WO2012112690A2

WO2012112690A2 - Targeting of therapeutic drugs and diagnostic agents employing collagen binding domains

Info

Publication number: WO2012112690A2
Application number: PCT/US2012/025261
Authority: WO
Inventors: Marcel E. Nimni; Bo Han; Peter BOASBERG
Original assignee: Fabius Biotechnology
Priority date: 2011-02-16
Filing date: 2012-02-15
Publication date: 2012-08-23
Also published as: WO2012112690A3

Abstract

A targeting composition comprising: (1) a therapeutic agent; (2) an intermediate release linker bound to the therapeutic agent; and (3) a targeting moiety bound to the intermediate release linker, the targeting moiety for binding the targeting composition to native collagen fibers, provides an improved means of targeting therapeutic agents to cells, particularly malignant cells. The therapeutic agents can be, but are not limited to, anti-neoplastic agents or anti-inflammatory agents. The peptide motif can be a motif derived from the sequence present in von Willebrand's factor. The invention also encompasses pharmaceutical compositions and methods of treatment, as well as diagnostic compositions.

Description

TARGETING OF THERAPEUTIC DRUGS AND DIAGNOSTIC AGENTS EMPLOYING

COLLAGEN BINDING DOMAINS by

Dr. Marcel E. Nimni, Dr. Bo Han, & Dr. Peter Boasberg

CROSS-REFERENCES

[0001] This application claims the benefit of United States Provisional Patent Application Serial No. 61/443,346 by Nimni et al., filed February 16, 201 1 and entitled "Targeting of Therapeutic Drugs and Diagnostic Agents Employing Collagen Binding Domains," which is incorporated herein in its entirety by this reference.

FIELD OF THE INVENTION

[0002] This invention is directed to compositions and methods for the targeted delivery of anticancer drugs, anti-inflammatory drugs, and other drugs to cancer cells and other cell types.

BACKGROUND OF THE INVENTION

[0003] Cancer still accounts for one in every four deaths in the USA every year and is exceeded only by heart disease in terms of the number of resultant deaths. From the drug-delivery perspective, this is a result, to some extent, of low drug bioavailability of cancer therapeutics in vivo within the cancer cells that constitute the tumors, combined with high toxicities in normal organs that limit the maximum administered doses of such anticancer drugs (T. Lammers et al., "Tumour-Targeted Nanomedicines: Principles and Practice," Br. J. Cancer 99: 392-397 (2008)). These high toxicities can produce a number of significant side effects, from minor but annoying side effects such as hair loss to major side effects such as cachexia, gastrointestinal problems, interference with normal maturation of blood cells, and disturbances of the immune system, leading to a seriously increased susceptibility to infection. Although potentially valuable information continues to accumulate on cancer cells, less progress has been made in cancer therapeutics. Five year survival rates for most metastatic cancers remain dismal. Cost, lack of efficacy, and systemic toxicity are often factors which cause patients to decline or discontinue chemotherapy.

[0004] Various approaches have been tried, including the conjugation of anticancer drugs to monoclonal antibodies, but these approaches have had significant deficiencies, including the need for specific conjugation reagents and methods, the risk that conjugating the anticancer drug to an appropriate monoclonal antibody may reduce the therapeutic efficacy of the drug or its bioavailability subsequent to delivery to the cancer cell, and the risk of immune reactions to the monoclonal antibody, especially if a non-fully human monoclonal antibody, such as a chimeric antibody or a humanized antibody, is used.

[0005] True targeting of medications to any significant extent has repeatedly failed because of inability to identify unique and specific markers in cancer cells that are not present in normal tissues. Essentially all the targeting systems used so far employ the so-called "passive targeting concept," an approach associated with increased extravasation of the medications into the interstitial fluid in the tumor area, relying primarily on the locally increased vascular permeability.

[0006] Accordingly, there exists a need for an improved route of delivery of anticancer medications that targets the cancer cells more specifically and improves bioavailability of anticancer medications, while at the same time reducing the toxicity typically associated with the administration of anticancer medications and not contributing any additional toxicity. The route of delivery of such anticancer medications should not interfere with the therapeutic efficacy of the medications.

[0007] Although the targeted delivery of cancer drugs to malignant cells is extremely important, there exist a large number of other serious and potentially fatal diseases and conditions for which potentially useful therapeutic drugs exist and for which targeting of the drugs to the cell type, tissue, or organ involved in the disease or condition would prove useful in increasing therapeutic efficacy while at the same time reducing toxicity or side effects caused by the administration of the drugs. Such diseases and conditions include, but are not limited to, inflammatory conditions, infections, allergies, diseases affecting the central nervous system such as depression, diseases affecting the gastrointestinal tract such as inflammatory bowel disease, and many other diseases and conditions.

[0008] In general, therefore, there is a need for an improved method of general applicability for drug delivery and targeting that can target the drug to a specific cell type, tissue, or organ in order to improve therapeutic efficacy and minimize toxicity and side effects.

SUMMARY OF THE INVENTION

[0009] It has become increasingly recognized that in order to decrease or eliminate the deleterious systemic effects of drugs used to treat cancer, as well as other diseases that are localized or that have localized effects on particular tissues, such compounds should be selective and/or able to be delivered selectively, in such a way the they are only lethal or inactivating to target cells. The present invention addresses that issue by providing a new route for delivery of a therapeutic drug involving the use of a peptide sequence and a linker to target the therapeutic drug to a specific tissue environment and thus to specific cells in which the therapeutic drug is pharmacologically active. In addition, such a targeting approach should be useful to recognize areas of active disease for diagnostic purpose.

[0010] Accordingly, one aspect of the present invention is a targeting

composition comprising:

( 1 ) a therapeutic agent;

(2) an intermediate release linker bound to the therapeutic agent; and

(3) a targeting moiety bound to the intermediate release linker, the targeting moiety for binding the targeting composition to native collagen fibers. [0011] The peptide motif can be selected from the group consisting of: Trp-Arg- Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser (WREPSFMALS) (SEQ ID NO: 1); Trp-Arg-Glu-Pro- Ser-Phe-Cys-Ala-Leu-Ser (WREPSFCALS) (SEQ ID NO: 2); and peptides related to SEQ ID NO: 1 or SEQ ID NO: 2 by one or more conservative amino acid substitutions.

[0012] Alternatively, the peptide motif can be a peptide related to SEQ ID NO: 1 or SEQ ID NO: 2 by one or more conservative amino acid substitutions, wherein the peptide is selected from the group consisting of: Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-Leu- Ser (WRDPSFMALS) (SEQ ID NO: 3); Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-Leu-Ser (WRDPSFCALS) (SEQ ID NO: 4); Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-lle-Ser

(WREPSFMAIS) (SEQ ID NO: 5); Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-lle-Ser

(WREPSFCAIS) (SEQ ID NO: 6); Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-lle-Ser

(WRDPSFMAIS) (SEQ ID NO: 7); and Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-lle-Ser (WRDPSFCAIS) (SEQ ID NO: 8).

[0013] In still another alternative, the peptide motif can be a peptide selected from the group consisting of: Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu- Ser-Gly-Pro-Pro-Gly (GPPGWREPSFMALSGPPG) (SEQ ID NO: 9); Gly-Pro-Pro-Gly- Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly

(GPPGWREPSFCALSGPPG) (SEQ ID NO: 10); Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser- Phe-Met-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFMALSGPPG) (SEQ ID NO: 11); Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly

(GPPGWRDPSFCALSGPPG) (SEQ ID NO: 12); Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser- Phe-Met-Ala-lle-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFMAISGPPG) (SEQ ID NO: 13); Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-lle-Ser-Gly-Pro-Pro-Gly

(GPPGWREPSFCAISGPPG) (SEQ ID NO: 14); Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser- Phe-Met-Ala-lle-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFMAISGPPG) (SEQ ID NO: 15); and Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-lle-Ser-Gly-Pro-Pro-Gly

(GPPGWRDPSFCAISGPPG) (SEQ ID NO. 16).

[0014] In still another alternative, the peptide motif is an elongated peptide structure of Formula (I): [Gly-Pro-Pro-Gly-Xi-Gly-Pro-Pro-Gly-X₂-Gly-Pro-Pro-Gly]n

(I) wherein: (1) and X₂ are one of peptide sequences SEQ ID NO: 1 through SEQ ID NO: 16; and (2) n is an integer from 1 to 15.

[0015] In yet another alternative, the targeting moiety can be a collagen binding site of a platelet collagen binding receptor, including, but not limited to, integrin α2β1 and glycoprotein VI.

[0016] In still another alternative, the targeting moiety can be a targeting moiety in which the peptide sequence WREPSFMALS (SEQ ID NO: 1) or WREPSFCALS (SEQ ID NO: 2) is incorporated into a molecule to generate a peptide of from about 2,000 daltons to about 10,000 daltons in molecular weight. In this alternative, the flanking sequences can mimic a sequence found in native collagen or native elastin; the targeting moiety can also include at least one reactive amino acid. The targeting moiety can include two or three collagen binding domains, with the collagen binding domains being separated by spacers. The spacers can provide laterally displaced equivalent sites with a lateral displacement of about 3 nm. The spacers can elongate in solution. The spacers can include alternating polar and nonpolar sequences; alternatively, the spacers can include polylysine or polyglycine residues.

[0017] The targeting moiety can be pegylated.

[0018] The targeting moiety can include a peptide sequence including an amino- terminal amino acid that is acetylated, or can include a peptide sequence including a carboxyi-terminal amino acid that is amidated. The targeting moiety can include a fluorescein moiety for labeling.

[0019] In yet another alternative, the targeting moiety includes the amino acid sequence GVMGFO (SEQ ID NO. 17).

[0020] In still another alternative, the targeting moiety includes a CBD from discoidin domain receptor DDR1 or DDR2, or includes a CBD incorporating the amino acids on the surface of the three-dimensional protein structure of DDR1 or DDR2 in which at least one of the amino acids not directly contacting collagen is replaced with a conservative amino acid substitution such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the native CBD from DDR1 or DDR2.

[0021] In still another alternative, the targeting moiety includes the amino acid sequence GTPGPGGIAGQRGW (SEQ ID NO: 19), or includes an amino acid sequence derived from GTPGPGGIAGQRGW (SEQ ID NO: 19) by one or more conservative amino acid substitutions such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the sequence GTPGPGGIAGQRGW (SEQ ID NO: 19).

[0022] In one alternative, the therapeutic agent is an anti-neoplastic therapeutic agent. Typically, the anti-neoplastic therapeutic agent is selected from the group consisting of mechlorethamine, cyclophosphamide, ifosfamide, melphalan,

chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, streptozocin, dacarbazine, temozolomide, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, 6- mercaptopurine, 6-thioguanine, pentostatin, vinblastine, vincristine, paclitaxel, docetaxel, topotecan, irinotecan, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitomycin C, L-asparaginase, interferon-alfa, interleukin-2, cisplatin, carboplatin, mitoxantrone, hydroxyurea, /V-methylhydrazine, mitotane, aminoglutethimide, imatinib, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, hydroxyprogesterone, medroxyprogesterone, megestrol acetate, diethy Isti Ibestrol , ethinyl estradiol, tamoxifen, anastrozole, testosterone propionate, fluoxymesterone, flutamine, leuprolide, trastuzumab, rituximab, alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ibritumomab, panitumumab, tositumomab, and an interferon.

[0023] In another alternative, the therapeutic agent is an anti-inflammatory therapeutic agent. The anti-inflammatory therapeutic agent can be, for example, a histamine receptor antagonist, a kinin receptor antagonist, a leukotriene receptor antagonist, a non-steroidal anti-inflammatory drug, or a steroid with anti-inflammatory activity.

[0024] In another alternative, the therapeutic agent can be selected from the group consisting of. (1) a muscarinic cholinergic receptor agonist;

(2) a muscarinic cholinergic receptor antagonist;

(3) an anticholinesterase agent;

(4) a ₂-selective adrenergic receptor agonist;

(5) a a₂-selective adrenergic receptor agonist;

(6) a sympathomimetic agonist;

(7) an on-selective adrenergic receptor antagonist;

(8) a β-selective adrenergic receptor antagonist;

(9) a 5-hydroxytryptamine receptor agonist;

(10 a 5-hydroxytryptamine receptor antagonist;

(11 a benzodiazepine;

(12 an antidepressant;

(13 an antipsychotic drug;

(14 an antiseizure drug;

(15 a drug that is an anti-parkinsonism drug or a drug effective against a neurodegenera ive disease;

(16: an opioid;

(1 a diuretic;

(18 an angiotensin converting enzyme inhibitor;

(19 a nonpeptide angiotensin II receptor antagonist;

(20 a calcium channel blocker;

(21 an antiarrhythmic drug;

(22 a drug having a therapeutic effect on hypercholesterolemia and/or dyslipidemia;

(23 an H₂ receptor antagonist;

(24 a drug having a therapeutic effect on disorders of bowel motility and/or water flux

(25 a drug having a therapeutic effect on inflammatory bowel disease; (26 an antimalarial;

(27 an anti protozoan agent; (28) an antihelminthic agent;

(29) an antibacterial agent;

(30) an antifungal agent;

(31) an antiviral agent;

(32) an antiretroviral agent;

(33) an immunomodulator;

(34) a growth factor;

(35) a hormone;

(36) a nucleic acid or nucleotide sequence for nonviral gene therapy

(37) an antiangiogenic compound;

(38) an antisense oligonucleotide; and

(39) an antibody that binds a pharmacologically significant molecule; and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof.

[0025] In another alternative, the composition can include two or more therapeutic agents; one possible combination is an anti-neoplastic therapeutic agent together with an anti-inflammatory therapeutic agent.

[0026] Typically, the intermediate release linker of the composition is a polymer that shields the therapeutic agent of the composition from clearance by macrophages. The polymer can be a protein or non-protein polymer. If the polymer is a protein polymer, it can be selected from the group consisting of albumin, gelatin, keyhole limpet hemocyanin, ferritin, and ovalbumin. Typically, the protein polymer is albumin or gelatin, such as bovine serum albumin. The protein polymer can also be a synthetic polypeptide. The protein polymer can be pegylated. Typically, the intermediate release linker does not interact with the therapeutic agent and does not bind to or otherwise interact with the targeting moiety. If the polymer is a non-protein polymer, it can be selected from the group consisting of polyethylene glycol and polypropylene glycol. Typically, the non-protein polymer is polyethylene glycol.

[0027] The linkages between the therapeutic agent and the intermediate release linker and between the intermediate release linker and the targeting moiety can be covalent linkages or non-covalent linkages. In one alternative, the linkages are peptide linkages formed by derivatization of the components involved with peptides and the formation of a peptide linkage between the peptides. If the linkages are non-covalent linkages, they can be, for example, biotin/avidin or biotin/streptavidin linkages or specific antigen/antibody or hapten/antibody linkages.

[0028] The targeting composition can bind to native collagen fibers that differ from other collagen fibers in an organism that is targeted by virtue of having their surface exposed as a consequence of the metabolic activity associated with metastasis and/or inflammation.

[0029] The intermediate release linker can be stabilized by crosslinking, such as by reaction with an aldehyde, or by a reaction catalyzed by a transglutaminase, in which case the intermediate release linker includes groups that are substrates for a

transglutaminase. In another alternative, the intermediate release linker can include a thiol-containing amino acid sequence derived from keratin or a biosynthesized thiol- containing amino acid sequence mimicking the properties of the thiol-containing amino acid sequence derived from keratin, or can include a hydrophobic amino acid sequence derived from elastin or a biosynthesized hydrophobic amino acid sequence mimicking the properties of the hydrophobic amino acid sequence derived from elastin.

[0030] In one alternative, the therapeutic agent of a targeting composition according to the present invention is for treating joint inflammation. The therapeutic agent can be a bisphosphonate or a bone morphogenetic protein or active portion thereof.

[0031] In another alternative, the therapeutic agent of a targeting composition according to the present invention is a growth factor. The growth factor can be selected from the group consisting of adrenomedullin (AM), autocrine mobility factor, bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatoma derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), novel neurotrophin-1 (NNT-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor a (TGF-a), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and placental growth factor (PGF). A preferred growth factor is GM-CSF.

[0032] In yet another alternative, the therapeutic agent is a polypeptide or protein and the targeting composition further comprises a growth factor bound either to the polypeptide or protein therapeutic agent or the intermediate release linker.

[0033] The targeting composition can further comprise a cell-penetrating peptide or a transcription-activating peptide.

[0034] Another aspect of the invention is a pharmaceutical composition comprising:

(1 ) a therapeutically effective quantity of a targeting composition according to the present invention; and

(2) a pharmaceutically acceptable carrier, diluent, or excipient in unit dosage form.

[0035] Yet another aspect of the invention is a method of treating a disease, disorder, or condition treatable by administration of a therapeutic agent comprising administration of a therapeutically effective quantity of a targeting composition according to the present invention or of a therapeutically effective quantity of a pharmaceutical composition according to the present invention to a subject in need of treatment.

[0036] Yet another aspect of the invention is a diagnostic composition comprising:

(1 ) a diagnostic agent;

(2) optionally, an intermediate release linker bound to the therapeutic agent; and

(3) a targeting moiety as described above bound to the intermediate release linker, if present, or to the diagnostic agent if the intermediate release linker is not present, the targeting moiety for binding the targeting composition to native collagen fibers.

[0037] The diagnostic agent can be a diagnostic agent usable in computed tomography (CT) imaging, or can be a diagnostic agent usable in magnetic resonance imaging (MRI). The diagnostic agent can be selected from the group consisting of iron oxide nanoparticles, gadolinium contrast agents with or without hyperpolarized substances, gadolinium-apoferritin, and manganese complexes. In another alternative, the diagnostic agent can be an iodinated contrast agent, such as an iodinated contrast agent selected from the group consisting of iohexol, iodixanol, ioversol, diatrizoate, metrizoate, ioxaglate, iopamidol, ioxilan, and iopromide.

[0038] If the diagnostic agent is attached to the targeting moiety, it can be attached covalently or non-covalently to the targeting moiety. The diagnostic agent can be attached in the form of individual molecules or ions to the targeting moiety.

Alternatively, the diagnostic agent can be attached in the form of a coating or other composite to the targeting moiety. If an intermediate release linker is used, the diagnostic agent is attached to the intermediate release linker.

[0039] Suitable targeting moieties and intermediate release linkers, if present, are as described above.

[0040] Yet another aspect of the present invention is a method for diagnostic imaging comprising the steps of:

(1 ) administering a quantity of a diagnostic agent according to the present invention to a patient in need of diagnosis sufficient to produce a degree of contrast sufficient for a diagnostic imaging procedure; and

(2) performing a diagnostic imaging procedure on the patient.

[0041] The diagnostic imaging procedure can be computerized tomography (CT) or magnetic resonance imaging (MRI).

[0042] Another aspect of the invention is a composition comprising a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers. The composition can further comprise a therapeutic agent. The therapeutic agent can be incorporated in the interior of the liposome or can be attached to the surface of the liposome. The liposome can further comprise a substance that can be identified by a radiological procedure selected from the group consisting of X-ray, MRI, and CT; the substance can be selected from the group consisting of a radio-opaque substance and a radioactive substance.

[0043] Yet another aspect of the invention is a pharmaceutical composition comprising:

(1) a therapeutically effective quantity of the composition comprising a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers and further comprising a therapeutic agent; and

[0044] In another alternative, the invention comprises a composition comprising a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers. The peptide motif is as described above. In one preferred alternative, the liposome is pegylated. Typically, the diameter of the liposome is from about 50 nm to about 2000 nm, preferably from about 200 nm to about 2000 nm, and more preferably about 1000 nm.

[0045] Yet another aspect of the invention is a targeting composition comprising:

(1) a therapeutically effective radionuclide;

(2) an intermediate release linker bound to the therapeutically effective radionuclide; and

(3) a targeting moiety bound to the intermediate release linker, the targeting moiety for binding the targeting composition to native collagen fibers.

[0046] The therapeutically effective radionuclide can be ¹³¹l, in which case the radionuclide is typically covalently bound to the intermediate release linker.

Alternatively, the therapeutically effective radionuclide can be selected from the group consisting of ⁹⁰Y and ¹¹¹ In, in which case the radionuclide is bound to the intermediate release linker by a chelator that bound to the intermediate release linker. The chelator can be selected from the group consisting of cyclic DPTA (diethylene triamine

pentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid), and tiuxetan. Diagnostically effective radionuclides include, but are not limited to, ^99mTc, ²⁰¹TI, and ⁶⁷Ga.

[0047] In yet another alternative, the targeting composition can include and deliver a diagnostically effective nucleotide for diagnosis by a technique such as scintigraphy. In general, in this alternative, the targeting composition comprises:

(1) a diagnostically effective radionuclide;

(2) an intermediate release linker bound to the diagnostically effective radionuclide; and

[0048] Diagnostically effective radionuclides include, but are not limited to, ^99mTc, ²⁰¹TI, and ⁶⁷Ga.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, appended claims, and accompanying drawings where:

[0050] Figure 1 shows a histological section of excised liver tumor showing a preponderance of fibrosis (fib) with pseudo-differentiated epithelioid tumor cells (tu) arrayed in columnar/ductal structures, seen in various stages of degeneration (A, hematoxylin-eosin (H&E) stain), as marked by a cytokeratin-17 immunostain (inset). Abundant fibrosis is observed throughout the tumor nodule, as shown by Masson's trichrome stain for ECM (blue stain, B). Remarkably, Rexin-G appears to have induced massive amounts of apoptosis of the pancreatic cancer cells (see TUN EL Stain in C, D, and negative control E), as well as visible karyorrhexis and fragmentation— which is evident all along the borders of the pseudo-differentiated structures.

[0051] Figure 2 shows immunohistochemical staining of the excised tumor for the gp70 envelope protein of the Rexin-G nanoparticle reveals an accumulation of immunoreactivity throughout the ECM-rich mass of the tumor (A versus B, negative control), particularly in the cellular components, including the diffuse islands (C) and ductal structures (D) comprised of cancer cells and the elongate endothelial cells lining the vessels of the tumor-associated vasculature (E).

[0052] Figure 3 shows a native collagen fiber stained with phosphotungstic acid, showing 68-nm periodicity and a schematic representation of collagen molecules measuring approximately 300 nm (adapted from . Nimni, ed., "Collagen", Vol. 1 , CRC Press, 1988).

[0053] Figure 4 shows the molecular packing of the Type I collagen fiber.

[0054] Figure 5 depicts a genetically engineered fusion protein consisting of TGF-βΙ with a collagen binding decapeptide. The purification tag comprises a hexapeptide of histidine, linked via a Gly-Gly link; it binds tightly to a Ni-NTA column. DNA constructs were transfected into Escherichia coli.

[0055] Figure 6 depicts the binding of the TGF-β with a collagen binding domain to collagen; the binding requires a high concentration of urea for dissociation. This is compared to the behavior of TGF-β without the collagen binding domain, which has poor affinity for collagen.

[0056] Figure 7 shows results for the binding of paclitaxel associated with albumin (Abraxane) to collagen. (A) Paclitaxel associated with albumin (Abraxane), with a covalently bound CBD, exhibits greater retention on a collagen matrix. (B) Abraxane with a CBD binds tighter to collagen and is more resistant to elution from collagen by 0.5 M urea. (C) The CBD bound to Abraxane, by targeting the site of the tumor, enhances chemotherapeutic effects in mice bearing colon cancer cells in a mouse model.

[0057] Figure 8 shows molecular modeling of discoidin, including the amino acids on the surface involved in binding to collagen. These amino acids and their distribution within the DS domain provide a three-dimensional view of the nature of the collagen-ligand interaction.

[0058] Figure 9 is a schematic drawing of molecular packing within a collagen fiber. (A) Axial view showing linear staggering; (B) Cross-sectional view showing the unit cell. (B) shows how particular segments are repeated on the surface of the fiber (b- b for instance is separated by 2 χ the diameter of a molecule or approximately 3 nm laterally, the distance that repeating CBDs should be set apart for optimal binding).

[0059] Figure 10 is a diagrammatic representation of a collagen targeting vector: (A) CBD; (B) peptide for facilitating drug (D) attachment (length of peptide and specific amino acids in peptide leading to suitable conformations in solution will vary); (C) reactive functional groups suitable for drug attachment (-SH, -CO₂H, -NH₂, or other groups); (D) drug; (E) additional site for identical or different CBD, separated by a suitable length of spacer (B) can be added.

[0060] Figure 11 shows the entire wild-type DDR2 amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

[0061] It has become increasingly recognized that in order to decrease or eliminate the deleterious systemic effects of drugs used to treat cancer or other diseases and conditions, that such compounds should be selective and/or able to be delivered selectively, in such a way the they are only lethal or inactivating to target cells and spare normal cells. The present invention addresses that need by the use of specific peptide targeting sequences linked to the therapeutic drug through a release domain.

[0062] Drug-delivery carriers aim to address these major issues in cancer therapy and therapy of other diseases and conditions and provide a more efficient means of drug delivery for anticancer medications and other therapeutic agents.

[0063] Targeted delivery of therapeutic agents to cancer cells aims to increase the therapeutic efficacy and to minimize nonspecific toxicities. Unfortunately, the surfaces of cancer cells do not seem to posses identifiable unique molecular targets. Although there is ongoing research to identify tumor-specific antigens or receptors for selective targeting of cancer cells, for many cases such targets might not exist.

However, it is common for cancer cells to overexpress antigens/receptors that are relatively downregulated in healthy cells, but such overexpressed antigens or receptors seem to be only of limited value in terms of specificity. [0064] For micrometastatic disease, direct targeting of molecular moieties on the cell surface is believed to be necessary to localize the therapeutics at the site of the cancer cells. In solid tumors, targeting of the vasculature and the cancer cells that constitute the tumor is being explored. In particular, both antiangiogenic and

antivascular targets are pursued to either prevent new blood vessel formation by targeting growth factor receptors on the vasculature endothelial cells ("antiangiogenic") or to damage and kill tumor cells by cutting the blood flow through the neovasculature, so as to deprive the tumor of growth factors ("antivascular"). Again this targeting is not highly specific and is considered a form of passive targeting.

[0065] A number of targeting methods are known, but, typically what is being targeted by such methods is a particular selected compound, present in tumor cells, but also always present, often in smaller amounts, in normal cells. Unfortunately the surface of a cancer cell does not have unique molecular targets. Such cells are too similar to normal cells. The present invention, therefore, targets in a unique way, the environment in which the cancer cells thrive, which has unique characteristics, different from that of cells in healthy tissues. The present invention addresses that problem.

[0066] Solid tumors are usually associated with considerable matrix production, at least in part by fibroblasts. Collagen fibers, once deposited, become rapidly coated by a layer of glycoproteins due to their high positive charge causing them to become "invisible" to cells and circulating factors. This plays a protective important physiological role, since the exposed triple-helical surface of a collagen molecule will initiate, among other things, clotting and tissue remodeling.

[0067] Metastatic tumor cells are associated with an abundance of proteolytic enzymes, which have the capacity to "uncoat" the collagen fibers making their surfaces accessible to recognition. The inventors of the present application have pioneered the use of peptide sequences, variants of a decapeptide motif present in Von Willebrand factor, a protein involved in clotting, to target certain growth factors to the surface of collagen. This same concept will be applied to chemotherapy. Peptides and variants with enhanced binding affinities will be synthesized and attached to the terminal ends of a carrier polypeptide to which chemotherapeutic agents will be coupled with the aid of selected biodegradable links. In addition, novel constructs composed of multiple peptides, selectively spaced to recognize repeating binding motifs present on the surface of collagen fibers, will be developed to enhance binding affinity. This approach could provide a novel modality to deliver bioactive molecules to sites of tumor metastasis, helping overcome two major setbacks of current chemotherapy: systemic toxicity and lack of specificity. Hopefully this approach can serve as a basis for a universal carrier for cancer drugs.

[0068] Although many potent anti-cancer drugs exist, unfortunately, the therapeutic index for these drugs is suboptimal for eliminating tumors, particularly micrometastases. The collagen peptides employed in the present invention have the potential to selectively deliver drugs, growth factors, and anti-inflammatory agents, as well as other therapeutic and diagnostic agents, to sites of tumor growth and

inflammation. The present invention describes approaches for improving the collagen binding domain and coupling it with an effective drug carrying backbone, to achieve better delivery and longer retention in the tumor niche.

