about 10 ⁇ M, more preferably less than about 1 ⁇ M, most preferably less than about 0.5 ⁇ M or even lower.
  • patient refers to any mammal, especially humans.
  • the term "pharmaceutically acceptable” carrier or excipient refers to a non-toxic carrier or excipient that may be administered to a patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof.
  • the term "target” or “target molecule” refers to any substance that a binding moiety or binding polypeptide can bind to, such as proteins or polypeptides, cells, receptors, carbohydrates, lipids, etc. As used herein, “target” also includes a family of receptors, such as, for example, protein-tyrosine kinase receptors.
  • therapeutic agent refers to a compound or an agent having a beneficial, therapeutic or cytotoxic effect in vivo.
  • Therapeutic agents include those compositions referred to as, for example, bioactive agents, cytotoxic agents, drugs, chemotherapy agents, radiotherapeutic agents, genetic material, etc.
  • the targeting constructs of the invention include two or more binding moieties which bind to different binding sites of a single target.
  • the binding moieties are specific for different sites on the same target. They may be peptidic, peptidomimetic, etc. and include binding polypeptides as defined herein. Additionally, binding moieties include small binding molecules. In a preferred embodiment the binding moieties comprise binding polypeptides.
  • targeting constructs are by definition dimeric or multimeric and may be referred to as “multivalent targeting constructs,” “heterodimers,” “heterodimeric constructs,” “heterodimeric peptides,” “heterotrimers, “heterotetramers,” “heteromultimers,” “hetermultimeric constructs” or “heteromers” for example.
  • These dimeric or multimeric constructs exhibit improved binding, as compared to a monomeric construct.
  • the polypeptide sequences 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 bromo acetamide derivatives), but is not so limited.
  • preferred dimers according to the invention can be constructed by connecting a first binding peptide to a branching group to a first spacer to a linker to second spacer and finally to the second binding peptide.
  • This linking scheme for the dimers can be represented by the following general structure: A-B-C-D-E-F where A and F are two different binding peptides which bind to different sites on the same target, B is a branching group, C and E are optional spacers, and D is a linker. Suitable spacers and linkers are known in the art and are also provided in the Examples below. In various embodiments, C, D and/or E may optionally be present or absent.
  • a reporter moiety or similar group may optionally be attached to the dimer via the branch group.
  • the exact arrangement of these components can vary depending, for example, on whether the peptides are linked from C-terminus to C-terminus, from N-terminus to C-terminus, or from N- terminus to N-terminus. Examples of these different attachment schemes are shown in FIGS. 37A-37E.
  • the present invention is directed to a method for preparing a multivalent compound comprising the formula
  • A-B-C-D-E-B'-F comprising the steps of attaching in any order any of said A, B, C, D, E, B' or F with any of its respective adjacent component or components in the formula A, B, C, D, E, B' and F until the compound A-B-C-D-E-B'-F is formed, wherein D is a linker comprising a derivative of a dicarboxylic acid.
  • the present invention is directed to a method for preparing a multivalent compound comprising the formula
  • A-B-C-D-E-B'-F comprising the steps of attaching in any order any of said A, B, C, D, E, B' or F with any of its respective adjacent component or components in the formula A, B, C, D, E, B' and F until the compound A-B-C-D-E-B'-F is formed, wherein D is a linker comprising a glutaric acid bis N-hydroxysuccinimidyl ester or a derivative therof.
  • the present invention is directed to a method for preparing a multivalent compound comprising the formula
  • A-B-C-D-E-B'-F comprising the steps of attaching in any order any of said A, B, C, D, E, B' or F with any of its respective adjacent component or components in the formula A, B, C, D, E, B' and F until the compound A-B-C-D-E-B'-F is formed, wherein D is a diamine linker or a derivative thereof.
  • A is a first peptide
  • B is a first branching group
  • C is an optional first spacer
  • D is a linker which comprises a derivative of dicarboxylic acid or a diamine or derivative thereof
  • E is an optional second spacer which may be the same as or different from said first spacer C
  • B' is an optional second branching group which may be the same as or different from said first branching group B
  • F is a second peptide which may be the same as or different from said first peptide A.
  • D is a glutaric acid bis
  • peptides A and F are compounds, including any of the monomers described herein, and specifically include those in Table 1 in Section 19. In a preferred embodiment, at least one of peptides A and F comprises two or more amino acids.
  • the peptides A and F may be different peptides, optionally targeting peptides, and may target different eptiopes of the same target or may target different epitopes of the same receptor.
  • the receptors may include KDR or cMET.
  • Branching groups B and B' provide a point of attachment for an optional reporter-sp group to the compound.
  • One or both of said first branching group B and said second branching group B' may be, but are not limited to, lysine, ornithine, aspartic acid or glutamic acid.
  • Optional spacers C and E may be used to attach the linker D to the other components A, B, B' or F of the compound while keeping those other components at a distance or "space" from the linker D.
  • B', C and/or E may optionally be absent.
  • a reporter moiety or similar group may optionally be attached to the dimer via the branching group.
  • a di-lysine derivative can be constructed on 2-chlorotrityl resin. See Barlos, K. and Gatos, D. "Convergent Peptide Synthesis” in Fmoc Solid Phase Peptide Synthesis, Chan, W.C. and White, P.D. eds., Chap 9: pp. 215-228 (Oxford University Press, New York, 2000), which is hereby incorporated by reference in its entirety.
  • 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 binding peptide as described above, provides a di-lysine construct having a single binding peptide attached. Removal of the second protecting group unmasks the remaining nitrogen. See, e.g., Mellor, S.L.
  • a binding peptide is first 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 N 8 -amino group of a lysine moiety, for example.
  • Cleavage of the protecting groups is accomplished by 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 glutaric acid bis-N-hydroxysuccinimide ester (commercially available from Tyger Scientific Inc., 324 Stokes Avenue, Ewing, NJ, 08638).
  • a bifunctional electrophile such as glutaric acid bis-N-hydroxysuccinimide ester (commercially available from Tyger Scientific Inc., 324 Stokes Avenue, Ewing, NJ, 08638).
  • 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 binding peptide. Purification of the resulting material by HPLC affords the desired homo- or hetero-dimeric construct bearing a suitable labeling group.
  • a modular scheme can be employed to prepare dimeric or higher multimeric constructs bearing suitable labeling groups as defined above.
  • fmoc-lysine(iVDde) Rink amide resin is treated with piperidine to remove the fmoc moiety.
  • 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 iVDde group.
  • the resin is treated with cyanuric chloride and a hindered base such as diisopropylethylamine in a suitable solvent such as DMF, NMP or dichloromethane to provide a monofunctionalized dichlorotriazine bound to the resin.
  • a suitable solvent such as DMF, NMP or dichloromethane
  • Subsequent successive displacement of the remaining chlorine atoms either by two equivalents of a binding peptide or one equivalent of a binding peptide, followed by a second binding peptide provides a resin-bound, hetero- or homo- dimeric, labeling group-functionalized construct. See, e.g., Falorni, M., et al., Tetrahedron Lett.
  • 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.
  • 2,3-diamino propionic acid 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 binding peptides, and to increase the rigidity of the construct (by constraining the motion and relative positions of the binding peptides relative to each other and the reporter) is entirely within the scope of the synthetic methods described herein.
  • Direct synthesis of the binding polypeptides 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 (1989), W. H. Freeman Co., San Francisco; 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 of 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, MA). Automated peptide synthesis machines, such as manufactured by Perkin- Elmer Applied Biosystems, also are available.
  • the polypeptide compound is preferably purified once it has been isolated or synthesized by either chemical or recombinant techniques.
  • purification purposes there are many standard methods that may be employed, including reverse-phase high-pressure liquid chromatography (RP-HPLC) using an alkylated silica column such as C 4 -, C 8 - or C 18 - silica.
  • RP-HPLC reverse-phase high-pressure liquid chromatography
  • a gradient mobile phase of increasing organic content is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid.
  • Ion-exchange chromatography can also be used to separate peptides based on their charge.
  • the degree of purity of the polypeptide may be determined by various methods, including identification of a major large peak on HPLC.
  • a polypeptide that produces a single peak that is at least 95% of the input material on an HPLC column is 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.
  • analysis of the peptide composition may be carried out. Such composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the peptide.
  • the amino acid content of the peptide can be confirmed by hydrolysis of the peptide in aqueous acid, separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer. Protein sequencers, which sequentially degrade the peptide and identify the amino acids in order, may also be used to determine the sequence of the peptide.
  • binding polypeptides also may be produced using recombinant
  • DNA techniques utilizing nucleic acids (polynucleotides) encoding the polypeptides of the 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 binding polypeptides.
  • Such procedures are within the capability of those skilled in the art (see Davis et ah, Basic Methods in Molecular Biology, (1986), which is hereby incorporated by reference in its entirety).
  • a determination of the affinity of the heteromultimer or a constituent binding moiety for the target relative to another protein or target is a useful measure, and is referred to as affinity for the target.
  • Standard assays for quantitating binding and determining affinity include, e.g., equilibrium dialysis, equilibrium binding, gel filtration, surface plasmon resonance 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 or modifications thereof measure the concentration of bound and free ligand as a function of ligand (or protein) concentration.
  • the concentration of bound heteromultimer or polypeptide is related to the concentration of free heteromultimer or polypeptide ([Free]) and the concentration of binding sites for the polypeptide, i.e., on KDR, VEGF/KDR complex, cMet, or the cMet/HGF complex (N), as described in the following equation:
  • heteromultimers of the invention and constituent binding polypeptides bind to the target, e.g., KDR, VEGF/KDR complex, cMet or cMet/HGF and have a K D for the target in the range of 1 nanomolar (nM) to 100 micromolar ( ⁇ M) and preferably have K D values less than 50 ⁇ M, more preferably less than 1 ⁇ M, even more preferably less than 50 nM, and most preferably less than 10 nM.
  • the target e.g., KDR, VEGF/KDR complex, cMet or cMet/HGF
  • K D for the target in the range of 1 nanomolar (nM) to 100 micromolar ( ⁇ M) and preferably have K D values less than 50 ⁇ M, more preferably less than 1 ⁇ M, even more preferably less than 50 nM, and most preferably less than 10 nM.
  • imaging agents operate in a dynamic system, in that binding of the imaging agent to the target (such as KDR or VEGF/KDR complex, e.g., on activated endothelium) is not in a stable equilibrium state throughout the imaging procedure.
  • the concentration of imaging agent and of agent-target complex rapidly increases.
  • 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.
  • heteromultimeric binding compounds such as those of the present invention, are that they generally possess very slow dissociation rates relative to their constituent monomers ⁇ see Tissot et al., J. Immunol. Methods 236(1-2):147-165 (2000)).
  • heteromultimeric compounds capable of binding to two distinct epitopes on a target molecule simultaneously can achieve multimeric binding regardless of the distance between target molecules on the cell surface.
  • Homomultimeric binding compounds on the other hand, depend on the presence of two or more target molecules being in close enough proximity such that the homomultimer can span the distance between them.
  • heteromultimeric binding compounds of the present invention are particularly well suited for binding to receptors and other cell surface molecules that are less abundant and therefore more distant from each other on the cell surface.
  • 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 and Miller, Clin. Chem., 34(7):1417-21 (1988)), surface plasmon resonance (see Malmborg et al., J. Immunol.
  • heteromultimers are within the scope of the present invention.
  • modified or optimized heteromultimers are included within the definition of "heteromultimers.”
  • modified or optimized binding polypeptides are included within the definition of "binding polypeptides” and the phrase “KDR and VEGF/KDR complex-binding polypeptides” includes modified or optimized KDR and VEGF/KDR-binding polypeptides, and the phrase “cMet and cMet/HGF complex-binding polypeptides” includes modified or optimized cMet and cMet/HGF binding polypeptides.
  • a polypeptide sequence for use in the heteromultimers of the invention can be modified to optimize its potency, pharmacokinetic behavior, stability and/or other biological, physical and chemical properties.
  • substitutions of amino acids within the same class are well known in the art. 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:
  • 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 1-10 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 d-C 4 )-substituted forms of the previous listed aromatic amino acids, illustrative examples of which are: 2-, 3-, or 4- aminophenylalanine, 2-, 3-, or 4-chlorophenylalanine, 2-, 3-, or 4-methylphenylalanine, 2-, 3- , or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2'
  • Substitution of amino acids containing basic functions Including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or aryl- substituted (from C 1 -C 10 branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example.
  • N-epsilon-isopropyl-lysine 3-(4- tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma'-diethyl- homoarginine.
  • amides formed from alkyl, aromatic, heteroaromatic where the heteroaromatic group has one or more nitrogens, oxygens or sulfur atoms singly or in combination
  • carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • lysine, ornithine, or 2,3- diaminopropionic acid any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives
  • Substitution of acidic amino acids Including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, aralkyl, and heteroaryl sulfonamides of 2,3- diaminopropionic 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.
  • amino acids within each of the categories listed above may be substituted for another of the same group.
  • amino acids may be substituted with analogs that have similar steric characteristics but whose acidic or basic functions are mimicked using groups that are sterically similar but whose acidic or basic properties may be altered, inverted, reduced or increased. Examples are: substitution of arginine by the analog wherein the guanidine group has a cyano group appended to one of the guanidine NH 2 groups, substitution of arginine by citrulline, the amino acids wherein the guanidine group is replaced with a ureido function, and substitution of lysine by glutamine or by glutamic acid delta hydrazide.
  • Another type of modification within the scope of the invention is the substitution of amide bonds within the backbone of a binding polypeptide.
  • substitution of amide bonds within the backbone of a binding polypeptide For example, to reduce or eliminate undesired proteolysis, or other degradation pathways which 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.
  • D-alanine or another D-amino acid, distal or proximal to a labile peptide bond
  • D-amino acid substitutions can, and at times, must be made, with D-amino acids whose side chains are not conservative replacements for those of the L-amino acid being replaced.
  • D-amino acids whose side chains are not conservative replacements for those of the L-amino acid being replaced. This is because of the difference in chirality and hence side-chain orientation, which may result in the accessing of a previously unexplored region of the binding site of the target which has moieties of different charge, hydrophobicity, steric requirements, etc., than that serviced by the side chain of the replaced L-amino acid.
  • such moieties affecting the pharmacokinetic and pharmacodynamic behavior 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, diaminopropionic acid, ornithine, or other amino acid in the peptide that possesses a pendant amine group or a pendant alkoxyamino or hydrazine group.
  • the moieties introduced may be groups that are hydrophilic, basic, or nonpolar alkyl or aromatic groups depending on the peptide of interest and the extant requirements for modification of its properties.
  • glycosylated amino acid residues e.g., serine, threonine or asparagine residues
  • Glycosylation which may be carried out using standard conditions, may be used to enhance solubility, alter pharmacokinetics and pharmacodynamics or to enhance binding via a specific or non-specific interaction involving the glycosidic moiety.
  • glycosylated amino acids such as O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl- ⁇ -D-glucopyranosyl) serine or the analogous threonine derivative (either the D- or L- amino acids) may be incorporated into the peptide during manual or automated solid phase peptide synthesis, or in manual or automated solution phase peptide synthesis.
  • D- or L-N ⁇ -(2-acetamido-2-deoxy-3,4,6-tri-O- acetyl- ⁇ -D-glucopyranosyl)-asparagine can be employed.
  • linkage of the amino acid to the glycoside is not limited to the formation of a bond to the anomeric carbon of the carbohydrate function. Instead, linkage of the carbohydrate moiety to the amino acid could be through any suitable, sufficiently reactive oxygen atom, nitrogen atom, carbon atom or other pendant atom of the carbohydrate function via methods employed for formation of C-heteroatom, C-C or heteroatom-heteroatom (examples are S-S, O-N, N- N, P-O, P-N) bonds known in the art.
  • Shortened cyclic peptides can be formed using disulfide bonds or amide bonds of suitably located carboxylic acid groups and amino groups.
  • D-amino acids can be added to the peptide sequence to stabilize turn features (especially in the case of glycine).
  • alpha, beta, gamma or delta dipeptide or turn mimics (such as ⁇ , ⁇ , ⁇ , or ⁇ turn mimics) some of which are shown in structures 1, 2 and 3, below, 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.
  • a region in a cyclic peptide that contains a pair of amino acids can be replaced with a single amino acid that spans the same distance or a similar distance.
  • Such amino acids may act as a mimetic of the structure of the pair of amino acids replaced or they may merely act as a spacer, especially when information is available (as from alanine scans, D-amino acid scans or other techniques designed to probe the necessity or contribution of particular amino acids in a structure to the biological activity or the desired conformation of the substance) that indicates that the pair of amino acids is non-critical to the activity of the compound.
  • spacer amino acids can be drawn from a pool of a vast array of compounds that have four intervening atoms between a carboxylic acid function and an amino group.
  • Examples of such compounds are: 4-aminobenzoic acid, 4-aminocyclohexane carboxylic acid, 5-aminovaleric acid, 5-hydrazinobutanoic acid, 3-aminomethylcyclobutanecarboxylic acid, l-aminocyclobutane-3 -acetic acid, 3-aminophenylacetic acid and N-aminoisonipecotic acid.
  • the invention also includes other compounds wherein the essential spacer element is preserved while other functionality that may enhance the binding activity of the peptide is included in such molecules.
  • disulfide mimetics for disulfide bonds within the binding polypeptides of the invention.
  • disulfide bonds might need to be replaced to avoid certain difficulties that are sometimes posed by the presence of a disulfide bond.
  • the presence of the disulfide bond can be a significant problem.
  • 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-compatible chelating groups. This is because the disulfide bond is rather easily reduced by the reducing agents commonly used in kits devised for one-step preparation of radiopharmaceuticals. Therefore, the ease with which the disulfide bond can be reduced during Tc chelation may require substitution with mimetics of the disulfide bonds.
  • the oxime moiety has been employed as a linker by investigators in a number of contexts. Of the most interest is the work by Wahl, F and Mutter, M, Tetrahedron Lett. (1996) 37, 6861-6864).
  • the amino acids containing an aminoalcohol function (4), and containing an alkoxyamino function (5a or 5b), are incorporated into the peptide chain, not necessarily at the end of the peptide chain.
  • the sidechain protecting groups are removed. The aldehyde group is unmasked and an oxime linkage is formed.
  • Lanthionines are cyclic sulfides, wherein the disulfide linkage (S-S) is replaced by a (C-S) linkage. Thus the lability to reduction is far lower and this linkage should be stable to stannous chloride.
  • Lanthionines may be prepared by a number of methods. i. Preparation of Lanthionines using Bromoacetylated Peptides
  • Lanthionines are readily prepared using known methods. See, for example,
  • a peptide may be prepared by fmoc peptide synthesis methodology on safety catch resin or on Rink-amide resin. The N-terminal amino group can be unmasked using piperidine/DMF or using 2% DBU in DMF.
  • the resin can be neutralized by treatment with mild acid such as acetic acid to remove traces of the strongly nucleophilic piperidine or strongly basic DBU. Subsequently the resin can be treated with DIEA to remove the acetic acid which is present as ion pairs with the N-terminal amino groups on the resin. Washing with DMF then removes traces of DIEA and DIEA acetate. A bromoacetyl group is appended by treatment of the resin with bromoacetic acid and DIC.
  • mild acid such as acetic acid
  • DIEA strongly nucleophilic piperidine
  • DBU strongly basic DBU
  • the crude precipitated peptide may also be dissolved in water or in a mixture of water and a suitable organic cosolvent such as DMSO, NMP, DMA, methanol, ethanol, n- propanol, isopropanol, n-butanol, isobutanol, glyme, diglyme, triglyme, tetraglyme, tetrahydrofuran or dioxane, the pH of the solution is adjusted to 7.0-8.0 by addition of a base such as N-methylglucamine or ammonium hydroxide and the mixture is stirred at ambient temperature. The formation of the lanthionine is followed using HPLC and mass spectroscopy. Upon completion of the reaction the desired peptide is purified by gel filtration, HPLC and other techniques for purification of peptides known to those skilled in the art. ii. Preparation of Lanthionines via Cysteine Thiol Addition to
  • Resin-bound serine can be employed to prepare the lanthionine ring on resin either using a bromination- dehydrobromination-thiol addition sequence or by dehydration with disuccinimidyl carbonate followed by thiol addition.
  • Ploinsky et al. M. J. Med. Chem., 35:4185-4194 (1992); Mayer et al., "Peptides, Frontiers of Peptide Science", in Proceedings of the 15 th American Peptide Symposium, Tarn & Kaumaya (eds.), June 14-19, 1995, Nashville, Tenn. (Kluwer Academic Pub. Boston) pp. 291-292.
