WO1995012414A1 - Nouvelles compositions a base de pf4 modifie et procedes d'utilisation - Google Patents

Nouvelles compositions a base de pf4 modifie et procedes d'utilisation Download PDF

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Publication number
WO1995012414A1
WO1995012414A1 PCT/US1994/012737 US9412737W WO9512414A1 WO 1995012414 A1 WO1995012414 A1 WO 1995012414A1 US 9412737 W US9412737 W US 9412737W WO 9512414 A1 WO9512414 A1 WO 9512414A1
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rpf4
conjugate
conjugated
angiogenesis
entity
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PCT/US1994/012737
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English (en)
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Theodore E. Maione
Chee Kong Lai
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Repligen Corporation
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Priority to AU11717/95A priority Critical patent/AU1171795A/en
Publication of WO1995012414A1 publication Critical patent/WO1995012414A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • Angiogenesis the development of new capillary blood vessels, is an important process in the developing fetus and growing human. However, in healthy adults, angiogenesis occurs significantly only during wound healing and in the menstrual cycle.
  • angiogenic dysfunctions include diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune inflammation (including rheumatoid arthritis), capillary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma.
  • angiogenic diseases Folkman, J., M. Klagsbrun [1987] Science 235:442-447.
  • endothelial cell proliferation is pathological or, at least, unwanted.
  • endometriosis is characterized by the abnormal proliferation and positioning of certain endothelial cells which normally line the inner wall of the uterus. Control of the angiogenic process could help to prevent or alleviate endometriosis. Also, prevention of endothelial cell growth in the uterus could be a means of birth control.
  • Endothelial cell growth is associated with wound healing. This growth is undesirable during extended surgical proceedings and where excessive scar formation may occur. Therefore, a means of controlling endothelial cell proliferation would help prevent or reduce unwanted scar formation.
  • protamine is a protein found only in sperm.
  • Clinical experiments with protamine have not been pursued because of the toxicity associated with protamine injection.
  • PF4 platelet factor 4
  • major basic protein has demonstrated heparin-binding activity but is of little practical utility because of its high toxicity.
  • Platelet factor 4 is a well-known protein which has been completely sequenced (Deuel, T.F., P.S. Keim, M. Farmer, R.L. Heinrikson [1977] Proc. Natl. Acad. Sci.
  • compositions which contain a compound described as "oncostatin A,” which appears to be the same, or similar to, native PF4, have been implicated as affecting the growth of tumors (U.S. Patent Nos. 4,645,828 and 4,737,580; both issued to
  • Twardzik et al the effects reported in these patents pertain to slowly growing human cancer cells in immunodeficient mice. The results of these experiments cannot be reliably extrapolated to predict the effect on rapidly growing tumors which are native to the host animal. Furthermore, the experiments reported in these patents in no way predict or disclose any angiostatic properties. Also, the preparations used by Twardzik et al. were purified from platelets and likely contained a mixture of proteins. These purified platelet compositions would be expected to contain other biologically-active proteins including compounds such as TGF- ⁇ which are known to inhibit angiogenesis.
  • Angiogenesis plays a major role in the initiation and progression of widespread catastrophic illnesses, including cancer.
  • An effective, non-toxic agent which can be administered locally and/or systemically to treat these illnesses would be highly advantageous and has long eluded identification.
  • angiogenesis and endothelial cell proliferation can be useful for surgical, radiotherapeutic, and chemotherapeutic approaches to control or eliminate the unwanted angiogenesis.
  • chemotherapeutic approaches to control or eliminate the unwanted angiogenesis.
  • monoclonal antibodies prepared against tumors have been proposed for use in the past as effective carrier molecules for the delivery of contrast and radionuclide agents.
  • the use of such monoclonal antibodies can be accompanied by disadvantages.
  • antibodies are very large molecules that also can carry cross-reactive antigenic determinants that may immunoreact in undesirable ways.
  • monoclonal antibodies that bind with particular tumor-specific antigens can have an important limitation of being too specific. Due to their specificity, these antibodies may not recognize many unwanted neovascularization sites. Thus, a monoclonal antibody specific to a particular tumor agent may be effective in directing an imaging agent to that particular type of tumor, but other sites of neovascularization may go undetected. Furthermore, the tumor to which the antibody is specifically directed may not be reliably detected if the antigen recognized by the antibody is not exposed or otherwise is inappropriately presented.
  • polypeptides such as polylysine which selectively binds to tumor cells
  • polylysine which selectively binds to tumor cells
  • carrier agents for therapeutic or imaging agents.