[0069] The present invention is focused on what is considered to be a unique approach for targeting metastases, based on changes in the extracellular matrix around areas of metastasis and tumor growth, namely the exposure of the surfaces of collagen fibers caused by proteolytic cleavage and detachment of absorbed proteins. Very rapidly after collagen molecules are secreted and deposited as fibers in the extracellular space, a variety of glycoproteins and circulating macromolecules, many secreted by these cells, coat these fibers. This essentially renders individual collagen molecules on the surface of these fibers invisible to other cells and circulating factors.

[0070] Inflammation, as well as localized metastatic activity, can lead to

"uncoating"Of the collagen fibers. MMPs, a family of zinc-dependent neutral

endopeptidases, play a significant role in this connection. In addition many metabolic processes are known to be associated with tumors, including new collagen deposition and turnover. We propose to use peptide sequences present in Von Willebrand's factor, which we have shown are able to cause polypeptide growth factors to bind tightly to collagen (Takagi, Asai et al. 1992) (Tuan, Cheung, Nimni et al. 1996), (Nimni 1997). We will continue to focus on these peptides as other sequences are being explored to optimize binding efficiency. ((Romijn, et a). 2003), (Farndale, Lisman et al. 2008) (Herr and Farndale 2009).

[0071] Collagen fibers are major constituents of tissue parenchyma or stroma that surround all cells. Such fibers contribute to the structural and functional properties of the majority of tissues. These fibers are normally not visible to cells or in direct contact with them as they are coated with a layer of proteoglycans, another major component of the connective tissues. This "coating" of the collagen fibers plays a key physiological role, since exposed collagen serves as a site for platelet attachment, and can initiate the blood clotting process. If collagen were exposed, abnormal hemostasis (blood clotting) would occur at multiple sites. At sites of inflammation and of tumor invasion or metastasis, enhanced enzymatic activity can degrade this protective coat, thus exposing collagen. This now allows it to become a target for recognition.

[0072] Collagen fibers are major constituents of tissue parenchyma. There are now over 30 distinct collagens. The first unique and distinct mammalian collagen, now known as type II collagen, was unique because it was not constructed from three identical polypeptides; rather, it was constructed of two identical polypeptides and one polypeptide that was slightly different (Strawich and Nimni, 1971). All these collagens have a characteristic repeating motif or a variation of this motif, typically a Gly-Pro- Hypro-Gly sequence, where Hypro is hydroxylated proline. Hydroxylated proline is not directly incorporated into the collagen molecule during polypeptide synthesis, but is produced by post-translational modification. Most important every fourth residue is by necessity glycine. Intervening amino acids can vary. The collagen molecules organize into a 3 dimensional structure, leading to fibers. As mentioned collagen fibers are not normally directly accessible to cells as these fibers are coated with a layer of proteins and proteoglycans. This has an important physiological function as it prevents, among other things platelets to attach and initiate the clotting cascade. It is only during the process of tissue damage (wound healing, release of inflammatory cytokines, and as we have now learned, tumor cell invasion) that metalloproteases and other related enzymes are released and remove such a coat, thereby exposing the surface of the collagen fibers.

[0073] Our working hypothesis is that if we link a therapeutic drug to a peptide or peptides which recognize such naked collagen, the peptide or peptides will target such a site. As discussed below, such a peptide can be a decapeptide sequence, identical or similar to the sequence present in von Willebrand's factor (VWF) used by platelets to attach to collagen, which can be used to generate fusion proteins or other proteins or polypeptides which have an ability to strongly bind to collagen.

[0074] Since the VWF collagen binding domain was first identified, many new collagen binding sites of platelet collagen binding receptors, such as integrin α2β1 , glycoprotein VI, and others, as well as more effective modifications of the VWF collagen binding domain, are being constantly mapped. These can provide binding sequences of increased affinity and specificity that can be incorporated into targeting compositions according to the present invention. Accordingly, the targeting composition can comprise a targeting moiety, as discussed further below, that is a collagen binding site of a platelet collagen binding receptor, including, but not limited to, integrin α2β1 and glycoprotein VI.

[0075] The collagen targeting approach designed in our laboratory has served as a basis for the generation of a pathotropically-targeted replication-incompetent viral vector encoding a dominant mutant form of the cyclin G1 gene, an essential component of the cell cycle control pathway (Hall FL, et al Oncology Reports 24: 829-833, 2010).

[0076] A collagen targeting mechanism has been used to generate a guided vector which accumulates at sites of tumor metastasis. This has given rise to a drug (Rexin-G), which incorporates a dominant negative mutant of cyclin-G). The efficacy of this approach is beginning to manifest itself. Rexin-G has been approved by the FDA as an orphan drug and is in phase III clinical trials. Accumulation of the vector at sites of excised single liver tumor metastasis in a patient with pancreatic cancer has recently been documented. Unfortunately this vector at this time is not able to deliver other chemotherapeutic agents. [0077] One improvement specific to this application is to target sites of metastatic or primary tumors by focusing on identifiable specific changes that occur in immediate proximity to these sites. For this purpose we have selected a unique event that occurs at these locations, the exposure of normally masked collagen fibers, which become visible to targets as a result of the active metabolic activity associated with tumor cell metabolism. We believe that this approach provides enhanced targeting by orders of magnitude to areas of tumor activity as compared to the passive targeting concept. Typically, the native collagen fibers to which a targeting composition according to the present invention is bound differ from other collagen fibers in the organism as they are clearly recognizable to the targeting moiety, by virtue of having their surface exposed as a consequence of the metabolic activity associated with metastasis and/or inflammation.

[0078] One particular aspect of this invention is a focus on the synergistic role of cancer progression and local inflammation. Both of these events are associated with recognizable exposure of local collagen fibers. This suggests the possibility of combining the use of anti-cancer drugs with anti-inflammatory drugs.

[0079] Accordingly, one advantage of the use of the targeting methods of the present invention, employing the linkage of a therapeutic drug to a peptide sequence targeting collagen, is that they have the ability to deliver a wide variety of

chemotherapeutic agents, as discussed further below.

[0080] Additionally, such peptide sequences targeting collagen can be used to deliver diagnostic agents such as are commonly used in CT or MRI scanning. Such diagnostic agents include, but are not limited to, iron oxide nanoparticles, gadolinium contrast agents with or without hyperpolarized substances, gadolinium-apoferritin, or manganese complexes. Such diagnostic agents are attached either covalently or non- covalently to the peptide sequences targeting collagen, either as individual molecules or ions, or in the form of a coating or other composite. Such diagnostic agents can be either directly bound to the peptide sequence targeting collagen, or can be bound to the peptide sequence targeting collagen through an intermediate release linker, if an intermediate release linker is used. [0081] In some alternatives, as discussed below, it may be desirable to insert multiple binding motifs on the surface of the targeting particle to assure good linking and to stabilize its attachment. On the other hand it is possible that protruding PEG chains may suffice to achieve this goal. In one alternative, the protruding peptides can be extended by inserting repeating sequences of glycine. Glycine provides maximum rotation around peptide bonds, and therefore maximal degree of motion. When such polyglycine extensions are employed, the polyglycine extensions typically range up to 50 repeating units. The displacement between the most adjacent binding sites among parallel oriented molecules is in the range of 2-4 nm. Free random movement of the glycine chains and the generation of as many attachments as possible are desirable. In another alternative, the extensions can be made more rigid, such as by using repeating Gly-Pro-Pro-Gly sequences) to generate a collagen like rigid triple helical extension radiating from the surface of the targeting particle.

[0082] For micrometastatic disease, direct targeting of molecular moieties on the cell surface is necessary to localize the therapeutic agent carried by the therapeutic composition to the cancer cells and, in some alternatives, also allow them to be internalized by the tumor cells so that they can act intracellularly. In solid tumors, targeting of the vasculature and the cancer cells that constitute the tumor is being explored. In particular, both antiangiogenic and antivascular targets can be utilized in compositions according to the present invention to either prevent new blood vessel formation by targeting growth factor receptors on the vasculature endothelial cells ("antiangiogenic") or to damage and kill tumor cells by cutting the blood flow through the neovasculature, so as to deprive the tumor of growth factors ("antivascular").

[0083] Depending on the nature of delivered therapeutic modalities, cellular internalization of the targeting ligand can be of great importance in cancer killing and can be utilized by compositions according to the present invention.

[0084] Other alternatives employing compositions according to the present invention are also within the scope of the present invention. In one such alternative, two separate preparations with different targeting sites are administered to the same organism in need of treatment. In another such alternative, known as pretargeting, a secondary targeting reagent that specifically binds to the targeting composition and directly targets the tumor, such as a suitable monoclonal antibody, is administered first. In one alternative, the monoclonal antibody or other secondary targeting reagent is conjugated to one of two binding partners that use the biotin-avidin link, while the targeting composition is conjugated to the other of the two binding partners that use the biotin-avidin link. In another alternative, the monoclonal antibody or other secondary targeting reagent and the targeting composition are both conjugated to biotin or a derivative or analogue of biotin, and a biotin-binding component is introduced to crosslink the monoclonal antibody or other secondary targeting reagent to the targeting composition. The avidin-binding component can be selected from the group consisting of avidin, streptavidin, a derivative or analogue of avidin or streptavidin, and a biotin- binding antibody. In still another approach, the targeting composition can contain a derivatized chemotherapeutic moiety that binds to a bispecific antibody, and, prior to the administration of the targeting composition, the bispecific antibody is administered to the organism to be treated. The bispecific antibody binds both to the derivatized

chemotherapeutic moiety of the targeting composition and to a tumor marker in the organism. In one example of this approach, the tumor marker is carcinoembryonic antigen (CEA), the derivatized chemotherapeutic moiety is indium-1 1 1- diethylenetriamine pentaacetic acid (DTPA)-derivatized phosphatidylethanolamine, and the bispecific antibody is a bispecific anti-CEA anti-indium DTPA antibody. Other alternatives for the tumor marker, the derivatized chemotherapeutic moiety, and the bispecific antibody are known in the art.

[0085] In still another approach, targeting compositions according to the present invention are used that include at least two antibodies, wherein each antibody is an antibody for a specific receptor on the surface of tumor cells in the organism to be treated, the receptors occurring in the same tumor cell. The rationale for this approach is based on the observation that several targeted receptors on tumor cells cluster on the surface of the membrane on binding to antibodies, thus, when clustered receptors are exposed to clustered antibodies (owing to their localization on the same liposomal platform), increased complex valency and avidity is expected to occur because of the decreased off-rates of multimerized antibodies or other targeting ligands. This provides more efficient targeting.

[0086] In yet another approach according to the present invention, a targeting composition according to the present invention has two functionalities in addition to the targeting moiety described above. These two functionalities are: (1) a binding

functionality to a noninternalizing receptor on the malignant cell's surface; and (2) an initially hidden functionality through a cell-penetrating peptide that is activated only after binding to the cell surface at slightly acidic conditions corresponding to the

approximately pH 6.0 of the tumor interstitium.

[0087] A particularly preferred targeting moiety that can be included in targeting compositions of the present invention to target them to tumor cells is a peptide motif identical or similar to that used by von Willebrand's factor to bind to collagen. Examples of such peptide motifs are described further below.

[0088] Accordingly, one aspect of the present invention is a targeting

composition comprising: (1) a therapeutic agent; (2) an intermediate release linker bound to the therapeutic agent; and (3) a targeting moiety bound to the intermediate release linker as described further below for binding the targeting composition to native collagen fibers, such as a peptide motif identical or similar to that used by von

Willebrand's factor to bind to collagen. The ingredients of this composition and the methods used to link them in the composition are described further below.

[0089] As indicated above, one of a number of peptide motifs can be used for binding the composition to native collagen fibers. Typically, such a peptide motif is based on the peptide motif used by von Willebrand's factor to bind to collagen.

[0090] Such sequences include, but are not limited to: (1) Trp-Arg-Glu-Pro-Ser- Phe- et-Ala-Leu-Ser (WREPSFMALS) (SEQ ID NO: 1); (2) Trp-Arg-Glu-Pro-Ser-Phe- Cys-Ala-Leu-Ser (WREPSFCALS) (SEQ ID NO: 2); (3) peptides related to (1) or (2) by one or more conservative amino acid substitutions, as defined below, including, but not limited to: (3a) Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-Leu-Ser (WRDPSFMALS) (SEQ ID NO: 3); (3b) Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-Leu-Ser (WRDPSFCALS) (SEQ ID NO. 4); (3c) Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-lle-Ser (WREPSFMAIS) (SEQ ID NO: 5); (3d) Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-lle-Ser (WREPSFCAIS) (SEQ ID NO: 6); (3e) Trp- Arg-Asp-Pro-Ser-Phe- et-Ala-lle-Ser (WRDPSFMAIS) (SEQ ID NO: 7); and (3f) Trp- Arg-Asp-Pro-Ser-Phe-Cys-Ala-lle-Ser (WRDPSFCAIS) (SEQ ID NO: 8).

[0091] Conservative amino acid substitutions are well known in the art. More specifically, in a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and may be made generally without altering the biological activity of the resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g. Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, Benjamin/Cummings, p. 224). In particular, such a

conservative variant has a modified amino acid sequence, such that the change(s) do not substantially alter the protein's (the conservative variant's) secondary or tertiary structure and/or activity, specifically binding activity in this context. Conservative amino acid substitution generally involves substitutions of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non- polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): Ala Gly or Ser; Arg/Lys; Asn/Gln or His; Asp/Glu; Cys/Ser; Gln/Asn;

Gly/Asp; Gly/Ala or Pro; His/Asn or Gin; lle/Leu or Val; Leu/lle or Val; Lys/Arg or Gin or Glu; Met/Leu or Tyr or lie; Phe/Met or Leu or Tyr; Ser/Thr; Thr/Ser; Trp/T yr; Tyr Trp or Phe; Val/lle or Leu. An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: (1) alanine (A or Ala), serine (S or Ser), threonine (T or Thr); (2) aspartic acid (D or Asp), glutamic acid (E or Glu); (3) asparagine (N or Asn), glutamine (Q or Gin); (4) arginine (R or Arg), lysine (K or Lys); (5) isoleucine (I or He), leucine (L or Leu), methionine (M or Met), valine (V or Val); and (6) phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp); (see also, e.g., Creighton (1984) Proteins, W. H. Freeman and Company; Schuiz and Schimer (1979) Principles of Protein Structure, Springer- Verlag). One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative.

[0092] Additionally, such sequences include the decapeptides of SEQ ID NOs: 1-8 extended at both the amino-terminus and the carboxyl-terminus by the addition of the sequences Gly-Pro-Pro-Gly (GPPG). Accordingly, these sequences are as follows: (4) Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser-Gly-Pro-Pro-Gly

(GPPGWREPSFMALSGPPG) (SEQ ID NO: 9); (5) Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro- Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFCALSGPPG) (SEQ ID NO: 10); (6) Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFMALSGPPG) (SEQ ID NO: 11); (7) Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro- Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFCALSGPPG) (SEQ ID NO: 12); (8) Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-lle-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFMAISGPPG) (SEQ ID NO: 13); (9) Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro- Ser-Phe-Cys-Ala-lle-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFCAISGPPG) (SEQ ID NO. 14); (10) Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-lle-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFMAISGPPG) (SEQ ID NO: 15); and (11) Gly-Pro-Pro-Gly-Trp-Arg-Asp- Pro-Ser-Phe-Cys-Ala-lle-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFCAISGPPG) (SEQ ID NO: 16).

[0093] In another alternative, peptide sequences SEQ ID NO: 1 through SEQ ID NO: 16 (i.e., SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, 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: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16) can be incorporated into an elongated peptide structure of Formula (I):

[Gly-Pro-Pro-Gly-Xi-Gly-Pro-Pro-Gly-X₂-Gly-Pro-Pro-Gly]_n

(I) wherein: (1) Xi and X₂ are one of peptide sequences SEQ ID NO. 1 through SEQ ID NO: 16, described above; and (2) n is an integer from 1 to 15. [0094] This repeating sequence would provide: (1) increased sites of attachment; and (2) an intervening sequence which resembles collagen. This sequence should increase the compatibility with the surface of collagen, since it is similar to the normally adjacent sequences which are normal constituents of the native collagen molecule.

[0095] Other such sequences which cover longer ranges, and therefore would increase interaction, can be used. Also, modified sequences that take into account the triple helical configuration of collagen can be designed to enhance the longer range contact and surface compatibility with the triple stranded configuration displayed on the surface of the fiber. In particular, peptide motifs that bind collagen with a binding affinity of at least 80% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen can be used and are encompassed by the invention. Preferably, such peptide motifs bind collagen with a binding affinity of at least 90% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen. More preferably, such peptide motifs bind collagen with a binding affinity of at least 95% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen. Such sequences can be deduced from

crystallography data or other techniques known in the art for determining protein- peptide interactions, including NMR.

[0096] The nature of this sequence can be readily determined by observing a collagen model which includes the individual collagen molecules packed into a 3- dimensional quarter-staggered array.

[0097] Other polypeptide sequences known to bind to collagen can alternatively be used.

[0098] In one alternative, the targeting moiety can be a targeting moiety in which the peptide sequences WREPSFMALS (SEQ ID NO: 1) or WREPSFCALS (SEQ ID NO: 2) can be incorporated into a variety of molecules of diverse nature to generate polypeptides which range from 2,000 to 10,000 daltons in size. The flanking sequences will vary but in general will mimic sequences found in native proteins, primarily collagen or elastin, with various degrees of hydrophilicity and hydrophobicity. As used herein, the term "mimics" refers to a sequence that results in the peptide including the sequence specifically binding to at least one target or receptor that the native protein intended to be mimicked by the sequence specifically binds to with an affinity that is at least 50% of the affinity of the native protein for the target or receptor. Inserted amino acids containing reactive groups will allow for drug coupling. In its simplest form one, two or three collagen binding domains are incorporated in a construct, separated by spacers with lead to various suitable conformations. These should be able to bridge the span between repeating domains on the surface of collagen, and therefore enhance the binding of the vector.

[0099] Multiple binding, to laterally displaced equivalent sites, on the surface of the collagen fiber should enhance binding affinity. Although the inventors do not intend to be bound by this theory, the inventors estimate a lateral displacement of about 3 nm, or twice the diameter of a collagen molecule to be in that range. Therefore spacers that maximally elongate in solution, i.e alternating sequences (polar/nonpolar) which contribute to a β-like sheet or polylysine or polyglycine which stretch out in solution may give rise to adequate spacers (Figure 9). This approach should provide (1) increased sites of attachment; and (2) an intervening sequence which resembles collagen.

[0100] In another alternative, the collagen binding domain sequences can be subject to pegylation (covalent conjugation with polyethylene glycol (PEG) moieties). These PEG polymers are nonionic, nontoxic, biocompatible, and highly hydrophilic. Their use provides increased solubility for hydrophobic therapeutic agents and increased bioavailability, as well as prolonged circulatory time within the host through reduced renal clearance. Such structures can be inserted at Site (C) in Figure 10.

[0101] In yet another alternative, if a peptide is selected from an internal sequence of a protein, terminal amidation at the carboxyl-terminus or acetylation at the amino-terminus will eliminate the charge at the termini. In addition, these modifications will make the resulting peptide more stable towards enzymatic degradation by exopeptidases. Biotin and fluorescein isothiocyanate (FITC) are activated precursors used for fluorescein labelings. For efficient N-terminal labeling, a seven-atom

aminohexanoyl spacer (NH₂-CH2-CH2-CH2-CH2-CH₂-COOH) can be inserted between the fluorophore (fluorescein) and the N-terminus of the peptide. One common means of conjugation involves the use of maleimide, which couples amino-terminal or carboxyl- terminal cysteine residues of the peptide to the carrier protein.

[0102] The decapeptides WREPSFMALS (SEQ ID NO: 1) or WREPSFCALS (SEQ ID NO: 2) involve a series of exposed amino acids, located strategically within the N-terminus, in an area extending from residues 570 (F) to 682 of VonWillebrand factor (Takagi, Asai et al. 1992). By binding competition this decapeptide was found to bind, on a molar basis, 20 times more efficiently to collagen than the intact WVF (Takagi, Asai et al. 1992). Further examination of the crystal structure of the collagen binding regions of WVF A-3 Domain (lchikawa, Osawa et al. 2007); (Romijn, Westein et al. 2003);

(Staelens, Hadders et al. 2006) as well as the complementary collagen exposed surface (Lisman, Raynal et al. 2006) is expected to yield collagen binding domains (CBDs) with an increased binding affinity.

[0103] Collagens are large, triple-helical proteins that form fibrils and networklike structures in the extracellular matrix. They have played a major role in the evolution of metazoans from their earliest origins. Cell adhesion receptors that interact with collagen, such as the integrins are at least as old as the collagens (Heino, Huhtala et al. 2009); (Whittaker and Hynes 2002) and instrumental in the evolution of bone, cartilage, and the immune system in chordates. In vertebrates collagen binding receptor tyrosine kinases send signals into cells after adhesion to collagen. Nevertheless, collagen continues to be seen primarily as an inert scaffold. To the inventors of the present application, the value of using it as a target became most relevant when they observed that it is only at sites of pathology or rapid tissue remodeling that collagen fibers become devoid of their normal proteoglycan coating, and therefore recognizable as such.

[0104] Other CBDs, such as the discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases known to be activated by native triple-helical collagen. The sequence on collagen that binds DDR2 with highest affinity has similarity to the binding site for von Willebrand's factor, GVMGFO (O is hydroxyproline) (SEQ ID NO: 17). (Konitsiotis, Raynal et al. 2008). The scattered amino acids on the binding site on the ligand are highlighted (Figure 8). The complete DDR2 amino acid sequence (SEQ ID NO: 18) is shown in Figure 11.

[0105] Accordingly, in drug delivery compositions according to the present inventions, the CBDs from DDR1 and DDR2 can be employed. These include: (1) the native CBDs from DDR1 and DDR2; and (2) CBDs incorporating the amino acids on the surface of the three-dimensional protein structure of DDR1 or DDR2 in which at least one of the amino acids not directly contacting collagen is replaced with a conservative amino acid substitution as described above such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the native CBD from DDR1 or DDR2.

[0106] Yet another alternative for a CBD is the synthetic peptide P-15, which is a synthetic 15-residue peptide that binds to collagen at the single mammalian collagenase cleavage site. This peptide has the sequence GTPGPGGIAGQRGW (SEQ ID NO: 19). The single unique collagenase site is particularly significant as it becomes exposed during periods of active collagen remodeling as occurs during fibrosis and metastasis. Additionally, the CBD can be a CBD derived from the sequence GTPGPGGIAGQRGW (SEQ ID NO: 19) by one or more conservative amino acid substitutions such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the sequence GTPGPGGIAGQRGW (SEQ ID NO: 19).

[0107] An increased number of CBDs can be employed, properly spaced from each other, as shown in Figure 9. Peptide (B) of the array shown in Figure 9 can be designed to match the profile of the therapeutic agent being carried; and amino acid sequences can be inserted and crosslinking mechanisms can be adjusted to the hydrophobic or electrostatic character of the therapeutic agent being carried. Collagen sequences, especially if repeated, will encourage collagen-like folding. Suitable sequences can be generated as well as cyanogen bromide peptides by cleavage of the native collagen molecule (Deshmukh and Nimni 1973). Such peptides fold and generate small size stable triple helical structures ("mini-collagens"), thermodynamically favored at 37° C, which should enhance binding to the fibers. [0108] Certain conservative amino acid substitutions, positive or negative, can improve binding affinity. Additionally, in another alternative, the CBDs can include one or more amino acids included in the collagen binding site for DDR2 and on the surface of DDR2 as shown in Figure 8.

[0109] The present invention is designed to result in minimal toxicities that can be achieved as long as the therapeutic agent is not released from targeting

compositions localized at normal organs or as long as inactive prodrugs included in the targeting composition are decomposed and removed from the body with minimal side effects.

[0110] A wide variety of therapeutic agents can be included in a targeting composition according to the present invention. These therapeutic agents include, but are not limited to, anti-neoplastic therapeutic agents and anti-inflammatory therapeutic agents, as well as nucleic acid and nucleotide sequences for nonviral gene therapy, hormones, growth factors, antiangiogenic compounds, antisense oligonucleotides, antibodies that specifically bind a pharmacologically significant molecule, such as a receptor, and others. Further details of the therapeutic agents are provided below.

[0111] Anti-neoplastic therapeutic agents that can be included in a targeting composition according to the present invention include, but are not limited to, therapeutic agents that inhibit cell replication or cause cell death. Examples of suitable anti-neoplastic therapeutic agents are listed below; however, the present invention encompasses other anti-neoplastic agents known in the art. For anti-neoplastic agents known in the art, one of ordinary skill in the art can determine appropriate dosages (i.e., concentration within a targeting composition according to the present invention), frequencies of administration, and durations of administration.

[0112] Mechlorethamine is a nitrogen mustard that has anti-neoplastic activity against Hodgkin's disease and non-Hodgkin's lymphomas. Cyclophosphamide and ifosfamide are nitrogen mustards that have anti-neoplastic activity against acute and chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, Wilms' tumor, cervical cancer, testicular cancer, and soft tissue sarcomas. Melphalan is a nitrogen mustard that has anti-neoplastic activity against multiple myeloma, breast cancer, and ovarian cancer. Chlorambucil is a nitrogen mustard that has anti-neoplastic activity against chronic lymphocytic leukemia, primary macroglobulinemia, Hodgkin's disease, and non-Hodgkin's lymphomas. Hexamethylmelamine is an alkylating agent that has anti-neoplastic activity against ovarian cancer. Thiotepa is an alkylating agent that has anti-neoplastic activity against bladder cancer, breast cancer, and ovarian cancer.

Busulfan is an alkyl sulfonate that has anti-neoplastic activity against chronic

granulocytic leukemia. Carmustine is a nitrosourea that has anti-neoplastic activity against , Hodgkin's disease, non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma, and malignant melanoma. Streptozocin is a nitrosourea that has antineoplastic activity against malignant pancreatic insulinoma and malignant carcinoid. Dacarbazine is a triazene that has anti-neoplastic activity against malignant melanoma, Hodgkin's disease, and soft-tissue sarcomas. Temozolomide is a triazene that has antineoplastic activity against glioma and malignant melanoma. Methotrexate is a folic acid analogue that has anti-neoplastic activity against acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast cancer, head and neck cancer, lung cancer, and osteogenic sarcoma. 5-Fluorouracil is a pyrimidine analogue antimetabolite that has anti-neoplastic activity against breast cancer, colon cancer, stomach cancer, pancreatic cancer, ovarian cancer, head and neck cancer, and urinary bladder cancer. Cytarabine is a pyrimidine analogue antimetabolite that has anti-neoplastic activity against acute granulocytic leukemia and acute lymphocytic leukemia. Gemcitabine is a pyrimidine analogue antimetabolite that has anti-neoplastic activity against pancreatic cancer and ovarian cancer. 6-Mercaptopurine is a purine analogue antimetabolite that has anti-neoplastic activity against acute lymphocytic leukemia, acute granulocytic leukemia, and chronic granulocytic leukemia. 6-Thioguanine is a purine analogue antimetabolite that has anti-neoplastic activity against acute lymphocytic leukemia, acute granulocytic leukemia, and chronic granulocytic leukemia. Pentostatin is a purine analogue that has anti-neoplastic activity against hairy cell leukemia, mycosis

fungoides, chronic lymphocytic leukemia, and small cell leukemia. Vinblastine is a vinca alkaloid that has anti-neoplastic activity against Hodgkin's disease, non-Hodgkin's lymphoma, breast cancer, and testicular cancer. Vincristine is a vinca alkaloid that has anti-neoplastic activity against acute lymphocytic leukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's lymphoma, and small- cell lung cancer. Paclitaxel is a taxane that has anti-neoplastic activity against ovarian cancer, lung cancer, breast cancer, and head and neck cancer. Docetaxel is a taxane that has anti-neoplastic activity against ovarian cancer, lung cancer, breast cancer, and head and neck cancer. Topotecan is a camptothecin that has anti-neoplastic activity against ovarian cancer and small-cell lung cancer. Irinotecan is a camptothecin that has anti-neoplastic activity against colon cancer. Dactinomycin is an antibiotic that has anti-neoplastic activity against choriocarcinoma, Wilms' tumor, rhabdomyosarcoma, testicular cancer, and Kaposi's sarcoma. Daunorubicin is an antibiotic that has antineoplastic activity against acute granulocytic and acute lymphocytic leukemias.