  • Another approach that may be employed involves intramolecular cyclization of suitably located vicinal amino mercaptan functions (usually derived from placement of a cysteine at a terminus of the linear sequence or tethered to the sequence via a side-chain nitrogen of a lysine, for example) and aldehyde functions to provide thiazolidines which result in the formation of a bicyclic peptide, one ring of which is that formed by the residues in the main chain, and the second ring being the thiazolidine ring.
  • Scheme 2 above provides an example.
  • the required aldehyde function can be generated by sodium metaperiodate cleavage of a suitably located vicinal aminoalcohol function, which can be present as an unprotected serine tethered to the chain by appendage to a side chain amino group of a lysine moiety.
  • the required aldehyde function is generated by unmasking of a protected aldehyde derivative at the C-terminus or the N-terminus of the chain.
  • An example of this strategy is found in: Botti, P.; Pallin, T.D. and Tarn, J.P. J. Am. Chem. Soc. 1996, /75, 10018-10034.
  • Macrocyclic peptides can be 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) ([Oxfoerd 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 Scheme 3 a, below.
  • 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.
  • Scheme 3b Lactam Surrogate for the Disulfide Bond via Quasiorthogonal Deprotection of Lys and Asp Using Allyl-based Protecting Groups followeded by On-Resin Cyclization and Cleavage from Resin
  • amino acids such as trans-4-(iVDde)methylaminocyclohexane carboxylic acid, trans-4- (iVDde)methylaminobenzoic acid, or their alloc congeners could be employed.
  • Yet another approach is to employ the safety catch method to intramolecular lactam formation during cleavage from the resin. 12. Cyclic Peptides Based on Olefin Metathesis
  • the Grubbs reaction involves the metathesis/cyclization of olefin bonds and is illustrated as shown below. See, e.g., Schuster et al., Angewandte. Chem. Int. Edn Engl, 36:2036-2056 (1997); Miller et al., J. Am.Chem. Soc 118:9606-9614 (1996).
  • the lysine ⁇ -amino group may be 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.
  • modifications within the scope of the invention include manipulations of peptide sequences which can be expected to yield peptides with similar or improved biological properties. These include, e.g., 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, retro-inverso peptoid sequences, and synthetic peptides. Structures wherein specific residues are peptoid instead of peptidic, which result in hybrid molecules, neither completely peptidic nor completely peptoid, are contemplated as well.
  • modifications within the invention include introduction of linkers or spacers between the targeting sequence of the binding moiety or binding polypeptide and the detectable label or therapeutic agent.
  • linkers/spacers may improve the relevant properties of the binding peptides (e.g., increase serum stability, etc.).
  • 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.
  • suitable linkers include homobifunctional and heterobifunctional cross-linking molecules.
  • the homobifunctional molecules have at least two reactive functional groups, which are the same.
  • the reactive functional groups on a homobifunctional molecule include, for example, aldehyde groups and active ester groups.
  • Homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and subaraldehyde.
  • Homobifunctional linker molecules having at least two active ester units include esters of dicarboxylic acids and N-hydroxysuccinimide.
  • O- succinimidyl esters include disuccinimidyl suberate and dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonic acid and bis-sulfonate salts such as their sodium and potassium salts.
  • Heterobifunctional linker molecules have at least two different reactive groups.
  • Some examples of heterobifunctional reagents containing reactive disulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate (Carlsson et al., Biochem J. 173:723- 737 (1978)), sodium S-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and 4- succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene. N-succinimidyl 3-(2- pyridyldithio)propionate is preferred.
  • Other heterobifunctional molecules include succinimidyl 3- (maleimido)propionate, sulfosuccinimidyl 4-(p-maleimido-phenyl)butyrate, sulfosuccinimidy 1 4-(N-maleimidomethyl-cyclohexane)- 1 -carboxylate, maleimidobenzoyl- 5N-hydroxy-succinimide ester.
  • linkers which are combinations of the molecules and/or 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.
  • peptide 1 (referred to above as A in the construct A-B-C-D-E-B '-F) may comprise 3-50 (D or L)-alpha amino acids, preferably 8-24 amino acids (with or without disulfide linkages), which can be joined via an amide bond to spacerl (referred to above as C in the construct A-B-C-D-E-B '-F) by an amino group, provided by the first branching group (referred to above as B in the construct A-B-C-D-E-B'- F), for example the epsilon amino group where lysine is the branching group B, or the gamma amino group of ornithine where ornithine is the branching group B, or the beta amino group of dianiinopropionic acid, where diaminopropionic acid is the branching group B at the N- or C-terminus of peptide 1.
  • a in the construct A-B-C-D-E-B '-F may comprise 3
  • Peptide 1 may also comprise other monomers described herein, such as those set forth in Table 1 in Section 19.
  • peptide 1 has specificity for a first binding site of a target.
  • peptide 1 is specific for a binding site on a receptor such as, for example, KDR or cMet.
  • the alpha amino group of branching group B can be employed to form the bond with the spacer 1, and the side chain amino groups of the branching group B can be the point of attachment of the branching group B to peptide 1.
  • the branching group B does not have to be limited to the aforementioned residues, which are provided as examples.
  • Peptide 1 can be attached to the amino acid(s) that constitute the branching group B, such as serine, cysteine, S-carboxymethylcysteine, S-2-thiopyridylcysteine, aspartic acid, glutamic acid, p-aminophenylalanine, propargyl or other alkynyl substituted glycine derivatives, and other amino acids which, after forming a peptide bond with peptide I, possess side chain hydroxy, carboxylic acid, thiol, disulfide, alkylcarboxylic acid, amino, alkylamino, alkynyl or other functions which can be employed to form a bond with spacerl . See FIG. 38A.
  • Preferred branching groups include lysine, ornithine, aspartic acid and glutamic acid.
  • the actual C-terminus of the peptide 1 or peptide2 construct is the acyl group of the branching group B, which may be present as a primary carboxamide or a carboxamido group NH-T.
  • T may be introduced to the nitrogen of a
  • Rink amide resin by reactions and techniques known in the art.
  • T refers to alkyl groups, aralkyl groups and/or heteroarylalkyl groups
  • these may be introduced by reaction of alkyl aldehydes with Rink amide resin under mildly dehydrating conditions in the presence of sodium borohydride or sodium cyanoborohydride.
  • the reaction of more complex derivatives such as bifunctional molecules where one of the two functional groups is an aldehyde with Rink amide resin in the presence of sodium cyanoborohydride at pH 5, will provide a monosubstituted alkyl Rink amide resin wherein the alkyl group bearing the second functional group is present.
  • the other functional group be compatible with sodium cyanoborohydride.
  • aryl groups may be conducted by preparation of the original Rink amide resin moiety employing an aromatic amine to introduce the nitrogen into the system by solvolytic means, or by the use of well established techniques of nucleophilic aromatic substitution on activated aromatic compounds.
  • Another method for the introduction of aryl groups on to the nitrogen of the ring amide resin is via the reaction of haloaromatics compounds in the presence of metal catalysts, that promote the coupling of amino groups.
  • Alkyl or functionalized alkyl groups may also be introduced by alkylation of the Rink amide resin amino group.
  • Hydroxyethyl groups may be introduced using the alkylation of the Rink amide resin amino group with suitably O-protected 2-haloethyl groups, or by alkylation with diethylcarbonate, for example.
  • reporter-sp groups may be introduced as part of the group T to the Rink amide resin, and further, that these, if sufficiently durable, could be carried through the entire synthesis to the end if desired.
  • peptide 1 -branching group B-spacerl and peptide2- branching group B'-spacer2 molecules can be assembled on a safety-catch resin, which, upon activation, can be treated with bifunctional amines bearing the group(s) T as described above to provide, in solution, constructs wherein the C-terminal amide of the branching group is amidated with the NH-T group.
  • branching group ultimately contained in the final construct will comprise the amino acids or other described moieties that have the leaving groups or protecting groups no longer present.
  • protecting groups or leaving groups are temporarily employed for the purposes of directing the specificity of the synthesis and that the descriptions of such functionalized amino acids as the branching groups is provided to facilitate the understanding of the synthetic paths or strategies that would employ them.
  • a branching group is described as Lys[fmoc-Lys(ivDde)], it is understood that the ultimate construct will contain the -Lys(Lys)- function devoid of the protecting groups, and that in their place will be the bonds between the formerly protected atoms and the other moieties such as peptide 1, peptide2 or the spacerl or spacer2 or appended reporter-sp groups, for example.
  • the branching group B has some group other than an amino group for bond formation with spacerl, those skilled in the art will understand that spacerl will be constituted with functionality suitable for bond formation with B.
  • spacerl can be 2-aminoethyl-2- pyridyldissulfide or an amine-protected version of that compound, which ultimately yields as the spacer, a 2-aminoethylthio moiety.
  • the amino group of the spacerl would be employed in reactions with the linker function D as described below.
  • peptide 1 can be joined to spacerl via the alpha amine of an amino acid at the N-terminus of peptide 1, or to a moiety at the N-terminus of peptide 1 which possesses any of the distal or side chain functions disclosed above as belonging to the branching group. Such methods are shown in FIG. 39.
  • Spacerl (referred to above as C in the construct A-B-C-D-E-B '-F) can be absent, or may comprise one or more amino acids, including, e.g., naturally occurring or unnatural alpha amino acids and naturally occurring or unnatural non-alpha amino acids.
  • a diverse set of spacers can be used to modify the distance between peptide 1 and the linker (referred to above as D in the construct A-B-C-D-E-B '-F), but also to provide a means to change the hydrophilicity or hydrophobicity of the dimeric construct.
  • Examples of spacerl that may be used in this invention include, without limitation, the following amino acids or linked series of amino acids or other bifunctional molecules:
  • Ser(S)-Gly-Ser(S) where the group S comprises a hydrophilic function such as a sugar moiety which may be selected from N-acetylgalactoseamine, D-(+)-allose, D-(+)-altrose, D-(+)-glucose, D-(+)- mannose, D-(-)-gulose, D-(-)-idose, D-(+)-galactose, D-(-)-talose, D-(-)-ribose, D-(-)- arabinose, D-(+)-xylose or D-(-)-lyxose and the linkage of the serine side-chain oxygen and the sugar is via the anomeric carbon;
  • a hydrophilic function such as a sugar moiety which may be selected from N-acetylgalactoseamine, D-(+)-allose, D-(+)-altrose, D-(+)-glucose,
  • spacerl is 8-amino-3,5-dioxaoctanoic acid.
  • the linker D (referred to above as D in the construct A-B-C-D-E-B ' -F), between spacerl and spacer2 is, in one embodiment, a derivative of a dicarboxylic acid that joins spacerl and spacer2 by amide bonds.
  • the derivative of a dicarboxylic acid used as linker D is a glutaric acid bis N- hydroxysuccinimidy ester or a derivative thereof.
  • the group Z can also contain N or O- substituted alkyl chains, which results in structures such as, e.g., those below:
  • the dicarboxylic acid employed to generate the linker D can also be a substituted aromatic or heterocyclic acid of the form HOOC-Z-COOH, where Z symbolizes the aromatic or heterocyclic nucleus which is the scaffold bearing the two carboxylic groups.
  • Examples of such molecules include, without limitation, benzene- 1,4-dicarboxylic acid, bipheny 1-1, 4 '-dicarboxylic acid, (4-carboxymethoxy-phenoxy)-acetic acid, (4' ⁇ carboxyrnethoxy-biphenyl-4-yloxy)-acetic acid, N-methylpyrrole-2,5-dicarboxylic acid, pyridine-2,6-dicarboxylic acid, N-methylpyrrole-2,3 -dicarboxylic acid, N-methylpyrrole-2,4- dicarboxylic acid, pyridine 2,3 -dicarboxylic acid, pyridine-3, 5 -dicarboxylic acid, pyridine 3,4-dicarboxylic acid or piperidine 3,5-dicarboxylic acid.
  • the group R in the linkers can be, e.g., acetyl, acylamino, acylaminoalkyl, amino, alkylamino, aminoalkyl, alkyl, alkenyl, alkoxycarbonyl, alkynyl, aryl, allyl, propargyl, heteroaryl, carboxyalkyl, carboxamido, carboxyamidoalkyl, formyl, hydrogen, hydroxy, hydroxyalkyl, polyhydroxyalkyl, polyethyleneoxy (meaning the structure (CH 2 CH 2 O) n -G where G is a terminating group selected from alkylamino, alkyl, alkylhydroxy, alkyl, carboxy, methylenecarboxy, alkylcarboxamido- or alkylcarboxy(alkyl)amido-, alkylformyl, 2-mercaptoethyl, or 2- pyridyldithioalkyl where the alkyl group contains from
  • the saturated derivatives of the aromatic compounds described above, where Z is now a saturated derivative of the aromatic or heterocyclic Z, can also be employed as the linker D with the same groups R and G.
  • Examples of the dicarboxylic acids, where Z is either aromatic or aliphatic, that can comprise linker D, are shown in FIG. 40.
  • activated acylating derivatives having the series of groups shown in FIG. 41 as X or Y, which may be the same or different, selected from the groups shown in FIG.
  • 41 may be acid chlorides, acid fluorides, acid bromides, active esters such as N-hydroxysuccinimide esters, N- hydroxybenzotriazolyl esters, 7-aza-l-hydroxy-benzotriazolyl esters, 5 or 6-chloro-l- hydroxy-lH-benzotriazolyl esters, substituted l-hydroxy-l,2,3-triazolyl esters, 2,3,4,5,6- pentachlorophenyl esters, 2,3,4,5,6-pentafluorophenyl esters, 2 or 4 nitrophenyl esters, N- hydroxyphthalimides, substituted-N-hydroxyphthalimides, N-acyl derivatives of substituted imidazoles, 1-acyl derivatives of substituted tetrazoles, 1-acyl substituted triazoles, S-acyl derivatives of pyridine 2-thiol, acyl cyanides, acyl azides, mixed alkyl carbonic anhydrides, mixed anhydrides of triflu
  • the linker D is preferably a dicarboxylic acid
  • the spacer 1 (referred to as C above) after appendage to the branching group B, contains a pendant function such as the 2-pyridydissulfide function
  • the linker D may be a bifunctional molecule, such that upon reaction with the mixed disulfide it can provide a pendant function (such as that of a carboxylic acid) that can be employed to form a peptide bond to an amino group of spacer2 to form the desired dimer.
  • the linker may also be, for example, 4-mercaptobutanoic acid, which may be employed to displace 2-thiopyridine from the branching group.
  • the pendant function of the linker D would be the carboxyl group, which could be activated to couple to the amino group of peptide2-branching group B'-spacer2-NH 2 (where NH 2 is an amino group and not the amido nitrogen of a carboxamide) to provide the assembled dimer.
  • the linker D is a diamine linker or derivative thereof.
  • Spacer2 (referred to above as E in the construct A-B-C-D-E-B ' -F) can be absent, or may comprise one or more amino acids, including, e.g., naturally occurring or unnatural alpha amino acids and naturally occurring or unnatural non-alpha amino acids, or other bifunctional molecules that can be appended to two discrete chemical entities.
  • a diverse set of spacers can be used to modify the distance between peptide2 and linker D but also to provide a means to change the hydrophilicity or hydrophobicity of the dimeric construct.
  • Examples of spacer2 that may be used in this invention include, without limitation, the following amino acids or linked series of amino acids or other bifunctional molecules:
  • Ser(S)-Gly-Ser(S) where the group S comprises a hydrophilic function such as a sugar moiety which may be selected from N-acetylgalactoseamine, D-(+)-allose, D-(+)-altrose, D-(+)-glucose, D-(+)- mannose, D-(-)-gulose, D-(-)-idose, D-(+)-galactose, D-(-)-talose, D-(-)-ribose, D-(-)- arabinose, D-(+)-xylose or D-(-)-lyxose and the linkage of the serine side-chain oxygen and the sugar is via the anomeric carbon;
  • a hydrophilic function such as a sugar moiety which may be selected from N-acetylgalactoseamine, D-(+)-allose, D-(+)-altrose, D-(+)-glucose,
  • spacer2 is 8-amino-3,5-dioxaoctanoic acid.
  • Peptide2 (referred to above as F in the construct A-B-C-D-E-B ' -F) may, in one embodiment, comprise 3-50 (D or L)-alpha amino acids, preferably 8-24 amino acids (with or without disulfide linkages), which can be joined via an amide bond to spacer2 (referred to as E in the construct A-B-C-D-E-B '-F) by an amino group of peptide2, provided by the epsilon amino group of lysine, or the gamma amino group of ornithine, or the beta amino group of 2,3-diaminopropionic acid at the N- or C-terminus of peptide2.
  • peptide2 can be joined to spacer2 via the alpha amine of an amino acid at the N-terminus of peptide2, or to a moiety at the N-terminus of peptide2 that possesses any of the side chain or distal functional groups as disclosed above as belonging to a branching group B or branching group B'.
  • Peptide2 may also comprise other monomers described herein, such as those set forth in Table 1 in Section 19.
  • peptide2 has specificity for a first binding site of a target.
  • peptide2 is a peptide of 3-50 amino acids and is specific for a binding site on a receptor such as, for example, KDR or cMet.
  • the alpha amino groups of the branching group B' may be employed to form the bond with the spacer2, and the side chain amino groups of each of the aforementioned branching groups may be the point of attachment of the branching group B' to peptide2.
  • the branching group is not limited to the aforementioned residues, which are only provided as examples.
  • Peptide2 may also be attached to amino acids of the branching group B', such as serine, cysteine, S-carboxymethylcysteine, S-2-thiopyridylcysteine, aspartic acid, glutamic acid, isonipecotic acid, p-aminophenylalanine, propargyl or other alkynyl substituted glycine derivatives and other amino acids which, after forming a peptide bond with peptide2, possess side chain hydroxy, carboxylic acid, thiol, disulfide, alkylcarboxylic acid, amino, alkylamino, alkynyl or other functions which can be employed to form a bond with spacer2.
  • amino acids of the branching group B' such as serine, cysteine, S-carboxymethylcysteine, S-2-thiopyridylcysteine, aspartic acid, glutamic acid, isonipecotic acid, p-aminopheny
  • spacer2 will be constituted with functionality suitable for bond formation with the branching group B'.
  • spacer2 could be chosen to be 2- aminoethyl-2-pyridyldissulfide or an amine-protected version of that compound. After formation of the disulfide bond the amino group would be employed in reactions with the linker D as described below.
  • peptide2 can be joined to spacer2 via the alpha amine of an amino acid at the N-terminus of peptide2, or to a moiety at the N-terminus of peptide2 that possesses any of the side chain functions disclosed above as belonging to the branching group B'.
  • a general synthesis can be initiated by the solid phase peptide synthesis of peptide 1 -branching group B and peptide2-branching group B' with internal lysines (or other amino acids having a side-chain amino groups, such as ornithine, 2,3-diaminopropionic acid or isonipecotic acid, for example) protected as ivDde or aloe, where needed.
  • the preparation of the peptidel construct is shown in FIG. 44.
  • the branching group B' may be absent or may comprise other amino acids such as serine, cysteine, S-carboxymethylcysteine, S-2-thiopyridylcysteine, aspartic acid, glutamic acid, p-aminophenylalanine, propargyl or other alkynyl substituted glycine derivatives as described for the branching group B, as well; but the illustration of this approach will be discussed in the context of the use of lysine residues as the branching group B'.
  • the corresponding spacers for each peptide may be appended by using amide bond-forming coupling reactions in a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane (the alpha amino group is protected as Fmoc then removed following coupling), in separate flasks to the branching groups B and B' of peptidel and peptide2 on a solid support such as Rink amide resin.
  • a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane
  • any disulfide linkages are formed by aerial oxidation or the use of other oxidative techniques known in the art for disulfide formation (e.g., HgCl 2 , T1(OOCCF 3 ) 3 , Me 3 SiCl/DMSO, Iodine) in aqueous solution, optionally using dimethylsulfoxide, acetonitrile, DMF, DMA, tetrahydrofuran, dioxane, ethanol, methanol, n-propanol, ethylene glycol, isopropanol, isobutanol, n-butanol, tert-butanol, 1,2-dimethoxyethane, 2-ethoxyethanol, 2-ethoxyethoxy-ethanol, diglyme, triglyme or tetraglyme singly or in combination as cosolvent with water.
  • other oxidative techniques known in the art for disulfide formation (e.g., HgCl 2 , T1
  • the nonaqueous solvent in some cases it is necessary to employ the nonaqueous solvent by itself or using a minimal amount ( ⁇ 15%) of water.
  • the disulfide bond forming reaction can also be carried out simultaneously with acidolytic cleavage and deprotection employing TFA-based solvent mixtures, (see Alberico, F.; Annis, L.; Royo, M. and Barany, G., "Preparation and handling of peptides containing methionine and cysteine,” Fmoc Solid Phase Peptide Synthesis — A Practical Approach (Chan, W.C. and White, P.D. Eds.) Oxford University Press, New York, NY Chap. 4, pp.