  • polypeptides can be highly toxic and, therefore, impractical for clinical use.
  • the subject invention relates to diagnostic and therapeutic compositions obtained through chemical modifications of platelet factor 4 (PF4) or recombinant PF4 (rPF4).
  • PF4 refers both to PF4 purified from platelets and to recombinant PF4 unless otherwise noted.
  • conjugates comprising PF4 can be made by chemically attaching a second entity to the PF4.
  • the subject invention further relates to methods for using the PF4 conjugates described herein.
  • compositions and methods of the subject invention are particularly useful in the detection and treatment of tumors and other sites or areas of unwanted neovascularization.
  • the compositions and methods of the subject invention can also be used to locate normal angiogenesis such as what may occur during pregnancy or during the formation of scar tissue.
  • the compositions and methods of the subject invention are also useful for the rapid, accurate, and safe detection of sites of angiogenesis and/or endothelial cell proliferation. Because PF4 is non-toxic, non-immunogenic, and can be used systemically, the compositions and methods of the subject invention can be used to safely locate and treat widely disseminated sites of neovascularization.
  • PF4 can be conjugated to a variety of entities and surprisingly retain its advantageous angiostatic properties.
  • PF4 can be conjugated to a second entity which will target the activity of PF4 to a particular location where angiostatic activity is needed.
  • Molecules which can be used in this manner to direct the activity of the PF4 include, but are not limited to, monoclonal antibodies, polyclonal antibodies, carrier proteins, carbohydrate lectins, cell receptor molecules, single chain antigen-binding proteins, and other binding protein sequences.
  • PF4 can be conjugated to molecules in order to prolong the half-life of the PF4 in the biological system.
  • a polymer can be conjugated to PF4 to increase the circulating half- life of the PF4.
  • the polymer may be, for example, a polyamino acid such as polyglutamate, a polysaccharide such as dextran, or a biocompatible polymer such as polyethylene glycol (PEG).
  • PF4 preferentially binds to sites of endothelial cell proliferation and angiogenesis. Therefore, PF4 can itself be used as a targeting agent and be conjugated to any of a variety of entities which are desired to be selectively and accurately delivered to sites of angiogenesis or endothelial cell proliferation.
  • PF4 is the molecule which directs the activity of a second entity to a specific location.
  • PF4 can be conjugated to toxins which will kill tumor cells.
  • PF4 can be conjugated to imaging agents which facilitate the detection and visualization of sites of angiogenesis and endothelial cell proliferation. This embodiment is based on applicants' discovery that PF4 is transported systemically through the body to areas of angiogenesis including new proliferating vascular beds.
  • this embodiment can be used to facilitate the accurate visualization of tumors and other sites of angiogenesis.
  • PF4 can also be used to detect sites of normal neovascularization such as may occur in pregnancy or during scar tissue formation.
  • One of the advantages of using PF4 as the targeting agent is that it detects a wide range of sites of angiogenesis and it can safely be used systemically in such a procedure without toxicity.
  • PF4 can be conjugated with a second entity which can be detected by means known to those skilled in the art.
  • This second entity can be, for example, a fluorescent compound or a radioisotope.
  • the PF4 conjugate comprising the detectable second entity is then administered such that the
  • PF4 can be biologically transported to the location at which knowledge of angiogenesis is desired.
  • this transport can occur systemically.
  • PF4 can be modified through its free amino groups with fluorescein-isothiocyanate (FITC) and retain the capability of inhibiting angiogenic activity and endothelial cell proliferation. Modification with FITC also makes it possible to visualize sites of angiogenesis and endothelial cell proliferation. Many other agents in addition to FITC can also be used for this purpose. As described in detail below, similar modifications can be made with PF4 analogs, mutants, or fragments.
  • FITC fluorescein-isothiocyanate
  • One embodiment of the subject invention concerns the discovery of a particular region of the PF4 molecule at which conjugation can occur without losing biological activity. Specifically, the approximately 15 amino acids at the N-terminus of the full- length PF4 molecule has been discovered to be a preferred location for conjugation. The discovery of this region of the PF4 molecule, referred to herein as the "Preferred
  • PCS Conjugation Site
  • Figure 1 shows the inhibition of angiogenesis resulting from treatment with rPF4 and various related peptides.
  • Figure 2 compares the amino acid sequence of rPF4 with rPF4-241.
  • Figure 3 depicts the ⁇ -helical configurations of rPF4 and rPF4-241.
  • Figure 4 compares the inhibition of angiogenesis resulting from treatment with rPF4 and rPF4-241.