Doxorubicin is an antibiotic that has anti-neoplastic activity against soft-tissue

sarcomas, osteogenic sarcomas, other sarcomas, Hodgkin's disease, non-Hodgkin's lymphoma, acute leukemias, breast cancer, genitourinary tract cancer, thyroid cancer, lung cancer, gastric cancer, and neuroblastoma. Bleomycin is an antibiotic that has anti-neoplastic activity against testicular cancer, head and neck cancer, skin cancer, esophageal cancer, lung cancer, genitourinary tract cancer, Hodgkin's disease, and non-Hodgkin's lymphoma. Mitomycin C is an antibiotic that has anti-neoplastic activity against gastric cancer, cervical cancer, colon cancer, breast cancer, pancreatic cancer, bladder cancer, and head and neck cancer. L-Asparaginase is an enzyme that has anti-neoplastic activity against acute lymphocytic leukemia. Interferon-alfa is a biological response modifier that has anti-neoplastic activity against hairy cell leukemia, Kaposi's sarcoma, malignant melanoma, carcinoid, renal cell cancer, ovarian cancer, bladder cancer, non-Hodgkin's lymphoma, mycosis fungoides, multiple myeloma, and chronic granulocytic leukemia, lnterleukin-2 is a biological response modifier that has anti-neoplastic activity against malignant melanoma and renal cell cancer. Cisplatin is a platinum coordination complex that has anti-neoplastic activity against testicular cancer, ovarian cancer, bladder cancer, head and neck cancer, lung cancer, thyroid cancer, cervical cancer, endometrial cancer, neuroblastoma, and osteogenic sarcoma. Carboplatin is a platinum coordination complex that has anti-neoplastic activity against testicular cancer, ovarian cancer, bladder cancer, head and neck cancer, lung cancer, thyroid cancer, cervical cancer, endometrial cancer, neuroblastoma, and osteogenic sarcoma. Mitoxantrone is an anthracenedione that has anti-neoplastic activity against acute granulocytic leukemia, breast cancer, and prostate cancer. Hydroxyurea is a substituted urea that has anti-neoplastic activity against chronic granulocytic leukemia, polycythemia vera, essential thrombocytosis, and malignant melanoma. N- Methylhydrazine is a substituted hydrazine that has anti-neoplastic activity against Hodgkin's disease. Mitotane is an adrenocortical suppressant that has anti-neoplastic activity against adrenal cortex cancer. Aminoglutethimide is an adrenocortical suppressant that has anti-neoplastic activity against breast cancer. Imatinib is a tyrosine kinase inhibitor that has anti-neoplastic activity against chronic myelocytic leukemia. Prednisone is an adrenocorticosteroid that has anti-neoplastic activity against acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and breast cancer. Prednisolone is an adrenocorticosteroid that has anti-neoplastic activity against acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and breast cancer.

Methylprednisolone is an adrenocorticosteroid that has anti-neoplastic activity against acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non- Hodgkin's lymphoma, and breast cancer. Dexamethasone is an adrenocorticosteroid that has anti-neoplastic activity against acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and breast cancer.

Betamethasone is an adrenocorticosteroid that has anti-neoplastic activity against acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and breast cancer. Triamcinolone is an adrenocorticosteroid that has antineoplastic activity against acute lymphocytic leukemia, chronic lymphocytic leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, and breast cancer. Hydroxyprogesterone caproate is a progestin that has anti-neoplastic activity against endometrial cancer and breast cancer. Medroxyprogesterone acetate is a progestin that has anti-neoplastic activity against endometrial cancer and breast cancer. Megestrol acetate is a progestin that has anti-neoplastic activity against endometrial cancer and breast cancer. Diethylstilbestrol is an estrogen that has anti-neoplastic activity against breast cancer and prostate cancer. Ethinyl estradiol is an estrogen that has anti-neoplastic activity against breast cancer and prostate cancer. Tamoxifen is an antiestrogen that has antineoplastic activity against breast cancer. Anastrozole is an antiestrogen that has antineoplastic activity against breast cancer. Testosterone propionate is an androgen that has anti-neoplastic activity against breast cancer. Fluoxymesterone is an androgen that has anti-neoplastic activity against breast cancer. Flutamine is an antiandrogen that has anti-neoplastic activity against prostate cancer. Leuprolide is an gonadotropin- releasing hormone analogue that has anti-neoplastic activity against prostate cancer. Trastuzumab is a monoclonal antibody that has anti-neoplastic activity against breast cancer. Rituximab is a monoclonal antibody that has anti-neoplastic activity against non-Hodgkin's lymphoma. Alemtuzumab is a monoclonal antibody that has antineoplastic activity against chronic lymphocytic leukemia. Bevacizumab is a monoclonal antibody that has anti-neoplastic activity against colorectal cancer. Cetuximab is a monoclonal antibody that has anti-neoplastic activity against colorectal cancer and head and neck cancer. Gemtuzumab is a monoclonal antibody that has anti-neoplastic activity against acute myelocytic leukemia. Ibritumomab is a monoclonal antibody that has anti-neoplastic activity against non-Hodgkin's lymphoma. Panitumumab is a monoclonal antibody that has anti-neoplastic activity against colorectal cancer.

Tositumomab is a monoclonal antibody that has anti-neoplastic activity against non- Hodgkin's lymphoma.

[0113] Another group of anti-neoplastic agents suitable for incorporation into a targeting composition according to the present invention is interferons. Interferon works in a different way toward cancer cells than it does toward viruses and there are numerous pathways that interferon activates to help treat cancers. It has an

antiproliferative effect on tumor cells, it stimulates the tumor cells to change their surfaces so that they are recognized by the immune system as abnormal cells, and it blocks the growth of new blood vessels and helps cut off the supply of nutrients. At this time there are 12 identified interferon alphas. [0114] Other anti-neoplastic agents are known in the art and can be

incorporated into targeting compositions according to the present invention.

[0115] An important factor for one of ordinary skill in the art to consider in determining the construction of the targeting composition is the relative hydrophobicity or hydrophilicity of the anti-neoplastic agent or other therapeutic agent, including its solubility in water or aqueous solutions. This can assist one of ordinary skill in the art in determining suitable intermediate release linkers and targeting moieties as well as appropriate techniques for linking the therapeutic agent, the intermediate release linker, and the targeting moiety, including the reactive groups to be used; suitable

combinations of reactive groups are described further below.

[0116] In another alternative, the targeting composition can further include an anti-inflammatory agent in addition to the anti-neoplastic agent. In other words, the targeting composition includes both an anti-neoplastic agent and an anti-inflammatory agent. In general, a targeting composition according to the present invention can include two or more therapeutic agents.

[01 7] Anti-inflammatory agents suitable for incorporation into a targeting composition of the present invention include, but are not limited to: (1) histamine receptor antagonists such as, but not limited to, doxepin hydrochloride,

carbinoxamine maleate, clemastine fumarate, diphenhydramine hydrochloride, dimenhydrinate, pyrilamine citrate, tripelennamine hydrochloride, tripelennamine citrate, chlorpheniramine mdialeate, brompheniramine maleate, hydroxyzine hydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizine lactate, meclizine hydrochloride, promethazine hydrochloride, cyproheptadine hydrochloride, phenindamine tartrate, acrivastine, cetirizine hydrochloride, azelastine hydrochloride, levocabastine

hydrochloride, loratidine, desloratidine, ebastine, mizolastine, and fexofenadine, and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof, as well as additional ethanolamines, alkylamines, ethylenediamines, piperazines, phenothiazines, and tricyclic piperidines that are histamine receptor Hi antagonists; (2) histamine receptor H₂ antagonists such as, but not limited to cimetidine, ranitidine, and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof; (3) histamine receptor H₃ antagonists and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof; and (4) histamine receptor H₄ antagonists, including but not limited to 5-chloro-2-[(4- methylpiperazin-1-yl)carbonyl]-1f -indole (JNJ7777120) and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof.

[0118] Additionally, anti-inflammatory agents suitable for incorporation into a targeting composition according to the present invention include kinin receptor antagonists, including, but not limited to, Bi or B₂ receptor antagonists and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof. A number of kinin receptor antagonists, including peptides containing one or more amino acids of the D-configuration and small molecules, are disclosed in United States Patent Application Publication No.

2008/0221039 by Gibson et al., incorporated herein by this reference.

[0119] Additionally, anti-inflammatory agents suitable for incorporation into a targeting composition according to the present invention include leukotriene receptor antagonists such as zafirlukast and montelukast, and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof.

[0120] Additionally, anti-inflammatory agents suitable for incorporation into a targeting composition according to the present invention include non-steroidal antiinflammatory drugs (NSAIDs). NSAIDs include, but are not limited to, acetylsalicylic acid (aspirin), sodium salicylate, choline magnesium trisalicylate, salsalate, diflunisal, sulfasalazine, olsalazine, acetaminophen, indomethacin, sulindac, tolmetin, diclofenac, ketorolac, ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofin, oxaprozin, mefenamic acid, meclofenamic acid, piroxicam, meloxicam, nabumetone, rofecoxib, celecoxib, etodolac, nimesulide, aceclofenac, alclofenac, alminoprofen, amfenac, ampiroxicam, apazone, araprofen, azapropazone, bendazac, benoxaprofen,

benzydamine, bermoprofen, benzpiperylon, bromfenac, bucloxic acid, bumadizone, butibufen, carprofen, cimicoxib, cinmetacin, cinnoxicam, clidanac, clofezone, clonixin, clopirac, darbufelone, deracoxib, droxicam, eltenac, enfenamic acid, epirizole, esflurbiprofen, ethenzamide, etofenamate, etoricoxib, felbinac, fenbufen, fenclofenac, fenclozic acid, fenclozine, fendosal, fentiazac, feprazone, filenadol, flobufen, florifenine, flosulide, flubichin methanesulfonate, flufenamic acid, flufenisal, flunixin, flunoxaprofen, fluprofen, fluproquazone, furofenac, ibufenac, imrecoxib, indoprofen, isofezolac, isoxepac, isoxicam, licofelone, lobuprofen, lomoxicam, lonazolac, loxaprofen,

lumaricoxib, mabuprofen, miroprofen, mofebutazone, mofezolac, morazone, nepafanac, niflumic acid, nitrofenac, nitroflurbiprofen, nitronaproxen, orpanoxin, oxaceprol, oxindanac, oxpinac, oxyphenbutazone, pamicogrel, parcetasal, parecoxib, parsalmide, pelubiprofen, pemedolac, phenylbutazone, pirazolac, pirprofen, pranoprofen, salicin, salicylamide, salicyisalicylic acid, satigrel, sudoxicam, suprofen, talmetacin, talniflumate, tazofelone, tebufelone, tenidap, tenoxicam, tepoxalin, tiaprofenic acid, tiaramide, tilmacoxib, tinoridine, tiopinac, tioxaprofen, tolfenamic acid, triflusal, tropesin, ursolic acid, valdecoxib, ximoprofen, zaltoprofen, zidometacin, and zomepirac, and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof.

[0121] Additionally, anti-inflammatory agents suitable for incorporation into a targeting composition according to the present invention include steroids with antiinflammatory activity. Steroids with anti-inflammatory activity include, but are not limited to, hydrocortisone, cortisone, beclomethasone dipropionate, betamethasone,

dexamethasone, prednisone, methylprednisolone, triamcinolone, fluocinolone

acetonide, and fludrocortisone.

[0122] Other anti-inflammatory agents are known in the art and can be incorporated into therapeutic compositions according to the present invention. In one alternative, the anti-inflammatory agent can be included in a therapeutic composition together with an anti-neoplastic agent; i.e., the two therapeutic agents are included in a single therapeutic composition according to the present invention. In another

alternative, the therapeutic composition includes only an anti-inflammatory agent. [0123] As detailed above, one of ordinary skill in the art typically considers the relative hydrophobicity or hydrophilicity of the anti-inflammatory agent, including its solubility in water or aqueous solutions. This can assist one of ordinary skill in the art in determining suitable intermediate release linkers and targeting moieties as well as appropriate techniques for linking the anti-inflammatory agent, the intermediate release linker, and the targeting moiety, including the reactive groups to be used; suitable combinations of reactive groups are described further below.

[0124] Targeting compositions according to the present invention are not limited to targeting compositions that include either anti-neoplastic therapeutic agents or antiinflammatory therapeutic agents. Targeting compositions according to the present invention can include one or more of the following categories or classes of therapeutic agents:

(1) muscarinic cholinergic receptor agonists, including bethanechol chloride, pilocarpine hydrochloride, and cevimeline;

(2) muscarinic cholinergic receptor antagonists, including tolterodine, trospium chloride, oxybutinin, darifenacin, pirenzipine, telenzipine, and propantheline bromide;

(3) anticholinesterase agents, including physostigmine, neostigmine, pyridostygmine, rivastigmine, edrophonium, tacrine, donepezil, and galantamine;

(4) 3₂-selective adrenergic receptor agonists, including metaprotenerol, terbutaline, formoterol, albuterol, isoetharine, pirbuterol, bitolterol, fenoterol, procaterol, salmeterol, and ritodrine;

(5) o^-selective adrenergic receptor agonists, including clonidine, apraclonidine, brimonidine, guanfacine, and guanabenz;

(6) sympathomimetic agonists, including methylphenidate and pemoline:

(7) oci -selective adrenergic receptor antagonists, including prasozin, terazosin, doxazosin, alfuzosin, and tamsulosin;

(8) β-selective adrenergic receptor antagonists, including nadolol, penbutolol, pindolol, propranolol, timolol, acebutolol, atenolol, bisoprolol, esmolol, metoprolol, carteolol, carvedilol, bucindolol, labetolol, betaxolol, celiprolol, and nebivolol; (9) 5-hydroxytryptamine receptor agonists, including sumatriptan, zolmitriptan, naratriptan, and rizatriptan;

(10) 5-hydroxytryptamine receptor antagonists, including ondansetron and dolasetron;

(11) benzodiazepines, including alprazolam, brotizolam,

chlordiazepoxide, clobazam, clonazepam, clorazepate, demoxepam, diazepam, estazolam, flumazenil, flurazepam, lorazepam, midazolam, nitrazepam, nordazepam, oxazepam, prazepam, quazepam, temazepam, triazolam, zolpicone, Zolpidem, and indiplon;

(12) antidepressants, including amitriptyline, clomipramine, doxepin, imipramine, trimipramine, amoxapine, desipramine, maproti!ine, nortriptyline,

protriptyline, citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, sertraline, venlafaxine, atomoxetine, buproprion, duloxetine, mirtazapine, nefazodone, trazodone, phenelzine, tranylcypromine, and seligiline;

(13) antipsychotic drugs, including chlorpromazine, mesoridazine, thioridazine, fluphenazine, perphenazine, trifluoperazine, chloroprothixene, thiothixene, aripiprazole, clozapine, haloperidol, loxapine, molindone, olanzapine, pimozide, quetiapine, risperidone, and ziprasidone;

(14) antiseizure drugs, including carbamazepine, ethoxsuximide, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, phenobarbital, phenytoin, primidone, tiagapine, topiramate, valproate, and zonisamide;

( 5) anti-parkinsonism drugs and drugs effective against

neurodegenerative diseases, including carbidopa, levodopa, bromocriptine, pergolide, ropinirole, pramipexole, entacapone, tolcapone, amantidine, riluzole, and

trihexylphenylhydrochloride;

(16) opioids, including morphine, etorphine, fentanyl, sufentanyl, codeine, oxycodone, tramadol, meperidine, loperamide, and propoxyphene;

(17) diuretics, including furosemide, bumetanide, ethacrynic acid, torsemide, bendroflumethazide, chlorothiazide, hydrochlorothiazide, hydroflumethazide, amiloride, triamterene, and spironolactone; (18) angiotensin converting enzyme inhibitors, including benazepril, captopril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril;

(19) nonpeptide angiotensin II receptor antagonists, including candesartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan;

(20) calcium channel blockers, including amlodipine, felodipine, isradipine, nicardipine, nifedipine, diltiazem, and verapamil;

(21) antiarrhythmic drugs, including dofetilide, digoxin, digitoxin, flecainide, ibutilide, procainamide, propafenone, quinidine, and sotalol;

(22) drugs having a therapeutic effect on hypercholesterolemia and/or dyslipidemia, including mevastatin, lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, choletyramine, colestipol, colesevelam, nicotinic acid, clofibrate, gemfibrozil, fenofibrate, ciprofibrate, bezafibrate, and ezetimbe;

(23) H2 receptor antagonists, including cimetidine, ranitidine, famotidine, and nizatidine;

(24) drugs having a therapeutic effect on disorders of bowel motility and/or water flux, including metoclopramide, alosetron, tegaserod, prucalopride, and cisapride;

(25) drugs having a therapeutic effect on inflammatory bowel disease, including sulfasalazine, olsalazine, balsalazide, and infliximab;

(26) antimalarials, including chloroquine, mefloquine, pyrimethamine, atovaquone, proguanil, and primaquine;

(27) antiprotozoan agents, including metronidazole, miltefosine, nitazoxanide, paromomycin, pentamidine, and suramin;

(28) antihelminthic agents, including benzimidazoles, ivermectin, and praziquantel;

(29) antibacterial agents, including sulfanilamide, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfacetamide, trimethoprim, nalidixic acid, cinoxacin, ciprofloxacin, norfloxacin, sparfloxacin, fleroxacin, pefloxacin, levofloxacin, garenoxacin, gemifloxacin, ofloxacin, tiamulin, tetracyclines, erythromycin, penicillins, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, bacampicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, cephalosporins, imipenem, aztreonam, gentamicin, tobramycin, amikacin, netilmycin, kanamycin, neomycin, clarithromycin, azithromycin, clindamycin, telithrocymin, quinupristin, dalfopristin, linezolid,

spectinomycin, vancomycin, teicoplanin, daptomycin, and rifamycins;

(30) antifungal agents, including amphotericin B, flucytosine, ketoconazole, itraconazole, fluconazole, voriconazole, terbinafine, clotrimazole, econazole, miconazole, tolnaftate, and naftifine;

(31) antiviral agents, including acyclovir, valacyclovir, cidofovir, famciclovir, foscarnet, fomivirsen, ganciclovir, amantidine, rimantidine, and oseltamivir;

(32) antiretroviral agents, including zidovudine, didanosine, stavudine, zalcitabine, lamivudine, abacavir, tenofovir, emtricitabine, nevarapine, efavirenz, delavirdine, saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, and enfuvirtide;

(33) immunomodulators, including cyclosporine, mycophenolate mofetil, sirolimus, tacrolimus, azathioprine, daclizumab, and basiliximab;

(34) growth factors, including adrenomedullin, autocrine mobility factor, bone morphogenetic proteins, epidermal growth factor, erythropoietin, fibroblast growth factor, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, growth differentiation factor 9, hepatocyte growth factor, insulin-like growth factor, migration stimulating factor, myostatin, nerve growth factor, platelet- derived growth factor, thrombopoietin, transforming growth factor-a, transforming growth factor β, vascular endothelial growth factor, and placental growth factor;

(35) hormones;

(36) nucleic acid and nucleotide sequences for nonviral gene therapy;

(37) antiangiogenic compounds;

(38) antisense oligonucleotides; and

(39) antibodies that specifically bind a pharmacologically significant molecule, such as a receptor; and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof. As used herein, the term "antibody" includes all antibody derivatives with appropriate binding specificity, including naturally occurring antibodies, monoclonal antibodies, chimeric antibodies, humanized

antibodies, and single-chain antibodies such as sFv antibody derivatives.

[0125] The intermediate release linker that holds the therapeutic agent in place via ionic, covalent, or hydrophobic linkages can be further stabilized by a variety of techniques. These techniques can include various crosslinking modalities, some of which may offer various degrees of resistance to biodegradation. These crosslinking modalities can include the use of aldehydes such as formaldehyde to generate reversible crosslinks or glutaraldehyde to generate irreversible crosslinks. Alternatively, these crosslinks can be generated biologically through the activity of transglutaminases or other enzymes, which would require the insertion of suitable amino acid moieties into the primary structure of the intermediate release linker, such as free amino groups, free carboxyl groups, or combinations of such groups.

[0126] In another alternative, the composition can include cell-penetrating peptides and protein transcription-activating peptides, such as oligo-arginine and transcription activator peptides, to enable the internalization of agents that otherwise would not be taken up effectively by cancer cells because of the lipophilic barrier generated by cell membranes.

[0127] Cell-penetrating peptides include, but are not limited to, the following alternatives.

[0128] One group of alternatives for cell-penetrating peptides are the cell- penetrating peptides disclosed in United States Patent No. 7,754,678 to Guo et al., including RRHHCRSKAKRSRHH (SEQ ID NO: 20), SRRHHCRSKAKRSRHH (SEQ ID NO: 21), SARHHCRSKAKRSRHH (SEQ ID NO: 22), SRAHHCRSKAKRSRHH (SEQ ID NO: 23), SRRAHCRSKAKRSRHH (SEQ ID NO. 24), SRRHACRSKAKRSRHH (SEQ ID NO: 25), SRRHHARSKAKRSRHH (SEQ ID NO: 26), SRRHHCRAKAKRSRHH (SEQ ID NO: 27), SRRHHCRSAAKRSRHH (SEQ ID NO: 28), SRRHHCRSKAARSRHH (SEQ ID NO: 29), SRRHHCRSKAKASRHH (SEQ ID NO: 30), SRRHHCRSKAKRARHH (SEQ ID NO: 31), SRRHHCRSKAKRSAHH (SEQ ID NO: 32), RRHHCRSKAKRSR (SEQ ID NO: 33), RKGKHKRKKLP (SEQ ID NO: 34), GRKGKHKRKKLP (SEQ ID NO: 35), and GRRHHCRSKAKRSRHH (SEQ ID NO: 36).

[0129] Another group of alternatives for cell-penetrating peptides are the peptides disclosed in United States Patent No. 7,709,606 to Jalinot et al., including NRKKRRQRRR (SEQ ID NO. 37), RRRRRRR (SEQ ID NO: 38), RRRRRRRR (SEQ ID NO: 39), and RRRRRRRRR (SEQ ID NO: 40).

[0130] Yet another group of alternatives for cell-penetrating peptides are the D- amino-acid containing peptides disclosed in United States Patent No. 7,704,954 to Szeto et al., including Tyr-D-Arg-Phe-Lys-NH₂, 2',6'-Dmt- D-Arg-Phe-Lys-NH₂, Phe-D- Arg-Phe-Lys-NH₂, D-Arg-2',6'-Dmt-Lys-Phe-NH₂, and 2',6'-Dmp-D-Arg-Phe-Lys-NH₂.

[0131] Yet another group of alternatives for cell penetrating peptides are the peptides disclosed in United States Patent No. 7,579,318 to Divita et al., including

(SEQ ID NO: 41), wherein Xi is selected from the group consisting of A, L, and G, X₂ is selected from the group consisting of W and a peptide bond, X3 is selected from the group consisting of R and K, X4 is selected from the group consisting of K, L, and S, X5 is selected from the group consisting of L and K, Χβ is selected from the group consisting of R and W, X₇ is selected from the group consisting of K and S, Xe is selected from the group consisting of A, V, and Q, and X9 is selected from the group consisting of W, F, Y, and a non-amino-acid aromatic group. Additional non-peptide moieties can be covalently bound to this peptide sequence, in order to improve the overall stability of the molecule, and/or to provide it with additional properties, such as targeting ability. For example, a moiety such as cysteamide, a cysteine, a thiol, an amide, a carboxyl moiety, a linear or branched Ci_-6 optionally substituted alkyl moiety, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, or a polyethylene glycol can be covalently linked to the carboxyl terminus of the peptide sequence. A moiety such as an acetyl moiety, a fatty acid moiety, a cholesterol moiety, or a polyethylene glycol can be covalently linked to the amino terminus of the peptide sequence. Additionally, in some alternatives, for example in the amino-terminal addition of cholesterol, a secondary peptide bridge can be used to bind a non-peptide molecule to the peptide sequence. Preferred examples of this alternative of the cell-penetrating peptide are

GLWRALWRLLRSLWRLLWKA (SEQ ID NO: 42), GLWRALWRALWRSLWKLKRKV (SEQ ID NO: 43), GLWRALWRALRSLWKLKRKV (SEQ ID NO: 44),

GLWRALWRGLRSLWKLKRKV (SEQ ID NO: 45), GLWRALWRGLRSLWKKKRKV (SEQ ID NO: 46), GLWRALWRLLRSLWRLLWKA (SEQ ID NO: 47),

GLWRALWRALWRSLWKLKWKV (SEQ ID NO: 48), GLWRALWRALWRSLWKSKRKV (SEQ ID NO: 49), GLWRALWRALWRSLWKKKRKV (SEQ ID NO: 50), and

GLWRALWRLLRSLWRLLWSQ (SEQ ID NO: 51).

[0132] Yet another group of alternatives for cell-penetrating peptides are the peptides disclosed in United States Patent No. 7,576,058 to Lin et al. These peptides include AAVALLPAVLLALLAPAAADQNQLMP (SEQ ID NO: 52) and

AAVALLPAVLLALLAPAAANYKKPKLMP (SEQ ID NO: 53).

[0133] Other cell-penetrating peptides are known in the art.

[0134] Such cell-penetrating peptides are typically covalently bound to the targeting composition by reactions such as those described below.

[0135] Transcription-activating peptides include, but are not limited, to peptides disclosed in United States Patent No. 7,087,711 to Ptashne et al. These peptides include QLPPWL (SEQ ID NO: 54), QFLDAL (SEQ ID NO: 55), LDSFYV (SEQ ID NO: 56), PPPPWP (SEQ ID NO: 57), SWFDVE (SEQ ID NO. 58), QLPDLF (SEQ ID NO: 59), PLPDLF (SEQ ID NO: 60), FESDDI (SEQ ID NO: 61), QYDLFP (SEQ ID NO. 62), LPDLIL (SEQ ID NO: 63), LPDFDP (SEQ ID NO: 64), LFPYSL (SEQ ID NO: 65), FDPFNQ (SEQ ID NO: 66), DFDVLL (SEQ ID NO: 67), HPPPPI (SEQ ID NO: 68), LPGCFF (SEQ ID NO: 69), QYDLFD (SEQ ID NO. 70), YPPPPF (SEQ ID NO: 71), PLPPFL (SEQ ID NO: 72), LPPPWL (SEQ ID NO: 73), VWPPAV (SEQ ID NO: 74), DPPWYL (SEQ ID NO: 75), LY (SEQ ID NO: 76), FDPFGL (SEQ ID NO: 77), PPSVNL (SEQ ID NO: 78), YLLPTCIP (SEQ ID NO: 79), LQVHNST (SEQ ID NO: 80),

VLDFTPFL (SEQ ID NO: 81), HHAFYEIP (SEQ ID NO: 82), PWYPTPYL (SEQ ID NO: 83), YPLLPFLPY (SEQ ID NO: 84), YFLPLLST (SEQ ID NO: 85), FSPTFWAF (SEQ ID NO. 86), and LIMNWPTY (SEQ ID NO: 87). [0136] Other transcription-activating peptides are known in the art.

[0137] Such transcription-activating peptides are typically covalently bound to the targeting composition by reactions such as those described below.

[0138] Suitable reagents for cross-linking many combinations of functional groups are known in the art. For example, electrophilic groups can react with many functional groups, including those present in proteins or polypeptides. Various combinations of reactive amino acids and electrophiles are known in the art and can be used. For example, N-terminal cysteines, containing thiol groups, can be reacted with halogens or maieimides. Thiol groups are known to have reactivity with a large number of coupling agents, such as alkyl halides, haloacetyl derivatives, maieimides, aziridines, acryloyl derivatives, arylating agents such as aryl halides, and others. These are described in G. T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), pp. 146-150, incorporated herein by this reference. The reactivity of the cysteine residues can be optimized by appropriate selection of the neighboring amino acid residues. For example, a histidine residue adjacent to the cysteine residue will increase the reactivity of the cysteine residue. Other combinations of reactive amino acids and electrophilic reagents are known in the art. For example, maieimides can react with amino groups, such as the ε-amino group of the side chain of lysine, particularly at higher pH ranges. Aryl halides can also react with such amino groups. Haloacetyl derivatives can react with the imidazolyl side chain nitrogens of histidine, the thioether group of the side chain of methionine, and the ε-amino group of the side chain of lysine. Many other electrophilic reagents are known that will react with the ε-amino group of the side chain of lysine, including, but not limited to, isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide esters, sulfonyl chlorides, epoxides, oxiranes, carbonates, imidoesters, carbodiimides, and anhydrides. These are described in G.T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), pp. 137-146, incorporated herein by this reference. Additionally, electrophilic reagents are known that will react with carboxylate side chains such as those of aspartate and glutamate, such as diazoalkanes and diazoacetyl compounds,

carbonydilmidazole, and carbodiimides. These are described in G. T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), pp. 152-154, incorporated herein by this reference. Furthermore, electrophilic reagents are known that will react with hydroxyl groups such as those in the side chains of serine and threonine, including reactive haloalkane derivatives. These are described in G. T.