  • spacer 1 or spacer2 can be added by solution coupling, in a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane, of the protected (where necessary) spacer followed by removal of the protecting group (for example, fmoc).
  • a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane
  • a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane
  • the solution is concentrated, and the residue is treated with an appropriate solvent such as ethyl acetate or ether, which precipitates the peptidic construct or largely sequesters it in a separate phase, for example, an oil phase, and allows the removal of excess activated linker, which is soluble in the solvent, by decanting.
  • the remaining residue is washed with the solvent used for the separation of the peptidic construct and the excess activated linker D to remove remaining traces of the latter material, and the residue is then dissolved in a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane.
  • Peptide2— branching group B'-spacer2 is then added to provide the assembled dimer peptide 1 -branching group B-spacerl -Iinker-spacer2-branching group '-peptide2.
  • the methods described herein may be used, for example, to prepare dimers such as those described in Example 9.
  • reporter-sp molecules may be attached to the branching groups as shown in FIGS. 37A-37E.
  • reporter-sp molecules will be understood to encompass both: (1) molecules which actually exercise a reporter-sp function; and (2) molecules appended to the system that have a special purpose or function in addition to, or other than, serving as a reporter system.
  • FIGS. 37B, 37C, 37D and 37E are graphical representations of the systems described above and within the following discussion.
  • reporter-sp molecules are molecules that either serve as reporters or special purpose molecules, or serve both functions.
  • reporter refers to molecules that have attributes that allow their detection by sensing techniques which measure a signal by absorption or emission measurements, or a change in the level of absorption of a signal, whether directly generated by the reporter system, or influenced by the presence of the reporter system, in a portion of the electromagnetic frequency spectrum.
  • Such molecules may provide a reflected signal or a different intensity of the input signal (attenuation or enhancement or time resolved methods), or may provide a signal of different frequency, a signal which is a harmonic of the stimulating signal or an emitted signal, or a signal which is affected by the input signal.
  • reporter molecules include, but are not limited to, diamagnetic or paramagnetic metal chelates, metal chelator systems, radiometal chelating systems, radioisotope-labeled molecules including metal chelators which bear the radiometal, dye molecules, fluorescent molecules, phosphorescent molecules, molecules absorbing in the UV spectrum, molecules capable of absorption of near or far infrared radiation, and molecules or supramolecular constructs that absorb and/or emit x-ray, gamma rays, beta particles, alpha particles, ultrasound, optical or magnetic signals.
  • SP molecules also known as special purpose molecules, are defined as molecules that may or may not have a direct role in the interaction with a detection system, but have a functional attribute that facilitates the use of the peptidic constructs in some way.
  • Such molecules when appended to the peptidic construct, for example, may enable the incorporation of the peptidic construct into larger arrays of molecules (such as supramolecular constructs) which can be detected by equipment or apparatus employed for detection of signals, whether by presence or absence of the signal, a change in signal intensity, reflection of a signal or a derivative signal during or after a period of irradiation by the equipment or apparatus.
  • phospholipid conjugated molecules may be incorporated into liposomes or into microbubbles.
  • Liposomes may be employed to facilitate delivery of encapsulated solutions of chemotherapeutic agents, and the phospholipid-peptide conjugates can be incorporated into liposomes, thereby enabling the liposome to target a specific biomolecular target and release the therapeutic in higher concentration and proximity to the targeted region.
  • phospholipids bearing specialized molecules, such as targeting peptides may be incorporated into microbubbles consisting of a bulk phospholipid and several other minor components which encapsulate a gas.
  • the peptide is presented on the surface of the microbubble, and if a sufficient number of such peptides are presented on the surface of the microbubble, the microbubble may bind to a cell surface or an intravascular surface which expresses the biomolecular target. If successfully bound to the biomolecular target addressed by the peptide, then the microbubble may be detected via ultrasound imaging techniques, thereby allowing diagnosis or prognosis of a disease condition.
  • Special purpose molecules may also have other roles. For example, they may possess the capability to act as therapeutics, or as toxins employed for selective killing and/or inhibiting the growth of cancer cells, or for inhibiting or promoting the growth of normal tissues when required.
  • a dual role as a reporter and as an SP molecule can be realized by radiometal chelates or other radioisotope-labeled molecules when they are appended to the peptidic construct; this is by virtue of the fact that the radiation emitted by the radioisotope, which performs a therapeutic role, may also be detected by imaging systems.
  • the branching group B emanating from peptide 1 may comprise a pair of amino acid residues, each of which has an amino group bearing side-chain, such as, e.g., Lys(Lys), where the epsilon amino group of the C-terminal (first) lysine is amidated with the carbonyl group of a second lysine which has different protecting groups on its alpha nitrogen and epsilon nitrogen, or at least one of those nitrogen atoms is protected whereas the other is unprotected and available for reaction.
  • Lys(Lys) amino group bearing side-chain
  • the ivDde group is removed from the branching group B of the assembled dimer by treatment with 10% hydrazine in DMF.
  • standard post synthesis processing which may include purification of the dimer by normal phase, reversed phase, hydrophilic interaction, ion exchange or other suitable forms of HPLC (singly or in combination), SPE (solid phase extraction), SEC (size exclusion chromatography), gel filtration, medium pressure or low pressure reverse or normal phase chromatography, or preparative thin layer chromatography techniques.
  • the reporter-sp molecule is then conjugated to the dimer by reaction of the remaining lysine free epsilon amino group in the branching group B of peptide 1. This is accomplished by activation of a carboxylic acid function of the reporter-sp molecule, using reagents such as DIC/HOBt, HBTU/DIEA, HATU/DIEA, BOP/DIEA, PyBOP/DIEA or other coupling agents known to those skilled in the art, or by the use of a preformed active acylating species in a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane and treatment of the dimer with the active acylating reporter-sp molecule to provide the reporter-sp-functionalized dimer ⁇ see FIG. 46).
  • the final product is purified by SEC (size exclusion chromatography), reverse phase HPLC, ion exchange chromatography or other chromatographic techniques (as described above for the penultimate dimer) sing
  • the pair of amino acids can be Lys-Lys, wherein the epsilon amino group of the C-terminal lysine is protected with, for example, an aloe or an ivDde group, and the pendant amino group of the preceding lysine of the branching group was employed in the dimer forming reaction described above.
  • Attachment of the reporter-sp function may also be carried out as follows:
  • the dimer bearing the free amino group at the C-terminal lysine of the branching group B of peptide 1 may be reacted with a bis-activated linker D molecule, preferably glutaric acid bis- NHS ester, in large excess, in a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane.
  • a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane.
  • a suitable solvent one that will dissolve the excess bis-activated linker compound while precipitating the dimer or sequestering it in an insoluble phase, preferably a solvent such as ethyl acetate or ether).
  • the resulting activated dimer which bears the mono-NHS ester of the glutaric acid half amide, is isolated by decanting of the supernatant solution, which serves to remove the excess glutaric acid bis-NHS ester. Subsequent washings may be required to complete the dissolution and removal of remaining bis-activated linker reactant.
  • the residue may now be dissolved in a suitable solvent such as DMSO, NMP, DMF, DMA or dichloromethane, and a reporter-sp molecule bearing an amino group can be added to the solution. Reaction of the amino group with the activated carboxyl group of the glutaryl half amide mono-NHS ester appended to the dimer will provide the reporter-sp-functionalized dimer ⁇ see FIG. 47).
  • the reporter-sp molecule can be attached to peptide 1 -branching group B before the assembly of the dimer.
  • the resulting compound may be further elaborated to the reporter-sp-functionalized peptide 1-branching group B-spacerl- NH 2 and finally reacted with a suitable species containing peptide2-branching group B'— spacer2— linker D to provide the desired reporter-sp-functionalized dimer construct.
  • An additional measure of flexibility in this approach is that the position of the reporter-sp on the dimer can be controlled completely. Both aspects of the approach are explained below and shown in FIG. 48.
  • peptide 1 -branching group B in which branching group B is
  • Lys[fmoc-Lys(ivDde]- at the C-terminus may be prepared by solid phase synthesis on, for example, Rink-amide resin. Removal of the fmoc group followed by reaction with an activated acylating reporter-sp (formed as described above) provides the resin-bound, reporter-sp functionalized peptide l-(N ⁇ -reporter-sp-branching group B)-RmIc amide resin, where the reporter-sp is appended to N ⁇ of the lysine residue bearing the ivDde group.
  • peptide l-(N ⁇ -reporter-sp-branching group B)-Rink amide resin can be treated with hydrazine in DMF or DMA or another suitable solvent, to liberate the C-terminal lysine epsilon amino group.
  • a suitable activating agent such as HBTU and a base such as DIEA, or DIC/HOBt
  • the invention includes dimers wherein there are two or even more reporter-sp groups.
  • moieties for the branching group B' that are described herein as employed for constructing the structure branching group B (which is associated with peptidel), such as, e.g., Lys(Lys) or other appropriate dipeptide constructs bearing side chain groups that can be orthogonally protected and liberated when needed, will allow facile attachment of a reporter-sp to the branching group B' .
  • the requirements of the synthesis taking into consideration that the lysine or other aforementioned related amino acid functions of the branching groups should be manipulated independently and selectively from others in the peptide, permits both aloe- and ivDde-protected residues to be employed in the same peptide or dimer construct to facilitate the selective manipulation of the amino groups of the amino acids in the branching groups B and B'.
  • the ivDde or aloe group of the lysines in the branching groups B or B' or the lysine residues within the sequence of the peptide 1 or peptide2 may be removed with hydrazine (ivDde) or reductive methods which employ palladium-based reagents (aloe) at the appropriate juncture in the synthesis, in order to facilitate the selective manipulation of the various lysine residues in the individual peptides peptide 1 and peptide2, or the dimer construct at any stage in its elaboration to the final product.
  • ivDde hydrazine
  • aloe palladium-based reagents
  • the final product may be purified by SEC (size exclusion chromatography), reverse phase HPLC, ion exchange chromatography or other chromatographic techniques singly or in combination as needed.
  • SEC size exclusion chromatography
  • HPLC reverse phase HPLC
  • ion exchange chromatography or other chromatographic techniques singly or in combination as needed.
  • orthogonal protection methodology is anticipated wherein more than one amino acid having side chain functions such as, e.g., carboxyl, thiol or hydroxyl groups or others mentioned in the descriptions of the branching groups B and B' or even of the linker D functions, should be specifically manipulated in the presence of the others in the molecule.
  • Dimers including reporter-sp groups and their preparation are described throughout the disclosure herein, e.g., in Example 9, FIG. 46, FIG. 47 and FIG. 48.
  • D is a diamine linker
  • the diamine linker D is preferably a diamine that joins spacer 1 and spacer2 via amide bonds.
  • the diamines employed as the diamine linker D can be of the form:
  • the diamine employed as diamine linker D may also be a substituted aromatic or heterocyclic diamine of the form NH 2 -Q- ZZ-Q'-NH 2 , where
  • ZZ symbolizes the aromatic or heterocyclic nucleus which is the scaffold bearing the two amino groups
  • Examples of such molecules include, without limitation, A- aminomethylbenzylamine, 4-aminomethyl-4'-aminomethylbiphenyl, N-methylpyrrole-2,5- dicarboxylic acid bis 2-aminoethylamide, pyridine-2,6-dicarboxylic acid bis-2- aminoethylamide, N-methylpyrrole-2,3-dicarboxylic acid bis-2-aminoethylamide, N- methylpyrrole-2,4-dicarboxylic acid bis-2-aminoethylamide, pyridine 2,3-dicarboxylic acid bis-2-aminoethylamide, pyridine-3,5-dicarboxylic acid bis-2-aminoethylamide and pyridine 3,4-dicarboxylic acid bis-2-aminoethylamide (FIG. 50).
  • ZZ is now a saturated derivative of the aromatic or heterocyclic ZZ, can also be employed as the diamine linker D with the same groups.
  • the group R in the linker D diamines can be, without limitation, acetyl, acylamino, acylaminoalkyl, amino, alkylamino, aminoalkyl, alkyl, alkenyl, alkoxycarbonyl, alkynyl, aryl, allyl, propargyl, heteroaryl, carboxyalkyl, carboxamido, carboxyamidoalkyl, formyl, hydrogen, hydroxy, hydroxyalkyl, polyhydroxyalkyl or polyethyleneoxy (meaning the structure (CH 2 CH 2 O) n -G where G is a terminating group selected from alkylamino, alkyl, alkylhydroxy, alkyl, carboxy, methylenecarbox
  • the diamine linker D may be derived from a D or
  • L amino acid or a D or L amino acid amide derived from lysine, ornithine or 2,3- diaminopropionic acid. These may be employed as described above as the diamine linker D.
  • the alpha and side chain amino groups may be used to make the bonds from linker D to spacer 1 and spacer2 of the final dimer construct.
  • the amino acid acyl group may be amidated by groups -N-R as described above for the aliphatic NH 2 -Q-ZZ-Q '-NH 2 linkers shown in FIG. 51.
  • the specificity of the reaction for the alpha or side-chain amino groups of the diamine linker derived from amino acids may be controlled by protection of one of the amino groups of the amino acid with, e.g., an.
  • ivDde group a benzyloxycarbonyl group, an aloe group, a 4-methyltrityl group or a 2-chlorotrityl group; this is analogous to the use of orthogonal protection methods to direct reaction of the branching groups described above.
  • Reaction of the amino acid derivative, such as a lysine derivative where one of the two amino groups is protected, with peptide 1 -branching group B-spacerl -leaving group provides the construct peptide 1-branching group B-spacerl-linker D-NH-PG, where PG is the protecting group for the second amino function of the lysine.
  • Dimers containing a diamine linker D are described in, e.g., Example 9 (D 16) and FIG. 49.
  • a preferred general synthetic method of preparing multivalent peptides of the invention in which a diamine linker D is used is one in which peptide 1 and peptide2, with their associated branching groups B and B', can, in separate flasks, be coupled to excess amounts of bis-activated dicarboxylic acid spacers; for example, 3,6,9-trioxa-undecan-l,l l- dicarboxylic acid bis N-succinimidyl ester (spacer 1-C) and glutaric acid-bis-NHS ester (spacer2-E); providing, respectively, peptide 1-branching group B-spacerl-NHS ester and peptide2-branching group B'-spacer2-NHS ester.
  • bis-activated dicarboxylic acid spacers for example, 3,6,9-trioxa-undecan-l,l l- dicarboxylic acid bis N-succinimidyl ester (spacer 1-C) and glutaric acid-
  • the excess bis-NHS ester derivatives of these respective dicarboxylic acids can be washed off by precipitation of the peptide 1- branching group B-spacerl -mono-NHS ester and the peptide2-branching group B'-spacer2- mono-NHS ester with a suitable solvent such as, e.g., ether or ethyl acetate, which selectively solubilizes the bis-NHS esters of spacer 1 and spacer2, allowing their removal by decanting the respective solutions.
  • a suitable solvent such as, e.g., ether or ethyl acetate
  • Peptide 1— branching group B'— spacerl— NHS ester can then be dissolved in a suitable solvent such as, e.g., DMSO, NMP, DMF, or DMA, and this solution can be added to an excess (typically 5-100 fold excess) of a diamine linker D, such as 4,7,10- trioxatridecane-l,13-diamme, a preferred diamine linker, dissolved in an appropriate solvent such as, e.g., DMF or DMA, to provide peptide 1-branching group B-spacerl-linker D monoamine.
  • a suitable solvent such as, e.g., DMSO, NMP, DMF, or DMA
  • the remaining diamine linker D can be removed either by evaporation or by selective dissolution in a solvent, such as, e.g., ether or ethyl acetate.
  • a solvent such as, e.g., ether or ethyl acetate.
  • the addition of ether or ethyl acetate results in precipitation or sequestration of the peptide 1- branching group B-spacerl— linker D-monoamine in a separate phase from that bearing the excess diamine linker D. Decanting the supernatant then removes the excess diamine linker D.
  • the excess diamine linker D may be removed by these two operations executed sequentially as well. It is also anticipated that the protocol may have to be repeated, possibly more than once, to remove all of the excess diamine linker D.
  • the peptidel- branching group B— spacer 1 -linker D-monoamine can then be dissolved in a suitable solvent such as, e.g., DMSO, NMP, DMF or DMA, and reacted with peptide2-branching group B' ⁇ spacer2-mono-NHS ester in a stoichiometric amount to provide the assembled dimer, namely peptide 1— branching group B-spacerl -linker D-spacer2-branching group B'- peptide2.
  • a stoichiometric amount of the reacting constituent monomeric peptide- branching group-spacer constructs and intervening intermediates is not mandatory, and it may be desirable to employ other than a stoichiometric amount of either of the reacting partners that result in the preparation of the desired heterodimeric peptide construct as outlined briefly following.
  • the amount of the second reacting partner, namely peptide2- branching group B'-spacer2— mono-NHS ester employed may optionally be either more or less than a stoichiometric amount.
  • the optional variation of the amount employed is a function of the reactivity of the two compounds, the relative ease of separation of the desired product dimer from the starting peptide2-branching group B'-spacer2-mono-NHS ester (or its hydrolysis product the carboxylic acid congener) or the peptide 1— branching group B- spacerl -linker D-monoamine construct and the availability of either of the constructs.
  • construct peptide2— branching group B'— spacer2-mono-NHS ester is difficult to separate from the dimer reaction product, but not from peptide 1-branching group B-spacerl -linker D-monoamine, then employing a limiting, substoichiometric amount of the construct peptide2-branching group B'-spacer2-mono- NHS ester is the best strategy to insure its complete consumption, thus obviating the problem of a difficult separation of this material from the product dimer.
  • expedients as described above can be employed to optimize all phases of the preparation and purification of compounds. The entire sequence is illustrated in FIG. 49.
  • the methods of the present invention are advantageous because of, e.g., their modular nature, their versatility and their ability to incorporate reporter-sp groups onto one or both ends of a molecule.
  • peptide 1 and peptide2 may be the same peptide;
  • spacerl and spacer2 may be the same spacer;
  • the combinations of the same peptides and different spacers or different peptides with the same spacers can be made; and finally that these possibilities are all contemplated and anticipated by the general description given above.
  • bond forming reactions include, e.g., the formation of ureas by coupling an amino group of one of the peptides, branching groups, spacers, or the linker with the appropriate partner bearing an isocyanate function or a mixed urea prepared by treatment of an amino group of the other peptide, branching group, spacer or linker with carbonyldiimidazole, phosgene or phosgene surrogates such as diphosgene or triphosgene.
  • linkages that may be formed include, e.g., dissulfide bonds, ethers, esters, carbamates, hydrazones, acylhydrazones, semicarbazones, thioamides or thioureas.
  • Those skilled in the art will readily recognize that facilitating these different coupling strategies will necessarily involve changing the carboxylic acid or an amino group of one of the above partners to, e.g., an aldehyde, amine, alcohol, hydrazine, acylhydrazine, isothiocyanate, mercaptan, 2- pyridyldisulfide.
  • Such changes in functional groups are required and expected by those skilled in the art.
  • This method of dimerization has excellent positional flexibility, and tolerates other linking groups.
  • N-N N-terminus to N-terminus
  • C-N C- terminus to N-terminus dimerization
  • N-C N-terminus to C-terminus dimerization
  • C-C C-terminus to C-terminus dimerization
  • dimerization at the C-terminus used the epsilon amine of lysine
  • the methods of the present invention are remarkably flexible in providing dimerized peptides without esterif ⁇ cation of tyrosine or threonine derivatives.
  • the guanidine group of argine is not acylated and the carboxylic acid groups of aspartic and glutamic acid are unaffected. Lysine groups can be selectively protected with the ivDde group to prevent acylation during dimer formation and later de-protected with hydrazine and, where necessary, used in further elaboration.
  • Cylic disulfide other cyclic peptides, such as, e.g., cyclic lactam peptides, cyclic lactone peptides, all of the cyclic peptide types described as disulfide mimetics, linear peptides and all of the other types of monomer peptides and other monomer types such as, e.g., small molecules, peptidomimetics other than merely disulfide mimetics, can be employed to form heterodimers and even homodimers.
  • heterodimer (or homodimer) constructs with or without reporters can be prepared from all of the monomer types described in this disclosure.
  • the following Table is illustrative of the combinations of monomers and the dimers that will result.
  • the row directly below the Title in Table 1 lists the monomer types available for Peptide 'A' in a Peptide 'A' -Peptide 'B' heterodimer construct.
  • the leftmost column lists the available monomer types for Peptide 'B'.