  • Figure 5 shows inhibition of human endothelial cell proliferation by rPF4 and rPF4-241.
  • Figure 6 compares the inhibition of human umbilical vein endothelial cell proliferation resulting from treatment with rPF4 or rPF4-241.
  • Figure 7 shows the ability of rPF4 to inhibit tumor growth.
  • Figure 8 depicts the possible chemical structure of the C-terminal end of FrPF4.
  • Figure 9 shows inhibition of human endothelial cell proliferation by FrPF4.
  • Figure 10 shows inhibition of human endothelial cell proliferation by FrPF4- 241.
  • FIG. 11 shows inhibition of tumor growth by FrPF4.
  • Figure 12 shows activity of PF4 and PF4 conjugated to glycine (Gly-PF4) in the HUVEC proliferation assay.
  • Figure 13 shows activity of PF4 and a PEG-PF4 conjugate in the HUVEC proliferation assay.
  • Figure 14 shows the activity of PF4 and a biotin-PF4 conjugate in the HUVEC proliferation assay.
  • Figure 15 shows the effect of intravenous administration of PEG-rPF4 or rPF4 on lung metastasis formation as measured by the total number of lung metastases.
  • Figure 16 shows the effect of intravenous administration of PEG-rPF4 or rPF4 as measured by lung weight.
  • Figure 17 shows inhibition of tumor growth achieved by intradermal injection of PEG-rPF4 or rPF4 as compared to controls.
  • Figure 18 shows pharmacokinetics of PEG-rPF4 compared to rPF4 after intravenous administration to Sprague-Dawley rats.
  • Figure 19 shows the biodistribution of intravenously administered PEG-rPF4 as compared to rPF4.
  • SEQ ED NO. 1 is the amino acid sequence of C-13-241.
  • SEQ ED NO. 2 is the mutant sequence designated rPF4-211.
  • SEQ ID NO. 3 is the mutant sequence designated rPF4-231.
  • SEQ ED NO. 4 is the mutant sequence designated rPF4-241.
  • SEQ ED NO. 5 is the mutant sequence designated rPF4-302.
  • SEQ ID NO. 6 is the mutant sequence designated rPF4-303.
  • SEQ ID NO. 7 is the mutant sequence designated rPF4-307.
  • SEQ ID NO. 8 is the mutant sequence designated rPF4-308.
  • SEQ ID NO. 9 is the mutant sequence designated rPF4-315.
  • SEQ ID NO. 10 is a generic sequence formula according to the subject invention.
  • SEQ ID NO. 11 is a more detailed sequence formula for practicing the subject invention.
  • the subject invention concerns the discovery that PF4, rPF4, and fragments and mutants of these compounds can be chemically modified to create conjugates with highly desirable characteristics.
  • chemical modification of PF4, its fragments, and its mutants has resulted in the discovery of compounds which show surprising ability to inhibit endothelial cell proliferation and angiogenic activity and they preferentially bind to proliferating endothelial cells.
  • PF4 recombinant PF4
  • PF4 purified from its natural source PF4
  • PF4 should also be understood to refer to variants, mutants, and fragments as described herein.
  • conjugation of PF4 to a second entity includes direct conjugation and indirect conjugation such as through a binder or chelation.
  • PF4 is conjugated to entities which enhance, direct, or prolong the biological activity of PF4.
  • PF4 may be conjugated to molecules that specifically recognize sites of unwanted angiogenesis. Examples of such molecules are monoclonal antibodies and cell receptors which direct the angiostatic PF4 activity to a specific location such as a tumor.
  • PF4 may be conjugated to molecules such as polyethylene glycol (PEG), which will prolong its half-life in the circulation of a treated animal.
  • PEG polyethylene glycol
  • PF4 is itself used to direct a second entity to certain locations within the body of an animal or in vitro.
  • This embodiment arises from applicants' discovery that PF4 preferentially binds to sites of endothelial cell proliferation characteristic of angiogenesis. Because PF4 binds to these sites, it can be used to direct other molecules with useful properties to these sites. Therefore, although PF4 is itself a potent inhibitor of angiogenesis, it can be conjugated with other angiogenesis inhibitors which can result in enhanced or expedited activity. For example, PF4 can be conjugated with toxins which destroy tumor cells, thereby producing an enhanced effect when used with PF4 which itself inhibits tumor growth.
  • Such a combination of PF4 with another active ingredient has the advantage of not only targeting these activities specifically to sites of neovascularization, but also such a combination can create a synergistic antitumor effect through the combined activities of the components of the conjugate.