Hermanson, "Bioconjugate Techniques," (Academic Press, San Diego, 1996), pp. 154- 158, incorporated herein by this reference. In another alternative embodiment, the relative positions of electrophile and nucleophile (i.e., a molecule reactive with an electrophile) are reversed so that, for example, if a protein is to be coupled to another molecule, the protein to be coupled has an amino acid residue with an electrophilic group that is reactive with a nucleophile and the molecule with which the protein to be coupled includes therein a nucleophilic group. This includes the reaction of aldehydes (the electrophile) with hydroxylamine (the nucleophile), described above, but is more general than that reaction; other groups can be used as electrophile and nucleophile. Suitable groups are well known in organic chemistry and need not be described further in detail.

[0139] Additional combinations of reactive groups for cross-linking are known in the art. For example, amino groups can be reacted with isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, alkylating agents, imidoesters, carbodiimides, and anhydrides. Thiol groups can be reacted with haloacetyl or alkyl haiide derivatives, maleimides, aziridines, acryloyl derivatives, acylating agents, or other thiol groups by way of oxidation and the formation of mixed disulfides. Carboxy groups can be reacted with diazoalkanes, diazoacetyl compounds, carbonyldiimidazole, carbodiimides.

Hydroxyl groups can be reacted with epoxides, oxiranes, carbonyldiimidazole, Ν,Ν'- disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, periodate (for oxidation), alkyl halogens, or isocyanates. Aldehyde and ketone groups can react with hydrazines, reagents forming Schiff bases, and other groups in reductive amination reactions or Mannich condensation reactions. Still other reactions suitable for cross- linking reactions are known in the art. In some cases, it may be desirable to introduce a specific functional group that can subsequently be cross-linked. Such functional groups that can be introduced for cross-linking purposes can include, for example, sulfhydryl groups, carboxylate groups, primary amine groups, aldehyde groups, and hydrazide groups. Such cross-linking reagents and reactions, including the introduction of suitable functional groups for cross-linking, are described in G.T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), incorporated herein in its entirety by this reference.

[0140] The intermediate release linker of the composition is typically a polymer that shields the therapeutic agent of the composition from clearance by macrophages. The polymer can be a protein polymer or a non-protein polymer. If the polymer is a protein polymer, the protein polymer can be, but is not limited to, a protein such as albumin or gelatin. Other suitable proteins are known in the art and include, but are not limited to, keyhole limpet hemocyanin, ferritin, and ovalbumin. If albumin is used, it is typically bovine serum albumin, although analogous serum albumin proteins from other species, such as rats, mice, or horses, can also be used. As used herein, the term "protein" includes synthetic polypeptides including polypeptides of random sequence or defined sequence, block synthetic polypeptides that contain multiple regions, each region being comprised of residues of the same amino acid, or synthetic polypeptides of alternating sequence (i.e., polymers of a defined dipeptide or tripeptide); the synthetic polypeptides can be linear or branched, and many variations are possible. Typically, such synthetic polypeptides are produced by polymerization of a-amino acid-/V- carboxyanhydrides (NCAs); the production and use of synthetic polypeptides are described in T.J. Deming, "Synthetic Polypeptides for Biomedical Applications," Prog. Polymer Sci. 32: 858-875 (2007), incorporated herein in its entirety by this reference. Typically, if the intermediate release linker is a protein, it possesses at least one metalloprotease cleavage site for better local delivery, especially to tumor cells, and less systemic toxicity. If the intermediate release linker is a protein, it can optionally be substituted with polyethylene glycol moieties (pegylation). When the protein

intermediate release linker is pegylated, typically, the length of the polyethylene glycol chains, expressed in terms of the number of ethylene glycol monomers, is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers. Preferably, the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers. More preferably, the length of the polyethylene glycol chains is about 32 ethylene glycol monomers (i.e., polyethylene glycol with a molecular weight of about 2000 daltons). The polyethylene glycol chains can be attached through reactive groups, including reactive groups in side chains of amino acids in the polypeptide sequence of the protein, such as hydroxyl groups in serine or threonine or the ε-amino groups of lysine. Procedures for coupling polyethylene glycol groups to protein molecules are well known in the art; for example, and not by way of limitation, coupling can be performed by the creation of a reactive electrophilic intermediate that is capable of spontaneously coupling to nucleophilic residues on another molecule. To preserve the specificity of binding, the polyethylene glycol groups can be blocked at one end (the end not bound to the protein) with a methyl ether group. Methods for pegylation of proteins and other polypeptide sequences are described in G.T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), pp. 605-618, incorporated herein by this reference. Preferably, the intermediate release linker does not interact with the therapeutic agent and does not bind to or otherwise interact with the targeting moiety.

[0141] If a natural protein rich in thiol groups is desired, peptides derived from keratin can be used, and if long segments of hydrophobic residues are to be used for the endothermic attachment of nonpolar drugs, peptides derived from elastin, or biosynthesized sequences which mimic such sequences, can be used.

[0142] If the intermediate release linker is a non-protein polymer, it is typically polyethylene glycol, although analogous polymers, such as polypropylene glycol, can be used. When the intermediate release linker is polyethylene glycol, typically, the length of the polyethylene glycol chains, expressed in terms of the number of ethylene glycol monomers, is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers. Preferably, the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers. More preferably, the length of the polyethylene glycol chains is about 32 ethylene glycol monomers (i.e., polyethylene glycol with a molecular weight of about 2000 daltons). [0143] Typically, the linkage between the therapeutic agent and the intermediate release linker is a covalent linkage, such as a covalent linkage involving the reactive moieties and the cross-linking agents described above, or other reactive moieties and cross-linking agents known in the art. In some alternatives, the therapeutic agent and the intermediate release linker can be derivatized by peptides, such as linkers such as TGEKP (SEQ ID NO: 85) and the longer linker TGGGGSGGGGTGEKP (SEQ ID NO: 86). Modifications of the longer linker of SEQ ID NO: 86 can also be used. For example, the polyglycine runs of four glycine (C) residues each can be of greater or lesser length (i.e., 3 or 5 glycine residues each). The serine residue (S) between the polyglycine runs can be replaced with threonine (T). The TGEKP (SEQ ID NO: 85) moiety that comprises part of the linker TGGGGSGGGGTGEKP (SEQ ID NO: 86) can be modified as described above for the TGEKP (SEQ ID NO: 85) linker alone. Still other linkers are known in the art and can alternatively be used. These include the linkers LRQKDGGGSERP (SEQ ID NO: 87), LRQKDGERP (SEQ ID NO: 88),

GGRGRGRGRQ (SEQ ID NO: 89), QNKKGGSGDGKKKQHI (SEQ ID NO: 90),

TGGERP (SEQ ID NO: 91), ATGEKP (SEQ ID NO. 92), and GGGSGGGGEGP (SEQ ID NO: 93), as well as derivatives of those linkers in which amino acid substitutions are made as described above for TGEKP (SEQ ID NO. 85) and TGGGGSGGGGTGEKP (SEQ ID NO: 86). For example, in these linkers, the serine (S) residue between the diglycine or polyglycine runs in QNKKGGSGDGKKKQHI (SEQ ID NO: 90) or

GGGSGGGGEGP (SEQ ID NO: 93) can be replaced with threonine (T). In

GGGSGGGGEGP (SEQ ID NO: 93), the glutamic acid (E) at position 9 can be replaced with aspartic acid (D). Other linkers such as glycine or serine repeats are well known in the art to link peptides such as single chain antibody domains. These linkers are described in United States Patent Application Publication No. 2007/0178499 by Barbas, III, incorporated herein in its entirety by this reference. Still other linkers are known in the art; some suitable linkers are described, for example in United States Patent No. 6,936,439 to Mann et al., incorporated herein by this reference. Such linkers typically comprise short oligopeptide regions that typically assume a random coil conformation. The linker typically consists of less than about 15 amino acid residues, more typically about 4 to 10 amino acid residues. When both the therapeutic drug and the intermediate release linker are derivatized by peptides, the linkage between the therapeutic agent and the intermediate release linker can then be a peptide (amide) bond formed between these peptides. In another alternative, the covalent linkage between the therapeutic agent and the intermediate release linker is a cleavable linker, such as, for example, cathepsin-cleavable linkers such as Val-Cit which are cleaved by intracellular cathepsins. Cleavable linkers include di-, tri- and tetrapeptide subunits of cathepsin B, D, and L. Other cleavable linkers include acid-cleavable groups such as hydrazones which may be cleaved by endocytosis or through intracellular interaction with lysosomes. Still other cleavable linkers include acid-labile linkers. Examples of acid-labile linkers include linkers containing an orthoester group, a hydrazone, a cis- acetonyl, an acetal, a ketal, a silyl ether, a silazane, an imine, a citriconic anhydride, a maleic anhydride, a crown ether, an azacrown ether, a thiacrown ether, a dithiobenzyl group, a cis-aconitic acid, a cis-carboxylic alkatriene, methacrylic acid, and mixtures thereof. Examples of acid-labile groups and linkers are provided in United States Patent No. 7,098,032 to Trubetskoy et al., United States Patent No. 6,897,196 to Szoka, Jr., et al., United States Patent No. 6,426,086 to Papahadjopolous et al., United States Patent No. 7,138,382 to Wolff et al., United States Patent No. 5,563,250 to Hylarides et al., and United States Patent No. 5,505,931 to Pribish, all of which are incorporated herein in their entirety by this reference. Additional cleavable linkers include, but are not limited to, protease sensitive cleavable peptide linkers, nuclease sensitive cleavable nucleic acid linkers, lipase sensitive cleavable lipid linkers, glycosidase sensitive cleavable carbohydrate linkers, pH sensitive cleavable linkers such as acid-labile cleavable linkers or base-labile cleavable linkers, photo-cleavable linkers, heat-labile cleavable linkers, cleavable linkers that are cleaved by the action of a hydrolytic enzyme (i.e., esterase cleavable linkers), and others. Cleavable linkers are described, for example, in United States Patent Application Publication No. 2010/0183727 by lannacone et al., United States Patent Application Publication No. 2010/0112042 by Polisky et al., United States Patent Application Publication No. 2010/0129392 by Shi et al., and United States Patent Application Publication No. 2010/0184831 by Hart et al., all of which are incorporated herein in their entirety by this reference. Cleavable linkers also include linkers containing disulfide groups, which can be cleaved by reduction, linkers containing glycols, which can be cleaved by periodate, linkers containing diazo groups, which can be cleaved by dithionite, linkers containing ester groups, which can be cleaved by hydroxylamine, and linkers containing sulfones, which can be cleaved by bases. Further details on such cleavable linkers are provided in G.T. Hermanson, "Bioconjugate Techniques" (Academic Press, San Diego, 1996), pp. 292-296, hereby incorporated in its entirety by this reference.

[0144] In another alternative, the linkage between the intermediate release linker and the therapeutic agent can be a non-covalent linkage. If the linkage is a non- covalent linkage, it must be sufficiently stable to withstand storage and delivery and not be disrupted until the composition reaches its target cell, tissue, or organ in order to insure that targeting is specific. If the linkage between the intermediate release linker is a non-covalent linkage, it is typically a biotin/avidin or biotin/streptavidin linkage. As used herein, the term "biotin" encompasses both biotin (hexahydro-2-oxo-1 -/-thieno[3,4- d]imidazole-4-pentanoic acid) itself, or its lysine derivative biocytin (e-W-biotinyl-L- lysine).

[0145] In yet another alternative, a specific antigen/antibody or hapten/antibody linkage can be used to couple the therapeutic drug and the intermediate release linker. As used herein, the term "antibody" includes all antibody derivatives with appropriate binding specificity, including naturally occurring antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and single-chain antibodies such as sFv antibody derivatives.

[0146] In a targeting composition according to the present invention, the intermediate release linker is further linked to the targeting moiety. Typically, the linkage between the intermediate release linker and the targeting moiety is a covalent linkage as described above; cleavable linkers can also be used in some alternatives, as described above. In some alternatives, either the intermediate release linker or the targeting moiety can be extended with a peptide linker such as described above. In some alternatives, the linkage between the intermediate release linker and the targeting moiety is a non-covalent linkage, such as a biotin/avidin or biotin/streptavidin. Other non-covalent linkages are possible alternatives, as described above, including antigen/antibody or hapten/antibody linkages.

[0147] In another alternative, the composition comprises a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers.

[0148] When a liposome is employed, because of the nature of the relatively large nanoparticle we contemplate using (around 100 nm diameter, which represents around 1/3 the length of a collagen molecule, it may be desirable to insert multiple binding motifs on the surface of such a sphere to assure good linking and to stabilize its attachment. On the other hand it is possible that the protruding PEG chains may suffice to achieve this goal. In one alternative, the protruding peptides can be extended by inserting repeating sequences of glycine. Glycine provides maximum rotation around peptide bonds, and therefore maximal degree of motion. When such polyglycine extensions are employed, the polyglycine extensions typically range up to 50 repeating units. The displacement between the most adjacent binding sites among parallel oriented molecules is in the range of 2-4 nm. Free random movement of the glycine chains and the generation of as many attachments as possible are desirable. In another alternative, the extensions can be made more rigid, such as by using repeating Gly-Pro-Pro-Gly sequences) to generate a collagen like rigid triple helical extension radiating from surface of the liposome.

[0149] The properties of the liposomes that are employed in compositions in this alternative of the present invention depend on the chemotherapeutic agent to be carried by the liposomes, the details of the targeting process, pharmacokinetic considerations related to the persistence of the liposomes in the circulation and their clearance by the body, especially by the reticuloendothelial system.

[0150] Independently of the localization of cancer throughout the body, i.e., its topology, and the topographic features of the cancer at the supracellular level, as expressed in the existence of solid tumors as opposed to micrometastatic clusters or circulating single malignant cells, it is desirable that liposomes remain undetectable by the host for as long as possible. This is to increase the probability of accumulating at the sites of disease and also to minimize the chance of localizing at sites of normal (healthy) organs, where liposomes will eventually decompose and release their therapeutic contents, thus resulting in increased toxicity to the host. Therefore, it is desirable to prevent localization of the liposomes in tissues other than malignant tissues.

[0151] In particular for vascularized tumors, the selective accumulation and retention of liposomes and other particles (with sizes on the order of several nm) compared with smaller compounds is a result of leaky tumor neovasculature, malfunctioning lymphatics and high interstitial pressures. The enhanced permeability and retention (EPR) effect that describes this mechanism has been verified

experimentally by several groups. Owing to their size, liposomes are ideal for selective tumor accumulation. For optimum clinical efficacy, liposomes are required to

extensively sustain their blood circulation times so as to increase the probability that they will extravasate into the tumor. Slow clearance of liposomes, to improve the probability of specific binding to cancer cells, is also required when the targets are avascular micrometastases that either circulate in the blood or are suspended in the peritoneal cavity.

[0152] One critical variable for therapeutic efficacy in the administration of liposomes to target cancer cells is the circulation time of the liposomal

chemotherapeutic drug carriers, and it is desirable to increase this if possible. It is understood that, although liposomes circulate in the bloodstream or diffuse in other biological fluids, they are attacked through several mechanisms that aim to eventually clear them from the system. The attacking mechanisms that result in liposome clearance involve insertion into or adsorption of proteins on the membrane or recognition of tumor-targeting antibodies or peptides on the liposome surface.

Lipoproteins destabilize the membrane by lipid removal, which causes leakage of the contents. Several plasma proteins are suggested to be responsible for the presentation of liposomes in a form recognizable by macrophages that transport them to the liver and spleen. Therefore, for therapeutic targeting employing liposomes, it is desirable to increase their circulation of time in the bloodstream and retard their clearance. [0153] Size is important in determining the rate of clearance of liposomes in vivo. It is well established that an increase in size enhances the splenic uptake of particles, thus reducing the expected lifespan of liposomes in circulation. The splenic accumulation of large liposomes containing ganglioside GM1 has been evaluated and it is reported that liposomes with sizes above approximately 300 nm in diameter will accumulate in the spleen quickly and to a large extent (almost 40%, or higher, of the injected dose), probably owing to physical entrapment of these large particles into the splenic sinusoids. An increase in liposome diameter has also been suggested to result in an increase of the surface area of liposomes that, after complement activation, might be recognized more easily, giving rise to faster clearance of increasingly large liposomes. Therefore, it is typically preferred to use smaller liposomes. However, the optimal size of liposomes depends as well on other factors, such as charge, rigidity, and the presence or absence of attachments such as polyethylene glycol (PEG) moieties, as described further below.

[0154] Liposome charge also influences the electrostatic adsorption of proteins and, therefore, the clearance times and interactions with cells and tissues. For cationic complexes containing liposomes used in gene therapy, pronounced cytokine-related toxicity has been observed and is suggested to occur owing to enhanced accumulation and uptake of complexes including liposomes by Kupffer cells that would in turn activate inflammatory responses. The effect of negative charge of liposomes on complement activation has also been studied extensively. In general, it is preferred to use neutral or negatively-charged liposomes rather than positively-charged liposomes.

[0155] For long circulation times, pegylation of drug-carrier surfaces has been explored and has revolutionized in vivo drug-delivery, including liposomes, micelles and proteins. Surface-grafted PEG chains, as a result of their high mobility and hydration in water, are thought to stabilize the liposome surface sterically and increase the circulation times of liposomes. In particular, pegylation results in long-circulating liposomes regardless of the liposome surface charge or the presence of cholesterol in the liposome membrane. [0156] Accordingly, in compositions of the present invention, it is generally preferred to employ pegylated liposomes. However, the present invention is not limited to pegylated liposomes; other types of liposomes can be used, particularly if other means known in the art to increase liposome stability and improve the circulation lifetimes of liposomes are employed.

[0157] It is suggested that PEG provides a steric barrier to protein adsorption on the liposome surface, that it alters liposome interactions with cells (including cells of the reticuloendothelial system [RES]) and/or that it results in reduced liposome aggregation. However, Applicants are not bound by this theory concerning the mechanism of increased stability liposomes that are derivatized with polyethylene glycol chains. The level of pegylation (PEG grafting density) does not seem to influence total protein adsorption from plasma but it does seem to influence the adsorption kinetics and the types or sizes of particular proteins that adsorb on the liposome surface. For short incubation times (2 minutes), low-grafted PEG densities in mushroom conformations (at concentrations not shielding the liposome surface entirely) almost abort specific binding between avidin and surface-grafted biotins and it was suggested that pegylation might have an effect on the diffusion of avidin on the liposome surface owing to the higher energy barrier presented by the grafted PEG chains, which, in a way, need to be

"pushed" by avidin in order for the latter to bind to biotin. For the effect of pegylation in vivo, it has also been suggested that it might be a result partly of the role of PEG as a steric barrier to approaching macrophages by decreasing liposome uptake by these cells, which results in delayed clearance of PEG-modified liposomes, although adsorption of serum proteins might still occur on the liposome surface; in such cases, the adsorption of serum proteins on the liposome surface may not adversely impact the circulatory lifespan of such liposomes. Recently, in a well designed study, very strong evidence shows that a major mechanism by which pegylation extends circulation time would be to prohibit not the adsorption of proteins on the liposome surface but the approximation and aggregation of liposomes into larger liposomal structures while in the circulation. Aggregation, leading to the formation of larger structures (not necessarily resulting in fusion) of non-pegylated liposomes can explain their short circulation times. [0158] Various approaches for surface pegylation have been pursued. Pegylated liposome surfaces with multiloop PEG chains containing hydrophobic

"anchoring chains" protect liposomes from complement binding more than do grafted linear PEG chains and exhibit surface protection of the liposomes for longer times. Other pegylated surface architectures include branched PEG chains and tiered surface strategies with mixed lengths of extended PEG chains (potentially to present a molecular sieve for the different shapes and sizes of serum proteins); in vivo, these do seem to result in longer circulation times and higher tumor uptake than single-length grafted PEG chains. Any of these alternatives can be used in compositions according to the present invention. Commercial formulations (e.g., STEALTH^® liposomes) contain grafted PEG at relatively high surface densities (5% mole for 2000 molecular weight [ W] PEG) that should result in extended brush conformations, with lengths that might extend beyond the surface of adsorbed proteins, providing the above-mentioned steric barrier. Pegylation, however, enhances accumulation of liposomes to the skin and, in the case of liposomal formulations containing doxorubicin, it might increase the incidence of palmar-plantar erythrodysesthesia.

[0159] Other methods of derivatizing liposomes to enhance their circulatory lifespan are known, including the use of monosialoganglioside (GM1) and

phosphatidylinositol (PI), and are within the scope of the present invention. However, in most applications, the use of pegylated liposomes is preferred.

[0160] For small pegylated liposomes, the lipid composition probably determines the extent and types of surface-adsorbed plasma proteins that, in turn, affect specific liposome-cell interactions, thus determining the type of major toxicity or, in other words, the types of macrophages (hepatic, splenic, or macrophages from the bone marrow) or hepatocytes that will take up liposomes to a greater extent.

[0161] Engineering of liposomes for potential therapy of solid tumors is of particular significance because solid tumors still account for more than 85% of cancer mortality. Liposome extravasation into the tumor insterstitium is a substantially random event and, therefore, long liposome circulation times would increase accumulation within the tumor insterstitium. In addition, extravasation depends on the tumor's vasculature "pore cut-off size," which seems to be a function of the type of cancer cells and the tumor's topology. After liposomal extravasation as a result of high intratumoral pressures and lack of convection, low penetration depths (20-30 μ ) have been reported for liposomes. These low penetration depths seem to depend greatly on the liposome charge and size. Studies of liposome penetration profiles in cancer-cell spheroid analogues showed that neutral liposomes exhibit greater penetration depths compared with cationic liposomes owing to a lack of electrostatic binding to cells and smaller liposomes have higher penetration than liposomes of sizes that are larger, relative to the intercellular distances within the spheroids. In these spheroid studies, pegylation inhibited the interaction of liposomes with cells (no significant adsorption of liposomes was observed on the spheroid surface) and therefore no liposome

penetration into the spheroids was reported. Interestingly, the increased rigidity of the liposome membrane aborted attractive interactions between gel-phase liposomes and spheroids, which suggests that the mechanism of liposome introduction into the interstitium of cells comprising a spheroid and of cells comprising a tumor in vivo might be different (because, in vivo, tumor extravasation of both pegylated and rigid liposomes is observed). Nevertheless, the spheroid penetration profiles provide a valuable correlation of basic physicochemical characteristics of liposomes and their intratumoral transport capabilities. In addition, in vivo, after extravasation, liposomes are reported to exhibit perivascular distributions that are heterogeneous along the blood vessels with vessel leakiness decreasing towards the advancing edge of the neovasculature, which could be related to the locally variant physiology of the neovasculature present in tumor cells.

[0162] To relax the extracellular matrix (ECM) of the tumor and enhance liposome penetration, different approaches have been followed. In one approach, hyaluronidase, the ECM-degrading enzyme, was administered intratumorally preceding the administration of liposomal doxorubicin (Caelyx^®). The adjuvant use of

hyaluronidase has already been found in Phase I and II trials to improve prevention of tumor regrowth when administered with chemotherapy. Interestingly, intravenous administration of the enzyme was not toxic to normal tissues. Such strategies of ECM pretreatment could, in principle, be applied to other types of macromolecular and nanosized delivery carriers to improve the carrier distribution within tumors and can be used in methods according to the present invention.

[0163] For liposomes with surface-conjugated tumor-targeting antibodies, it would be expected that the penetration depth is also influenced by the targeting antibody's binding-site barrier. For intact immunoglobulin G molecules, for example, in breast cancer spheroids, the penetration depth of externally added fluorescence-labeled antibodies do not exceed 40 μ (after 5 hours of incubation) measured from the spheroid rim, with cancer cells that express higher levels of the IgG receptors exhibiting higher concentrations of bound antibody. The relative amount of tumor-extravasated liposomes with surface-conjugated antibodies might also be decreased owing to their faster clearance. In targeted cancer therapy by immunoliposomes, the Fc region of intact tumor-targeting IgGs that are used to increase targeting specificity might potentially be identified by phagocytic cells and might be removed from circulation, thereby reducing their lifetime in circulation. Advances in antibody engineering have been used for the development of antibody fragments (e.g., Fab', scFv) that lack the Fc region. Such fragments, when conjugated to pegylated liposomes, exhibit blood- clearance kinetics that are identical to nontargeted liposomes. However, for smaller fragments, tumor binding is decreased compared with the complete antibodies. In alternatives employing antibodies, it is possible to optimize both tumor binding and lifetime in circulation of the targeted liposomes.

[0164] For micrometastatic disease, direct targeting of molecular moieties on the cell surface, potentially followed by internalization of liposomes, is necessary to localize the therapeutic agent carried by the liposomes to the cancer cells and also allow them to be internalized by the tumor cells so that they can act intracellularly. In solid tumors, targeting of the vasculature and the cancer cells that constitute the tumor is being explored. In particular, both antiangiogenic and antivascular targets can be utilized in compositions according to the present invention to either prevent new blood vessel formation by targeting growth factor receptors on the vasculature endothelial cells ("antiangiogenic") or to damage and kill tumor cells by cutting the blood flow through the neovasculature, so as to deprive the tumor of growth factors ("antivascular").

[0165] Depending on the nature of delivered therapeutic modalities, cellular internalization of the targeting ligand and, therefore, of the targeted liposome, can be of great importance in cancer killing and can be utilized by compositions according to the present invention. For chemotherapeutic drugs whose molecular targets are located intracellular^, liposomal internalization would be expected to have a critical role in determining the drug's bioavailability. Comparison of immunoliposomes labeled with internalizing anti-CD19 antibodies and with non internalizing anti-CD20 antibodies for the delivery of doxorubicin into human B-lymphoma cells showed killing superiority in vitro and in vivo of internalizing liposomes. These liposomes were suggested to be decomposed by the hydrolytic action of endosomal and lysosomal enzymes, whereas the anti-CD20 liposomes were suggested to just bind to the cancer cells and to release the encapsulated drug over time with some of it diffusing away from the cell.

Internalization can have an important role even for liposomes that are used as delivery carriers in internal radiotherapy of cancer. Internalization of the carrier might not be critical for the delivery of β-particle emitters (recoil ranges of the order of a few mm), although, when a-particle or Auger-electron emitters are delivered that have shorter recoil lengths (100 pm and a few tens of nm, respectively), then internalization and close approximation to the nucleus would become critical. Accordingly, in one embodiment of the present invention, the targeted liposomes are internalized.

[0166] Three different immobilization architectures of targeting ligands on liposomes exist. These immobilization architectures are: (1) type A liposomes, in which ligands are conjugated directly on the phospholipid headgroups of non-pegylated liposomes; (2) type B liposomes, in which ligands are conjugated directly on the phospholipid headgroups of pegylated liposomes; and (3) type C liposomes, in which ligands are conjugated on the free termini of pegylated chains. Type A liposomes bind to target cells specifically and exhibit fast blood clearance that is restored by the addition of grafted PEG chains and the formation of type B liposomes, which, however, exhibit reduced targeting owing to steric hindrance of the targeting ligand by the neighboring polymer chains. These observations lead to type C liposomes resulting in minimal screening of the ligand by the neighboring grafted PEG chains, however, in vivo, their circulation time was inversely proportional to their conjugated ligand-grafting density. In the case of type C liposomes in which the ligands are antibodies, removal of the antibody's Fc region can, in principle, restore longer circulation times.