  • the cells forming the intersection of the members of the topmost row and the leftmost column describe the types of heterodimer Peptide 'A' -Peptide 'B' or even the types of homodimers Peptide 'A' -Peptide 'A' or Peptide 'B'-Peptide 'B' that may be formed in terms of the constituent monomer types.
  • Peptide A and Peptide B are broad terms that denote peptides with associated spacer(s) and reporter-sp molecules, and that the dimer formation may require the use of a linker function as well.
  • Peptide A and Peptide B bind to different epitopes on a single target.
  • Peptide A and Peptide B bind to different epitopes on the KDR- or cMET- receptors.
  • the methods of the present invention allow preparation of homodimers and heterodimers, allow control of the position of linkage of both peptides, and also allow control of the hydrophilicity, length and rigidity of the linker, and hence optimization of, the physical and binding properties of the compounds. Further, the methods of the present invenion rely on acylation and provide the desired products irreversibly vs some chemical ligation approaches (cf above), and are compatible with protected and unprotected peptides. [00262] By very minor modification of the monomer peptides and the overall approach, the methods of the present invention allow for positionally controlled incorporation of labeling groups such as fluorophores, biotin and metal chelators. Furthermore, the introduction of such groups can be controlled with respect to when they are incorporated - either before or after dimerization. These present methods form the basis of the preparation of high affinity homodimers and heterodimers with superior biological properties.
  • Heteromultimeric constructs of the present invention can be used in a multitude of applications, including immunoassays (e.g., ELISA), as pharmaceuticals useful for treatments of various diseases, as well as in in vivo diagnosis and therapy.
  • immunoassays e.g., ELISA
  • the heteromultimeric constructs described herein will be extremely useful for detection and/or imaging of target containing tissue in vitro or in vivo.
  • KDR- or VEGF/KDR complex-binding heteromultimeric constructs will be extremely useful for detection and/or imaging of KDR or VEGF/KDR complex-containing tissue, 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.
  • cMet or HGF/cMet complex-binding heteromultimeric constructs will be extremely useful for detection and/or imaging of cMet or HGF/cMet complex containing tissue, and particularly for detection and/or imaging tumors or other sites of hyperproliferation, in which HGF and cMet are intimately involved, as explained above.
  • Any suitable method of assaying or imaging cMet or HGF/cMet complex may be employed.
  • the compounds of the invention also have utility in the treatment of a variety of disease states, whether used alone or in combination with another therapeutic agent.
  • a compound of the invention that inhibits a biological process that contributes to a disease state may itself be used as a therapeutic or pharmaceutical composition.
  • a compound of the invention may include one or more additional therapeutic agents.
  • the invention includes heteromultimers including KDR- or VEGF/KDR complex-binding moieties which 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, or those associated with a number of pathogens.
  • the invention includes heteromultimers including cMet or HGF/cMet complex- binding moieties which 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 cMet-expressing cells, including tumors, sites of hyperproliferation or sites of angiogenesis.
  • therapeutic agents e.g., a chemotherapeutic, a radiotherapeutic, genetic material, etc.
  • the heteromultimeric constructs of the present invention are particularly useful as therapeutic agents for treating conditions that involve endothelial cells. Because an important function of endothelial cells is angiogenesis (the formation of blood vessels), the heteromultimers of the invention are particularly useful for treating conditions that involve angiogenesis, which 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, cancer of the breast, lung, prostate, colon, head and neck, and ovary. Additional tumors and related disorders are listed in Table 1 of U.S. Patent No.
  • Benign tumors include, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas.
  • angiogenesis includes for example, rheumatoid arthritis, psoriasis, and ocular disease, such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasias, rebeosis, Osier- Webber syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma and wound granulation.
  • Other relevant diseases or conditions that involve blood vessel growth include intestinal adhesions, atherosclerosis, scleroderma, and hypertropic scars, and ulcers.
  • the heteromultimers of the present invention can be used to reduce or prevent uterine neovascularization required for embryo implantation, for example, as a birth control agent.
  • a heteromultimer according to the invention can be detectably labeled, e.g., fluorophore-labeled, enzymatically labeled, or labeled with a radionuclide or paramagnetic metal or attached to bubbles, then contacted with the solution, and thereafter formation of a complex between the heteromultimer and the target can be detected.
  • detectably labeled e.g., fluorophore-labeled, enzymatically labeled, or labeled with a radionuclide or paramagnetic metal or attached to bubbles
  • a fluorophore-labeled KDR- or VEGF/KDR complex- binding heteromultimeric construct may be used for in vitro KDR or VEGF/KDR complex detection assays, wherein the heteromultimeric construct is added to a solution to be tested for KDR or VEGF/KDR complex under conditions allowing binding to occur.
  • the complex between the fluorophore-labeled KDR- or VEGF/KDR complex-binding heteromultimer 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 heteromultimer relative to that of the free heteromultimer.
  • Heteromultimers comprising cMet binding moieties may be used similarly.
  • a sandwich-type "ELISA" assay may be used, wherein a heteromultimeric construct is immobilized on a solid support such as a plastic tube or well, then the solution suspected of containing the target is contacted with the immobilized heteromultimeric construct, non-binding materials are washed away, and complexed target is detected using a suitable detection reagent, such as a monoclonal antibody recognizing the target.
  • a suitable detection reagent such as a monoclonal antibody recognizing the target.
  • 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 fluorophore-labeled.
  • heteromultimers of the invention can be immobilized on a solid substrate such as a chromatographic support or other matrix material, then the immobilized heteromultimer can be loaded or contacted with the solution under conditions suitable for formation of a heteromultime ⁇ target complex.
  • the solution which contains unbound target and non- binding substances, can be removed and the complex may be detected, e.g., using an antibody against the target, such as an anti-binding polypeptide antibody (e.g., anti-KDR, anti- VEGF/KDR complex, anti-cMet, or anti-cMet/HGF complex antibody), or the heteromultime ⁇ target complex may be released from the binding moiety under appropriate elution conditions.
  • an anti-binding polypeptide antibody e.g., anti-KDR, anti- VEGF/KDR complex, anti-cMet, or anti-cMet/HGF complex antibody
  • heteromultimeric constructs of the present invention may be used in in vivo diagnostic applications to image specific tissues or cellular disorders.
  • a particularly preferred use for the heteromultimeric constructs according to the present invention is for creating visually readable images of tissues expressing or containing the biomolectular target.
  • the heteromultimers of the invention are conjugated with a label appropriate for diagnostic detection, optionally via an interposed linker.
  • Suitable linkers can be substituted or unsubstituted alkyl chains, amino acid chains (e.g., polyglycine), polyethylene glycols, polyamides, and other simple oligomeric or polymeric linkers known in the art.
  • linker may be employed, singly or in combination, to form the actual linker structure.
  • a heteromultimer exhibiting much greater specificity for the target than for other serum proteins is conjugated or linked to a label appropriate for the detection methodology to be employed.
  • heteromultimers of the invention 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 if necessary a chelator, such as those described herein, for a radioactive metal) with an ultrasound contrast agent (e.g., a stabilized microbubble, a microballoon, a microsphere or what has been referred to as a gas filled "liposome”) suitable for ultrasound detection, or with an optical imaging dye.
  • MRI magnetic resonance imaging
  • KDR- or VEGF/KDR complex-binding heteromultimeric constructs of the invention or cMet- or HGF complex-binding heteromultimeric constructs of the invention may be used to image neoplastic tumors, which require angiogenesis for survival and metastasis, or other sites of angiogenic activity.
  • heteromultimeric constructs including KDR- and VEGF/KDR complex-binding polypeptides or cMet- or HGF/cMet complex-binding polypeptides are converted to imaging reagents by conjugation with a label appropriate for diagnostic detection, optionally via a linker, as described herein.
  • the technique of using a detectably labeled heteromultimeric construct is based on the premise that the label generates a signal that is detectable outside the patient's body.
  • a detectably labeled heteromultimer of the invention when administered to the patient in which angiogenesis, e.g., due to a tumor, is occurring, the high affinity of the KDR- or VEGF/KDR complex-binding moieties included in the heteromultimeric constructs for KDR or VEGF/KDR complex causes the heteromultimeric construct to bind to the site of angiogenesis and accumulate label at the site of angiogenesis. Sufficient time is allowed for the labeled heteromultimeric construct to localize at the site of angiogenesis.
  • the signal generated by the labeled peptide is detected by a scanning device which will vary according to the type of label used, and the signal is then converted to an image of the site of angiogenesis.
  • heteromultimers 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.
  • heteromultimers can be conjugated to streptavidin or avidin for in vivo binding to target-containing or target-expressing cells. After the unbound heteromultimer has cleared from the body, a biotinylated detectable label or radiotherapeutic construct (e.g., a chelate molecule complexed with a radioactive metal) which will rapidly concentrate at the site where the targeting construct is bound, can be infused.
  • a biotinylated detectable label or radiotherapeutic construct e.g., a chelate molecule complexed with a radioactive metal
  • this approach can reduce the time required until imaging, after administering the detectable label, can take place. It can also increase the 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 Goodwin et al., ("Advances in pretargeting biotechnology") Biotechnology Advances 19(6), 435-450 (2001), incorporated by reference herein; and Rosebrough, Q. J. Nucl. Med.
  • heteromultimeric constructs including KDR or VEGF/KDR-binding moieties are used.
  • heteromultimeric constructs including cMet or HGF/cMet binding moieties are used.
  • the heteromultimers of the present invention may advantageously be conjugated with one or more paramagnetic metal chelates in order to form a contrast agent for use in MRI.
  • Preferred paramagnetic metal ions have atomic numbers 21-29, 42, 44, or 57-83. This includes ions of the transition metal or lanthanide series which have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1.7 Bohr magneton.
  • Preferred paramagnetic metals include, but are not limited to, chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (H), 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). Additionally, heteromultimers of the present invention may also be conjugated with one or more superparamagnetic particles.
  • Gd(III) is particularly preferred for MRI due to its high relaxivity and low toxicity, and the availability of only one biologically accessible oxidation state. 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. [00276]
  • One skilled in the art will select a metal according to the dose required to detect target containing tisssue and considering other factors such as toxicity of the metal to the subject. See Tweedle et al., Magnetic Resonance Imaging (2nd ed ⁇ vol. 1, pp. 796-97, Partain et al., eds. (W.B. Saunders Co. 1988).
  • the desired dose for an individual metal will be proportional to its relaxivity, modified by the biodistribution, pharmacokinetics and metabolism of the metal.
  • 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 metabolization of the metal by a patient.
  • Another useful metal is Cr 3+ , which is relatively inexpensive.
  • the paramagnetic metal chelator is a molecule having one or more polar groups that act as a ligand for, and complex with, a paramagnetic metal or metal ion.
  • Suitable chelators are known in the art and include acids with methylene phosphonic acid groups, methylene hydroxamic acid groups, carboxyethylidene groups, or carboxymethylene groups.
  • chelators include, but are not limited to, diethylenetriamine pentaacetic acid (DTPA), 1 ,4,7,10-tetraazacyclotetradecane-1, 4,7,10-tetraacetic acid (DOTA), 1- substituted 1,4,7,-tricarboxymethyl 1,4,7,10 teraazacyclododecane triacetic acid (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,1 l-tetraazacyclotetradecane-1,4,8, 11- tetraacetic acid (TETA).
  • DTPA diethylenetriamine pentaacetic acid
  • DOTA 1- substituted 1,4,7,-tricarboxymethyl 1,4,7,10 teraazacyclododecane triacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • TETA 1,4,8,1 l-tetraazacyclotetradecane-1,4,8, 11- tetraacetic acid
  • Additional chelating ligands are ethylenebis-(2-hydroxy- phenylglycine) (EHPG), and derivatives thereof, including 5-Cl-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl DTPA, bis-2
  • HBED hydroxybenzyl-ethylene-diaminediacetic acid
  • 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-l,4,8,ll-(methyl tetraacetic acid); derivatives of 1,3-propylenediaminet
  • a preferred chelator for use in the present invention is DTPA.
  • 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, PCTfUS 98/01473, PCT/US98/20182, and U.S. 4,899,755, U.S. 5,474,756, U.S. 5,846,519 and U.S. 6,143,274, each of which is hereby incorporated by reference in its entirety.
  • Use of the chelate DO3A is particularly preferred.
  • the chelator(s) of the MRI contrast agent is coupled to a heteromultimer, such as, for example one comprised of KDR- or VEGF/KDR complex-binding polypeptides or cMet- or HGF/cMet complex-binding polypeptides.
  • a heteromultimer such as, for example one comprised of KDR- or VEGF/KDR complex-binding polypeptides or cMet- or HGF/cMet complex-binding polypeptides.
  • the positioning of the chelate(s) should be selected so as not to interfere with the binding affinity or specificity of the heteromultimeric construct.
  • 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.
  • a chelator having a free central carboxylic acid group makes it easy to attach the chelators to the N-terminus of a binding peptide by forming an amide bond.
  • the chelate(s) could also be attached at the C-terminus with the aid of a linker.
  • isothiocyanate conjugation chemistry could be employed as a way of linking the appropriate isothiocyante group bearing DTPA to a free amino group anywhere within the peptide sequence.
  • the heteromultimer can be bound directly or covalently to the metal chelator(s) (or other detectable label), or it may be coupled or conjugated to the metal chelator(s) 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 binding moieties); derivatized or underivatized polyethylene glycol, polyoxyethylene, or polyvinylpyridine chains; substituted or unsubstituted polyamide chains; derivatized or underivatized poly
  • the molecular weight of the linker can be tightly controlled.
  • the molecular weights can range in size from less than 100 to greater than 1000.
  • the molecular weight of the linker is less than 300.
  • biodegradable functionalities can include ester, double ester, amide, phosphoester, ether, acetal, and ketal functionalities.
  • known methods can be used to couple the metal chelate and a heteromultimer of the invention using such linkers.
  • a heteromultimer can be linked through the N- or C-terminus of a component-binding moiety 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(s) on any position, provided the metal chelate retains the ability to bind the metal tightly in order to minimize toxicity.
  • a component-binding moiety of a heteromultimer 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 the target.
  • MRI contrast reagents prepared according to the disclosures herein may be used in the same manner as conventional MRI contrast reagents.
  • certain MR techniques and pulse sequences may be preferred to enhance the contrast of the site to the background blood and tissues.
  • 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)).
  • fast spin echo sequences ⁇ see, e.g., Alexander et al., Magnetic Resonance in Medicine, 40(2): 298-310 (1998)
  • flow- spoiled gradient echo sequences ⁇ see, e.g., Edelman et al., Radiology, 177(1): 45-50 (1990)
  • 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 target containing tissue, such as an angiogenic tumor, and background tissues.
  • the labeled reagent is administered to the patient in the form of an injectable composition.
  • the method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecally, interstitially, or intracavitarilly.
  • intravenous or intraarterial administration is preferred.
  • the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the target ⁇ e.g., a site of angiogenesis) at least 10%.
  • heteromultimeric constructs including the MRI reagent After injection of the heteromultimeric construct including the MRI reagent, the patient is scanned in the MRI machine to determine the location of any sites containing the target. In therapeutic settings, upon target localization, a cytotoxic or therapeutic agent can be immediately administered, if necessary, and the patient can be subsequently scanned to visualize the therapeutic effect.
  • heteromultimers including KDR- or VEGF/KDR complex-binding moieties are conjugated to one or more paramagnetic metal chelates or one or more superparamagnetic particles, optionally via a linker.
  • heteromultimeric constructs including cMet or HGF/cMet complex-binding moieties are used.
  • Such heteromultimeric constructs are complexed with one or more paramagnetic metal and adminitered in a dose sufficient to enhance the MR signal at the site of angiogenesis at least 10%.
  • the patient is scanned to determine the location of any sites of angiogenesis (e.g., angiogenic tumors, etc.) or hyperproliferative tissue. If necessary, upon location of an angiogenic or hyperproliferative site, an anti-angiogenic or tumoricidal agent, such as, for example, an inhibitor of VEGF (or VEGF activation of KDR) may be administered. If necessary, the patient may be scanned again to visualize/track the tumor regression, arrest of angiogenesis, etc.
  • angiogenesis e.g., angiogenic tumors, etc.
  • an anti-angiogenic or tumoricidal agent such as, for example, an inhibitor of VEGF (or VEGF activation of KDR) may be administered. If necessary, the patient may be scanned again to visualize/track the tumor regression, arrest of an
  • 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.
  • the heteromultimeric constructs may include a material that is useful for ultrasound imaging.
  • heteromultimers of the invention may be linked to materials employed to form vesicles (e.g., microbubbles, microballoons, microspheres, etc.), or emulsions containing a liquid or gas which functions as the detectable label (e.g., an echogenic gas or material capable of generating an echogenic gas).
  • vesicles 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.
  • contrast agents comprising suspensions of stabilized microbubbles (a preferred embodiment), phospholipids, and particularly saturated phospholipids are preferred.
  • the preferred gas-filled microbubbles can be prepared by means known in the art, such as, for example, by a method described in any one of the following patents: EP 554213, US 5,413,774, US 5,578,292, EP 744962, EP 682530, US 5,556,610, US 5,846,518, US 6,183,725, EP 474833, US 5,271,928, US 5,380,519, US 5,531,980, US 5,567,414, US 5,658,551, US 5,643,553, US 5,911,972, US 6,110,443, US 6,136,293, EP 619743, US 5,445,813, US 5,597,549, US 5,686,060, US 6,187,288, and US 5,908,610, each of which is incorporated by reference herein in its entirety.
  • 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.
  • 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.
  • phospholipids are phosphatide 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.
  • phosphatide 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
  • 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.
  • PC phosphatidylcholine
  • 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
  • DPPC dipalmitoyl-phosphatidylcholine
  • DAPC diarachidoylphosphatidylcholine
  • DSPC distearoyl-phosphatidylcholine
  • MPPC l-myristoyl-2-palmitoylphosphatidylcholine
  • PMPC l-palmitoyl-2- myristoylphosphatidylcholine
  • PSPC 1 -palmitoyl-2-stearoylphosphatid-ylcholine
  • SPPC dioleoylphosphatidylycholine
  • DOPC 1,2 distearoyl-sn-glycero-3-ethylphosphocholine
  • Ethyl-DSPC dilauryloyl-phosphatidylglycerol
  • compositions may also 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, such as CF 4 , C 3 F 8 and C 4 F 1 O, are preferred.
  • the preferred microbubble suspensions may be prepared from phospholipids using known processes such as a freeze-drying or spray-drying solutions of the crude phospholipids in a suitable solvent or using the processes set forth in EP 554213, US 5,413,774, US 5,578,292, EP 744962, EP 682530, US 5,556,610, US 5,846,518, US 6,183,725, EP 474833, US 5,271,928, US 5,380,519, US 5,531,980, US 5,567,414, US 5,658,551, US 5,643,553, US 5,911,972, US 6,110,443, US 6,136,293, EP 619743, US 5,445,813, US 5,597,549, US 5,686,060, US 6,187,288, and US 5,908,610, each of which is incorporated by reference herein in its entirety.
  • the phospholipids are dissolved in an organic solvent and the solution is dried without going through a liposome formation stage.
  • This can be done by dissolving the phospholipids in a suitable organic solvent together with a hydrophilic stabilizer substance or a compound soluble both in the organic solvent and water and freeze-drying or spray-drying the solution.
  • the criteria used for selection of the hydrophilic stabilizer is its solubility in the organic solvent of choice.
  • hydrophilic stabilizer compounds soluble in water and the organic solvent are e.g., a polymer, like polyvinyl 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 by spray drying, freeze-drying (lyophilization) or other drying techniques known to those of ordinary skill in the art.
  • Typical organic solvents include, for example, dioxane, cyclohexanol, tertiary butanol, tetrachlorodifluoro ethylene (C 2 Cl 4 F 2 ) or 2-methyl-2- butanol; however, 2-methyl-2-butanol and C 2 Cl 4 F 2 are preferred.
  • the freeze-dried or spray-dried phospholipid powders Prior to formation of the suspension of microbubbles by dispersion in an aqueous carrier, the freeze-dried or spray-dried phospholipid powders are contacted with air or another gas. When contacted with the aqueous carrier, the powdered phospholipid 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.
  • microbubbles can be prepared by suspending a gas into an aqueous solution at high agitation speed, as disclosed in e.g., 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.
  • 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% by weight.
  • microballoons refers to gas-filled bodies with a material boundary or envelope. More information on microballoon formulations and methods of preparation may be found in, e.g., EP-A-O 324 938 US 4,844,882; US 5,711,933; US 5,840,275; US 5,863,520; US 6,123,922; US 6,200,548; US 4,900,540; US 5,123,414; US 5,230,882; 5,469,854; 5,585,112; US 4,718,433; US 4774,958; WO 9501187; US 5,529,766; US 5,536,490 and US 5,990,263, each of which is incorporated herein by reference in its entirety.
  • 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, US 5,711,933, US 5,840,275, EP 554213, US 5,413,774 and US 5,578,292, the entire contents of which are incorporated herein by reference.
  • the polymer can be selected from biodegradable physiologically compatible polymers, such as polysaccharides of low water solubility, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as ⁇ -caprolactone, ⁇ - valerolactone and polypeptides.
  • biodegradable physiologically compatible polymers such as polysaccharides of low water solubility, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as ⁇ -caprolactone, ⁇ - valerolactone and polypeptides.
  • suitable polymers include poly(ortho)esters (see, e.g., US 4,093,709; US 4,131,648; US 4,138,344; US 4,180,646); polylactic and polyglycolic acid and their copolymers, for instance DEXON (see J.
  • 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.
  • 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.
  • microballoons may employ any of the gases disclosed herein or 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
  • any of these ultrasound compositions should also be, as far as possible, isotonic with blood.
  • small amounts of isotonic agents may be added to any of above ultrasound contrast agent suspensions.
  • the isotonic agents are physiological solutions commonly used in medicine and they comprise aqueous saline solution (0.9% NaCl), 2.6% glycerol solution, 5% dextrose solution, etc.
  • the ultrasound compositions may include standard pharmaceutically acceptable additives, including, for example, emulsifying agents, viscosity modifiers, cryoprotectants, lyoprotectants, bulking agents etc.
  • gas includes any substances (including mixtures) substantially in gaseous form at the normal human body temperature.
  • the gas may thus include, for example, air; nitrogen; oxygen; CO 2 ; argon; xenon or krypton, fluorinated gases (including for example, perfluorocarbons, SF 6 , SeF 6 ) a low molecular weight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), for example, an alkane such as methane, ethane, propane, butane or pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene, propene, propadiene or butene, or an alkyne such as acetylene or propyne and/or mixtures thereof.
  • fluorinated gases including for example, perfluorocarbons, SF 6 , SeF 6
  • a low molecular weight hydrocarbon e
  • Fluorinated gases include materials which contain at least one fluorine atom. Examples include, but are not limited to compounds 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 Fg, C 4 F 8 , C 4 F 10 , CBrF 3 , CCI 2 F 2 , C 2 CIF 5 , and CBrClF 2 ) and perfluorocarbons.
  • freons organic compounds containing one or more carbon atoms and fluorine, i.e. CF 4 , C 2 F 6 , C 3 Fg, C 4 F 8 , C 4 F 10 , CBrF 3 , CCI 2 F 2 , C 2 CIF 5 , and CBrClF 2
  • perfluorocarbon refers to compounds containing only carbon and fluorine atoms. Such compounds include saturated, unsaturated, and cyclic perfluorocarbons
  • the saturated perfluorocarbons which are preferred, have the formula C n F n+2 , where n is from 1 to 12, preferably from 2 to 10, more preferably from 3 to 8 and most preferably from 3 to 6.
  • Suitable perfluorocarbons include, but are not limited to, CF 4 , C 2 F 6 , C 3 F 8 , C 4 F 8 , C 4 F 10 , C 5 F 12 , C 6 F 12 , C 7 F 14 , C 8 F 18 and C 9 F 20 .
  • the gas or gas mixture comprises SF 6 or a perfluorocarbon selected from the group consisting Of C 3 F 8 , C 4 F 8 , C 4 F 10 , C 5 F 12 , C 6 F 12 , C 7 F 14 , C 8 F 18 , with C 4 F 10 being particularly preferred.
  • 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.
  • 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, and WO 98/17324 the contents of which are incorporated herein in their entirety.
  • a precursor to a gaseous substance e.g., a material that is capable of being converted to a gas in vivo, often referred to as a "gas precursor"
  • gas precursor a material that is capable of being converted to a gas in vivo
  • the gas precursor and the gas it produces are physiologically acceptable.
  • the gas precursor may be pH-activated, photo-activated, temperature-activated, etc.
  • certain perfluorocarbons may be used as temperature-activated gas precursors.
  • perfluorocarbons such as, e.g., perfluoropentane
  • the gas can include a mixture of gases.
  • 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 and 41% by vol., has a molecular weight greater than 80 daltons and is a fluorinated gas, and (A) is selected from the group consisting of air, oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance of the mixture being gas A.
  • ultrasound vesicles may be larger than the other detectable labels described herein, they may be linked or conjugated to a plurality of heteromultimeric constructs in order to increase the targeting efficiency of the agent.
  • Attachment to the ultrasound contrast agents described above may be done via direct covalent bond between a binding polypeptide and the material used to make the vesicle or via a linker, as described previously.
  • a linker for example, see generally WO 98/53857 for a description of the attachment of a peptide to a bifunctional PEG linker, which is then reacted with a liposome composition. See also Lanza et al., Ultrasound in Med. & Bio., 23(6): 863-870 (1997).
  • a number of methods may be used to prepare suspensions of microbubbles conjugated to heteromultimers. 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-maleirnido-phenyl butyramide), (Avanti Polar-Lipids, Inc) in the phospholipid formulation.
  • N-MPB-PE 1-, 2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-4-(p-maleirnido-phenyl butyramide), (Avanti Polar-Lipids, Inc)
  • mercaptoacetylated heteromultimers (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 then added to the maleimide-activated microbubble suspension. After incubation in the dark, under gentle agitation, the heteromultimer 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 vesicle; said portion typically represented by a lipid moiety (e.g., dipalmitin, distearoyl); and (b) a spacer (typically PEGs of different molecular weights), which are optional in some cases (microbubbles may, for instance, be difficult to freeze dry 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).
  • a hydrophobic portion compatible with the material
  • heteromultimers conjugated to microbubbles may be prepared using the biotin/avidin system.
  • 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 heteromultimers (prepared as described herein), are then added to the suspension of avidin-conjugated microbubbles, yielding a suspension of microbubbles conjugated to the heteromultimers.
  • the lyophilized residue may be stored and transported without the need for temperature control of its environment; in particular it may be supplied to hospitals and physicians for on-site formulation into a ready-to-use administrable suspension without requiring such users to have special storage facilities.
  • a hyperpolarized gas which is known to require special storage conditions
  • the lyophilized residue may be stored and transported without the need for temperature control of its environment; 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.
  • it can be supplied in the form of a two-component kit, which can include two separate containers or a dual-chamber container.
  • the container is a conventional septum-sealed vial, wherein the vial containing the lyophilized residue of step b) is sealed with a septum through which the carrier liquid may be injected using an optionally prefilled syringe.
  • the syringe used as the container of the second component is also used then for injecting the contrast agent.
  • the dual-chamber container is a dual-chamber syringe, and once the lyophilizate has been reconstituted and then suitably mixed or gently shaken, the container can be used directly for injecting the contrast agent.
  • means for directing or permitting application of sufficient bubble forming energy into the contents of the container are provided.
  • the size of the gas microbubbles is substantially independent of the amount of agitation energy applied to the reconstituted dried product. Accordingly, no more than gentle hand shaking is generally required to give reproducible products with consistent microbubble size.
  • aqueous phase can be interposed between the water-insoluble gas and the environment, to increase shelf life of the product.
  • 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.
  • 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 may allow undesirable substances to enter the vial.
  • vacuum retention is essential for products stoppered at ambient or reduced pressures to assure safe and proper reconstitution.
  • the stopper it may be a compound or multicomponent formulation based on an elastomer, such as, e.g., poly(isobutylene) or butyl rubber.
  • Ultrasound imaging techniques that may be used in accordance with the present invention include known techniques, such as color Doppler, power Doppler, Doppler amplitude, stimulated acoustic imaging, and two- or three-dimensional imaging techniques. Imaging may be done in harmonic (resonant frequency) or fundamental modes, with the second harmonic preferred.
  • 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 ⁇ g/kg body weight, preferably from about 0.1 to 30 ⁇ g/kg body weight.
  • Microballoon-containing contrast agents are typically administered in doses such that the amount of wall-forming polymer or lipid is from about 10 ⁇ g/kg to about 20 mg/kg of body weight.
  • the ultrasound contrast agents described herein are conjugated to one or more heteromultimers comprising KDR- or VEGF/KDR complex- binding moieties, and target tissue expressing KDR. As shown in the Examples, these targeted ultrasound contrast agents will localize at sites of angiogenesis and other tissue expressing KDR and may demonstrate superior imaging of angiogenic tissue. In another preferred embodiment illustrated in the Examples, the ultrasound contrast agents described herein are conjugated to one or more heteromultimers comprising or cMet- or HGF/cMet complex-binding moieties, and, target tissue expressing cMet. These targeted ultrasound contrast agents will localize at sites of hyperproliferation or angiogenesis (including tumors) and other tissue expressing cMet, and demonstrate superior imaging of such tissue.
  • Optical Imaging, Sonoluminescence or Photoacoustic Imaging may be employed to determine the location of a target, such as a KDR, VEGF/KDR complex, cMet or HGF/cMet complex, with in vivo light imaging after injection of the subject with an optically-labeled heteromultimeric construct.
  • Optical parameters to be detected in the preparation of an image may include, e.g., transmitted radiation, absorption, fluorescent or phosphorescent emission, light reflection, changes in absorbance amplitude or maxima, and elastically scattered radiation.
  • biological tissue is relatively translucent to light in the near infrared (NIR) wavelength range of 650-1000 nm.
  • heteromultimeric constructs comprised of KDR-, VEGF/KDR complex-, cMet-, or HGF/cMet-binding polypeptides may be used for optical imaging of KDR, VEGF/KDR complex, cMet, or HGF/cMet complex in vivo.
  • the heteromultimeric constructs of the invention may be conjugated with photolabels, such as optical dyes, including organic chromophores or fluorophores, having extensively conjugated and hence delocalized ring systems and having absorption or emission maxima in the range of 400-1500 nm.
  • the compounds of the invention 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.
  • photoabsorption labels have large molar absorptivities, e.g., > 10 5 Cm -1 M -1 , while fluorescent optical dyes will have high quantum yields.
  • optical dyes include, but are not limited, to those described in WO 98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO 98/47538, and references cited therein.
  • the photolabels may be covalently linked directly to heteromultimers of the invention, such as, e.g., heteromultimers comprising KDR- or VEGF/KDR complex-binding peptides, or linked to such a heteromultimers via a linker, as described previously.
  • the patient is scanned with one or more light sources (e.g., a laser) in the wavelength range appropriate for the photolabel employed in the agent.
  • the light used may be monochromatic or polychromatic and continuous or pulsed. Transmitted, scattered, or reflected light is detected via a photodetector tuned to one or multiple wavelengths to determine the location of target- containing tissue (e.g., tissue containing KDR, VEGF/KDR complex, cMet, or HGF/cMet complex) in the subject. Changes in the optical parameter may be monitored over time to detect accumulation of the optically-labeled reagent at the target site (e.g., the site of angiogenesis). Standard image processing and detecting devices may be used in conjunction with the optical imaging reagents of the present invention.
  • optical imaging reagents described above may also be used for acousto- optical or sonoluminescent imaging performed with optically-labeled imaging agents (see U.S. 5,171,298, WO 98/57666, and references therein).
  • acousto-optical imaging ultrasound radiation is applied to the subject and affects the optical parameters of the transmitted, emitted, or reflected light.
  • sonoluminescent imaging the applied ultrasound actually generates the light detected. Suitable imaging methods using such techniques are described in WO 98/57666, which is hereby incorporated by reference in its entirety.
  • Heteromultimers of the invention may be conjugated with a radionuclide reporter appropriate for scintigraphy, SPECT or PET imaging or with a radionuclide appropriate for radiotherapy. Constructs in which the heteromultimers of the invention are conjugated with both a chelator for a radionuclide useful for diagnostic imaging and a chelator for a radionuclide useful for radiotherapy are within the scope of the invention.
  • a heteromultimer may be 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 heteromultimeric constructs can also be labeled by halogenation using radionuclides, such as 18 F, 124 1, 125 1, 131 1, 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 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, 117m Sn, 149 Pm, 161 Tb, 177 Lu, 198 Au and 199 Au.
  • the preferred radionuclides include 64 Cu, 67 Ga, 68 Ga, 99m Tc, and 111 In.
  • the preferred radionuclides include 64 Cu, 90 Y, 105 Rh, 111 In, 117ra Sn, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 1867188 Re, and 199 Au.
  • m 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 99m Tc 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, e.g. , linear, macrocyclic, terpyridine, and N 3 S, N 2 S 2 , or N 4 chelants (see also U.S. 5,367,080, U.S. 5,364,613, U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see also U.S. 5,720,934).
  • N 4 chelators are described in U.S. Patent Nos.
  • N 3 S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Patent Nos. 5,662,885; 5,976,495; and 5,780,006.
  • the chelator may also include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N 3 S, and N 2 S 2 systems such as MAMA (monoamidemonoaminedithiols), DADS (N 2 S 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. [00324] The chelator may also include complexes containing ligand atoms that are not donated to the metal in a tetradentate array.
  • MAG3 chelating ligand mercapto-acetyl-acetyl-glycyl-glycine
  • disulfide bonds of a binding polypeptide of the invention are used as two ligands for chelation of a radionuclide such as 99m Tc.
  • a radionuclide such as 99m Tc.
  • Tc peptide-S-S-peptide changed to peptide-S-Tc-S-peptide.
  • the other chelating groups for Tc can be supplied by amide nitrogens of the backbone, another cystine amino acid or other modifications of amino acids.
  • Particularly preferred metal chelators include those of Formula 20, 21, and 22
  • R is alkyl, preferably methyl.
  • X is either CH 2 or O
  • Y is either C 1 -C 10 branched or unbranched alkyl
  • Y is aryl, aryloxy, arylamino, arylaminoacyl
  • Y is arylkyl - where the alkyl group or groups attached to the aryl group are C 1 -C 10 branched or unbranched alkyl groups, C 1 -C 10 branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or polyhydroxy- polyalkoxyalkyl groups
  • Other variants of these structures are described, for example, in U.
  • the chelators may be covalently linked directly to the heteromultimers or linked to heteromultimers via a linker, as described previously, and then directly labeled with the radioactive metal of choice ⁇ see WO 98/52618, U.S. 5,879,658, and U.S. 5,849,261).
  • Complexes of radioactive technetium are particularly useful for diagnostic imaging and complexes of radioactive rhenium are particularly useful for radiotherapy.
  • the technetium complex preferably a salt of 99m Tc 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 99m Tc.
  • the complex may be formed by reacting a heteromultimer of this invention, which is 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.
  • transfer ligands as tartrate, citrate, gluconate or mannitol, for example.
  • m Tc 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 as starting materials rhenium compounds wherein the metal is in the +5 or +7 oxidation state.
  • rhenium 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 ( ⁇ yridine) 4 + (Ph is phenyl; 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 must contain a suitable amount of radioactivity. In forming 99m Tc complexes, it is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 millicurie (mCi) to 100 mCi per mL.
  • the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi.
  • the solution to be injected at unit dosage is from about 0.01 mL to about 10 mL.
  • Typical doses of a radionuclide-labeled heteromultimeric construct imaging agent of the invention provide 10-50 mCi.
  • a PET camera or 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, in hours or even longer, after the radiolabeled peptide is injected into a patient.
  • Radiotherapeutic compounds of the present invention 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 which include, but are not limited to, a single or multiple IV or IP injections, using a quantity of radioactivity that is sufficient to cause damage or ablation of the targeted 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 that is used, the degree of uptake and clearance of the agent from the body and the mass of the tumor.
  • doses can range from a single dose of about 30- 50 mCi to a cumulative dose of up to about 3 Curies.
  • the radiotherapeutic compositions of the invention can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection.
  • Radiation stabilizers are known to those skilled in the art, and may include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.
  • a single- vial kit preferably contains a chelating ligand (if a metal radionuclide is used), a source of a stannous salt (if reduction is required, e.g., when using technetium), 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.
  • a chelating ligand if a metal radionuclide is used
  • a source of a stannous salt if reduction is required, e.g., when using technetium
  • other pharmaceutically acceptable reducing agent if reduction is required, e.g., when using technetium
  • the quantity and type of reducing agent used would depend highly on the nature of the exchange complex to be formed. The proper conditions are well known to those that are skilled in the art. It is preferred that the kit contents be in lyophilized form.
  • Such a single vial kit may optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraacetic acid (EDTA), or ⁇ -, ⁇ -, or ⁇ -cyclodextrin that serve to improve the radiochemical purity and stability of the final product.
  • the kit may also contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.
  • a multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical.
  • one vial may contain all of the ingredients that are required to form a labile Tc(V) complex on addition of pertechnetate (e.g., the stannous source or other reducing agent).
  • pertechnetate e.g., the stannous source or other reducing agent.
  • Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the ligand, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized.
  • reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. may be present in either or both vials.
  • the radiotherapeutic and radiodiagnostic agents described herein are conjugated to one or more heteromultimers comprised of KDR- or VEGF/KDR complex-binding moieties, and target tissue expressing KDR. As shown in the Examples, these targeted radiopharmaceuticals will localize at sites of angiogenesis and other tissue expressing KDR, and may be used to treat or image angiogenic tissue or other tissue expressing KDR. In another preferred embodiment illustrated in the Examples, the radiotherapeutic and radiodiagnostic agents described herein are conjugated to one or more heteromultimers comprised of cMet- or HGF/cMet complex-binding moieties, and, target tissue expressing cMet. These targeted radiopharmaceuticals will localize at sites of hyperproliferation or angiogenesis (including tumors) and other tissue expressing cMet, and will enable imaging and treatment of such tissue.
  • heteromultimeric constructs of the present invention can be used to improve the activity and/or efficacy of therapeutic agents by, for example, improving their affinity for or residence time at the target.
  • heteromultimers are conjugated with the therapeutic agent.
  • a liposome or bubble containing a therapeutic agent may be conjugated to heteromultimers of the invention.
  • the therapeutic agent may be a radiotherapeutic, discussed above, a drug, chemotherapeutic or tumorcidal agent, genetic material, or a gene delivery vehicle, etc.
  • the heteromultimer portion of the conjugate causes the therapeutic to "home" to the sites of target expression/localization and to improve the affinity of the conjugate for these sites, so that the therapeutic activity of the conjugate is more localized and concentrated at the target sites.
  • heteromultimers including KDR- or VEGF/KDR complex- binding polypeptides 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 the VEGF/KDR complex and their residence time at a KDR or VEGF/KDR complex on endothelium undergoing angiogenesis.
  • therapeutic agents such as anti- angiogenic or tumorcidal agents
  • angiogenesis such as occurs in neoplastic tumors
  • hybrid agents are provided by conjugating KDR- or VEGF/KDR complex-binding heteromultimers with a therapeutic agent.
  • heteromultimeric constructs will be useful in treating angiogenesis associated diseases, especially neoplastic tumor growth and metastasis, in mammals, including humans.
  • the method of treatment comprises administering to a mammal in need thereof an effective amount of a heteromultimeric construct comprising KDR- or VEGF/KDR complex-binding polypeptides 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.
  • Heteromultimeric constructs of the invention comprising cMet- or HGF/cMet complex-binding moieties may be used similarly to treat disease associated with hyperproliferation or angiogenesis.
  • 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, a,LPAM or phenylalanine mustard), mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin (actinomycin D), daunorubcin, hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide, estramustine phosphate sodium, flu
  • the heteromultimeric constructs of the present invention may also be used to target genetic material to specific cells.
  • the heteromultimeric constructs of the present invention may be used to localize genetic material to cells or tissue containing the desired target.
  • 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 (YACs) 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.
  • the heteromultimeric constructs of the invention are utilized in gene therapy for treatment of diseases associated with angiogenesis.
  • genetic material, or one or more delivery vehicles containing genetic material, e.g. , useful in treating an angiogenesis-related disease may be conjugated to one or more KDR- or VEGF/KDR complex-binding heteromultimers or cMet- or HGF/cMET complex- binding heteromultimers of the invention and administered to a patient.
  • Constructs including genetic material and the KDR-binding heteromultimers 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 heteromultimers of the invention can be linked or fused in known ways, using the same type of linkers discussed herein.
  • Preferred linkers will be substituted or unsubstituted alkyl chains, amino acid chains, polyethylene glycol chains, and other polymeric linkers known in the art, which may be employed singly or in combination with each other.
  • the therapeutic agent is itself a protein, for which the encoding DNA sequence is known, the therapeutic protein and a binding polypeptide of the invention may be coexpressed from the same synthetic gene, created using recombinant DNA techniques, as described above.
  • the coding sequence for a 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 binding polypeptide.
  • a particular advantage of this general approach is that concatamerization of multiple, tandemly arranged binding polypeptides is possible, thereby increasing the number and concentration of binding sites associated with each therapeutic protein. In this manner binding peptide 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 binding polypeptide may be useful in imaging or therapeutic applications.