[0167] Other alternatives employing compositions according to the present invention are also within the scope of the present invention. In one such alternative, two separate liposomal preparations with different targeting sites are administered to the same organism in need of treatment. In another such alternative, known as

pretargeting, a reagent that specifically binds to the liposomes and directly targets the tumor, such as a suitable monoclonal antibody, is administered first. In one alternative, the monoclonal antibody or other targeting reagent is conjugated to one of two binding partners that use the biotin-avidin link, while the liposome is conjugated to the other of the two binding partners that use the biotin-avidin link. In another alternative, the monoclonal antibody or other targeting reagent and the liposome are both conjugated to biotin or a derivative or analogue of biotin, and a biotin-binding component is introduced to cross-link the monoclonal antibody or other targeting reagent to the liposome. The avidin-binding component can be selected from the group consisting of avidin, streptavidin, a derivative or analogue of avidin or streptavidin, and a biotin-binding antibody. In still another approach, the liposomes can contain a derivatized

chemotherapeutic moiety that binds to a bispecific antibody, and, prior to the

administration of the liposomes, the bispecific antibody is administered to the organism to be treated. The bispecific antibody binds both to the derivatized chemotherapeutic moiety of the liposome and to a tumor marker in the organism. In one example of this approach, the tumor marker is carcinoembryonic antigen (CEA), the derivatized chemotherapeutic moiety is indium-111-diethylenetriamine pentaacetic acid (DTPA)- derivatized phosphatidylethanolamine, and the bispecific antibody is a bispecific anti- CEA anti-indium DTPA antibody. Other alternatives for the tumor marker, the

derivatized chemotherapeutic moiety, and the bispecific antibody are known in the art. [0168] In still another approach, liposome compositions according to the present invention are used that include at least two antibodies, wherein each antibody is an antibody for a specific receptor on the surface of tumor cells in the organism to be treated, the receptors occurring in the same tumor cell. The rationale for this approach is based on the observation that several targeted receptors on tumor cells cluster on the surface of the membrane on binding to antibodies, thus, when clustered receptors are exposed to clustered antibodies (owing to their localization on the same liposomal platform), increased complex valency and avidity is expected to occur because of the decreased off-rates of multimerized antibodies or other targeting Iigands. This provides more efficient targeting.

[0169] In yet another approach according to the present invention, a liposome composition according to the present invention has two functionalities in addition to the targeting moiety described above. These two functionalities are: (1) a binding functionality to a noninternalizing receptor on the malignant cell's surface; and (2) an initially hidden functionality through a cell-penetrating peptide that is activated only after binding to the cell surface at slightly acidic conditions corresponding to the

approximately pH 6.0 of the tumor interstitium.

[0170] One targeting moiety that can be included in liposomes to target them to tumor cells, according to the present invention, is a peptide motif as described above that includes one or more CBDs as described above.

[0171] Accordingly, one aspect of the present invention is a composition comprising a liposome having attached to its surface a peptide motif (CBD) for binding the liposome to native collagen fibers.

[0172] Typically, the liposome used in the composition of the present invention is one that has a substantial circulation time after administration and allows efficient binding of the collagen-binding polypeptide described above.

[0173] Increasing the circulation time of liposomal drug carriers is of great significance, since they are recognized through several mechanisms that aim to eventually clear them from the system. Common organ-accumulation sites for liposomes are the liver, spleen and bone marrow. Depending on their sensitivity to the administered anticancer therapeutics, these organs could determine the dose-limiting toxicities. This could potentially prohibit administration of high doses with tumor- regressive responses. Liposome properties that seem to affect circulation include liposome size, lipid membrane rigidity, liposome charge and liposomal surface architecture (pegylation).

[0174] For long circulation times, pegylation of drug-carrier surfaces has been explored and has revolutionized in vivo drug-delivery, including liposomes, micelles, and proteins. Surface-grafted PEG chains, as a result of their high mobility and hydration in water, are thought to stabilize the liposome surface sterically and increase the

circulation times of liposomes. In particular, pegylation results in long-circulating liposomes regardless of the liposome surface charge or the presence of cholesterol in the liposome membrane.

[0175] Recent evidence shows that a major mechanism by which pegylation extends circulation time would be to prohibit not the adsorption of proteins on the liposome surface but the approximation and aggregation of liposomes into larger liposomal structures while in the circulation.

[0176] Targeted liposomal delivery to cancer cells aims to increase the therapeutic efficacy and to minimize nonspecific toxicities. Unfortunately, the surface of cancer cells does not seem to possess identifiable unique molecular targets. Although there is ongoing research to identify tumor-specific antigens or receptors for selective targeting of cancer cells, for many cases such targets might not exist. However, it is common for cancer cells to overexpress antigens/receptors that are relatively

downregulated in healthy cells, but they seem to be only of limited value in terms of specificity.

[0177] For micrometastatic disease, direct targeting of molecular moieties on the cell surface, potentially followed by internalization of liposomes, is believed to be necessary to localize the therapeutics at the site of the cancer cells. Although the inventors are not bound by this theory of the operation of the invention, the inventors believe that it only takes a small number of rapidly dividing cancer cells to alter the extracellular matrix in such a way as to expose naked collagen, thus providing the target for activated liposomes to attach. In solid tumors, targeting of the vasculature and the cancer cells that constitute the tumor is being explored. In particular, both

antiangiogenic and antivascular targets are pursued to either prevent new blood vessel formation by targeting growth factor receptors on the vasculature endothelial cells ("antiangiogenic") or to damage and kill tumor cells by cutting the blood flow through the neovasculature, so as to deprive the tumor of growth factors ("antivascular"). Again this targeting is not highly specific.

[0178] Unfortunately, it appears as if that, in vivo, immunolabeled liposomes exhibit the same tumor accumulation as nonimmunolabeled liposomes, although, at the cellular level, targeted liposomes were internalized to a much greater extent. Because high tumor interstitial pressure that increases the probability the extravasated liposomes will escape back into the bloodstream, targeted liposomes might exhibit enhanced tumor retention owing to liposome binding to cancer cells after extravasation.

[0179] Release of encapsulated contents is desired to occur after localization of liposomes within the tumor interstitium or after cellular uptake and accumulation into subcellular compartments to result in enhanced drug bioavailability. Release of liposome contents after their localization in the tumor interstitial space appears to take advantage of environmental stimuli that are endogenous to the tumor interstitial space (slightly acidic pH or excess of particular enzymes). Tumor interstitial pH is measured to be as low as 6.5 and has been used as the stimulus to activate content release.

[0180] These "localized therapies" aim to result in minimal toxicities that can be achieved as long as the encapsulated therapeutics are not released from liposomes localized at normal organs or as long as inactive encapsulated prodrugs are

decomposed and removed from the body with minimal side effects.

[0181] Accordingly, in one alternative of a composition according to the present invention, the diameter of the liposome is from about 50 nm to about 2000 nm.

Typically, the diameter of the liposome is from about 200 nm to about 2000 nm.

Preferably, the diameter of the liposome is about 1000 nm.

[0182] Accordingly, in another alternative of a composition according to the present invention, the liposome has polyethylene glycol (PEG) chains on its surface, i.e., is pegylated. Typically, the length of the polyethylene glycol chains, expressed in terms of the number of ethylene glycol monomers, is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers. Preferably, the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers. More preferably, the length of the polyethylene glycol chains is about 32 ethylene glycol monomers (i.e., polyethylene glycol with a molecular weight of about 2000 daltons).

[0183] When a composition according to the present invention has polyethylene glycol chains on the surface of the liposome, the polyethylene glycol chains can have either a free amino group or a free carboxyl group available for reaction. Alternatively, the polyethylene glycol chains can be derivatized with another reactive group that can be linked to a polypeptide or a therapeutic agent, such as a hydroxyl group or a carbonyl group.

[0184] In one preferred alternative, when a composition according to the present invention has polyethylene glycol chains on the surface of the liposome, and the polyethylene glycol chains have a free amino group, the peptide motif as described above can be linked to the polyethylene glycol chains through the formation of a peptide (amide) bond between the free amino group of the polyethylene glycol group and the free carboxyl group of the peptide motif. Alternatively, when the polyethylene glycol chains have free carboxyl groups, the peptide motif can be linked to the polyethylene glycol chains through the formation of a peptide (amide) bond between the free carboxyl group of the polyethylene glycol group and the free amino group of the peptide motif. These alternatives results in opposite orientations of the peptide motif.

[0185] The composition of the liposome is not critical; suitable proportions of ingredients for the preparation of liposomes are known in the art and are described, for example, in European Patent Application Publication No. EP 1332755 by Weng et al., incorporated herein in its entirety by this reference. In one alternative, the liposome can comprise from about 25% to about 35% of cholesterol, from about 60% to about 70% of dipalmitoylphosphatidylcholine (DPPC), and from about 2% to about 5% of a reactive pegylated lipid. The reactive pegylated lipid can be, but is not limited to, 1 ,2-distearoyl- sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol-2000). Other reactive pegylated lipids can be used. In this alternative, preferably, the liposome comprises from about 27.5% to about 32.5% of cholesterol. In this alternative, more preferably, the liposome comprises about 30% of cholesterol.

[0186] The liposome can also include a small proportion (less than about 1%, typically less than 0.1 %) of a standard fluorophore as a fluorescent marker to assist in determining the binding of the liposome to collagen. The fluorescent marker can be, but is not limited to, markers such as NDB II, which provides a green color, or rhodamine, which provides a pink color. The use of the fluorophore is optional and its omission does not interfere with the activity of the liposome. The fluorophore can be used for identifying and localizing liposomes clinically and in experimental studies to determine efficacy of the procedure.

[0187] Liposome compositions according to the present invention can be prepared according to standard liposome preparation techniques known in the art, such as those described in European Patent Application Publication No. EP 1332755 by Weng et al., supra. The peptide motif as described above can be either attached after liposome synthesis or attached to one of the ingredients of the liposome prior to liposome assembly. Typically, the peptide motif is attached to the reactive pegylated lipid as described above, although the peptide motif can alternatively be attached to one of the other components of the liposome. Typically, as described above, the peptide motif is attached through a free carboxyl group on the peptide motif to an amino group on the reactive PEG moiety, forming a peptide (amide) bond. In another alternative, the peptide motif is attached through a free amino group on the peptide motif to a carboxyl group on the reactive PEG moiety, again, forming a peptide bond. In other alternatives, other cross-linking methods as known in the art can be used for cross-linking the peptide motif to the liposome. These cross-linking methods are discussed below.

[0188] Additionally, a composition according to the present invention comprising a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers, as described above, can further comprise a therapeutic agent. The therapeutic agent can be either incorporated in the interior of the liposome or attached to the surface of the liposome.

[0189] Therefore, when liposomes are used, another aspect of the present invention is a composition comprising a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers. The peptide motif is one of the collagen binding domains described above. The composition can further comprise a therapeutic agent; alternatives for therapeutic agents are also described above. The therapeutic agent can be incorporated in the interior of the liposome;

alternatively, the therapeutic agent can be attached to the surface of the liposome. The liposome can further comprise a substance that can be identified by a radiological procedure selected from the group consisting of X-ray, MRI, and CT. The substance can be selected from the group consisting of a radio-opaque substance and a radioactive substance. When liposomes are employed, a pharmaceutical composition can comprise:

(1) a therapeutically effective quantity of the composition of the present invention with liposomes; and

[0190] Another aspect of the present invention is a pharmaceutical composition comprising:

(1) a therapeutically effective quantity of at least one targeting composition according to the present invention as described above; and

[0191] The targeting composition included in the pharmaceutical composition can, for example, include an anti-neoplastic therapeutic agent, an anti-inflammatory therapeutic agent, both an anti-neoplastic therapeutic agent and an anti-inflammatory therapeutic agent, or a therapeutic agent selected from the group consisting of:

(1 ) a muscarinic cholinergic receptor agonist;

(2) a muscarinic cholinergic receptor antagonist; (3) an anticholinesterase agent;

(4) a P2-selective adrenergic receptor agonist;

(5) a oc2-selective adrenergic receptor agonist;

(6) a sympathomimetic agonist;

(7) an on -selective adrenergic receptor antagonist;

(8) a β-selective adrenergic receptor antagonist;

(9) a 5-hydroxytryptamine receptor agonist;

(10 a 5-hydroxytryptamine receptor antagonist;

(1 1 a benzodiazepine;

(12 an antidepressant;

(13 an antipsychotic drug;

(14 an antiseizure drug;

(16 an opioid;

(17 a diuretic;

(18 an angiotensin converting enzyme inhibitor;

(19 a nonpeptide angiotensin II receptor antagonist;

(20 a calcium channel blocker;

(21 an antiarrhythmic drug;

(23 an H₂ receptor antagonist;

(24 a drug having a therapeutic effect on disorders of bowel motility and/or water flux;

(27 an antiprotozoan agent;

(28 an antihelminthic agent;

(29 an antibacterial agent; (30) an antifungal agent;

(31) an antiviral agent;

(32) an antiretroviral agent;

(33) an immunomodulator;

(34) a growth factor;

(35) a hormone;

(36) a nucleic acid or nucleotide sequence for nonviral gene therapy;

(37) an antiangiogenic compound;

(38) an antisense oligonucleotide; and

[0192] In another alternative, the pharmaceutical composition can include a first targeting composition that includes an anti-neoplastic therapeutic agent and a second targeting composition that includes an anti-inflammatory therapeutic agent. Other combinations of two or more targeting compositions in a pharmaceutical composition according to the present invention are also possible. In another alternative, a

pharmaceutical composition according to the present invention can include: (i) at least one targeting composition according to the present invention as described above; and (ii) at least one additional therapeutic agent. Various combinations are possible. For example, and not by way of limitation, the pharmaceutical composition can include: (i) a targeting composition including a first anti-neoplastic therapeutic agent; and (ii) a second anti-neoplastic therapeutic agent. As another alternative, the pharmaceutical composition can include: (i) a targeting composition including an anti-neoplastic therapeutic agent; and (ii) an anti-inflammatory therapeutic agent. As still another alternative, the pharmaceutical composition can include: (i) a targeting composition including a first anti-inflammatory therapeutic agent; and (ii) a second anti-inflammatory therapeutic agent. As yet another alternative, the pharmaceutical composition can include: (i) a targeting composition including an anti-inflammatory agent; and (ii) an antineoplastic therapeutic agent. Still other combinations are possible. [0193] Another aspect of the present invention is directed to targeting compositions intended to treat joint inflammation, including, but not limited to, joint inflammation associated with arthritis or cartilage or bone loss and repair. These conditions are typical connective tissue diseases which result in accelerated

degradation of collagen and decrease in collagen deposition. The exposed collagen, which can reach a maximum in these areas, can clearly serve as a target for the local delivery of growth factors, nutrients and bioactive compounds which will block or reverse these changes. Cartilage collagen is rapidly exposed during the inflammatory process associated with joint diseases, and bone collagen is also exposed to the targeting mechanism as mineral is lost during periods of bone resorption or active remodeling. Accordingly, in this alternative, the targeting composition comprises: : (1) a therapeutic agent for treating joint inflammation; (2) an intermediate release linker bound to the therapeutic agent; and (3) a targeting moiety bound to the intermediate release linker. Suitable intermediate release linkers and targeting moieties for use in this alternative of compositions according to the present invention are as described above.

[0194] Accordingly, using this approach, drugs such as bisphosphonates, which are so useful in inhibiting osteoclastic activity associated with bone resorption, could be targeted preferentially to areas of exposed collagen which are so apparent at such sites. Such bisphosphonates include, but are not limited to, medronate, clodronate,

etidronate, tiludronate, pamidronate, alendronate, ibandronate, risedronate, and zoledronate. Such bisphosphonates are useful in inhibiting osteoclastic activity associated with bone resorption and could be targeted preferentially to areas of exposed collagen which are so apparent at such sites. Accordingly, in this alternative, the therapeutic agent can be a bisphosphonate.

[0195] In another alternative, bone morphogenetic proteins (BMPs) or active portions thereof can be targeted to such areas of exposed collagen. Bone

morphogenetic proteins are described further below. Therefore, in this alternative, the therapeutic agent can be a bone morphogenetic protein (BMP) or an active portion thereof. [0196] The BMPs are described in further detail in the following publications: (1) F. P. Luyten et al., "Purification and Partial Amino Acid Sequence of Osteogenin, a Protein Initiating Bone Differentiation," J. Biol. Chem. 264: 13377-13380 (1989); (2) E. Ozkaynak et al., "Murine Osteogenic Protein (OP-1): High Levels of mRNA in Kidney," Biochem. Biophvs. Res. Commun. 179: 116-123 (1991); (3) R. M. Harland et al., "The Transforming Growth Factor β Family and Induction of the Vertebrate Mesoderm: Bone Morphogenetic Proteins are Ventral Inducers," Proc. Natl. Acad. Sci. USA 91 : 10243- 10246 (1994); (4) S. K. Maiti & G. R. Singh, "Bone Morphogenetic Proteins-Novel Regulators of Bone Formation," Ind. J. EXP. Biol. 36: 237-244 (1998); (5) J. M. Wozney et al., "Novel Regulators of Bone Formation: Molecular Clones and Activities," Science 242: 1528-1534 (1988); (6) D. M. Kingsley et al., "What Do BMPs Do in Mammals? Clues from the Mouse Short-Ear Mutation," Trends Genet. 10: 16-21 (1994); (7) C. Scheufler et al., "Crystal Structure of Human Bone Morphogenetic Protein-2 at 2.7 A Resolution," J. Mol. Biol. 287: 103-1 15 (1999); (8) J. Q. Feng et al., "Structure and Sequence of Mouse Bone Morphogenetic Protein-2 Gene (BMP-2): Comparison of the Structures and Promoter Regions of BMP-2 and BMP-4 Genes," Biochim. Biophvs. Acta 1218: 221-224 (1994); (9) N. Ghosh-Choudhury et al., "Expression of the BMP 2 Gene During Bone Cell Differentiation," Crit. Rev. Eukarvot. Gene Expr. 4: 345-355 (1994);

(10) B. L. Rosenzweig et al., "Cloning and Characterization of a Human Type II

Receptor for Bone Morphogenetic Proteins," Proc. Natl. Acad. Sci. USA 92: 7632-7636;

(1 1) L. J. Jonk et al., "Identification and Functional Characterization of a Smad Binding Element (SBE) in the JunB Promoter That Acts as a Transforming Growth Factor-β, Activin, and Bone-Morphogenetic-Protein-lnducible Enhancer," J. Biol. Chem. 273: 21 145-21152 (1998); and (12) M. Kawabata et al., "Signal Transduction by Bone Morphogenetic Proteins," Cytokine Growth Factor Rev. 9: 49-61 (1998). The BMPs represent a family of proteins that initiate, promote, and maintain cartilage and bone morphogenesis, differentiation and regeneration in both the developing embryo and the adult. There are more than 30 known BMPs, of which 15 are found in mammals. BMPs belong to the transforming growth factor β (ΤΰΡβ) superfamily, which includes ΤβΡβε, activins/inhibins, Mullerian-inhibiting substance (MIS) and glial cell line-derived neurotrophic factor. Comparison and alignment of the amino acid sequences of BMPs reveal that BMPs, except for BMP-1 , share a common structural motif that is distinct from the structure of BMP-1. These BMPs include BMP-2, BMP-3, BMP-3b, BMP4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8B, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF- 9, GDF-10, and nodal. In this specification, the term "BMP," without further qualification, is to be taken to include BMP-1 ; the term "BMP sharing a common structural motif is to be taken to include BMPs other than BMP-1. These BMPs sharing a common structural motif are disulfide-linked dimeric proteins. BMP-1 is not properly a BMP family member; rather it is a procollagen C proteinase related to Drosophila tolloid and which is postulated to regulate BMP activity through proteolysis of BMP antagonists/binding proteins. These growth factors can exist in multiple forms, such as: (1) splicing variants produced from mRNAs generated by spicing from alternative sites; (2) variants produced by proteolysis, such as the cleavage of signal peptides or propeptides; (3) variants produced by the presence or lack of glycosylation, typically N-linked

glycosylation; (4) naturally-occurring isoforms; (5) naturally-occurring mutations or allelic variants; and (6) artificial variants produced by genetic engineering in which one or more amino acids in the primary sequence are altered by techniques such as site- specific mutagenesis; such artificial variants are frequently designated muteins. In general, these multiple forms are within the scope of the present invention when they exist or can be produced for a particular growth factor. Additionally, growth factors useful in compositions according to the present invention can be incorporated into fusion proteins. Examples of fusion proteins incorporating bone morphogenetic proteins are disclosed in U.S. Pat. No. 6,352,972 to Nimni et al., incorporated herein by this reference. In general, such fusion proteins are also within the scope of the present invention when they exist or can be generated. These fusion proteins can incorporate multiple domains or domains from more than one naturally-occurring growth factor. They can also incorporate elements such as reporter genes or detection tags.

Therefore, the use of such fusion proteins incorporating growth factors, including but not limited to BMPs, is within the scope of the invention. The BMPs can be BMPs sharing a common structural motif and that are disulfide-linked dimeric proteins, such as, but not limited to, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8B, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, and nodal. A particularly preferred BMP for incorporation into a fusion protein is BMP-3.

[0197] In yet another alternative, a composition according to the present invention can include a growth factor. Growth factors suitable for incorporation into compositions according to the present invention include, but are not limited to, adrenomedullin (AM), autocrine mobility factor, bone morphogenetic proteins (BMPs) (considered to be growth factors and covered above), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatoma derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), novel neurotrophin-1 (NNT-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor a (TGF-a), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and placental growth factor (PGF); other growth factors are known in the art. A particularly suitable growth factor is GM-CSF.

[0198] In one alternative of a composition according to the present invention, a growth factor is the therapeutic agent.

[0199] In yet another alternative of a therapeutic composition according to the present invention, the therapeutic composition comprises: (1) a therapeutic agent as described above that is a polypeptide or protein; (2) an intermediate release linker bound to the therapeutic agent; (3) a targeting moiety bound to the intermediate release linker; and (4) a growth factor bound either to the polypeptide or protein therapeutic agent or the intermediate release linker. The growth factor that is bound either to the polypeptide or protein therapeutic agent or to the intermediate release linker is covalently linked to the polypeptide or protein therapeutic agent or to the intermediate release linker by one of the coupling reactions described above, depending on the functional groups available on the growth factor and the polypeptide for crosslinking as described above.

[0200] In this alternative, a particularly preferred growth factor is GM-CSF. In this alternative, the therapeutic agent is typically an anti-neoplastic therapeutic agent as described above.

[0201] In another alternative of the present invention, the therapeutic agent is a therapeutically effective radionuclide. In this alternative of the present invention, the targeting composition comprises:

(1 ) a therapeutically effective radionuclide;

[0202] The therapeutically effective radionuclide can be ¹³¹1. Typically, when the therapeutically effective radionuclide is ¹³¹1, the ¹³¹1 is covalently bound to the

intermediate release linker. Methods for radioiodination of proteins and polypeptides are well known in the art and are described, for example, in E. Harlow & D.Lane, "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1988), ch. 9, pp. 324-339, incorporated herein by this reference. Briefly, in one method, radioiodine can be converted to radioactive I², which then attacks tyrosyl and histidyl side chains of the protein or polypeptide. In another method, an iodinated reagent carrying a reactive coupling group is bound to the protein; a frequently used iodinated reagent is the Bolton-Hunter reagent, iodinated /V-succinimidyl 3(4- hydroxyphenyl) propionate. Still another method involves the use of iodine

monochloride.

[0203] Alternatively, the radionuclide can be selected from the group consisting of ⁹⁰Y and ^mln. When the radionuclide is selected from the group consisting of ⁹⁰Y and ¹¹¹ In, typically, the radionuclide is bound to the intermediate release linker by a chelator that bound to the intermediate release linker. A number of suitable chelators are known in the art, including cyclic DPTA (diethylene triamine pentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10-tetraazacyclododecane- 1 ,4,7,10-tetraacetic acid), and tiuxetan.

[0204] Still other therapeutically effective radionuclides are known in the art, including ¹³⁷Cs, ⁶⁰Co, ^{1 2}lr, ¹²⁵l, ⁰³Pd, and ¹⁰⁵Ru. These radionuclides are suitable for brachytherapy.

[0205] In yet another alternative, the targeting composition can include and deliver a diagnostically effective nucleotide for diagnosis by a technique such as scintigraphy, SPECT, or positron emission tomography; these techniques are well known in the art. In general, in this alternative, the targeting composition comprises:

(1) a diagnostically effective radionuclide;

[0206] Diagnostically effective radionuclides include, but are not limited to, 99mj_c 201 j| _anc| 67Q₃ Q_tner diagnostically effective radionuclides are known in the art and are in use.

[0207] Still another aspect of the present invention is a method of treating a disease, disorder, or condition treatable by administration of a therapeutic agent as described above comprising administration of a therapeutically effective quantity of either: (1) a targeting composition according to the present invention as described above; or (2) a pharmaceutical composition according to the present invention including a targeting composition according to the present invention as described above to a subject in need of treatment.

[0208] In these methods, the disease, disorder, or condition to be treated can be cancer, although treatment of other diseases, disorders, or conditions is also

contemplated. In particular, the disease, disorder, or condition can involve an inflammatory process. Additionally, the disease, disorder, or condition can include a local release of enzymes that degrade components of the extracellular matrix without affecting the molecular structure of collagen. The collagen fibers can be exposed in such a way as to make them accessible to circulating molecules or groups of molecules; the circulating molecules can be the targeting compositions of the present invention.

[0209] As used herein, terms such as "treatment," "treating," and similar terminology includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, condition, or disorder (e.g., a malignancy), alleviating the symptoms of the disease, condition, or disorder, or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include patients already suffering from the disease or disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. As used herein, the terms "treating," or similar terminology, do not imply a cure for a malignancy or any other disease or condition; rather, this terminology is used to refer to any clinically detectable improvement in the disease, disorder, or condition being treated or alleviated, including, but not limited to, in the case of a malignancy, reduction of tumor burden, reduction of tumor size, reduction of tumor spread, reduction of development of metastases, killing of tumor cells, arrest of growth or division of tumor cells, improved susceptibility of tumor cells to anti-neoplastic agents, reduction of pain, improvement in subjective well-being experienced by the patient, or any other clinically detectable improvement. Analogous parameters can be used for determining effective treatment of conditions other than malignancies and are well known in the art. As used herein, the term "therapeutically effective quantity" refers to the amount of a therapeutic agent which is sufficient to treat the disease, disorder, or condition treatable by the therapeutic agent incorporated in the targeting composition, as described above.

[0210] The targeting compositions comprising a therapeutic agent described above can be administered directly to subjects in need of treatment. However, targeting compositions according to the present invention comprising a therapeutic agent are preferably administered to the subjects in pharmaceutical compositions which comprise the targeting composition comprising the therapeutic agent, and, optionally, other therapeutically active agents in a therapeutically effective dose along with a

pharmaceutically acceptable carrier, diluent or excipient in unit dosage form. Pharmaceutically acceptable carriers are agents which are not biologically or otherwise undesirable, i.e., the agents can be administered to a subject along with the targeting composition comprising the therapeutic agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained. The compositions can additionally contain other therapeutic agents, as described above, that are suitable for treating or preventing the disease, disorder, or condition treatable by the therapeutic agent as described above. Pharmaceutically acceptable carriers enhance or stabilize the composition, or can facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutically acceptable carrier should be suitable for various routes of administration described herein.

[0211] A pharmaceutical composition containing a therapeutic composition incorporating a therapeutic agent and/or other therapeutic agents can be administered by a variety of methods known in the art. The routes and/or modes of administration vary depending upon the desired results. Depending on the route of administration, the targeting composition or other therapeutic agent may be coated in a material to protect the targeting composition or other therapeutic agent from the action of acids and other compounds that may inactivate the agent. Conventional pharmaceutical practice can be employed to provide suitable formulations or compositions for the administration of such targeting compositions or pharmaceutical compositions to subjects. Any appropriate route of administration can be employed, for example, but not limited to, intravenous, parenteral, intraperitoneal, intravenous, transcutaneous, subcutaneous, intramuscular, intraurethral, or oral administration. Depending on the severity of the malignancy or other disease, disorder, or condition to be treated, as well as other conditions affecting of the subject to be treated, either systemic or localized delivery of the targeting composition or pharmaceutical composition can be used in the course of treatment. The targeting composition or pharmaceutical composition as described above can be administered together with additional therapeutic agents intended to treat a particular disease or condition, which may be the same disease or condition that the therapeutic agent incorporated in the targeting composition or pharmaceutical composition is intended to treat, which may be a related disease or condition, or which even may be an unrelated disease or condition. For example, and not by way of limitation, the targeting composition or pharmaceutical composition can include an antineoplastic therapeutic agent and an additional anti-neoplastic therapeutic agent can be administered separately. As another example, and again not by way of limitation, the targeting composition or pharmaceutical composition can include an anti-neoplastic therapeutic agent and an anti-inflammatory therapeutic agent can be administered separately. The additional therapeutic agent or agents can be administered

simultaneously or at different times.

[0212] In some embodiments, local administration of the targeting composition or pharmaceutical composition is desired in order to achieve the intended therapeutic effect. Many methods of localized delivery of therapeutic agents can be used in the practice of the invention. For example, a targeting composition or therapeutic

composition according to the present invention can be administered directly to the site of the malignancy by direct injection or infusion or by other means known in the art.