  • the coding sequence for a KDR-, VEGF/KDR complex-, cMet-, or HGF/cMet-binding peptide may be fused in frame to a sequence encoding an antibody (or an antibody fragment or recombinant DNA construct including an antibody, etc.) which, for example, binds to a chelator for a radionuclide (or another detectable label).
  • the antibody expressing the KDR-, VEGF/KDR complex-, cMet-, or HGF/cMet-binding polypeptide is then administered to a patient and allowed to localize and bind to KDR- or cMet-expressing tissue.
  • the chelator-radionuclide complex or other detectable label, which the antibody recognizes, is administered, permitting imaging of or radiotherapy to the KDR- or cMet-expressing tissues.
  • the coding sequence for a binding peptide may be fused in frame to a sequence encoding, for example, serum proteins or other proteins that produce biological effects (such as, e.g., apoptosis, coagulation, internalization, differentiation, cellular stasis, immune system stimulation or suppression, or combinations thereof).
  • serum proteins or other proteins that produce biological effects (such as, e.g., apoptosis, coagulation, internalization, differentiation, cellular stasis, immune system stimulation or suppression, or combinations thereof).
  • biological effects such as, e.g., apoptosis, coagulation, internalization, differentiation, cellular stasis, immune system stimulation or suppression, or combinations thereof.
  • heteromultimers of the present invention may themselves be used as therapeutics to treat a number of diseases.
  • a protein or other molecule e.g., a growth factor, hormone etc.
  • a binding moiety inhibits such binding
  • heteromultimers including such binding moieties may be useful as therapeutics.
  • binding of a binding moiety itself inhibits a disease process heteromultimers containing such binding moieties may also be useful as therapeutics.
  • heteromultimers including KDR- or VEGF/KDR complex-binding polypeptides that inhibit the binding or inhibit VEGF to KDR (or otherwise inhibit activation of KDR) may be used as anti-angiogenic agents.
  • Certain heteromultimers of the invention that inhibit activation of KDR are discussed in the Examples.
  • a particularly preferred heteromultimer is the heterodimer-containing construct D1 (structure shown below in Example 9).
  • Other preferred heterodimer constructs include D4, D5, D6, D7, D10, D13, D17, D23, D24, D25, and D26 (structures provided in the Examples below).
  • heteromultimers may be useful in the treatment of cancer or other diseases associated with inappropriate or excessive angiogenesis, such as, for example, arthritis and atherosclerotic plaques, trachoma, corneal graft neovascularization, psoriasis, scleroderma, hemangioma and hypertrophic scarring, vascular adhesions, angiofibroma, and ocular diseases, such as, e.g., diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neo vascular glaucoma, retrolental fibroplasia, rebeosis, Osier- Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma and wound granulation.
  • angiogenesis such as, for example, arthritis and atherosclerotic plaques, trachoma, corneal graft n
  • tumors and related disorders include, for example, melanoma, central nervous system tumors, neuroendocrine tumors, sarcoma, multiple myeloma as well as cancer of the breast, lung, prostate, colon, head and neck, and ovaries. Additional tumors and related disorders are listed in Table I of U.S. Patent No. 6,025,331, issued February 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.
  • heteromultimers 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. See, e.g., Example 30. hi renal failure it has been shown that anti-VEGF antibodies can reverse damage and in a similar way the compounds of the invention can reverse renal permeability pathogenesis in, for example, diabetes.
  • the heteromultimers include cMet- or HGF/cMet complex-binding polypeptides that inhibit the binding of cMet to HGF (or otherwise inhibit the activation of cMet) may be used to treat tumors and other hyperproliferative disorders.
  • Particular heteromultimers that inhibit cMet are discussed in the Examples.
  • a preferred heteromultimer is D28 (structure shown below in Example 9).
  • heteromultimers of the present invention may be useful in treating diseases associated with certain pathogens, including, for example, malaria, HIV, SrV, 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 have been noted between KDR- and VEGF/KDR complex-binding polypeptides and proteins from various malaria strains, HIV, SIV, simian hemorrhagic fever virus, and an enterohemorrhagic E. coli strain.
  • homologous proteins such as PfEMPl and EBL-I
  • PfEMPl and EBL-I hypermutable adhesion proteins known to play roles in virulence. These proteins possess multiple binding sites that are capable of binding to more than one target molecule on the host's surface. Their high mutation and recombination rates allow them to develop new binding sites quickly to promote survival and/or invasion.
  • proteins such as gpl20 of HIV which also has homology to some of the KDR-binding peptides disclosed herein
  • heteromultimeric constructs including the KDR-binding peptide sequences disclosed herein, may be useful in blocking infection with the pathogen species that possesses the homology. Indeed, a 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 SE, et al., "Synthetic peptides representing discontinuous CD4 binding epitopes of HIV-I gpl20 that induce T cell apoptosis and block cell death induced by gpl20," FASEB J 12(ll):991-8 (Aug. 1998). Thus, KDR may represent a previously unknown target for a number of pathogens, and the heteromultimeric constructs including KDR- or VEGF/KDR complex-binding peptides may therefore be useful in treating the diseases associated with these pathogens.
  • the compounds may be administered by any convenient route customary for therapeutic agents; e.g., parenterally, enterally or intranasaly, and preferably by infusion or bolus injection, or by depot or slow release formulation.
  • the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • compositions include, but are not limited to, sterile water, saline solution, buffered saline (including buffers like phosphate or acetate), alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, paraffin, etc.
  • the composition may also include a solubilizing agent and a local anaesthetic such as, e.g., lidocaine to ease pain at the site of the injection; and further may include preservatives, stabilizers, wetting agents, emulsifiers, salts, lubricants, etc. as long as they do not react deleteriously with the active compounds.
  • the composition may comprise conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal application which do not deleteriously react with the active compounds.
  • the ingredients will be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as, e.g., an ampoule or sachette indicating the quantity of active agent in activity units.
  • a hermetically sealed container such as, e.g., an ampoule or sachette indicating the quantity of active agent in activity units.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade "water for injection'Or saline.
  • an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.
  • the quantity of material administered will depend on the seriousness of the condition. For example, for treatment of anangiogenic 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 heteromultimer/therapeutic agent conjugate will follow the dosages that are routine for the therapeutic agent alone, although the improved affinity of a 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.
  • 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.
  • the heteromultimers can be administered to an individual over a suitable time course, depending on the nature of the condition and the desired outcome.
  • the heteromultimeric constructs can be administered prophylactically, i.e., before the condition is diagnosed or to an individual predisposed to a condition.
  • the heteromultimers of the invention can be administered while the individual exhibits symptoms of the condition, or after the symptoms have passed or otherwise been relieved (such as after removal of a tumor).
  • the heteromultimers of the present invention can be administered as a part of a maintenance regimen, for example to prevent or lessen the recurrence, symptoms or condition.
  • the heteromultimers of the present invention can be administered systemically or locally.
  • the term "therapeutic" includes at least partial alleviation of symptoms of a given condition.
  • the heteromultimeric constructs of the present invention need not produce a complete alleviation of symptoms to be useful.
  • treatment of an individual can result in a decrease in the size of a tumor or diseased area, or prevention of an increase in size of the tumor or diseased area or partial alleviation of other symptoms.
  • 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 metastic outgrowths of the main rumor(s).
  • symptoms that can be alleviated include, e.g., physiological characteristics such as VEGF receptor activity and migration ability of endothelial cells.
  • the heteromultimers of the present invention can inhibit activity of VEGF receptors, including VEGF-2/KDR, VEGF-l/Flt-1 and VEGF-3/Flt-4. Such inhibition can also 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. 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 measured before and after treatment.
  • 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 heteromultimers may depend on the age, sex, health, and weight of the individual, a well as the nature of the condition and overall treatment regimen.
  • the biological effects of the multimers are described herein. Therefore, based on the biological effects of the heteromultimers provided herein, and the desired outcome of treatment, the preferred dosage is determinable by one of ordinary skill in the art through route optimization procedures.
  • the daily regimen is in the range of about 0.1 ⁇ g/kg to about 1 mg/kg.
  • the heteromultimers 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 a combination thereof.
  • the heteromultimers can be conjugated to therapeutic agents, for example, to improve specificity, residence time in the body, or therapeutic effect.
  • therapeutic agents include, for example, other antiangiogenic compounds, and tumoricidal compounds.
  • the therapeutic agent can also include antibodies.
  • the heteromultimers of the present invention can be used as an endothelial cell homing device.
  • the heteromultimeric constructs can be conjugated to nucleic acids encoding, for example, a therapeutic polypeptide, in order to target the nucleic acid to endothelial cells. Once exposed to the nucleic acid, thereby delivering the therapeutic peptide to the target cells.
  • the therapeutic agent can be associated with an ultrasound contrast agent composition, said ultrasound contrast agent including the KDR-, VEGF/KDR complex-, cMet, or HGF/cMet-binding peptides of the invention linked to the material employed to form the vesicles (particularly microbubbles or microballoons) comprised in the contrast agent, as previously described.
  • said contrast agent/therapeutic agent association can be carried out as described in US 6,258,378, herein incorporated by reference.
  • 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 cause the bursting of micro vesicles, as disclosed for instance in the above cited U.S. Patent No. 6,258,378.
  • an energy beam preferably ultrasonic, e.g., with a frequency of from 0.3 to 3 MHz
  • the therapeutic effect of the therapeutic agent can thus be advantageously enhanced by the energy released by the burst of the micro vesicles, in particular causing an effective delivery of the therapeutic agent to the targeted pathogenic site.
  • the heteromultimers can be administered by any suitable route.
  • Suitable routes of administration include, but are not limited to, topical application, transdermal, parenteral, gastrointestinal, intravaginal, and transalvcolar.
  • Compositions for the desired route of administration can be prepared by any of the methods well known in the pharmaceutical arts. Details concerning dosages, dosage forms, modes of administration, composition and the like are further discussed in a standard pharmaceutical text, such as Remington's Pharmaceutical Sciences, 18th ed., Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), which is hereby incorporated by reference.
  • the heteromultimers can be suspended, for example, in a cream, gel or rinse which allows the polypeptides or constructs to penetrate the skin and enter the blood stream, for systemic delivery, or contact the are of interest, for localized delivery.
  • Compositions suitable for topical application include any pharmaceutically acceptable base in which the polypeptides are at least minimally soluble.
  • the heteromultimers can be applied in pharmaceutically acceptable suspension together with a suitable transdermal device or "patch.” Examples of suitable transdermal devices for administration of the heteromultimers of the present invention are described, for example, in U.S. Patent No. 6,165,458, issued December 26, 2000 to Foldvari, et al., and U.S. Patent No.
  • the heteromultimers can be suspended, for example, in a pharmaceutically acceptable sterile isotonic solution, such as saline and phosphate buffered saline.
  • a pharmaceutically acceptable sterile isotonic solution such as saline and phosphate buffered saline.
  • the constructs of the invention can then be injected intravenously, intramuscularly, intraperitoneally, or subcutaneously.
  • the heteromultimers can be incorporated into pharmaceutically acceptable powders, pills or liquids for ingestion, and suppositories for rectal or vaginal administration.
  • the heteromultimers can be suspended in a pharmaceutically acceptable excipient suitable for aerosolization and inhalation or as a mouthwash.
  • a pharmaceutically acceptable excipient suitable for aerosolization and inhalation or as a mouthwash such as atomizers and vaporizes are also included within the scope of the invention.
  • Suitable formulations for aerosol delivery of polypeptides using buccal or pulmonary routes can be found, for example in U.S. Patent No. 6,312,665Bl, issued November 6, 2001 to Pankaj Modi, the teachings of which are incorporated herein by reference.
  • heteromultimers of the present invention can be administered nasally or ocularly, where the heteromultimers are suspended in a liquid pharmaceutically acceptable agent suitable for dropwise dosing.
  • the heteromultimers of the present invention can be administered such that the polypeptide is released in the individual over an extended period of time (sustained or controlled release).
  • the heteromultimers can be formulated into a composition such that a single administration provides delivery of the constructs of the invention for at least one week, or over the period of a year or more.
  • Controlled release systems include monolithic or reservoir-type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches and iontophoretic devices.
  • the heteromultimers of the present invention are encapsulated or admixed in a slow degrading, non-toxic polymer. Additional formulations suitable for controlled release of constructs of the invention are described in U.S. Patent No. 4,391,797, issued July 5, 1983, to Folkman, et al., the teachings of which are incorporated herein by reference.
  • Another suitable method for delivering the heteromultimers of the present invention to an individual is via in vivo production of the polypeptides.
  • Genes encoding the polypeptides can be administered to the individual such that the encoded polypeptides are expressed.
  • the genes can be transiently expressed.
  • the genes encoding the polypeptide are transfected into cells that have been obtained from the patient, a method referred to as ex vivo gene therapy. Cells expressing the polypeptides are then returned to the patient's body. Methods of ex vivo gene therapy are well known in the art, and are described, for example, in U.S. Patent No.
  • the present invention features multivalent constructs which bind to a target of interest, as well as various methods related to the use of these constructs.
  • the present invention uses small targeting moieties which bind to different binding sites of the same target, allowing for improved localization to the desired target, and providing an improved means for detecting, imaging and/or treating the target site.
  • multivalent targeting constructs which include two or more targeting moieties, for example binding polypeptides, specific for different binding sites of the same target are described herein.
  • These targeting constructs may be linked or conjugated to a detectable label and/or a therapeutic agent (as defined herein) and used to deliver the detectable label and/or therapeutic agent to the target of interest.
  • the invention includes diagnostic imaging agents and therapeutic agents useful in diagnostic imaging and treating various disease states.
  • the invention includes use of the targeting constructs of the invention themselves to treat disease.
  • the present invention features a compound having a plurality of binding moieties, wherein at least two binding moieties have specificity for different binding sites on the same target.
  • the plurality of binding moieties includes a polypeptide.
  • the targeting moieties are all binding polypeptides which bind to different sites on the desired target.
  • the target is a protein, a receptor, or a receptor/ligand complex and the binding polypeptides bind to different epitopes on the protein, the receptor, or the receptor/ligand complex.
  • the target is a receptor involved in angiogenesis, hyperproliferative disorders or wound healing.
  • the target includes a family of receptors, such as, for example, protein-tyrosine kinase receptors.
  • the target is KDR or the KDR/VEGF complex, and the binding moieties, particularly binding peptides, bind to different epitopes on KDR or the KDR/VEGF complex.
  • the target is the hepatocyte growth factor
  • the affinity constant of a compound of the invention for its target is greater than the affinity constant of a constituent polypeptide for the target.
  • the compounds of the invention include a labeling group or a therapeutic agent.
  • the compounds of the invention include a linker between a binding moiety and the labeling group.
  • the linker may include a substituted allcyl chain, an unsubstituted alkyl chain, a polyethylene glycol derivative, an amino acid spacer, a sugar, an aliphatic spacer, an aromatic spacer, a lipid molecule, or combination thereof.
  • Preferred labeling groups include a radionuclide, a paramagnetic metal ion, an ultrasound contrast agent, and/or a photolabel.
  • preferred paramagnetic metal ions used in compounds of the invention include Mn , Cu , Fe , Co , Ni , Gd , Eu 3+ , Dy 3+ , Pr 3+ , Cr 3+ , Co 3+ , Fe 3+ , Ti 3+ , Tb 3+ , Nd 3+ , Sm 3+ , Ho 3+ , Er 3+ , Pa 4+ , and Eu 2+ .
  • Radionuclides are also preferred detectable labels and therapeutic agents. The choice of radionuclide will be determined based on the desired therapeutic or diagnostic application.
  • the compounds of the invention include a chelator or chelating group.
  • Preferable chelators include DTPA, DOTA, DO3A, EDTA, TETA, EHPG, HBED, NOTA, DOTMA, TETMA, PDTA, TTHA, LICAM, or MECAM.
  • a peptide may be 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 heteromultimeric constructs can also be labeled by halogenation using radionuclides, such as 18 F, 124 1, 125 1, 131 1, 123 1, 77 Br, and 76 Br.
  • Preferred metal radionuclides for scintigraphy or radiotherapy include 99m Tc, 51 Cr, 7 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 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 preferred radionuclides include 64 Cu, 67 Ga, 68 Ga, 99m Tc, and 111 In.
  • the preferred radionuclides include 64 Cu, 90 Y, 105 Rh, 111 In, 117m Sn, 149 Pm, 153 Sm, 161 Tb, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186/188 Re, and 199 Au.
  • a most preferred chelator used in compounds of the invention is 1 -substituted 4,7,10-tricarboxymethyl 1,4,7,10 tetraazacyclododecane triacetic acid (DO3A).
  • a radioactive lanthanide such as, for example, 177 Lu, 90 Y, 153 Sm, 111 In, or 166 Ho is used with DOTA or DO3A in compounds of the invention.
  • a radioactive lanthanide such as, for example, 177 Lu, 90 Y, 153 Sm, 111 In, or 166 Ho is used with DOTA or DO3A in compounds of the invention.
  • Compounds of the invention include chelators having the following structure:
  • X is CH 2 or O
  • Y is C 1 -C 10 branched or unbranched alkyl, aryl, aryloxy, arylamino, arylaminoacyl, or aralkyl comprising C 1 -C 10 branched or unbranched alkyl groups, C 1 -C 10 branched or unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups
  • n is 1-100.
  • the compounds further include 99m Tc, 186 Re, or 188 Re.
  • compounds of the invention include a chelator having the following structure:
  • the compound further includes 9 y 9 y I m tIrT T c n , 18 ⁇ 6 n Re, or 1 i 8 ⁇ 8 R ⁇ e
  • the chelator comprises a compound having the following structure:
  • the compound further includes 99m Tc.
  • compounds of the invention include a chelator having the following structure:
  • R is an alkyl group, such as CH 3 .
  • the compound further includes
  • compounds of the invention include a chelator having the following structure:
  • R is an alkyl group, such as CH 3.
  • the compound further includes
  • the compounds of the invention include a chelator having the following structure:
  • the compound further includes 177 Lu, 90 Y, 153 Sm, 111 In, or
  • Preferred ultrasound contrast agents for use in compounds of the invention include phospholipid stabilized microbubbles or microballoons comprising a fluorinated gas.
  • One preferred embodiment of the invention includes compounds comprising at least two binding moieties with specificity for different binding sites on a target.
  • the target is a single receptor or receptor/ligand complex such as, for example, KDR or the KDR/VEGF complex or cMet of the cMet/VEGF complex.
  • the binding moieties bind to different epitopes on the receptor or receptor/ligand complex.
  • the binding moieties include a polypeptide.
  • a compound of the invention includes a polypeptide having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29.
  • the invention also provides a compound having one or more of the foregoing amino acid sequences that have been modified to include one or more amino acid substitutions, amide bond substitutions, D-amino acid substitutions, glycosylated amino acids, disulfide mimetic substitutions, amino acid translocations, or has been modified to include a retroinverso peptide, a peptoid, a retro-inverso peptoid, and/or a synthetic peptide.
  • the compound of the invention comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 12, SEQ ID NO:26, and/or SEQ ID NO:27.
  • such compounds further include a labeling group or therapeutic agent as described herein.
  • the invention features diagnostic imaging methods using compounds of the invention that include a labeling group.
  • Methods of the invention include the steps of administering to a patient a pharmaceutical preparation that includes a compound of the invention having a labeling group, and imaging the compound after administration to the patient.
  • the imaging step includes magnetic resonance imaging, ultrasound imaging, optical imaging, sonoluminescence imaging, photoacoustic imaging, or nuclear imaging.
  • the administering step may include, e.g., inhalation, transdermal absorbtion, intramuscular injection, subcutaneous injection, intravenous injection, intraperitoneal injection, intraarterial injection or parenteral administration.
  • the compounds of the invention serve as therapeutic agents themselves and/or include a therapeutic agent.
  • the compounds of the invention include a linker between a binding moiety and the therapeutic agent.
  • the linker may include a substituted alkyl chain, an unsubstituted alkyl chain, a polyethylene glycol derivative, an amino acid or peptide spacer, a sugar, an aliphatic spacer, an aromatic spacer, a lipid molecule, or combination thereof.
  • Preferred therapeutic agents for use with compounds of the invention include a bioactive agent, a cytotoxic agent, a drug, a chemotherapeutic agent, or a radiotherapeutic agent.
  • the invention features a method of treating a disease by administering to a patient a pharmaceutical preparation including a compound of the invention.
  • a pharmaceutical preparation including a compound of the invention.
  • the compound may be administered to treat that disease state.
  • the binding moieties may inhibit the biological process by preventing or diminishing the activity of the receptor (e.g., by competition with the natural ligand for the receptor, by directly inhibiting the receptor activity whether or not the natural ligand is bound, or by a combination of the two).