[0213] Pharmaceutical compositions according to the present invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Formulations for parenteral

administration may, for example, contain excipients, sterile water, or saline,

polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or

hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, and implantable infusion systems. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, e.g., polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or can be oily solutions for administration or gels.

[0214] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular therapeutic agent, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the severity of the condition, other health considerations affecting the subject, and the status of liver and kidney function of the subject. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular therapeutic agent employed, as well as the age, weight, condition, general health and prior medical history of the subject being treated, and like factors. Methods for determining optimal dosages are described in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000. Typically, a

pharmaceutically effective dosage would be between about 0.001 and 100 mg/kg body weight of the subject to be treated. Similar considerations apply if additional therapeutic agents are administered as described above.

[0215] The targeting composition or pharmaceutical composition that includes the targeting composition, and, if desired, other therapeutic agents described above, are usually administered to the subjects on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by therapeutic response or other parameters well known in the art. Alternatively, the targeting composition or pharmaceutical composition can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life in the subject of the therapeutic agent and the other drugs included in a pharmaceutical composition, as well as the lifespan of the targeting composition in the circulation of the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime.

[0216] For the purposes of the present application, treatment can be monitored by observing one or more of the improving symptoms associated with the disease, disorder, or condition being treated, or by observing one or more of the improving clinical parameters associated with the disease, disorder, or condition being treated, as described above.

[0217] Preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions

contemplated by the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0218] Pharmaceutical formulations for parenteral administration can include aqueous solutions or suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters, such as ethyl oleate or

triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or modulators which increase the solubility or dispersibility of the composition to allow for the preparation of highly concentrated solutions. Pharmaceutical preparations for oral use can be obtained by combining the compositions with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating modulators may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0219] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different doses of therapeutic agent.

[0220] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the liposome composition may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0221] Other ingredients such as stabilizers, for example, antioxidants such as sodium citrate, ascorbyl palmitate, propyl gallate, reducing agents, ascorbic acid, vitamin E, sodium bisulfite, butylated hydroxytoluene, BHA, acetylcysteine,

monothioglycerol, phenyl-a-naphthylamine, or lecithin can be used. Also, chelators such as EDTA can be used. Other ingredients that are conventional in the area of pharmaceutical compositions and formulations, such as lubricants in tablets or pills, coloring agents, or flavoring agents, can be used. Also, conventional pharmaceutical excipients or carriers can be used. The pharmaceutical excipients can include, but are not necessarily limited to, calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Other pharmaceutical excipients are well known in the art. Exemplary pharmaceutically acceptable carriers include, but are not limited to, any and/or all of solvents, including aqueous and non-aqueous solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents, and/or the like. The use of such media and/or agents for

pharmaceutically active substances is well known in the art. Except insofar as any conventional medium, carrier, or agent is incompatible with the active ingredient or ingredients, its use in a composition according to the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions, particularly as described above. For administration of any of the compounds used in the present invention, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biologies Standards or by other regulatory organizations regulating drugs.

[0222] Sustained-release formulations or controlled-release formulations are well-known in the art. For example, the sustained-release or controlled-release formulation can be (1) an oral matrix sustained-release or controlled-release

formulation; (2) an oral multilayered sustained-release or controlled-release tablet formulation; (3) an oral multiparticulate sustained-release or controlled-release formulation; (4) an oral osmotic sustained-release or controlled-release formulation; (5) an oral chewable sustained-release or controlled-release formulation; or (6) a dermal sustained-release or controlled-release patch formulation.

[0223] The pharmacokinetic principles of controlled drug delivery are described, for example, in B.M. Silber et al., "Pharmacokinetic/Pharmacodynamic Basis of

Controlled Drug Delivery" in Controlled Drug Delivery: Fundamentals and Applications (J.R. Robinson & V.H.L. Lee, eds, 2d ed., Marcel Dekker, New York, 1987), ch. 5, pp. 213-251 , incorporated herein by this reference.

[0224] One of ordinary skill in the art can readily prepare formulations for controlled release or sustained release comprising a targeting composition according to the present invention by modifying the formulations described above, such as according to principles disclosed in V.H.K. Li et al, "Influence of Drug Properties and Routes of Drug Administration on the Design of Sustained and Controlled Release Systems" in Controlled Drug Delivery: Fundamentals and Applications (J.R. Robinson & V.H.L Lee, eds, 2d ed., Marcel Dekker, New York, 1987), ch. 1 , pp. 3-94, incorporated herein by this reference. This process of preparation typically takes into account physicochemical properties of the targeting composition, such as aqueous solubility, partition coefficient, molecular size, stability, and nonspecific binding to proteins and other biological macromolecules. This process of preparation also takes into account biological factors, such as absorption, distribution, metabolism, duration of action, the possible existence of side effects, and margin of safety, for the targeting composition. Accordingly, one of ordinary skill in the art could modify the formulations into a formulation having the desirable properties described above for a particular application.

[0225] As detailed above, another aspect of the present invention is a diagnostic composition comprising: (1) a diagnostic agent; (2) optionally, an intermediate release linker bound to the diagnostic agent; and (3) a targeting moiety as described above, bound to the intermediate release linker, if present, or to the diagnostic agent if the intermediate release linker is not present.

[0226] Suitable targeting moieties and intermediate release linkers are

described above.

[0227] Typically, the diagnostic agent is a diagnostic agent usable in

computerized tomography (CT) or magnetic resonance imaging (MRI). Diagnostic agents suitable for use in diagnostic compositions according to the present inventions, include, but are not limited to, iron oxide nanoparticles, gadolinium contrast agents with or without hyperpolarized substances, gadolinium-apoferritin, or manganese complexes. Other diagnostic agents known in the art include iodinated contrast agents such as iohexol, iodixanol, ioversol, diatrizoate, metrizoate, ioxaglate, iopamidol, ioxilan, and iopromide. Such diagnostic agents can be attached either covalently or non-covalently to the peptide sequences targeting collagen (the targeting moiety), either as individual molecules or ions, or in the form of a coating or other composite. Such diagnostic agents can be either directly bound to the peptide sequence targeting collagen (the targeting moiety), or can be bound to the peptide sequence targeting collagen to the intermediate release linker. Other diagnostic agents are known in the art. [0228] Therefore, another aspect of the present invention is a method for diagnostic imaging comprising the steps of:

(2) performing a diagnostic imaging procedure on the patient.

[0229] Typically, the diagnostic procedure is computed tomography (CT) or magnetic resonance imaging ( RI). Principles of CT are described in J.D. Bronzino, ed., "The Biomedical Engineering Handbook" (CRC Press, Boca Raton, FL, 1995), ch. 64, pp. 990-1005, incorporated herein by this reference. Principles of MRI are described in J.D. Bronzino, ed., "The Biomedical Engineering Handbook" (CRC Press, Boca Raton, FL, 1995), ch. 65, pp. 1006-1045, incorporated herein by this reference.

[0230] The invention is illustrated by the following Examples. These Examples are included for illustrative purposes only, and are not intended to limit the invention.

Example 1

[0231] An ideal therapy for cancer in particular, as well as for inflammation associated with diseases of the joints, should include the possibility of targeting therapeutic agents exclusively to primary tumors, metastasis and/or specific

inflammatory sites.

[0232] Such agents should be able concentrate their effects on abnormal cells while not affecting normal ones. We believe that we have a way to achieve this objective, by either inhibiting the growth and migration of metastatic cells or by blocking changes associated with acute or chronic inflammation.

[0233] The rationale behind this novel approach is that enzymes secreted during these processes expose the surface of collagen fibers, normally invisible to cells because they are covered with a coat of glycoproteins. In our laboratory we developed a unique approach which selectively recognizes such "naked" collagen at sites of inflammation or metastasis. Our current plan is to prepare targeting compositions comprising: (1) a therapeutic agent; (2) an intermediate release linker bound to the therapeutic agent; and (3) a targeting moiety. The targeting moiety is to incorporate variants of a decapeptide we designed, to enable these nanoparticles carrying chemotherapeutic agents to such sites.

[0234] The emphasis of the research relates to a novel modality for drug delivery targeted to patients requiring chemotherapy, but may nevertheless also address the need of patients with active inflammation of the joints and patients with other conditions, especially inflammatory conditions. The technology developed in our laboratory in 1965 was aimed initially to target tissues such as cartilage and bone to aid in their repair. Since the basic biology of the inflammatory response is nevertheless associated also with tumor metastasis, the approach is proving to be very useful in cancer therapeutics.

[0235] Prior research resulted in the identification of the first distinct type of collagen in mammalian tissues (there are now close to 30 known types), and the unraveling of metabolic differences between normal and osteoarthritic cartilage. The research of the present example is intended to demonstrate the feasibility of using peptide sequences to target vectors incorporating pharmacologically active compounds, especially anti-neoplastic therapeutic agents.

[0236] The objective of this work is to develop a simple, universally acceptable delivery system for drugs used in cancer chemotherapy, targeted to the site of primary or metastatic lesions, with minimal side effects on normal tissues. Nanoscale drug delivery systems incorporating targeting compositions as described above are very likely to provide potential solutions for improved cancer therapeutics.

[0237] Background

[0238] Day after day it is becoming increasingly apparent that drug delivery systems, including polymers and other nanoparticles, have the potential to provide solutions for improving cancer therapeutics (Park et al 2004). A number of forms of drug delivery, such as proteins and smaller peptides, polymeric and ceramic

nanoparticles, micelles, chitosan and its derivatives, among others, show significant promise. (Rezier et al. 2007, Park et al 2009, MacKay 2009). [0239] Reticuloendothelial system-targeted formulations significantly reduce systemic exposure to high peak levels of free drug, but do not facilitate targeting tumors. Therefore, another approach to targeting tumors is required.

[0240] Given the extremely narrow therapeutic indices characteristic of all current cytotoxic chemotherapy drugs, it is obvious why and targeting of these drugs is of utmost importance. Stromal targeting coupled with enhancers of drug release at the target site would be very productive (Cheong et al. 2009). As a consequence of these findings combinatorial approaches to molecularly targeting with other forms of site recognition and novel modalities for drug release, offers significant promise.

[0241] Preliminary Studies

[0242] Collagen fibers are major constituents of tissue parenchyma. There are now 29 distinct collagens, and our laboratory was the first to describe the molecular structure of a unique and distinct kind of collagen in cartilage, known as type II collagen (Strawich and Nimni, 1971). All these collagens have a characteristic repeating motif or a variable of such, typically a Gly-Pro-Hypro-Gly. Most importantly, every 4^th residue is by necessity glycine. Intervening amino acids can vary. The collagen molecules organize into a 3 dimensional structure, leading to fibers, as shown in the diagrams below. As mentioned collagen fibers are not normally directly accessible to cells as these fibers are coated with a layer of proteins and proteoglycans. This has an important physiological function as it prevents, among other things, platelets from attaching and initiating the clotting cascade. It is only during process of tissue damage (wound healing, release of inflammatory cytokines, and as we have now learned, tumor cell invasion) that metalloproteases and other related enzymes are released and remove such a coat, thereby exposing the surface of the collagen fibers.

[0243] Our working hypothesis is that if we construct a targeting composition as described above, including a peptide or peptides which recognize such naked collagen, the targeting composition will target such a site. Our previous work has shown that a decapeptide sequence, identical or similar to the sequence present in von Willebrand's factor used by platelets to attach to collagen, can be used to generate fusion proteins which have an ability to strongly bind to collagen. (T.-L. Tuan et al., "Engineering, Expression, and Renaturation of Targeted TGF-β Fusion Proteins," Connect. Tiss. Res_. 34: 1-9 (1996); M.E. Nimni, "Review: Polypeptide Growth Factors, Targeted Delivery Systems," Biomaterials 18: 1201-1225 (1997); B. Han et al., "Refolding of a

Recombinant Collagen-Targeted TGF^₂," Prot. Exp. Purif. 168-178 (1997); B. Han, Ph.D. Thesis (1998)).

[0244] Subsequently Fred Hall, and oncologist Maria Gordon, in a very ingenious way adapted this collagen targeting mechanism to generate a guided vector which accumulates at sites of tumor metastasis. This has given rise to a drug (Rexin- G), now manufactured by Epeius Biotechnology, in San Marino, California, which incorporates a dominant negative mutant of cyclin-G (cloned by Hall). The efficacy of this approach, first outlined in a paper in Human Gene Therapy, Hall, Gordon et al 1 : 983-93, Molecular engineering of matrix-targeted retroviral vectors incorporating a surveillance function inherent in von Willebrand factor, is beginning to manifest itself clinically with great success : Advanced Phase l/l I studies of targeted gene delivery in vivo: Intravenous Rexin-G for Gemcitabine-resistant metastaic pancreatic cancer , Chawla, Gordon, Hall et al , Molecular Therapy, 18: 435-441 (2010). (Rexin-G has been approved by the FDA as an orphan drug and is in phase III clinical trials.

Accumulation of the vector at sites of excised single liver tumor metastasis in a patient with pancreatic cancer has recently been documented. (Pathotropic targeting advances clinical oncology: Tumor-targeted localization of therapeutic gene delivery, Hall FL, Gordon, et al , Oncology Reports 24: 829-833 (2010). Unfortunately this vector at this time, and in its present form, is not able to deliver other chemotherapeutic agents. Therefore, targeting compositions according to the present invention are designed to be able to deliver a wide range of chemotherapeutic anti-neoplastic agents as well as other therapeutic agents.

[0245] Figures 1 and 2 reveal the histological features of an excised metastatic liver lesion showing vector localization in the cytoplasm of pseudo-differentiated glandular cancer cells and associated tumor endothelial cells within the metastatic liver lesion (confirmatory evidence of the tumor-targeting property of Rexin-G), resulting in apoptosis of the cancer cells (by Tunel assay) and reparative fibrosis (as evidenced by Trichrome staining for collagen [blue-staining material]) (Hall, Chawla, Gordon, et al., Pathotropic targeting advances clinical oncology: Tumor-targeted localization of therapeutic gene delivery, Oncology Reports 24: 829-833 2010).

[0246] Figure 1 shows a histological section of excised liver tumor showing a preponderance of fibrosis (fib) with pseudo-differentiated epithelioid tumor cells (tu) arrayed in columnar/ductal structures, seen in various stages of degeneration (A, hematoxylin-eosin (H&E) stain), as marked by a cytokeratin-17 immunostain (inset). Abundant fibrosis is observed throughout the tumor nodule, as shown by Masson's trichrome stain for ECM (blue stain, B). Remarkably, Rexin-G appears to have induced massive amounts of apoptosis of the pancreatic cancer cells (see TUNEL Stain in C, D, and negative control E), as well as visible karyorrhexis and fragmentation— hich is evident all along the borders of the pseudo-differentiated structures.

[0247] Figure 2 shows immunohistochemical staining of the excised tumor for the gp70 envelope protein of the Rexin-G nanoparticle reveals an accumulation of immunoreactivity throughout the ECM-rich mass of the tumor (A versus B, negative control), particularly in the cellular components, including the diffuse islands (C) and ductal structures (D) comprised of cancer cells and the elongate endothelial cells lining the vessels of the tumor-associated vasculature (E).

[0248] Experimental Approach

[0249] Earlier studies in our laboratory with targeted peptides used the decapeptide sequence: Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser (WREPSFMALS) (SEQ ID NO: 1). It was linked to either TGF-β or BMP-3 at the C-terminus of the corresponding growth factors. These covalently linked growth factors retained significant biological activities and developed a strong binding affinity towards native collagen. This is the same peptide expressed on the surface of Rexin-G.

[0250] Because of the nature of the relatively large nanoparticle we contemplate using for the targeting composition (around 100 nm diameter, which represents around 1/3 the length of a collagen molecule (see Figure 3), we believe that we may have to insert multiple binding motifs on the surface of such a sphere to assure good linking and to stabilize its attachment. On the other hand it is possible that protruding PEG chains may suffice to achieve this goal. If we have to do so we will extend the protruding peptides by inserting repeating sequences of glycine (also shown in Figure 3). Glycine provides maximum rotation around peptide bonds because of the small size of the side chain (a hydrogen atom) and minimum steric hindrance, and therefore maximal degree of motion. We have selected polyglycine extensions ranging from zero and 50 repeating units. The displacement between the most adjacent binding sites among parallel oriented molecules is in the range of 2-4 nm. We want to allow for free random movement of the glycine chains, and of course generate as many attachments as possible. Initially we will experiment with the lower molecular weight enhanced extensions. In one alternative, the extension can be made more rigid (i.e., by using repeating Gly-Pro-Pro-Gly sequences) to generate a collagen-like rigid triple helical extension radiating from the targeting conjugate.

[0251] Figure 3 shows a native collagen fiber stained with phosphotungstic acid, showing 68-nm periodicity and a schematic representation of collagen molecules measuring approximately 300 nm (adapted from M. Nimni, ed., "Collagen", Vol. 1 , CRC Press, 1988).

[0252] Figure 4 shows the molecular packing of the Type I collagen fiber.

Example 2

[0253] This Example includes prospective work which is described in the future tense as appropriate.

[0254] The "magic bullet'" that will recognize only tumor cells and destroy them remains elusive, probably because tumor cells do not differ sufficiently in terms of markers from normal cells and no unique targets have been identified (Ruoslahti, Bhatia et al.). Leukemia and lymphoma treatments using antibodies conjugated to

radioisotopes have been in use for several years; however such approach has proven to be of limited success in solid tumors (Sharkey and Goldenberg 2005). The challenge is to find a rather simple, generic carrier which can selectively deliver, with a high degree of specificity, a host of cytotoxic drugs to primary or metastatic tumors. We have selected what we believe is a unique target for this purpose, namely, collagen devoid of its glycoprotein coating (naked collagen) seen at such sites.

[0255] The feasibility of our approach is substantiated by work done earlier in our laboratory. Polypeptide growth factors, of the TGF-β family, and others, were constructed to contain, at the C-terminal end, a decapeptide with high affinity for collagen (Andrades, Nimni et al. 1996; Tuan, Cheung et al. 1996; Andrades, Han et al. 1999; Hall, Han et al. 2001 ; Han, Perelman et al. 2002; Romijn, Westein et al. 2003). It became apparent to us some time ago that in order for many growth factors, such as TGF-β, to be used therapeutically with a minimum of undesirable side effects, a way had to be found to concentrate them where needed. In addition the slow release of the active molecule, and protection against rapid degradation seemed desirable. In our earlier work we genetically engineered a TGF-β fusion protein which contained, in addition to the active human growth factor and a purification tag, a binding domain with affinity for collagen, a major extracellular matrix component (Figure 5). The backbone peptides that we will use, and will be discussed later in this Example, either be produced in our laboratory or custom synthesized. In our earlier studies the purification tag and the matrix-binding domains were linked to the active TGF-β domain through Gly-Gly linkers.

[0256] The 3 initial fusion proteins constructed in our laboratory (TGF-P1-F1 , TGF-P1-F2 , BMP-3) required designing DNA constructs, transfecting into Escherichia coli, solubilizing in urea and renaturing in a redox-coupled refolding buffered system, to yield the active TGF-β fusion proteins (Figure 5). The purification tag includes a hexapeptide, (His which binds tightly to a Ni-NTA column which can be dissociated with acidic buffered imidazole. As seen in Figure 5 the TGF-βΙ contains only active TGF-βΙ and the purification tag, which was very effective in providing a quick, one-step purification process and pure TGF-β . All three recombinant fusion proteins were found to be biologically active judged by an in vitro mink lung epithelial (MV1 Lu) cell inhibition assay and the stimulation of mouse NIH 3T3 fibroblast proliferation. We have also demonstrated since that this construct not only binds to native collagen but also to gelatin (Figure 6). [0257] Figure 5 depicts a genetically engineered fusion protein consisting of TGF-βΙ with a collagen binding decapeptide. The purification tag comprises a hexapeptide of histidine, linked via a Gly-Gly link; it binds tightly to a Ni-NTA column. DNA constructs were transfected into Escherichia coli.

[0258] Figure 6 depicts the binding of the TGF-β with a collagen binding domain to collagen; the binding requires a high concentration of urea for dissociation. This is compared to the behavior of TGF-β without the collagen binding domain, which has poor affinity for collagen.

[0259] The feasibility and efficacy of the proposed approach to target tumors is supported by animal and human clinical studies (Chawla, Chua et al. ; Chawla, Chua et al. 2009; Gordon and Hall 2009), since viral particles coated with this decaptide accumulate at a site of metastasis (Figures 1 and 2). The construct of human cyclin G1 (Chawla, Chua et al. 2009) completed a phase l-ll clinical study. Rexin-G, is a vector produced by the transfection of 3 separate plasmids and demonstrated to have a high growth inhibitory activity on target cancer cells. The initial construct of this vector was developed at the University of Southern California, and includes the targeting

decapeptide which we previously attached to TGF-β.

[0260] The histology revealed a clear response to chemotherapy. Reaching this tumor through the systemic circulation, the collagen-targeting decapeptide containing nanoparticles appear to have transited the tumor vasculature and spread throughout the mass of the tumor (Chawla, Chua et al. 2009).

[0261] The findings so far described clearly demonstrate, both in animals as well as in patients, the ability of the VWF derived decapeptide to target exposed collagen at tumor sites and not normal tissues. In a separate relevant example, a cyclic

nanopeptide, LyP-10, was shown to bind preferentially to lymphatic vessels, providing the first evidence for a unique difference between these types of vasculatures (data not shown).

[0262] Fluorescent labeled Abraxane (paclitaxel-albumin) conjugated with such a peptide was able to accumulate in significant amounts in the tumor site of mice bearing a human cancer xenograft (Karmali, Kotamraju et al. 2009). We obtained similar results when nude mice bearing tumors derived from a colon cancer cell line (HT29) were treated with Abraxane conjugated with the CBD (Figure 7). Also, as we observed earlier with TGF-β, the CBD increased its binding affinity for collagen reflected by resistance to detachment by 0.5 M urea. This preliminary study is inserted as proof of principle. It will be optimized for maximum targeting as discussed below.

[0263] Figure 7 shows results for the binding of paclitaxel associated with albumin (Abraxane) to collagen. (A) Paclitaxel associated with albumin (Abraxane), with a covalently bound CBD, exhibits greater retention on a collagen matrix. (B) Abraxane with a CBD binds tighter to collagen and is more resistant to elution from collagen by 0.5 M urea. (C) The CBD bound to Abraxane, by targeting the site of the tumor, enhances chemotherapeutic effects in mice bearing colon cancer cells in a mouse model.

[0264] Initially we will use the original von Willebrand derived polypeptide binding sequence, namely WREPSFCALS (SEQ ID NO: 1) or a slight variant. We will compare this with the fibronectin binding sequence Gly-Gly-Trp-Ser-His-Trp

(GGWSHW) (SEQ ID NO: 94) derived from thrombospondin, as well as variants of the CBP with insertions, permutations, and modifications and, if practical, combinations separated by suitable spacers as will be described.

[0265] In exploratory studies we also tagged the surface of liposomes with the CBD. We conjugated fluorescent lipid-Dil-PEG-NH2 liposomes with the -COOH terminal of an extended CBD, with similar results. The work will continue to focus on polypeptide drug carriers, since we have more expertise in this area and believe it more likely to readily yield positive results. This specific aim will include evaluating a spectrum of collagen binding domains attached to polypeptides which will serve as carriers for drugs. The decapeptide we are using involves a series of exposed amino acids, located strategically within the N-terminus, in an area extending from residues 570 (F) to 682 of VonWillebrand factor (Takagi, Asai et al. 1992). By binding

competition this decapeptide was found to bind, on a molar basis, 20 times more efficiently to collagen than the intact VWF (Takagi, Asai et al. 1992). Further

examination of the crystal structure of the collagen binding regions of VWF A-3 Domain (lchikawa, Osawa et al. 2007); (Romijn, Westein et al. 2003); (Staelens, Hadders et al. 2006) as well as the complementary collagen exposed surface (Lisman, Raynal et al. 2006) is expected to yield CBDs with increased binding affinity.

[0266] Collagens are large, triple-helical proteins that form fibrils and networklike structures in the extracellular matrix. They have played a major role in the evolution of metazoans from their earliest origins. Cell adhesion receptors that interact with collagen such as the integrins are at least as old as the collagens (Heino, Huhtala et al. 2009); (Whittaker and Hynes 2002) and instrumental in the evolution of bone, cartilage, and the immune system in chordates. In vertebrates collagen binding receptor tyrosine kinases send signals into cells after adhesion to collagen. Nevertheless, collagen continues to be seen primarily as an inert scaffold. To us the value of using it as a target became most relevant when we observed that it is only at sites of pathology or rapid tissue remodeling that collagen fibers become devoid of their normal proteoglycan coating, and therefore recognizable as such.

[0267] Other CBD, such as the discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases known to be activated by native triple-helical collagen. The sequence on collagen that binds DDR2 with highest affinity has similarity to the binding site for von Willebrand's factor, GVMGFO (O is hydroxyproline). (Konitsiotis, Raynal et al. 2008). The scattered amino acids on the binding site on the ligand are highlighted (Figure 8). (Brondijk, de Ruiter et al.). The complete amino acid sequence of wild-type human DDR2 is shown in Figure 11. A peptide discovered in the process of mapping the topography of collagen is P-15, a synthetic 15 residue peptide which binds to collagen at the single mammalian collagenase cleavage city (Gough and Bhatnagar 1999). The P-15 peptide, characterized as GTPGPGGIAGQRGW (SEQ ID NO: 19) has found clinical application in the area of bone mineralization. The single unique collagenase cleavage site may be particularly interesting since it becomes exposed during periods of active collagen remodeling as occurs during fibrosis and metastasis.

[0268] Figure 8 shows molecular modeling of discoidin, including the amino acids on the surface involved in binding to collagen. These amino acids and their distribution within the DS domain provide a three-dimensional view of the nature of the collagen-ligand interaction.

[0269] As part of our intention to enhance binding affinities we will increase the number of CBD's, properly spaced from each other. (Figure 9). Peptide (B) will be designed to match the profile of the drug it carries and amino acid sequences inserted and crosslinking mechanisms will adjusted to the hydrophobic or electrostatic character of such a drug. The basic motifs will be selected from the group consisting of: Trp-Arg- Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser (WREPSFMALS) (SEQ ID NO: 1); Trp-Arg-Glu-Pro- Ser-Phe-Cys-Ala-Leu-Ser (WREPSFCALS) (SEQ ID NO: 2) and peptides related by one or more conservative amino acid substitutions. Alternatively we will expand to a group consisting of: GPPGWREPSFMALSGPPG) (SEQ ID NO: 9),

GPPGWREPSFCALSGPPG (SEQ ID NO: 10), and GPPGWRDPSFMALSGPPG (SEQ ID NO: 1 1), thus adding a "collagen like" sequence at one or both ends. Some of these sequences have already been synthesized and ready to be tested in vitro and eventually in vivo (see Figure 7). Collagen sequences, particularly if repeated, will encourage collagen-like folding. In the past our laboratory generated such sequences as well as CNBr peptides by cleavage of the native collagen molecule (Deshmukh and Nimni 1973). Such peptides fold and generate small size stable triple helical structures ("mini-collagens"), thermodynamically favored at 37° C, which should enhance binding to the fibers.

[0270] Figure 9 is a schematic drawing of molecular packing within a collagen fiber. (A) Axial view showing linear staggering; (B) Cross-sectional view showing the unit cell. (B) shows how particular segments are repeated on the surface of the fiber (b- b for instance is separated by 2 χ the diameter of a molecule or approximately 3 nm laterally, the distance that repeating CBDs should be set apart for optimal binding).

[0271] Conservative amino substitution will also be explored. These can include (original residue followed by possible substitution): Ala/Gly or Ser; Arg/Lys; Asp/Glu; Cys/Ser; Gln/Asn; Gly/Asp; Gly/Ala or Pro. (Creighton (1984) Proteins, W. H. Freeman and Company; Schuiz and Schimer (1979) Principles of Protein Structure, Springer- Verlag). Certain conservative substitutions, positive or negatively charged, may improve binding affinity. Ideally we would like to include in our CBDs amino acids such as those highlighted in Figure 8.

[0272] Also, modified sequences that take into account the triple helical configuration of collagen can be designed to enhance the longer range linear

interactions. Examples are, among others, GPPGWREPSFMALSGPPG) (SEQ ID NO: 9), GPPGWREPSFCALSGPPG (SEQ ID NO: 10), and GPPGWRDPSFMALSGPPG (SEQ ID NO: 11). Other chemical modifications to be contemplated in the future, include PEGylation, amidation, acetylation and introduction of identification markers for the targeted peptides.

[0273] PEGylation, the covalent conjugation of macromolecules (antibody, peptide, etc.) with polyethylene glycol (PEG), polymers that are nonionic, nontoxic, biocompatible and highly hydrophilic will be explored for their increased solubility (for hydrophobic drugs) and bioavailability and prolonged circulatory time within host through reduced renal clearance. Such structures will be inserted at site (C) in Figure 10.

[0274] Figure 10 is a diagrammatic representation of a collagen targeting vector: (A) CBD; (B) peptide for facilitating drug (D) attachment (length of peptide and specific amino acids in peptide leading to suitable conformations in solution will vary); (C) reactive functional groups suitable for drug attachment (-SH, -CO₂H, -NH₂, or other groups); (D) drug; (E) additional site for identical or different CBD, separated by a suitable length of spacer (B) can be added.

[0275] If a peptide is selected from an internal sequence of a protein, terminal amidation (C-terminus) or acetylation (N-terminus) will remove its charge. In addition, this modification makes the resulting peptide more stable towards enzymatic

degradation by exopeptidases. Biotin and fluorescein isothiocyanate (FITC) are activated precursor used for fluorescein labeling. For efficient N-terminal labeling, a seven-atom aminohexanoyl spacer (NH2-CH2-CH2-CH2-CH2-CH2-COOH) will be inserted between the fluorophore (fluorescein) and the N-terminus of the peptide. One common means of conjugation involves the use of maleimide, which couples N or C termins cysteine residues of the peptide to the carrier protein. [0276] In vitro binding studies will initially be carried out as described in Figure 6 and our earlier publications. In addition, in order to generate a more biocompatible and representative model, we will generate surfaces of native collagen, collagen/PG composites, reconstituted collagen fibers coated with supernatants of tissue

homogenates, and other collagen-containing constructs to resemble the "masked" collagen present in tissues. In the past we plated reconstituted fibrous collagen

(monomeric collagen assembled into fibers by heating to 37° C) in petri dishes for the purpose of evaluating agents that inhibited crosslinking and or/enzymatic degradation (Nimni 1968). Selected areas of a reconstituted collagen fibrous network coated with proteoglycans will be enclosed by removable cylindrical inserts. Some will be treated with MMP's to expose "naked" collagen molecules on the fiber surface, to simulate what happens at sites of inflammation or metastasis. Others will remain masked by the surface deposited non-collagen extracellular matrix glycoproteins. The selective affinity of the various CBD's towards the exposed collagen will be evaluated using specific histochemical stains, as well as built in fluorescent or other markers. The sequences to be evaluated will be derived from sequences derived from conformational analysis, and will include the simplest CBD now in use and variables with collagen compatible peptide conformations, spacers to bridge repeating motifs on the surface of collagen, separated by distances estimated from the pattern of molecular assembly, coiling conformation, and other molecular parameters. Binding constants will be quantified, and the constructs with highest binding affinities as drug carriers will be selected. Evaluation of binding constants will be aided by coupling fluorescent markers to the peptides. First the peptides alone and then the peptides carrying a variety of chemotherapeutic molecules, starting with Paclitaxel, a drug we have already been working with. In a later stage, collagen surfaces previously exposed to targeted drugs will be coated with cultured tumor cells (initially HT29) and cell proliferation will be correlated with drug accumulation. We expect both fluorescence and cell growth inhibition to correlate with each other.

[0277] A final set of ex vivo studies will involve the use of frozen sections from tumor-bearing animals. Such sections should contain areas of newly deposited and peri-tumoral collagen, partially devoid of proteoglycan coating, due to the MMP activity in such areas. The binding of the collagen targeted constructs will be assayed as described above. If necessary, the effects of adding MMPs to such specimens will also be evaluated to assess the relative degree of collagen masking, and the maximum possible collagen exposure. For purposes of comparison, the anti-cyclin G collagen targeted nano-particles (Rexin-G) described in Figures 7 and 8, already evaluated clinically in patients, will be tested. Rexin-G will be provided by Epeius Biotechnology, San Marino, CA. These studies will satisfy the major objectives of this application, namely to generate a functional universal carrier able to target chemotherapeutic agents to areas of tumor growth, based on recognition of exposed collagen molecules.

[0278] Animal studies shall also be undertaken. The approach will be similar to that described in Figure 9, where Paclitaxel was bound enzymaticlly to albumin containing a CBD and injected into nude mice bearing a colon cancer cell line (HT29).

[0279] We believe that have accumulated significant data, enhanced by relevant clinical findings, that supports the concept that effective targeting of exposed collagen fibers can be achieved for the purpose of selective drug delivery to tumor sites.

Alternate CBD which have enhanced affinity for collagen will be investigated and their binding efficacy quantitated. Using standard and newly designed biochemical procedures we will quantify the binding efficacy. The ability of drugs carried by the targeted vector to inhibit tumor cell growth will also be determined. These in vitro studies will include, for comparison, anti-cyclin-G targeted nanoparticles (Rexin-G), which have completed phase II clinical trials. Further animal studies will use nude mice with implanted colon cancer cells (HT29) or other available cell lines.

REFERENCES

[0280] The following references are cited, particularly in the Examples, and are hereby incorporated herein by this reference.

Andrades, J. A., B. Han, et al. (1999). "A recombinant human TGF-beta1 fusion protein with collagen-binding domain promotes migration, growth, and differentiation of bone marrow mesenchymal cells." Exp Cell Res 250(2): 485-98. Andrades, J. A., M. E. Nimni, et al. (1996). "Type I collagen combined with a recombinant TGF-beta serves as a scaffold for mesenchymal stem cells." Int J Dev Biol Suppl 1. 107S-108S.

Brondijk, T. H., T. de Ruiter, et al. (2010). "Crystal structure and collagen-binding site of immune inhibitory receptor LAIR-1 : unexpected implications for collagen binding by platelet receptor GPVI." Blood 115(7): 1364-73.

Chawla, S. P., V. S. Chua, et al. (2010). "Advanced phase l/ll studies of targeted gene delivery in vivo: intravenous Rexin-G for gemcitabine-resistant metastatic pancreatic cancer." Mol Ther 18(2): 435-41.

Chawla, S. P., V. S. Chua, et al. (2009). "Phase l/ll and phase II studies of targeted gene delivery in vivo: intravenous Rexin-G for chemotherapy-resistant sarcoma and osteosarcoma." Mol Ther 17(9): 1651-7.

Deshmukh, K. and M. E. Nimni (1973). "Isolation and characterization of cyanogen bromide peptides from the collagen of bovine articular cartilage." Biochem J 133(4): 615-22.

Famdale, R. W., T. Lisman, et al. (2008). "Cell-collagen interactions: the use of peptide Toolkits to investigate collagen-receptor interactions." Biochem Soc Trans 36(Pt 2): 241-50.

Gordon, E. M. and F. L. Hall (2009). "The 'timely' development of Rexin-G: first targeted injectable gene vector (review)." Int J Oncol 35(2): 229-38.

Gough, C. A. and R. S. Bhatnagar (1999). "Differential stability of the triple helix of (Pro-Pro-Gly)10 in H2O and D2O: thermodynamic and structural explanations." J Biomol Struct Dvn 17(3): 481-91.

Hall, F. L, B. Han, et al. (2001). "Phenotypic differentiation of TGF-beta1- responsive pluripotent premesenchymal prehematopoietic progenitor (P4 stem) cells from murine bone marrow." J Hematother Stem Cell Res 10(2): 261-71.

Han, B. (1998). "Collagen targeting TGF-beta: expression, characterization, and applications." Ph.D. Thesis. Han, B., N. Perelman, et al. (2002). "Collagen-targeted BMP3 fusion proteins arrayed on collagen matrices or porous ceramics impregnated with Type I collagen enhance osteogenesis in a rat cranial defect model." J Orthop Res 20(4): 747-55.

Heino, J., M. Huhtala, et al. (2009). "Evolution of collagen-based adhesion systems." Int J Biochem Cell Biol 41(2): 341-8.

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Ichikawa, O., M. Osawa, et al. (2007). "Structural basis of the collagen-binding mode of discoidin domain receptor 2." EMBO J 26(18): 4168-76.

Karmali, P. P., V. R. Kotamraju, et al. (2009). "Targeting of albumin-embedded paclitaxel nanoparticles to tumors." Nanomedicine 5(1): 73-82.

Konitsiotis, A. D., N. Raynal, et al. (2008). "Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen." J Biol Chem

283(11): 6861-8.

Lisman, T., N. Raynal, et al. (2006). "A single high-affinity binding site for von Willebrand factor in collagen III, identified using synthetic triple-helical peptides." Blood 108(12): 3753-6.

Nimni, M. E. (1968). "A defect in the intramolecular and intermolecular cross- linking of collagen caused by penicillamine. I. Metabolic and functional abnormalities in soft tissues." J Biol Chem 243(7): 1457-66.

Nimni, M. E. (1997). "Polypeptide growth factors: targeted delivery systems." Biomaterials 18(18): 1201-25.

Romijn, R. A., E. Westein, et al. (2003). "Mapping the collagen-binding site in the von Willebrand factor-A3 domain." J Biol Chem 278(17): 15035-9.

Ruoslahti, E., S. N. Bhatia, et al. (2010). "Targeting of drugs and nanoparticles to tumors." J Cell Biol 188(6): 759-68.

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Takagi, J., H. Asai, et al. (1992). "A collagen/gelatin-binding decapeptide derived from bovine propolypeptide of von Willebrand factor." Biochemistry 31(36): 8530-4.

Tuan, T. L, D. T. Cheung, et al. (1996). "Engineering, expression and

renaturation of targeted TGF-beta fusion proteins." Connect Tissue Res 34(1): 1-9.

Whittaker, C. A. and R. O. Hynes (2002). "Distribution and evolution of von Willebrand/integrin A domains: widely dispersed domains with roles in cell adhesion and elsewhere." Mol Biol Cell 13(10): 3369-87.

ADVANTAGES OF THE INVENTION

[0281] The present invention provides an improved method for targeting therapeutic agents, especially anti-neoplastic therapeutic agents, to cellular targets, as well as compositions for such targeting. The method and compositions can be employed for targeting of a wide range of therapeutic agents and does not depend critically on chemical reactivity or physical properties of the therapeutic agents to be targeted. By targeting to collagen molecules, methods and compositions according to the present invention provide a more efficient way of targeting that will reduce delivery of the therapeutic agents to undesired sites, reduce the quantity of therapeutic agents required, and reduce the frequency and severity of side effects. These advantages are especially significant for the delivery of anti-neoplastic agents.

[0282] Compositions according to the present invention possess industrial applicability as therapeutically useful compositions, as well as for preparation of a medicament for the treatment of diseases, disorders, and conditions treatable by the therapeutic agents incorporated into targeting compositions according to the present invention.

[0283] With respect to ranges of values, the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Moreover, the invention encompasses any other stated intervening values and ranges including either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

[0284] Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of ordinary skill in the art to which this invention belongs. One of ordinary skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test this invention.

[0285] The publications and patents discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0286] All the publications cited are incorporated herein by reference in their entireties, including all published patents, patent applications, and literature references, as well as those publications that have been incorporated in those published

documents. However, to the extent that any publication incorporated herein by reference refers to information to be published, applicants do not admit that any such information published after the filing date of this application to be prior art.

[0287] As used in this specification and in the appended claims, the singular forms include the plural forms. For example the terms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Additionally, the term "at least" preceding a series of elements is to be understood as referring to every element in the series. The inventions illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the future shown and described or any portion thereof, and it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions herein disclosed can be resorted by those skilled in the art, and that such modifications and variations are considered to be within the scope of the inventions disclosed herein. The inventions have been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised materials specifically resided therein. In addition, where features or aspects of an invention are described in terms of the Markush group, those schooled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of in the art upon reviewing the above description. The scope of the invention should therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. Those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A targeting composition comprising:

(a) a therapeutic agent;

(b) an intermediate release linker bound to the therapeutic agent; and

(c) a targeting moiety bound to the intermediate release linker, the targeting moiety for binding the targeting composition to native collagen fibers.

2. The targeting composition of claim 1 wherein the targeting moiety is selected from the group consisting of: (i) Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-Leu-Ser (WREPSFMALS) (SEQ ID NO: 1); (ii) Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-Leu-Ser (WREPSFCALS) (SEQ ID NO: 2); and (iii) peptides related to (i) or (ii) by one or more conservative amino acid substitutions.

3. The targeting composition of claim 2 wherein the targeting moiety is a peptide related to SEQ ID NO: 1 or SEQ ID NO: 2 by one or more conservative amino acid substitutions, and wherein the peptide is selected from the group consisting of: Trp- Arg-Asp-Pro-Ser-Phe-Met-Ala-Leu-Ser (WRDPSFMALS) (SEQ ID NO: 3); Trp-Arg-Asp- Pro-Ser-Phe-Cys-Ala-Leu-Ser (WRDPSFCALS) (SEQ ID NO: 4); Trp-Arg-Glu-Pro-Ser- Phe-Met-Ala-lle-Ser (WREPSF AIS) (SEQ ID NO: 5); Trp-Arg-Glu-Pro-Ser-Phe-Cys- Ala-lle-Ser (WREPSFCAIS) (SEQ ID NO: 6); Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-lle-Ser (WRDPSFMAIS) (SEQ ID NO: 7); and Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-lle-Ser (WRDPSFCAIS) (SEQ ID NO: 8).

4. The targeting composition of claim 1 wherein the targeting moiety is a peptide selected from the group consisting of: Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser- Phe-Met-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFMALSGPPG) (SEQ ID NO: 9); Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly

(GPPGWRDPSFCAISGPPG) (SEQ ID NO: 16).

5. The targeting composition of claim 1 wherein the targeting moiety is an elongated peptide structure of Formula (I):

[Gly-Pro-Pro-Gly-Xi-Gly-Pro-Pro-Gly-X₂-Gly-Pro-Pro-Gly]_n

(I) wherein: (1) Xi and X₂ are one of peptide sequences SEQ ID NO: 1 through SEQ ID NO: 16; and (2) n is an integer from 1 to 15.

6. The targeting composition of claim 1 wherein the targeting moiety is a peptide motif that binds collagen with a binding affinity of at least 80% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen.

7. The targeting composition of claim 6 wherein the targeting moiety is a peptide motif that binds collagen with a binding affinity of at least 90% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen.

8. The targeting composition of claim 7 wherein the targeting moiety is a peptide motif that binds collagen with a binding affinity of at least 95% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen.

9. The targeting composition of claim 1 wherein the targeting moiety is a collagen binding site of a platelet collagen binding receptor.

10. The targeting composition of claim 9 wherein the collagen binding site of the platelet collagen binding receptor is selected from the group consisting of integrin α2β1 and glycoprotein VI.

1. The targeting composition of claim 1 wherein the targeting moiety is a targeting moiety in which the peptide sequence WREPSFMALS (SEQ ID NO: 1) or WREPSFCALS (SEQ ID NO: 2) is incorporated into a molecule to generate a peptide of from about 2,000 daltons to about 10,000 daltons in molecular weight.

12. The targeting composition of claim 1 wherein the targeting moiety of the composition includes a flanking sequence that mimics a sequence found in native collagen.

13. The targeting composition of claim 11 wherein the targeting moiety of the composition includes a flanking sequence that mimics a sequence found in native elastin.

14. The targeting composition of claim 1 wherein the targeting moiety of the composition includes at least one reactive amino acid.

15. The targeting composition of claim 1 wherein the targeting moiety of the composition includes two or three collagen binding domains, with the collagen binding domains being separated by spacers.

16. The targeting composition of claim 15 wherein the spacers provide laterally displaced equivalent sites with a lateral displacement of about 3 nm.

17. The targeting composition of claim 15 wherein the spacers elongate in solution.

18. The targeting composition of claim 17 wherein the spacers include alternating polar and nonpolar sequences.

19. The targeting composition of claim 17 wherein the spacers include polylysine or polyglycine residues.

20. The targeting composition of claim 1 wherein the targeting moiety is pegylated.

21. The targeting composition of claim 1 wherein the targeting moiety includes a peptide sequence including an amino-terminal amino acid that is acetylated.

22. The targeting composition of claim 1 wherein the targeting moiety includes a peptide sequence including a carboxyl-terminal amino acid that is amidated.

23. The targeting composition of claim 1 wherein the targeting moiety includes a fluorescein moiety for labeling.

24. The targeting composition of claim 1 wherein the targeting moiety includes the amino acid sequence GVMGFO (SEQ ID NO. 17).

25. The targeting composition of claim 1 wherein the targeting moiety includes a CBD from discoidin domain receptor DDR1.

26. The targeting composition of claim 1 wherein the targeting moiety includes a CBD from discoidin domain receptor DDR2.

27. The targeting composition of claim 1 wherein the targeting moiety includes a CBD incorporating the amino acids on the surface of the three-dimensional protein structure of DDR1 or DDR2 in which at least one of the amino acids not directly contacting collagen is replaced with a conservative amino acid substitution such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the native CBD from DDR1 or DDR2.

28. The targeting composition of claim 1 wherein the targeting moiety includes the amino acid sequence GTPGPGGIAGQRGW (SEQ ID NO: 19).

29. The targeting composition of claim 1 wherein the targeting moiety includes an amino acid sequence derived from GTPGPGGIAGQRGW (SEQ ID NO: 19) by one or more conservative amino acid substitutions such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the sequence GTPGPGGIAGQRGW (SEQ ID NO. 19).

30. The targeting composition of claim 1 that binds to native collagen fibers that differ from other collagen fibers in an organism that is targeted by virtue of having their surface exposed as a consequence of the metabolic activity associated with metastasis and/or inflammation.

31. The targeting composition of claim 1 wherein the intermediate release linker is stabilized by crosslinking.

32. The targeting composition of claim 31 wherein the crosslinking is produced by reaction with an aldehyde.

33. The targeting composition of claim 32 wherein the aldehyde is formaldehyde and the crosslinking is reversible.

34. The targeting composition of claim 32 wherein the aldehyde is glutaraldehyde and the crosslinking is irreversible.

35. The targeting composition of claim 31 wherein the intermediate release linker includes groups that are substrates for a transglutaminase and the crosslinking is produced by a reaction catalyzed by a transglutaminase.

36. The targeting composition of claim 1 further comprising a cell- penetrating peptide.

37. The targeting composition of claim 36 wherein the cell-penetrating peptide is selected from the group consisting of RRHHCRSKAKRSRHH (SEQ ID NO: 20), SRRHHCRSKAKRSRHH (SEQ ID NO: 21), SARHHCRSKAKRSRHH (SEQ ID NO: 22), SRAHHCRSKAKRSRHH (SEQ ID NO: 23), SRRAHCRSKAKRSRHH (SEQ ID NO: 24), SRRHACRSKAKRSRHH (SEQ ID NO. 25), SRRHHARSKAKRSRHH (SEQ ID NO. 26), SRRHHCRAKAKRSRHH (SEQ ID NO: 27), SRRHHCRSAAKRSRHH (SEQ ID NO: 28), SRRHHCRSKAARSRHH (SEQ ID NO: 29), SRRHHCRSKAKASRHH (SEQ ID NO: 30), SRRHHCRSKAKRARHH (SEQ ID NO: 31), SRRHHCRSKAKRSAHH (SEQ ID NO. 32), RRHHCRSKAKRSR (SEQ ID NO: 33), RKGKHKRKKLP (SEQ ID NO: 34),

GRKGKHKRKKLP (SEQ ID NO: 35), and GRRHHCRSKAKRSRHH (SEQ ID NO. 36).

38. The targeting composition of claim 36 wherein the cell-penetrating peptide is selected from the group consisting of NRKKRRQRRR (SEQ ID NO: 37), RRRRRRR (SEQ ID NO: 38), RRRRRRRR (SEQ ID NO: 39), and RRRRRRRRR (SEQ ID NO: 40).

39. The targeting composition of claim 36 wherein the cell-penetrating peptide is selected from the group consisting of Tyr-D-Arg-Phe-Lys-NH2, 2',6'-Dmt- D- Arg-Phe-Lys-NH₂, Phe-D-Arg-Phe-Lys-NH₂, D-Arg-2',6'-Dmt-Lys-Phe-NH₂, and 2',6'- Dmp-D-Arg-Phe-Lys-NH₂.

40. The targeting composition of claim 36 wherein the cell-penetrating peptide is selected from the group consisting of:

(i)

(SEQ ID NO: 41), wherein Xi is selected from the group consisting of A, L, and G, X₂ is selected from the group consisting of W and a peptide bond, X3 is selected from the group consisting of R and K, X is selected from the group consisting of K, L, and S, X₅ is selected from the group consisting of L and K, X₆ is selected from the group consisting of R and W, X₇ is selected from the group consisting of K and S, X₈ is selected from the group consisting of A, V, and Q, and X₉ is selected from the group consisting of W, F, Y, and a non- amino-acid aromatic group;

(ii) a peptide of SEQ ID NO: 41 wherein a non-peptide group selected from the group consisting of cysteamide, a cysteine, a thiol, an amide, a carboxyl moiety, a linear or branched Ci-6 optionally substituted alkyl moiety, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, and a polyethylene glycol is covalently linked to the carboxyl terminus of the peptide sequence; and

(iii) a peptide of SEQ ID NO: 41 wherein a non-peptide group selected from the group consisting of an acetyl moiety, a fatty acid moiety, a cholesterol moiety, and polyethylene glycol.

41. The targeting composition of claim 40 wherein the cell-penetrating peptide is selected from the group consisting of GLWRALWRLLRSLWRLLWKA (SEQ ID NO: 42), GLWRALWRALWRSLWKLKRKV (SEQ ID NO: 43),

GLWRALWRALRSLWKLKRKV (SEQ ID NO: 44), GLWRALWRGLRSLWKLKRKV (SEQ ID NO: 45), GLWRALWRGLRSLWKKKRKV (SEQ ID NO: 46),

GLWRALWRLLRSLWRLLWKA (SEQ ID NO: 47), G LWRALWRALWRS LWKLKWKV (SEQ ID NO: 48), GLWRALWRALWRSLWKSKRKV (SEQ ID NO: 49),

GLWRALWRALWRSLWKKKRKV (SEQ ID NO: 50), and

GLWRALWRLLRSLWRLLWSQ (SEQ ID NO: 51).

42. The targeting composition of claim 36 wherein the cell-penetrating peptide is selected from the group consisting of AAVALLPAVLLALLAPAAADQNQLMP (SEQ ID NO: 52) and AAVALLPAVLLALLAPAAANYKKPKLMP (SEQ ID NO: 53).

43. The targeting composition of claim 1 further comprising a

transcription-activating peptide.

44. The targeting composition of claim 43 wherein the transcription- activating peptide is selected from the group consisting of QLPPWL (SEQ ID NO: 54), QFLDAL (SEQ ID NO: 55), LDSFYV (SEQ ID NO:56), PPPPWP (SEQ ID NO: 57), SWFDVE (SEQ ID NO: 58), QLPDLF (SEQ ID NO: 59), PLPDLF (SEQ ID NO: 60), FESDDI (SEQ ID NO: 61), QYDLFP (SEQ ID NO: 62), LPDLIL (SEQ ID NO: 63), LPDFDP (SEQ ID NO: 64), LFPYSL (SEQ ID NO: 65), FDPFNQ (SEQ ID NO: 66), DFDVLL (SEQ ID NO: 67), HPPPPI (SEQ ID NO: 68), LPGCFF (SEQ ID NO: 69), QYDLFD (SEQ ID NO: 70), YPPPPF (SEQ ID NO: 71), PLPPFL (SEQ ID NO: 72), LPPPWL (SEQ ID NO: 73), VWPPAV (SEQ ID NO: 74), DPPWYL (SEQ ID NO: 75), LY (SEQ ID NO: 76), FDPFGL (SEQ ID NO: 77), PPSVNL (SEQ ID NO: 78), YLLPTCIP (SEQ ID NO: 79), LQVHNST (SEQ ID NO: 80), VLDFTPFL (SEQ ID NO: 81),

HHAFYEIP (SEQ ID NO: 82), PWYPTPYL (SEQ ID NO: 83), YPLLPFLPY (SEQ ID NO: 84), YFLPLLST (SEQ ID NO: 85), FSPTFWAF (SEQ ID NO: 86), and LIMNWPTY (SEQ ID NO: 87).

45. The targeting composition of claim 1 wherein the therapeutic agent is an anti-neoplastic therapeutic agent.

46. The targeting composition of claim 45 wherein the anti-neoplastic therapeutic agent is selected from the group consisting of mechlorethamine,

cyclophosphamide, ifosfamide, melphalan, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, streptozocin, dacarbazine, temozolomide, methotrexate, 5-fluorouracil, cytarabine, gemcitabine, 6-mercaptopurine, 6-thioguanine, pentostatin, vinblastine, vincristine, paclitaxel, docetaxel, topotecan, irinotecan, dactinomycin, daunorubicin, doxorubicin, bleomycin, mitomycin C, L-asparaginase, interferon-alfa, interleukin-2, cisplatin, carboplatin, mitoxantrone, hydroxyurea, /V-methylhydrazine, mitotane, aminoglutethimide, imatinib, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, hydroxyprogesterone,

medroxyprogesterone, megestrol acetate, diethylstilbestrol, ethinyl estradiol, tamoxifen, anastrozole, testosterone propionate, fluoxymesterone, flutamine, leuprolide,

trastuzumab, rituximab, alemtuzumab, bevacizumab, cetuximab, gemtuzumab, ibritumomab, panitumumab, tositumomab, and an interferon.

47. The targeting composition of claim 1 wherein the therapeutic agent is an anti-inflammatory therapeutic agent.

48. The targeting composition of claim 47 wherein the antiinflammatory therapeutic agent is a histamine receptor antagonist selected from the group consisting of histamine receptor Hi antagonists, histamine receptor H½ antagonists, histamine receptor H₃ antagonists, and histamine receptor H₄ antagonists.

49. The targeting composition of claim 48 wherein the antiinflammatory therapeutic agent is a kinin receptor antagonist.

50. The targeting composition of claim 49 wherein the kinin receptor antagonist is a kinin receptor antagonist selected from the group consisting of Bi and B2 receptor antagonists.

51. The targeting composition of claim 47 wherein the antiinflammatory therapeutic agent is a leukotriene receptor antagonist.

52. The targeting composition of claim 51 wherein the leukotriene receptor antagonist is a leukotriene receptor antagonist selected from the group consisting of zafirlukast and montelukast.

53. The targeting composition of claim 47 wherein the antiinflammatory therapeutic agent is a non-steroidal anti-inflammatory drug.

54. The targeting composition of claim 53 wherein the non-steroidal anti-inflammatory drug is a non-steroidal anti-inflammatory drug selected from the group consisting of acetylsalicylic acid (aspirin), sodium salicylate, choline magnesium trisalicylate, salsalate, diflunisal, sulfasalazine, olsalazine, acetaminophen,

indomethacin, sulindac, tolmetin, diclofenac, ketorolac, ibuprofen, naproxen,

flurbiprofen, ketoprofen, fenoprofin, oxaprozin, mefenamic acid, meclofenamic acid, piroxicam, meloxicam, nabumetone, rofecoxib, celecoxib, etodolac, nimesulide, aceclofenac, alclofenac, alminoprofen, amfenac, ampiroxicam, apazone, araprofen, azapropazone, bendazac, benoxaprofen, benzydamine, bermoprofen, benzpiperylon, bromfenac, bucloxic acid, bumadizone, butibufen, carprofen, cimicoxib, cinmetacin, cinnoxicam, clidanac, clofezone, clonixin, clopirac, darbufelone, deracoxib, droxicam, eltenac, enfenamic acid, epirizole, esflurbiprofen, ethenzamide, etofenamate, etoricoxib, felbinac, fenbufen, fenclofenac, fenclozic acid, fenclozine, fendosal, fentiazac, feprazone, filenadol, flobufen, florifenine, flosulide, flubichin methanesulfonate, flufenamic acid, flufenisal, flunixin, flunoxaprofen, fluprofen, fluproquazone, furofenac, ibufenac, imrecoxib, indoprofen, isofezolac, isoxepac, isoxicam, licofelone, lobuprofen, lomoxicam, lonazolac, loxaprofen, lumaricoxib, mabuprofen, miroprofen, mofebutazone, mofezolac, morazone, nepafanac, niflumic acid, nitrofenac, nitroflurbiprofen,

nitronaproxen, orpanoxin, oxaceprol, oxindanac, oxpinac, oxyphenbutazone,

pamicogrel, parcetasal, parecoxib, parsalmide, pelubiprofen, pemedolac,

phenylbutazone, pirazolac, pirprofen, pranoprofen, salicin, salicylamide, salicylsalicylic acid, satigrel, sudoxicam, suprofen, talmetacin, talniflumate, tazofelone, tebufelone, tenidap, tenoxicam, tepoxalin, tiaprofenic acid, tiaramide, tilmacoxib, tinoridine, tiopinac, tioxaprofen, tolfenamic acid, triflusal, tropesin, ursolic acid, valdecoxib, ximoprofen, zaltoprofen, zidometacin, and zomepirac.