  • a heteromultimeric compound of the invention may inhibit the activity of, for instance KDR or cMet, and thus inhibit angiogenesis and/or hyperproliferation and consequently the diseases these processes contribute to. Therefore, the invention features a method of treating a disease by administering to a patient a pharmaceutical preparation including a compound of the invention alone or attached or linked to a separate therapeutic agent. In preferred embodiments, the invention features a method of treating a disease associated with angiogenesis or hyperproliferation. In a most preferred embodiment, the disease is neoplastic tumor growth. [00395] The invention also features a method of screening for heteromultimeric compounds having improved binding affinity.
  • This method includes the steps of preparing a labeled heteromultimeric compound comprising a plurality of binding moieties, wherein at least two binding moieties bind to different binding sites of a target; contacting the labeled heteromultimeric compound with a target; determining a binding strength of the labeled heteromultimeric compound (for example, by determining the dissociation constant); and comparing the binding strength (e.g., dissociation constant) of the labeled heteromultimeric compound with the binding strength (e.g., dissociation constant) of one or more individual binding moieties.
  • one of the binding moieties includes a polypeptide.
  • the target is KX)R or a KDR/VEGF complex.
  • one of the polypeptides used in this method is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.
  • the method includes the step of identifying a labeled heteromultimeric compound having a binding strength (for example, as measured by a decreased dissociation constant) that is greater than the binding strength of a constituent binding moiety.
  • the invention features dimeric or multimeric targeting constructs which include two or more KDR or VEGF/KDR complex-binding polypeptides which bind to different binding sites of KDR or the VEGF/KDR complex.
  • KDR or VEGF/KDR complex-binding polypeptides which bind to different binding sites of KDR or the VEGF/KDR complex.
  • Such polypeptides are described in detail in U.S.S.N. 60/360,851 and U.S.S.N. 60/440,441, both of which are incorporated by reference herein in their entirety, and in copending application U.S.S.N. 10/661,156, entitled "KDR and VEGF/KDR-binding peptides and their use in diagnosis and therapy," in the name of Aaron Sato et al., filed on September 11, 2003 and incorporated by reference herein in its entirety.
  • KDR-targeting constructs exhibit improved binding to KDR (e.g., increased specificity and/or affinity and/or avidity) compared to monomeric KDR- or VEGF/KDR complex-binding polypeptides, and compared to dimeric or multimeric constructs of a single KDR-binding polypeptide.
  • KDR-targeting constructs exhibit improved binding to KDR (e.g., increased specificity and/or affinity and/or avidity) compared to monomeric KDR- or VEGF/KDR complex-binding polypeptides, and compared to dimeric or multimeric constructs of a single KDR-binding polypeptide.
  • These preferred compounds may be linked or conjugated to a detectable moiety and used to target these compositions to KDR- expressing cells, permitting imaging of KDR-expressing tissue.
  • the invention features dimeric or multimeric targeting constructs which include two or more cMet or HGF/cMet complex-binding polypeptides which bind to different binding sites of cMet or the HGF/cMet complex.
  • cMet or HGF/cMet complex-binding polypeptides which bind to different binding sites of cMet or the HGF/cMet complex.
  • cMet-targeting constructs exhibit improved binding to cMet (e.g., increased specificity and/or affinity and/or avidity) compared to monomeric cMet or HGF/cMet complex -binding polypeptides, and compared to dimeric or multimeric constructs of a single cMet-binding polypeptide.
  • the cMet and KDR-targeting constructs of the invention may be linked or conjugated to a therapeutic agent and used to localize the therapeutic agent to cMet or KDR expressing tissue.
  • the cMet or KDR-targeting constructs of the invention may also be used as therapeutics themselves, as described herein.
  • the KDR-targeting constructs of the invention include two or more of the following KDR and VEGF/KDR complex-binding polypeptides: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, or SEQ ID NO:12.
  • the cMet-targeting constructs of the invention include two or more of the following binding polypeptides: SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and/or SEQ ID NO:29.
  • the invention provides a novel method for screening the KDR-targeting constructs for the ability to bind the target, and thus, identify multimeric constructs of KDR-binding polypeptides with improved binding (as determined, for example, by dissociation constants), as compared to binding of the constituent polypeptides. Additionally, the method of the invention allows for rapid determination of whether the multimeric targeting constructs will be stable in the presence of serum in vivo.
  • 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.
  • 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 a shown by a decrease in K D .
  • constructs comprising two or more binding polypeptides specific for different epitopes of KDR and/or KDR/VEGF (e.g., "heteromeric" constructs) were made.
  • Constructs comprising two or more binding polypeptide provided herein are expected to block multiple sites on KDR or VEGF/KDR.
  • the heteromeric constructs show superior binding ability over both the corresponding monomers, as well as tetrameric constructs comprising multiple copies of the same binding polypetide.
  • heteromeric constructs comprising two or more binding peptides specific for different epitopes were also able to efficiently bind KDR-transfected 293H cells.
  • 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.
  • 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 are expected to inhibit the function of receptor tyrosine kinases.
  • such constructs are expected to inhibit the function of VEGFR-2/KDR, VEGFR-l/Flt-1 and VEGFR-3/Flt-4.
  • heterornultimeric constructs of the invention comprising two or more binding moieties specific for different epitopes of cMet and/or cMet/HGF are expected to inhibit the function of receptor tyrosine kinases and, in particular the function of cMet.
  • receptor tyrosine kinase function can include any one of: oligomerization of the receptor, receptor phosphorylation, kinase activity of the receptor, recruitment of downstream signaling molecules, induction of genes induction of cell proliferation, induction of cell migration, or combination thereof.
  • heteromeric constructs of binding polypeptides provided herein inhibit VEGF- induced KDR receptor inactivation in human endothelial cells, as demonstrated by the inhibition of VEGF-induced phosphorylation of the KDR receptor.
  • heteromeric constructs of binding peptides provided herein inhibit VEGF-stimulated endothelial cell migration.
  • 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.
  • heteromultimers of this invention can also be useful for treating vascular permeability events that can result when VEGF binds KDR. See, e.g. , Example 30.
  • vascular permeability events that can result when VEGF binds KDR.
  • Example 30 In renal failure it has been shown that anti-VEGF antibodies can reverse damage, and in a similar way the compounds of the invention can reverse renal permeability pathogenesis in, for example, diabetes.
  • constructs comprising two or more binding polypeptides specific for different epitopes of cMet were made.
  • Constructs containing two or more cMet-binding polypeptides provided herein are expected to block multiple sites on cMet. These heteromeric cMet-targeting constructs show superior binding ability over the corresponding monomers.
  • the present invention is drawn to constructs comprising two or more binding polypeptides.
  • 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 a target, for example, KDR and/or KDR/VEGF and cMet and/or cMet/HGF.
  • These constructs are also referred to herein as "heteromeric constructs,” “heterodimeric constructs,” “heterodimers,” “heteromultimers” and/or “heteromultimeric constructs.”
  • the constructs of the present invention can also include an unrelated, or control peptide or peptides.
  • 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. Furthermore, the methods and teachings provided herein have been shown to allow for the improved binding to a variety of different targets (e.g., KDR and cMet), thus demonstrating the wide applicability of the present invention.
  • targets e.g., KDR and cMet
  • Peptides with selected lysine residues were protected, with the l-(4,4-dimethyl-2,6-dioxocyclohex-l- ylidene)-3-methybutyl (ivDde) protecting group, which allows selective coupling to the C- terminal lysine, and this group was not removed during peptide cleavage, but was removed after coupling with 2% hydrazine in DMF or 0.5 M hydroxylamine, at pH 8, in water.
  • ivDde l-(4,4-dimethyl-2,6-dioxocyclohex-l- ylidene)-3-methybutyl
  • Each peptide was labeled with a fluorescein-containing moiety on the C- terminal lysine using 5-carboxy- or 6-carboxy- or a mixture of 5-carboxy and 6-carboxy- fluorescein (as the N-hydroxysuccinimide ester derivative) or fluorescein isothiocyanate (FITC) in a 2% solution of DIEA in DMF. If the peptide contained an ivDde protected lysine, the reaction was quenched by the addition of 2% hydrazine, a compound that reacts with any remaining NHS -fluorescein and removes the internal protecting group.
  • 5-carboxy- or 6-carboxy- or a mixture of 5-carboxy and 6-carboxy- fluorescein as the N-hydroxysuccinimide ester derivative
  • FITC fluorescein isothiocyanate
  • the reaction was quenched by the addition of an equal volume of 0.5 M hydroxylamine, pH 8.
  • the quenched reaction mixtures were then diluted with water until the composition contained less than 10% DMF, and then they were purified using Cl 8 reverse phase chromatography, usually semipreparative or preparative C18-reverse phase HPLC.
  • the peptides were characterized for purity and correct mass on an LC-MS system (HPl 100 HPLC with an in-line SCIEX API 50 single quadrapole mass spectrometer).
  • Fluorescence anisotropy measurements were performed in 384-well microplates in a volume of 10 ⁇ L in binding buffer (PBS, 0.01% Tween-20, pH 7.5) using a Tecan Polarion fluorescence polarization plate reader. In some cases, heparin (0.5 ⁇ g/mL) or 10% human serum was added to the binding buffer. 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 Equation (1) below via nonlinear regression to obtain the apparent KQ. Equation (1) assumes that the synthetic peptide and HSA form a reversible complex in solution with 1 : 1 stoichiometry:
  • a typical amino acid coupling cycle (not including wash steps) was to dispense piperidine solution (2.4 mL) to each well and mix for 4 min, then empty all wells. NMP (320 ⁇ L), HOBt solution (320 ⁇ L, 4eq), amino acid (640 ⁇ L, 4eq) and DIC (320 ⁇ L, 4eq) 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.
  • the resin bound peptide was capped with 1.0 M 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 first with methanol and then with dichloromethane and dried. Cleavage of the peptides from the resin and side-chain deprotection was accomplished using Reagent B for 4.5h.
  • the cleavage solutions were collected and the resins were washing with an additional aliquot of Reagant 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.
  • 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 7.5-8.5 with aqueous ammonia, aqueous ammonium carbonate or aqueous ammonium bicarbonate. The mixture was stirred vigorously in air for 24-48 h. In the case of non-DMSO containing solvent systems, the pH of the solution was adjusted to 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 Cl 8, or C8 reverse phase semipreparative or preparative HPLC column.
  • 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.
  • 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 peptides synthesis on 0.01 mmol scale synthesis.
  • 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 or in aqueous DMSO, and treated with DIEA and stirred for 4-6 h to effect disulfide cyclization. The crude mixture was purified by preparative HPLC.
  • 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 4-24 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 reverse phase C- 18 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 provide the required biotinylated peptide.
  • ABI-433 A Synthesizer and bearing an ivDde-protected lysine was treated with 10% hydrazine in DMF (2 x 20 niL). The resin was washed with DMF (2 x 20 mL) and DCM (1 x 20 mL). The resin was resuspended in DMF (10 mL) and treated with Fmoc- aminodioxaoctanoic acid (0.4 mmol), HOBt (0.4 mmol), DIC (0.4 mmol), DIEA (0.8 mmol) with mixing for 4 h. After the reaction, the resin was washed with DMF (2 x 10 ml) and with DCM (Ix 10 mL).
  • the resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc-diaminodioxaoctanoic acid and removal of the Fmoc protecting group were repeated once more.
  • the resulting resin containing a peptide with a free amino group, was treated with a solution of Biotin- NHS ester (0.4 mmol, 5 equivalent) and DIEA (0.4 mmol, 5 equivalents) in DMF for 2 h.
  • 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.
  • Method 9 for Formation of 5-Carboxyfluorescein Labeled Peptides Peptide-resin obtained via from Method 5, containing an ivDde protecting group on the epsilon nitrogen of lysine, was mixed with a solution of hydrazine in DMF (10% hydrazine/DMF, 2 x 10 mL, 10 min) to remove the ivDde group.
  • the epsilon nitrogen of the lysine was labeled with fluorescein-5-isothiocyanate (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 x 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 (lmg/mL) and the pH of the mixture was adjusted to 8 with 10% aqueous meglumine.
  • N-Hydroxysuccinimide (1.5 equiv, 17.2 mg) and 1,3- diisopropylcarbodiimide (1.5 equiv, 24 ⁇ L) 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 (5x) to remove the unreacted NHS. The residue was dried to provide Compound B, which was used directly without further treatment or purification.
  • Method 13 for 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-hydoxysuccinimidyl ester. The progress of the reaction was monitored by HPLC analysis and mass spectroscopy. At completion of the reaction, the volatiles were removed in vacuo and the residue was washed with ethyl acetate to remove the unreacted bis-NHS ester.
  • KDR and VEGF/KDR Complex-binding Polypeptides [00439] Utilizing the methods described above, the KDR and VEGF/KDR complex- binding polypeptides in Table Ia were prepared. As used in Table Ia, the letter “J" in the peptide sequences refers to the spacer or linker group, 8-amino-3,6-dioxaoctanoyl. Also as used in Table 1, the designation "C*" refers to a cysteine residue that contributes to a disulfide bond. The ability of the biotinylated polypeptides to bind to KDR was assessed using the assay set described below.
  • P13-XB K D 1.81 nM +/- 0.27
  • P5-XB K D 14.87 +/- 5.07 nM, four experiment average
  • P6-XB K D 10.00 +/- 2.36 nM, four experiment average
  • P12-XB K D 4.031 +/- 0.86 nM, three experiment average
  • P6-F-XB K D 6.94 +/- 1.94 nM, one experiment
  • P12-F-XB K D 3.02 +/- 0.75 nM, one experiment).
  • KDR KDR
  • human Trail R4 #633-TR-100
  • VEGF 165 was purchased in carrier-free form from Peprotech (Rocky Hill, NJ).
  • Protein A Magnetic Beads were purchased from Dynal (Oslo, Norway).
  • Heparin was purchased from Sigma Chemical Company (St. Louis, MO).
  • a 2- component tetramethyl benzidine (TMB) system was purchased from KPL (Gaithersburg, MD).
  • Ml 3 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, TNl 2/1, and MTN13/I, and a linear display library, Lin20. The design of these libraries has been described, supra.
  • the DNA encoding the library was synthesized with constant DNA on either side so that the DNA could be PCR amplified using Taq DNA polymerase (Perkin-Elmer, Wellesley, MA), cleaved with Ncol and Pstl, and ligated to similarly cleaved phage display vector.
  • Taq DNA polymerase Perkin-Elmer, Wellesley, MA
  • cleaved with Ncol and Pstl cleaved with Ncol and Pstl
  • ligated to similarly cleaved phage display vector XLl -Blue MFR' E. coli cells were transformed with the ligated DNA. All of the libraries were constructed in same manner.
  • 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 TNl 2/1 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 ⁇ L PBSTH were pooled together; e.g., 3 pooled libraries would result in a total volume of -350 ⁇ l in PBSTH.
  • To prepare the irrelevant Fc fusion beads 500 ⁇ l of Trail R4-Fc fusion
  • KDR-Fc fusion 0.1 ⁇ g/ ⁇ L stock in PBST (no heparin)
  • the KDR-Fc fusion was allowed to bind overnight with agitation at 4 °C.
  • the beads were washed 5 times with PBSTH.
  • Each depleted library pool was added to 100 ⁇ L of KDR-Fc beads and allowed to incubate on a LabQuake shaker for 1 hour at RT. Beads were then washed as rapidly as possible with 5 x 1 mL PBSTH using a magnetic stand (Promega) to separate the beads from the wash buffer.
  • Phage still bound to beads after the washing were eluted once with 250 ⁇ l of VEGF (50 ⁇ g/mL, ⁇ 1 ⁇ M) in PBSTH for 1 hour at RT on a LabQuake shaker. The 1-hour elution was removed and saved. After the first elution, the beads were incubated again with 250 ⁇ l of VEGF (50 ⁇ g/mL, ⁇ 1 ⁇ M) overnight at RT on a LabQuake shaker. The two VEGF elutions were kept separate and a small aliquot taken from each for titering. Each elution was mixed with an aliquot of XLl -Blue MRF' (or other F' cell line) E.
  • coli cells 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).
  • the phage/cell mixtures were spread onto Bio-Assay Dishes (243 x 243 x 18 mm, Nalge Nunc) containing 250 mL of NZCYM agar with 50 ⁇ g/mL of ampicillin. The plate was incubated overnight at 37 °C.
  • each pool yielded three amplified eluates. These eluates were panned for 2-3 more additional rounds of selection using ⁇ 10 10 input phage/round according to the same protocol as described above. For each additional round, the KDR-Fc beads were prepared the night before the round was initiated.
  • the amplified elution re-screen on KDR-Fc beads was always eluted in the same manner, and all other elutions were treated as washes.
  • the amplified elution recovered by using the still-bound beads to infect E. coli the 1-hour and overnight VEGF elutions were performed and then discarded as washes. Then the beads were used to again infect E. coli and produce the next round amplified elution.
  • each library pool only yielded three final elutions at the end of the selection. Two pools and one linear library, therefore, yielded a total of 9 final elutions at the end of the selection.
  • TNl 1/1 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).
  • TNl 1/1 alone was used in the selection and screening. For selected peptides, see [FIGS. 40A-40R.]
  • KDR-binding peptides P5-B and P5-XB and P6-B and P6-XB were conjugated to fluorescent beads and their ability to bind to KDR-expressing 293H cells was assessed.
  • the experiments show that both peptide sequences can be used to bind particles such as beads to KDR-expressing sites.
  • the P6 peptides exhibited better binding to the KDR-expressing cells than P5.
  • the binding of both peptides improved with the addition of a spacer.
  • Preparation of peptide-conjugated fluorescent beads [00454] 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.
  • 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.
  • 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 (with no DNA) and those 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; otherwise the assay was aborted.
  • 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.
  • Biotinylated P5 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 enhanced binding to KDR cells without increasing the binding to mock-transfected cells.
  • Biotinylated P6 beads showed greater binding to KDR-transfected cells.
  • adding a hydrophilic spacer between the peptide portion and the biotin of the molecule significantly improved the specific binding to KDR in the transfected cells.
  • the peptide sequences of both P5 and P6 can be used to bind particles such as beads to KDR-expressing sites.
  • KDR-binding peptides assessed for the ability of KDR-binding peptides to compete with 125 I-labeled VEGF for binding to KDR expressed by transfected 293H cells. While KDR-binding polypeptide P4 did not compete significantly with 125 I-labeled VEGF, P5-XB, P6 and P 12-XB competed very well with 125 I-labeled VEGF, inhibiting 96.29 ⁇ 2.97% and 104.48 ⁇ 2.07% of 125 I-labeled VEGF binding.
  • 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 (with no DNA) and those 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; otherwise the assay was aborted. 2. Preparation of M 199 media
  • M199 medium packet (GIBCO, cat. # 31100-035), 20 rnL of 1 niM HEPES (GIBCO, cat. #15630-080), and 2 g of DIFCO Gelatin (DIFCO, cat. # 0143-15-1) were added to 950 mL of double distilled (dd) H 2 O and the pH of the solution was adjusted to 7.4 by adding approximately 4 mL of IN NaOH.
  • the Ml 99 medium was warmed to 37 °C in a water bath for 2 h to dissolve the gelatin, then filter sterilized using 0.2 ⁇ m filters (Corning, cat. # 43109), and stored at 4 °C to be used later in the assay.
  • solubilizing solution 2% Triton X-100, 10% Glycerol, 0.1% BSA
  • solubilizing solution 100 ⁇ L 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 ⁇ L 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 x 10 cm tube to be counted in an LKB Gamma Counter ( 125 I window for 1 minute).
  • the binding of 125 I- labeled VEGF to sites other than KDR is not included when calculating the inhibition of 125 I-labeled VEGF binding to 293H cells by KDR-binding peptides.
  • FIG. 2 shows the percentage inhibition of 125 I-labeled VEGF binding by peptides (P6, P4, P5-XB, and P12-XB) at two different concentrations (30 ⁇ M and 0.3 ⁇ M) to KDR-transfected 293H cells. Percentage inhibition was calculated using formula [(Yl- Y2)xl00/Yl], where Yl is specific binding to KDR-transfected 293H cells in the absence of peptides, and Y2 is specific binding to KDR-transfected 293H cells in the presence of peptides or DMSO (vehicle).
  • the percentage inhibition with P5-X- B was 47.95+5.09% of 125 I-labeled VEGF binding at 30 ⁇ M and 24.41+8.43% at 0.3 ⁇ M (FIG. 2).
  • This assay should also 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.
  • 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, including P5-D, P6-D, P10-D and Pl 1-D. As discussed above, peptides that inhibit activation of KDR may be useful as anti-angiogenic agents.