55. The targeting composition of claim 47 wherein the antiinflammatory therapeutic agent is a steroid with anti-inflammatory activity.

56. The targeting composition of claim 55 wherein the steroid with antiinflammatory activity is a steroid with anti-inflammatory activity selected from the group consisting of hydrocortisone, cortisone, beclomethasone dipropionate, betamethasone, dexamethasone, prednisone, methylprednisolone, triamcinolone, fluocinolone

acetonide, and fludrocortisone.

57. The targeting composition of claim 1 wherein the therapeutic agent is selected from the group consisting of:

(a) a muscarinic cholinergic receptor agonist;

(b) a muscarinic cholinergic receptor antagonist;

(c) an anticholinesterase agent;

(d) a P2-selective adrenergic receptor agonist;

(e) a a₂-selective adrenergic receptor agonist;

(f) a sympathomimetic agonist;

(g) an ai -selective adrenergic receptor antagonist;

(h) a β-selective adrenergic receptor antagonist;

(i) a 5-hydroxytryptamine receptor agonist;

G) a 5-hydroxytryptamine receptor antagonist;

(k) a benzodiazepine;

(I) an antidepressant; (m) an antipsychotic drug;

(n) an antiseizure drug;

(o) a drug that is an anti-parkinsonism drug or a drug effective against a neurodegenerative disease;

(p) an opioid;

(q) a diuretic;

(r) an angiotensin converting enzyme inhibitor;

(s) a nonpeptide angiotensin II receptor antagonist;

(t) a calcium channel blocker;

(u) an antiarrhythmic drug;

(v) a drug having a therapeutic effect on hypercholesterolemia and/or dyslipidemia;

(w) an H₂ receptor antagonist;

(x) a drug having a therapeutic effect on disorders of bowel motility and/or water flux;

(y) a drug having a therapeutic effect on inflammatory bowel disease;

(z) an antimalarial;

(aa) an antiprotozoan agent;

(ab) an antihelminthic agent;

(ac) an antibacterial agent;

(ad) an antifungal agent;

(ae) an antiviral agent;

(af) an antiretroviral agent;

(ag) an immunomodulator;

(ah) a growth factor;

(ai) a hormone;

(aj) a nucleic acid or nucleotide sequence for nonviral gene therapy;

(ak) an antiangiogenic compound;

(al) an antisense oligonucleotide; and

(am) an antibody that binds a pharmacologically significant molecule; and the salts, solvates, analogues, congeners, bioisosteres, hydrolysis products, metabolites, precursors, and prodrugs thereof.

58. The targeting composition of claim 1 comprising two or more therapeutic agents.

59. The targeting composition of claim 1 wherein the intermediate release linker is a polymer that shields the therapeutic agent of the composition from clearance by macrophages.

60. The targeting composition of claim 59 wherein the polymer is a protein.

61. The targeting composition of claim 60 wherein the protein is selected from the group consisting of albumin, gelatin, keyhole limpet hemocyanin, ferritin, and ovalbumin.

62. The targeting composition of claim 61 wherein the protein is selected from the group consisting of albumin and gelatin.

63. The targeting composition of claim 62 wherein the protein is albumin.

64. The targeting composition of claim 63 wherein the albumin is bovine serum albumin.

65. The targeting composition of claim 60 wherein the protein is a synthetic polypeptide.

66. The targeting composition of claim 60 wherein the protein possesses at least one metalloprotease cleavage site.

67. The targeting composition of claim 60 wherein the protein is pegylated.

68. The targeting composition of claim 67 wherein the length of the polyethylene glycol chains is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers.

69. The targeting composition of claim 68 wherein the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers.

70. The targeting composition of claim 69 wherein the length of the polyethylene glycol chains is about 32 ethylene glycol monomers.

71. The targeting composition of claim 67 wherein the polyethylene glycol chains are blocked at the end not bound to the protein with a methyl ether group.

72. The targeting composition of claim 1 wherein the intermediate release linker does not interact with the therapeutic agent and does not bind to or otherwise interact with the targeting moiety.

73. The targeting composition of claim 72 wherein the intermediate release linker is a non-protein polymer.

74. The targeting composition of claim 73 wherein the non-protein polymer is selected from the group consisting of polyethylene glycol and polypropylene glycol.

75. The targeting composition of claim 74 wherein the non-protein polymer is polyethylene glycol.

76. The targeting composition of claim 75 wherein the length of the polyethylene glycol chains is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers.

77. The targeting composition of claim 76 wherein the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers.

78. The targeting composition of claim 77 wherein the length of the polyethylene glycol chains is about 32 ethylene glycol monomers.

79. The targeting composition of claim 1 wherein the intermediate release linker includes a thiol-containing amino acid sequence derived from keratin or a biosynthesized thiol-containing amino acid sequence mimicking the properties of the thiol-containing amino acid sequence derived from keratin.

80. The targeting composition of claim 1 wherein the intermediate release linker includes a hydrophobic amino acid sequence derived from elastin or a biosynthesized hydrophobic amino acid sequence mimicking the properties of the hydrophobic amino acid sequence derived from elastin.

81. The targeting composition of claim 1 wherein the linkage between the therapeutic agent and the intermediate release linker is a covalent linkage.

82. The targeting composition of claim 61 wherein each of the therapeutic agent and the intermediate release linker is derivatized by a peptide and the linkage between the therapeutic agent and the intermediate release linker is a peptide linkage.

83. The targeting composition of claim 81 wherein the covalent linkage between the therapeutic agent and the intermediate release linker is a cleavable linker.

84. The targeting composition of claim 1 wherein the linkage between the therapeutic agent and the intermediate release linker is a non-covalent linkage.

85. The targeting composition of claim 84 wherein the non-covalent linkage is a biotin/avidin or biotin/streptavidin linkage.

86. The targeting composition of claim 84 wherein the non-covalent linkage is a specific antigen/antibody or hapten/antibody linkage.

87. The targeting composition of claim 1 wherein the linkage between the intermediate release linker and the targeting moiety is a covalent linkage.

88. The targeting composition of claim 87 wherein each of the targeting moiety and the intermediate release linker is derivatized by a peptide and the linkage between the intermediate release linker and the targeting moiety is a peptide linkage.

89. The targeting composition of claim 87 wherein the covalent linkage between the intermediate release linker and the targeting moiety is a cleavable linker.

90. The targeting composition of claim 1 wherein the linkage between the intermediate release linker and the targeting moiety is a non-covalent linkage.

91. The targeting composition of claim 90 wherein the non-covalent linkage is a biotin/avidin or biotin/streptavidin linkage.

92. The targeting composition of claim 90 wherein the non-covalent linkage is a specific antigen/antibody or hapten/antibody linkage.

93. The targeting composition of claim 1 wherein the therapeutic agent is for treating joint inflammation.

94. The targeting composition of claim 93 wherein the therapeutic agent is a bisphosphonate.

95. The targeting composition of claim 94 wherein the bisphosphonate is selected from the group consisting of medronate, clodronate, etidronate, tiludronate, pamidronate, alendronate, ibandronate, risedronate, and zoledronate.

96. The targeting composition of claim 93 wherein the therapeutic agent is a bone morphogenetic protein or active portion thereof.

97. The targeting composition of claim 96 wherein the bone morphogenetic protein is selected from the group consisting of BMP-1 , BMP-2, BMP-3, BMP-3b, BMP4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8B, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, and nodal.

98. The targeting composition of claim 1 wherein the therapeutic agent is a growth factor.

99. The targeting composition of claim 98 wherein the growth factor is selected from the group consisting of adrenomedullin (AM), autocrine mobility factor, bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatoma derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), novel neurotrophin-1 (NNT-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor a (TGF-a), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and placental growth factor (PGF).

100. The targeting composition of claim 99 wherein the growth factor is

GM-CSF.

101. The targeting composition of claim 1 , wherein the therapeutic agent is a polypeptide or protein, and wherein the targeting composition further comprises a growth factor bound either to the polypeptide or protein therapeutic agent or the intermediate release linker.

102. The targeting composition of claim 101 wherein the growth factor is covalently bound to the polypeptide or protein therapeutic agent.

103. The targeting composition of claim 101 wherein the growth factor is covalently bound to the intermediate release linker.

104. The targeting composition of claim 101 wherein the growth factor is selected from the group consisting of adrenomedullin (AM), autocrine mobility factor, bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth differentiation factor-9 (GDF-9), hepatocyte growth factor (HGF), hepatoma derived growth factor (HDGF), insulin-like growth factor (IGF), migration stimulating factor (MSF), myostatin (GDF-8), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), novel neurotrophin-1 (NNT-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor a (TGF-a), transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and placental growth factor (PGF).

105. The targeting composition of claim 104 wherein the growth factor is

GM-CSF.

106. The targeting composition of claim 101 wherein the therapeutic agent is an anti-neoplastic therapeutic agent.

107. A pharmaceutical composition comprising:

(a) a therapeutically effective quantity of at least one targeting composition of claim 1 ; and

(b) a pharmaceutically acceptable carrier, diluent, or excipient in unit dosage form.

108. The pharmaceutical composition of claim 107 wherein the targeting composition includes an anti-neoplastic therapeutic agent.

109. The pharmaceutical composition of claim 107 wherein the targeting composition includes an anti-inflammatory therapeutic agent.

110. The pharmaceutical composition of claim 107 wherein the targeting composition includes both an anti-neoplastic therapeutic agent and an anti-inflammatory therapeutic agent.

111. The pharmaceutical composition of claim 107 wherein the pharmaceutical composition includes a first targeting composition that includes an antineoplastic therapeutic agent and a second targeting composition that includes an antiinflammatory therapeutic agent.

112. The pharmaceutical composition of claim 107 wherein the targeting composition includes a therapeutic agent selected from the group consisting of.

(a) a muscarinic cholinergic receptor agonist;

(b) a muscarinic cholinergic receptor antagonist;

(c) an anticholinesterase agent;

(d) a βΣ-εβΙβοίι β adrenergic receptor agonist;

(e) a oc2-selective adrenergic receptor agonist;

( ) a sympathomimetic agonist;

(g) an ai -selective adrenergic receptor antagonist;

(h) a β-selective adrenergic receptor antagonist;

(i) a 5-hydroxytryptamine receptor agonist;

(j) a 5-hydroxytryptamine receptor antagonist;

(k) a benzodiazepine;

(I) an antidepressant;

(m) an antipsychotic drug;

(n) an antiseizure drug;

(p) an opioid;

(q) a diuretic;

(r) an angiotensin converting enzyme inhibitor; (S) a nonpeptide angiotensin II receptor antagonist;

(t) a calcium channel blocker;

(u) an antiarrhythmic drug;

(w) an H₂ receptor antagonist;

(y) a drug having a therapeutic effect on inflammatory bowel disease;

(z) an antimalarial;

(aa) an antiprotozoan agent;

(ab) an antihelminthic agent;

(ac) an antibacterial agent;

(ad) an antifungal agent;

(ae) an antiviral agent;

(af) an antiretroviral agent;

(ag) an immunomodulator;

(ah) a growth factor;

(ai) a hormone;

(aj) a nucleic acid or nucleotide sequence for nonviral gene therapy; (ak) an antiangiogenic compound;

(al) an antisense oligonucleotide; and

13. The pharmaceutical composition of claim 107 wherein the pharmaceutical composition includes at least one additional therapeutic agent.

114. The pharmaceutical composition of claim 113 wherein the targeting composition includes an anti-neoplastic therapeutic agent and the pharmaceutical composition includes an additional anti-neoplastic therapeutic agent.

115. The pharmaceutical composition of claim 113 wherein the targeting composition includes an anti-neoplastic therapeutic agent and the pharmaceutical composition includes an anti-inflammatory therapeutic agent.

116. The pharmaceutical composition of claim 113 wherein the targeting composition includes an anti-inflammatory therapeutic agent and the pharmaceutical composition includes an anti-neoplastic therapeutic agent.

117. The pharmaceutical composition of claim 113 wherein the targeting composition includes an anti-neoplastic therapeutic agent and the pharmaceutical composition includes an additional anti-neoplastic therapeutic agent.

118. A method of treating a disease, disorder, or condition treatable by administration of a therapeutic agent comprising administration of a therapeutically effective quantity of the composition of claim 1 to a subject in need of treatment.

119. A method of treating a disease, disorder, or condition treatable by administration of a therapeutic agent comprising administration of a therapeutically effective quantity of the pharmaceutical composition of claim 88 to a subject in need of treatment.

120. The method of claim 118 wherein the disease, disorder, or condition is cancer.

121. The method of claim 119 wherein the disease, disorder, or condition is cancer.

122. The method of claim 118 wherein the disease, disorder, or condition involves an inflammatory process.

123. The method of claim 119 wherein the disease, disorder, or condition involves an inflammatory process.

124. The method of claim 118 wherein the disease includes a local release of enzymes that degrade components of the extracellular matrix without affecting the molecular structure of collagen.

125. The method of claim 119 wherein the disease includes a local release of enzymes that degrade components of the extracellular matrix without affecting the molecular structure of collagen.

126. The method of claim 118 wherein the collagen fibers are exposed in such a way as to make them accessible to circulating molecules or groups of molecules.

127. The method of claim 119 wherein the collagen fibers are exposed in such a way as to make them accessible to circulating molecules or groups of molecules.

128. The method of claim 118 wherein the method comprises the administration of an additional therapeutic agent.

129. The method of claim 119 wherein the method comprises the administration of an additional therapeutic agent.

130. A diagnostic composition comprising:

(a) a diagnostic agent;

(b) optionally, an intermediate release linker bound to the therapeutic agent; and

(c) a targeting moiety bound to the intermediate release linker, if present, or to the diagnostic agent if the intermediate release linker is not present, the targeting moiety for binding the targeting composition to native collagen fibers.

31. The diagnostic composition of claim 130 wherein the diagnostic agent is a diagnostic agent usable in computed tomography (CT) imaging.

132. The diagnostic composition of claim 130 wherein the diagnostic agent is a diagnostic agent usable in magnetic resonance imaging (MRI).

133. The diagnostic composition of claim 130 wherein the diagnostic agent is selected from the group consisting of iron oxide nanoparticles, gadolinium contrast agents with or without hyperpolarized substances, gadolinium-apoferritin, and manganese complexes.

134. The diagnostic composition of claim 130 wherein the diagnostic agent is an iodinated contrast agent.

35. The diagnostic composition of claim 134 wherein the iodinated contrast agent is selected from the group consisting of iohexol, iodixanol, ioversol, diatrizoate, metrizoate, ioxaglate, iopamidol, ioxilan, and iopromide.

136. The diagnostic composition of claim 130 wherein the diagnostic agent is attached to the targeting moiety.

137. The diagnostic composition of claim 136 wherein the diagnostic agent is attached covalently to the targeting moiety.

138. The diagnostic composition of claim 136 wherein the diagnostic agent is attached covalently to the targeting moiety.

139. The diagnostic composition of claim 136 wherein the diagnostic agent is attached in the form of individual molecules or ions to the targeting moiety.

140. The diagnostic composition of claim 136 wherein the diagnostic agent is attached in the form of a coating or other composite to the targeting moiety.

141. The diagnostic composition of claim 130 wherein the composition comprises an intermediate release linker and the diagnostic agent is attached to the intermediate release linker.

142. The diagnostic composition of claim 130 wherein the targeting moiety is selected from the group consisting of: (i) Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala- Leu-Ser (WREPSFMALS) (SEQ ID NO: 1); (ii) Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-Leu- Ser (WREPSFCALS) (SEQ ID NO: 2); and (iii) peptides related to (i) or (ii) by one or more conservative amino acid substitutions.

143. The diagnostic composition of claim 130 wherein the targeting moiety is a peptide related to SEQ ID NO: 1 or SEQ ID NO: 2 by one or more

conservative amino acid substitutions, and wherein the peptide is selected from the group consisting of: Trp-Arg-Asp-Pro-Ser-Phe-Met-Ala-Leu-Ser (WRDPSFMALS) (SEQ ID NO: 3); Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-Leu-Ser (WRDPSFCALS) (SEQ ID NO. 4); Trp-Arg-Glu-Pro-Ser-Phe-Met-Ala-lle-Ser (WREPSFMAIS) (SEQ ID NO: 5); Trp-Arg- Glu-Pro-Ser-Phe-Cys-Ala-lle-Ser (WREPSFCAIS) (SEQ ID NO: 6); Trp-Arg-Asp-Pro- Ser-Phe-Met-Ala-lle-Ser (WRDPSFMAIS) (SEQ ID NO: 7); and Trp-Arg-Asp-Pro-Ser- Phe-Cys-Ala-lle-Ser (WRDPSFCAIS) (SEQ ID NO: 8).

144. The diagnostic composition of claim 130 wherein the targeting moiety is a peptide selected from the group consisting of: Gly-Pro-Pro-Gly-Trp-Arg-Glu- Pro-Ser-Phe-Met-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFMALSGPPG) (SEQ ID NO: 9); Gly-Pro-Pro-Gly-Trp-Arg-Glu-Pro-Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWREPSFCALSGPPG) (SEQ ID NO: 10); Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser- Phe-Met-Ala-Leu-Ser-Gly-Pro-Pro-Gly (GPPGWRDPSFMALSGPPG) (SEQ ID NO: 11); Gly-Pro-Pro-Gly-Trp-Arg-Asp-Pro-Ser-Phe-Cys-Ala-Leu-Ser-Gly-Pro-Pro-Gly

(GPPGWRDPSFCAISGPPG) (SEQ ID NO: 16).

45. The diagnostic composition of claim 130 wherein the targeting moiety is an elongated peptide structure of Formula (I):

[Gly-Pro-Pro-Gly-Xi-Gly-Pro-Pro-Gly-X₂-Gly-Pro-Pro-Gly]_n

146. The diagnostic composition of claim 130 wherein the targeting moiety is a peptide motif that binds collagen with a binding affinity of at least 80% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen.

147. The diagnostic composition of claim 146 wherein the targeting moiety is a peptide motif that binds collagen with a binding affinity of at least 90% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen.

148. The diagnostic composition of claim 146 wherein the targeting moiety is a peptide motif that binds collagen with a binding affinity of at least 95% of the binding affinity of either SEQ ID NO: 1 or SEQ ID NO: 2 for collagen.

149. The diagnostic composition of claim 130 wherein the targeting moiety is a collagen binding site of a platelet collagen binding receptor.

150. The diagnostic composition of claim 149 wherein the collagen binding site of the platelet collagen binding receptor is selected from the group consisting of integrin α2β1 and glycoprotein VI.

151. The diagnostic composition of claim 130 wherein the peptide sequence WREPSF ALS (SEQ ID NO. 1) or WREPSFCALS (SEQ ID NO: 2) is incorporated into a molecule to generate a peptide of from about 2,000 daltons to about 10,000 daltons in molecular weight.

152. The diagnostic composition of claim 151 wherein the targeting moiety of the composition includes a flanking sequence that mimics a sequence found in native collagen.

153. The diagnostic composition of claim 151 wherein the targeting moiety of the composition includes a flanking sequence that mimics a sequence found in native elastin.

154. The diagnostic composition of claim 151 wherein the targeting moiety of the composition includes at least one reactive amino acid.

155. The diagnostic composition of claim 151 wherein the targeting moiety of the composition includes two or three collagen binding domains, with the collagen binding domains being separated by spacers.

156. The diagnostic composition of claim 155 wherein the spacers provide laterally displaced equivalent sites with a lateral displacement of about 3 nm.

157. The diagnostic composition of claim 55 wherein the spacers elongate in solution.

158. The diagnostic composition of claim 157 wherein the spacers include alternating polar and nonpolar sequences.

159. The diagnostic composition of claim 157 wherein the spacers include polylysine or polyglycine residues.

160. The diagnostic composition of claim 130 wherein the targeting moiety is pegylated.

161. The diagnostic composition of claim 130 wherein the targeting moiety includes a peptide sequence including an amino-terminal amino acid that is acetylated.

162. The diagnostic composition of claim 130 wherein the targeting moiety includes a peptide sequence including a carboxyl-terminal amino acid that is amidated.

163. The diagnostic composition of claim 130 wherein the targeting moiety includes a fluorescein moiety for labeling.

164. The diagnostic composition of claim 130 wherein the targeting moiety includes the amino acid sequence GVMGFO (SEQ ID NO. 17).

165. The diagnostic composition of claim 130 wherein the targeting moiety includes a CBD from discoidin domain receptor DDR1.

166. The diagnostic composition of claim 130 wherein the targeting moiety includes a CBD from discoidin domain receptor DDR2.

167. The diagnostic composition of claim 130 wherein the targeting moiety includes a CBD incorporating the amino acids on the surface of the three- dimensional protein structure of DDR1 or DDR2 in which at least one of the amino acids not directly contacting collagen is replaced with a conservative amino acid substitution such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the native CBD from DDR1 or DDR2.

168. The diagnostic composition of claim 130 wherein the targeting moiety includes the amino acid sequence GTPGPGGIAGQRGW (SEQ ID NO: 19).

169. The diagnostic composition of claim 130 wherein the targeting moiety includes an amino acid sequence derived from GTPGPGGIAGQRGW (SEQ ID NO: 19) by one or more conservative amino acid substitutions such that the CBD binds collagen with a binding affinity of at least 80% of the binding affinity of the sequence GTPGPGGIAGQRGW (SEQ ID NO: 19).

170. The diagnostic composition of claim 141 wherein the intermediate release linker is a polymer that shields the therapeutic agent of the composition from clearance by macrophages.

171. The diagnostic composition of claim 170 wherein the polymer is a protein.

172. The diagnostic composition of claim 171 wherein the protein is selected from the group consisting of albumin, gelatin, keyhole limpet hemocyanin, ferritin, and ovalbumin.

173. The diagnostic composition of claim 172 wherein the protein is selected from the group consisting of albumin and gelatin.

174. The diagnostic composition of claim 173 wherein the protein is albumin.

175. The diagnostic composition of claim 174 wherein the albumin is bovine serum albumin.

176. The diagnostic composition of claim 171 wherein the protein is a synthetic polypeptide.

177. The diagnostic composition of claim 171 wherein the protein possesses at least one metalloprotease cleavage site.

178. The diagnostic composition of claim 171 wherein the protein is pegylated.

79. The diagnostic composition of claim 178 wherein the length of the polyethylene glycol chains is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers.

180. The diagnostic composition of claim 179 wherein the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers.

81. The diagnostic composition of claim 180 wherein the length of the polyethylene glycol chains is about 32 ethylene glycol monomers.

182. The diagnostic composition of claim 178 wherein the polyethylene glycol chains are blocked at the end not bound to the protein with a methyl ether group.

83. The diagnostic composition of claim 141 wherein the intermediate release linker is a non-protein polymer.

184. The diagnostic composition of claim 183 wherein the non-protein polymer is selected from the group consisting of polyethylene glycol and polypropylene glycol.

185. The diagnostic composition of claim 184 wherein the non-protein polymer is polyethylene glycol.

186. The diagnostic composition of claim 185 wherein the length of the polyethylene glycol chains is from about 10 ethylene glycol monomers to about 60 ethylene glycol monomers.

187. The diagnostic composition of claim 186 wherein the length of the polyethylene glycol chains is from about 20 ethylene glycol monomers to about 40 ethylene glycol monomers.

188. The diagnostic composition of claim 187 wherein the length of the polyethylene glycol chains is about 32 ethylene glycol monomers.

189. The diagnostic composition of claim 141 wherein the intermediate release linker includes a thiol-containing amino acid sequence derived from keratin or a biosynthesized thiol-containing amino acid sequence mimicking the properties of the thiol-containing amino acid sequence derived from keratin.

190. The diagnostic composition of claim 141 wherein the intermediate release linker includes a hydrophobic amino acid sequence derived from elastin or a biosynthesized hydrophobic amino acid sequence mimicking the properties of the hydrophobic amino acid sequence derived from elastin.

191. The diagnostic composition of claim 141 wherein the linkage between the diagnostic agent and the intermediate release linker is a covalent linkage.

192. The diagnostic composition of claim 191 wherein each of the diagnostic agent and the intermediate release linker is derivatized by a peptide and the linkage between the diagnostic agent and the intermediate release linker is a peptide linkage.

193. The diagnostic composition of claim 91 wherein the covalent linkage between the diagnostic agent and the intermediate release linker is a cleavable linker.

194. The diagnostic composition of claim 1 1 wherein the linkage between the diagnostic agent and the intermediate release linker is a non-covalent linkage.

195. The diagnostic composition of claim 94 wherein the non-covalent linkage is a biotin/avidin or biotin/streptavidin linkage.

96. The diagnostic composition of claim 194 wherein the non-covalent linkage is a specific antigen/antibody or hapten/antibody linkage.

197. The diagnostic composition of claim 141 wherein the linkage between the intermediate release linker and the targeting moiety is a covalent linkage.

198. The diagnostic composition of claim 197 wherein each of the targeting moiety and the intermediate release linker is derivatized by a peptide and the linkage between the intermediate release linker and the targeting moiety is a peptide linkage.

99. The diagnostic composition of claim 197 wherein the covalent linkage between the intermediate release linker and the targeting moiety is a cleavable linker.

200. The diagnostic composition of claim 141 wherein the linkage between the intermediate release linker and the targeting moiety is a non-covalent linkage.

201. The diagnostic composition of claim 200 wherein the non-covalent linkage is a biotin/avidin or biotin/streptavidin linkage.

202. The diagnostic composition of claim 200 wherein the non-covalent linkage is a specific antigen/antibody or hapten/antibody linkage.

203. A method for diagnostic imaging comprising the steps of:

(a) administering a quantity of the diagnostic agent of claim 203 to a patient in need of diagnosis sufficient to produce a degree of contrast sufficient for a diagnostic imaging procedure; and

(b) performing a diagnostic imaging procedure on the patient.

204. The method of claim 203 wherein the diagnostic imaging procedure is computerized tomography (CT).

205. The method of claim 203 wherein the diagnostic imaging procedure is magnetic resonance imaging (MRI).

206. A composition comprising a liposome having attached to its surface a peptide motif for binding the liposome to native collagen fibers.

207. The composition of claim 206 wherein the composition further comprises a therapeutic agent.

208. The composition of claim 207 wherein the therapeutic agent is incorporated in the interior of the liposome.

209. The composition of claim 207 wherein the therapeutic agent is attached to the surface of the liposome.

210. The composition of claim 206 wherein the liposome further comprises a substance that can be identified by a radiological procedure selected from the group consisting of X-ray, MRI, and CT.

211. The composition of claim 210 wherein the substance that can be identified by the radiological procedure is selected from the group consisting of a radio- opaque substance and a radioactive substance.

212. A pharmaceutical composition comprising:

(a) a therapeutically effective quantity of the composition of claim 207; and

a pharmaceutically acceptable carrier, diluent, or excipient in unit dosage form,

213. A targeting composition comprising:

(a) a therapeutically effective radionuclide;

(b) an intermediate release linker bound to the therapeutically effective radionuclide; and

214. The targeting composition of claim 213 wherein the radionuclide is

131 j

215. The targeting composition of claim 214 wherein the I¹³¹ is covalently bound to the intermediate release linker.

216. The targeting composition of claim 213 wherein the radionuclide is selected from the group consisting of ⁹⁰Y and ¹¹¹ln.

217. The targeting composition of claim 216 wherein the radionuclide is bound to the intermediate release linker by a chelator that bound to the intermediate release linker.

2 8. The targeting composition of claim 217 wherein the chelator is selected from the group consisting of cyclic DPTA (diethylene triamine pentaacetic acid) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid), and tiuxetan.

219. A targeting composition comprising:

(a) a diagnostically effective radionuclide;

(b) an intermediate release linker bound to the diagnostically effective radionuclide; and

220. The targeting composition of claim 219 wherein the diagnostically effective radionuclide is selected from the group consisting of ^99mTc, ²⁰¹ Tl, and ⁶⁷Ga.