  • EGM-MV medium Biowhittaker Cat No. CC-3125
  • Cells seeded into 100 mm dishes were allowed to become confluent, then cultured overnight in basal EBM medium lacking serum (Biowhittaker Cat No. CC-3121).
  • the next morning, the medium in the dishes was replaced with 10 mL fresh EBM medium at 37 °C containing either no additive (negative control), 5 ng/mL VEGF (Calbiochem Cat No. 676472 or Peprotech Cat No.
  • VEGF vascular endothelial growth factor
  • a neutralizing anti-KDR antibody Cat No. AF357, R&D Systems
  • 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.
  • D-PBS Dulbecco's phosphate buffered saline
  • the first dish of a set was drained and 0.5 mL of Triton lysis buffer was added (20 mM Tris base pH 8.0, 137 niM 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 ⁇ g/mL leupeptin, 10 ⁇ g/mL aprotinin).
  • the cells were quickly scraped into the lysis buffer using a cell scraper (Falcon, Cat No.
  • the lysates either freshly prepared or frozen and thawed, were precleared by adding 20 ⁇ L of protein A-sepharose beads (Sigma 3391, preswollen in D-PBS), washed three times with a large excess of D-PBS, reconstituted with 6 mL D-PBS to generate a 50% slurry) and rocked at 4 °C for 30 min.
  • the beads were pelleted by centrifugation for 2 min in a Picofuge (Stratgene, Cat No. 400550) at 2000xg and the supernatants transferred to new 1.5 mL tubes.
  • 20 ⁇ g of anti-Flk-1 antibody (Santa Cruz Biotechnology, Cat No.
  • sc-504 was added to each tube, and the tubes were incubated overnight (16-18 h) at 4°C on a rotator to immunoprecipitate KDR. The next day 40 ⁇ L of protein A-sepharose beads were added to the tubes, which were then incubated at 4°C for 1 h 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).
  • 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 h at room temp with 5% Blotto-TBS with 0.1% Tween 20 (Blotto-TBST), and reprobed with an anti-Flk-1 polyclonal antibody (Cat No. sc-315 from Santa Cruz Biotech), 1:200 in 5% Blotto-TBST with 1% normal goat serum (Life Tech Cat No. 16210064) for 2 h at room temp. The unbound antibody was removed by washing the blots four times with D-PBST, 5 min per wash.
  • the blots were probed with an HRP- conjugated donkey anti-rabbit secondary antibody (Amersham Biosciences Cat No. NA934) diluted 1 : 10,000 in 5% Blotto-TBST for 1 h at room temperature, 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.
  • the ability of the 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 (Cat No. AF-357 from R&D Systems) was combined with the VEGF, the extent of KDR phosphorylation was greatly reduced (FIG. 4, upper panel), indicating that the antibody was able to interfere with the ability of VEGF to bind to and activate KDR.
  • a neutralizing anti-KDR antibody Cat No. AF-357 from R&D Systems
  • P2 and P6 were the most potent compounds in the assay, producing at least a
  • XB (prepared as described above and discussed in Example 6, infra) produced at least a 50% inhibition of VEGF-induced KDR phosphorylation at 10 nM.
  • Tc-labeled P 12-C the ability of Tc-labeled P 12-C to bind KDR was assessed using KDR-transfected 293H cells.
  • the results show that Tc-labeled Pl 2-C bound significantly better to KDR transfected 293H cells than to mock transfected 293H cells, and binding increased with concentration of the Tc-labeled polypeptide in a linear manner.
  • the resin was filtered and washed with DMF (3 x 80 mL), CH 2 Cl 2 (3 x 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.
  • a stannous gluconate solution was prepared by adding 2 mL of a 20 ⁇ g/mL
  • the resulting radiochemical purity (RCP) was 10-47% when analyzed using a Vydac Cl 8 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 Cl 8 column (4.6 mm x 250 mm) at a flow rate of 1 mL/min, using 0.1% TFA in water as aqueous phase and 0.085% TFA in acetonitrile as the organic phase.
  • the following gradient was used; 29.5% org. for 35 min., ramp to 85% org. over 5 min, hold for 10 min.
  • the fraction containing 99m Tc-P12-C (which no longer contained the ACM protecting group) was collected into 500 ⁇ L of a stabilizing buffer containing 5 mg/niL ascorbic acid and 16 mg/mL hydroxypropyl- ⁇ -cyclodextrin in 50 niM phosphate buffer.
  • the mixture was concentrated using a centrifugal evaporation apparatus to remove acetonitrile, and 200 ⁇ L of 0.1% HSA in 50 mM pH 5 citrate buffer was added.
  • the resulting product had an RCP of 100%.
  • the compound Prior to injection into animals, the compound was diluted to the desired radioconcentration with normal saline.
  • 293H cells were transfected using the protocol described in avidin HRP example. Transfection was done in black/clear 96-well plates (Becton Dickinson, cat. # 354640). The cells in one half of the plates (48 wells) were mock-transfected (with no DNA) and the cells in the other 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.
  • Opti-MEMI was obtained from Invitrogen (cat. # 11058-021 ) and human serum albumin (HSA) was obtained from Sigma (cat. # A-3782).
  • HSA human serum albumin
  • Cells were used 24 h after transfection, and to prepare the cells for the assay, they were washed IX with 100 ⁇ L 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 ⁇ L of 1.17, 2.34, 4.68 and 11.7 ⁇ Ci/mL of 99m Tc-labeled P12-C (prepared as above). Each dilution was added to three separate wells of mock and KDR transfected cells.
  • Experiment C demonstrates the use of this assay to assess the effect of serum on binding of peptides of the invention to KDR and VEGF/KDR complex.
  • the binding of P5-XB, P6-XB and P13-XB was not significantly affected by the presence of serum, while the binding of P 12-XB was reduced more than 50% in the presence of serum.
  • Experiment D demonstrates that this assay is useful in evaluating distinct combinations of KDR and VEGF/KDR complex-binding polypeptides for use in multimeric targeting constructs which contain more than one KDR and VEGF/KDR complex-binding polypeptide.
  • Experiments D and E establish that heteromeric constructs, which have two or more KDR-binding peptides that bind to different binding sites, exhibited superior binding to "homotetrameric" constructs of the targeting peptides alone.
  • HUVEC cells were grown to almost 80% confluence in 175 cm tissue culture flasks (Becton Dickinson, Biocoat, cat # 6478), and then 10 ng/mL of bFGF (Oncogene, cat # PF003) was added for 24 h to induce expression of KDR.
  • mRNA was isolated using the micro-fast track 2.0 kit from Invitrogen (cat. # Kl 520-02). 12 ⁇ g of mRNA (measured by absorbance at 260 nM) was obtained from two flasks (about 30 million cells) following the kit instructions. Reverse transcription to generate cDNA was performed with 2 ⁇ g of mRNA, oligo dT primer (5'-(T) 25 GC-3') and/or smart II oligo
  • MMLV Moloney Murine Leukemia Virus reverse transcriptase
  • the reaction was performed in a total volume of 20 ⁇ L and the reaction mix contained 2 ⁇ L of RNA, 1 ⁇ L smart II oligo, 1 ⁇ L of oligo dT primer, 4 ⁇ L of 5x first-strand buffer (250 mM Tris HCl pH 8.3, 375 mM KCl, 30 mM MgCl 2 ) 1 ⁇ L DTT (20 mM, also supplied with reverse transcriptase), 1 ⁇ L dNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP in ddH 2 O, Stratagene, cat.
  • 5x first-strand buffer 250 mM Tris HCl pH 8.3, 375 mM KCl, 30 mM MgCl 2
  • DTT 20 mM, also supplied with reverse transcriptase
  • 1 dNTP mix 10 mM each of dATP
  • the PCR reaction was done in total volume of 50 ⁇ L and the reaction mix contained 2 ⁇ L 5' RACE ready cDNA library, 1 ⁇ L 5' oligo (10 ⁇ M), 1 ⁇ L 3' oligo (10 ⁇ M), 5 ⁇ L 1OX PCR buffer [PCR buffer (200 mM Tris-HCl pH 8.8, 20 mM MgSO 4 , 100 mM KCl, 100 mM (NH 4 ) 2 SO 4 ) supplied with pfu enzyme plus 1% DMSO and 8% glycerol], 1 ⁇ L dNTP mix (10 mM) and 40 ⁇ L ddH 2 O.
  • PCR buffer 200 mM Tris-HCl pH 8.8, 20 mM MgSO 4 , 100 mM KCl, 100 mM (NH 4 ) 2 SO 4
  • the PCR reaction was performed by using a program set for 40 cycles of 1 minute at 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 ⁇ L of ddH 2 O, the 2 ⁇ L of 1OX Taq polymerase buffer (100 mM Tris-HCl pH 8.8, 500 mM KCl, 15 mM MgCl 2 , 0.01% gelatin) and 1 ⁇ L of Taq polymerase (Stratagene, cat.
  • the TOPO vector allows easy cloning of PCR products because of the A-overhang in Taq (PCR enzyme)-treated PCR products.
  • the extra-cellular domain and the cytoplasmic domain were amplified by PCR separately from TOPO-sKDR and TOPO-CYTO respectively and ligated later to create the full-length receptor.
  • An oligo with a Notl site at the 5' end of the extracellular domain (A TAA GAA TGC GGC CGC AGG ATG GAG AGC AAG GTG CTG CTG G) and an oligo complimentary to the 3' end of the extracellular domain (TTC CAA GTT CGT CTT TTC CTG GGC ACC) were used to amplify by PCR the extracellular domain from TOPO-sKDR.
  • the 5' oligo (ATC ATT ATT CTA GTA GGC ACG GCG GTG) and the 3 1 oligo, with a Notl site (A TAA GAA TGC GGC CGC AAC AGG AGG AGA GCT CAG TGT GGT C), were used to amplify by PCR the cytoplasmic domain of KDR (with transmembrane domain) from TOPO-CYTO. Both PCR products were digested with Notl and ligated together to create the full-length receptor. The cDNA encoding the full-length receptor was purified on an agarose gel and ligated into the Notl site of the pcDNA6/V5- HisC vector.
  • 293H cells were obtained from Invitrogen (cat. # 11631) and grown as monolayer cultures in their recommended media plus lmL/L pen/strep (Invitrogen, cat. # 15140-148). All of 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.
  • E. coli. bacteria DH5 ⁇ containing pf-KDR was streaked onto LB with 50 ⁇ g/mL ampicillin (LB agar from US biologicals, cat. # 75851 and ampicillin from Sigma, cat. #A2804) plates from a glycerol stock, and plates were left in a 37 °C incubator to grow overnight. The next morning, a single colony was picked from the plate and grown in 3 mL of LB/ampicillin media (LB from US biologicals, cat. # US75852) at 37 °C. After 8 hours, 100 ⁇ L of bacterial culture from the 3 mL tube was 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.
  • Ml 99 media was prepared as described above.
  • 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.
  • Biotinylated peptides P6-XB, P5-XB, P12-XB, P13-XB and the biotinylated control peptide, Pl-XB, (prepared as described above) were used to prepare 250 ⁇ M stock solutions in 50% DMSO and a 33 ⁇ M stock solution of Neutravidin HRP was prepared by dissolving 2 mg of Neutravidin HRP (Pierce, cat. # 31001) in 1 mL of ddH 2 O. Peptide stock solutions were stored at -20 °C, whereas the Neutravidin HRP stock solution was stored at - 80 °C.
  • the structures of the biotinylated peptides are shown in Table Ia.
  • peptide/neutravidin HRP complexes 10 ⁇ L of 250 ⁇ M biotinylated peptide stock solution and 10 ⁇ L of 33 ⁇ M Neutravidin HRP were added to 1 ml of M 199 medium. This mixture was incubated on a rotator at 4 °C for 60 minutes, followed by addition of 50 ⁇ L of soft release avidin-sepharose (50% slurry in ddH 2 0) to remove excess peptides and another incubation for 30 minutes on a rotator at 4 °C.
  • Blocking solution was prepared by adding 20 mL of Ml 99 medium to 10 mg of lyophilized unlabeled neutravidin (Pierce, cat. # 31000). Fresh blocking solution was used for each experiment.
  • each well of the 293H cells was washed IX with 100 ⁇ L of M199 medium and incubated with 80 ⁇ L of blocking solution at 37 °C. After one hour, cells were washed 2X with 100 ⁇ L of Ml 99 media and incubated with 70 ⁇ L of peptide/neutravidin HRP dilutions of Pl-XB, P6-XB, P5-XB, P 12-XB and P 13-XB for two and half hours at room temperature. Each dilution was added to three separate wells of mock as well as KDR-transfected 293H cells (two plates were used for each saturation binding experiment).
  • Binding of peptide/ neutravidin HRP to KDR-transfected cells [00509] In this assay, complexes of P6-XB, P5-XB, P12-XB, P13-XB peptides, and the control peptide, Pl-XB, 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 ensure that all four biotin binding sites on neutravidin were occupied.
  • binding constants are, as expected, lower than those measured by FP against the KDRFc construct for the related monodentate peptides P6 (69 nM) and P5 (280 nM) (fluoresceinated) but similar for the monodentate peptide P12 (3 nM). As expected, no saturation of binding for the control Pl-X-B peptide/neutravidin HRP-complex was observed. As shown in FIG.
  • P 13-XB was not significantly affected by 40% rat serum, whereas binding of P 12-XB dropped more than 50% in presence of 40% rat serum. More than an 80% drop in the binding of Tc-labeled P12-C (P 12 with Tc-chelate), prepared by the method described in Example 5 above, was observed in the presence of 40% rat serum (data shown in FIG. 25). Because the serum effect on the binding of Tc-labeled P12-C is mimicked in the avidin HRP assay disclosed herein, this assay may be used to rapidly evaluate the serum effect on the binding of peptide(s) to KDR.
  • Experiment D was designed to evaluate the binding of tetrameric complexes of KDR and VEGF/KDR complex-binding polypeptides P6-XB and P5-XB, particularly where the constructs included at least two KDR-binding polypeptides.
  • the KDR-binding peptides and control binding peptide (Pl-XB) 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.
  • peptide/neutravidin HRP complexes To prepare peptide/neutravidin HRP complexes, a total 5.36 ⁇ L of 250 ⁇ M biotinylated peptide stock solution (or a mixture of peptide solutions, to give peptide molecules four times the number of avidin HRP molecules) and 10 ⁇ L of 33 ⁇ M Neutravidin HRP were added to 1 mL of Ml 99 medium. This mixture was incubated on a rotator at 4 °C for 60 minutes, followed by addition of 50 ⁇ L of soft release avidin-sepharose (50% slurry in ddH 2 0) to remove excess peptides and another incubation for 30 minutes on a rotator at 4 °C.
  • soft release avidin-sepharose 50% slurry in ddH 2 0
  • Pl -XB is a biotinylated derivative of P 1 , a control peptide that does not bind to KDR.
  • Pl-XB a biotinylated derivative of P 1 , a control peptide that does not bind to KDR.
  • a tetrameric complex of Pl-XB with avidin-HRP did not show enhanced binding to KDR-transfected cells.
  • tetrameric complexes of P6-XB or P5-XB bound to KDR-transfected cells significantly better than to mock- transfected cells.
  • P6-XB tetramers however, bound much better than P5-X tetramers.
  • Pl-XB was added to the peptide mixture used to form the tetrameric complexes, the binding to the KDR-transfected cells was decreased.
  • the ratios of specific binding of tetramer to monomer, dimer and trimer were calculated by dividing the specific binding (obtained by subtracting the binding to mock transfected cells from KDR transfected cells) of tetramer, trimer & dimer with that of monomer. Results suggest that there is co-operative effect of multimerization of P5-XB, P6-XB and P13-XB on the binding to KDR transfected cells.
  • XB bound quite well to KDR-transfected cells relative to mock-transfected cells, indicating that there is a great advantage to targeting two binding sites on the same target molecule. Furthermore, it was noted that tetrameric complexes containing different ratios of P6-XB and P5-XB (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.
  • the ratios of specific binding of heterotetramer, heterotrimer and heterodimer to monomer were 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. Monomer, which was used to calculate the ratios, for each set of heteromers is recorded at the end of each heteromer listing in the table and given the ratio of 1.
  • Experiment E was designed to confirm that P6-XB and P5-XB bind to distinct binding sites on KLDR. If the peptides bind to the same site on KDR, they would likely compete with each other for binding to KDR, whereas if the peptides bind to different sites, there should be no competition between the peptides for binding to KDR.
  • This experiment was performed using a single concentration of P5-XB/avidin HRP (3.33 nM) solution in each well and adding a varying concentration (0-2.5 ⁇ M) of Pl-XB, P5-XB and P6-XB, none of which were complexed with avidin.
  • each individual peptide monomer was modified at either the C-terminal lysine (to make the tail-to-tail dimer) or N-terminal amino (to make the head-to-head dimer) with a monodispersed PEG-based amino acid linker (Fmoc-NH-PEG 4 -CO 2 H).
  • tail-to-tail and head-to-head heterodimers were formed in solution and purified to homogeneity by standard reverse phase protocols.
  • a more detailed description of this linkage chemistry is found in K. Rose, et al. JACS, 1999, 121 : 7034-7038, which is hereby incorporated by reference in its entirety.
  • each heterodimer was assayed for binding using a surface plasmon resonance instrument (Biacore 3000). Soluble KDR receptor was cross-linked to the dextran surface of a CM5 sensor chip by the standard amine coupling procedure. A 0.5mg/mL solution was diluted 1 :40 with 5OmM acetate, pH 6.0 to immobilize a total R L of 12721. Experiments were performed in PBST buffer (5.5mM phosphate, pH 7.65, 0.15M NaCl, 0.1% Tween-20 (v/v)).
  • Peptide solutions quantified by extinction coefficient were diluted to produce 1000, 500, 250, 125, 62.5 and 31.3nM solutions.
  • peptides were injected at 20 ⁇ L/min for 2 minutes using the kinject program. Following a 3 minute dissociation period, any remaining peptide was stripped from the KDR surface with a quickinject of 5OmM NaOH, IM NaCl for 15s at 75 ⁇ L/min. Monomeric P9 and P10 were run as standards. Sensorgrams were analyzed by global analysis using BIAevaluation software 3.1.
  • the resin was washed with DMF (2 x 10 mL) and with DCM (I x 10 mL). The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc-8-amino-3,6-dioxaoctanoic acid and the Fmoc protecting group removal were repeated once more.
  • the resin was washed with DMF (2 x 10 mL) and with DCM (1 x 10 mL). The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc-8-amino-3,6-dioxaoctanoic acid and removal of the Fmoc protecting group were repeated once more.
  • the mixture was stirred for 4-6 h to effect the disulfide cyclization which was monitored by analytical HPLC.
  • the reaction mixture was diluted with 25% acetonitrile in water and applied directly to a reverse phase C-18 column. Purification was effected using a gradient of acetonitrile into water (both containing 0.1% TFA). Fractions were analyzed by HPLC and those containing the pure product were collected and lyophilized to provide the required biotinylated peptide.
  • the resin was washed with DMF (2 x 10 mL) and with DCM (1 x 10 mL). The resin was then treated with 20% piperidine in DMF (2 x 15 mL) for 10 min each time. The resin was washed and the coupling with Fmoc-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-l,4,7,10-tetraacetic acid,-l,4,7-tris-t-butyl ester (DOTA-tris-t-butyl ester, 0.4 mmol, 5 equiv.), HOBt (0.4 mmol), DIC (0.4 mmol) and DIEA (0.8 mmol) in DMF (10 mL) with mixing for 4 h.
  • DMF 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid,-l,4,7-tris-t-butyl ester
  • HOBt 0.4 mmol
  • DIC 0.4 mmol
  • DIEA 0.8 mmol
  • the resin was washed with DMF (2 x 10 mL) and with DCM (1 x 10 mL) and treated with Reagent B (20 mL) for 4 h. The mixture was filtered and the filtrate concentrated to dryness. The residue was 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.
  • C* refers to a cysteine residue that contributes to a disulfide bond
  • the monomeric peptides described herein are prepared as cyclic disulfide peptides and then linked together to form dimers. Consequently, even if a cysteine residue lacks the "C*" designation, the presence of a disulfide bond to the nearest cysteine in the monomer can generally be assumed.
  • the monomer components of the dimers will also generally contain such disulfide bonds, regardless of whether the cysteine residues contain the "C*" designation or not.
  • dimers and other heteromultimers of the present invention could alternatively be prepared by performing the cyclization of di-cysteine peptides after the monomers are linked to form dimers, and the present invention is not intended to be limiting with respect to the presence or absence of such disulfide bonds.
  • a and B refer to the constructs of monomer peptides A and F already functionalized within the branching group B and a spacer C and E, as defined previously in this text.
  • 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.
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