  • a further illustration of the second embodiment of the subject invention is the use of PF4 conjugates in diagnostic procedures whereby the PF4 is used to direct a detectable label to sites of angiogenesis or cellular proliferation.
  • sites of angiogenesis can be pathological, as in the case or tumors, or can be normal, such as in the case of pregnancy or scar tissue formation.
  • PF4 may be conjugated to radioisotopes or fluorescent compounds which can readily be detected using procedures which are well known to those skilled in the art.
  • PF4 conjugates as angiogenesis inhibiting agents.
  • the PF4 conjugates of the subject invention can be used in the treatment of angiogenic diseases.
  • angiogenic disease refers to pathological events such as growth of solid tumors, and other conditions involving angiogenic dysfunctions including diabetic retinopathy, retrolental fibroplasia, ocular neovascularization, corneal neovascularization (including that which is induced by trauma, infection or irritation), neovascular glaucoma, macular degeneration, psoriasis, angiofibromas, immune and non-immune inflammation (including rheumatoid arthritis), capillary proliferation within atherosclerotic plaques, unwanted scar tissue formation, hemangiomas, and Kaposi's sarcoma.
  • the PF4 conjugates described herein for the treatment of angiogenic diseases include conjugates of PF4 fragments, analogs, and variants.
  • the types of solid tumors that can be treated by PF4 conjugates of the subject invention include all types of lung tumors, including small cell lung carcinoma; tumors of the breast, colon/rectum, prostate, head and neck, stomach, bladder, kidney, pancreas, liver, ovary, and uterus; sarcomas; melanoma and other metastatic skin cancers; nonmetastatic skin cancers (e.g., Kaposi's sarcoma, basal cell carcinoma); and tumors of the brain.
  • lung tumors including small cell lung carcinoma; tumors of the breast, colon/rectum, prostate, head and neck, stomach, bladder, kidney, pancreas, liver, ovary, and uterus; sarcomas; melanoma and other metastatic skin cancers; nonmetastatic skin cancers (e.g., Kaposi's sarcoma, basal cell carcinoma); and tumors of the brain.
  • the types of tumors that can be treated using the compositions and methods of the invention include tumors that are not surgically accessible and/or are resistant to chemotherapy and radiation therapy.
  • Advanced invasive malignancy with or without surgery, can be treated using the compositions and methods of the invention.
  • the invention can be used as an adjuvant therapy following surgical resection and can be used to treat known metastatic disease and nonmetastatic cancer.
  • Treatment of the tumors and diseases described above can be either systemic, regional, or local (intralesional), depending upon the type and severity of the disease as well as the accessibility of the disease site.
  • Systemic treatment includes intravenous bolus injections and infusions, subcutaneous injections, implants, refillable reservoirs, and sustained release depots and intramuscular injections.
  • Regional treatment includes intraarterial for the treatment of primary liver tumors and liver metastases, and for the treatment of kidney, brain, and pancreatic tumors.
  • Regional intraperitoneal treatment can be used for the treatment of tumors of the ovary.
  • Local treatment can be used for tumors of the brain, uterus, bladder, head, and neck, for
  • the PF4 conjugates of the subject invention can be administered as an alternative to known procedures such as surgery, chemotherapy, or radiation therapy. Also, the PF4 conjugates can be used concurrently with, or following, such procedures in order to enhance the effectiveness of these procedures by, for example, preventing further growth of any tumor tissue which may remain after an initial surgical, chemical, or radiation treatment.
  • PF4 Conjugation of PF4 to targeting agents specific to certain sites of neovascularization can be used to increase the accumulation of PF4 at the site where inhibition of angiogenesis is desired.
  • targeting agents are preferably specific for the type of tissue or tumor for which PF4 activity is desired.
  • Specific examples of such targeting agents include proteins, antibodies, and binding fragments of antibodies.
  • other protein sequences which are known to bind to proliferating endothelial cells can be conjugated to PF4 according to the subject invention.
  • proteins which bind to proliferating cells include tumor necrosis factor (TNF), transforming growth factor- ⁇ (TGF- ⁇ ), antibodies to tumor-specific antigens, basic fibroblast growth factor (bFGF), prostate-specific antigen (PSA), and chorioembryonic antigen (CEA).
  • TNF tumor necrosis factor
  • TGF- ⁇ transforming growth factor- ⁇
  • bFGF basic fibroblast growth factor
  • PSA prostate-specific antigen
  • CEA chorioembryonic antigen
  • Such a conjugate will direct the PF4 activity in a highly specific manner to a precise location where angiostatic activity is desired.
  • carbohydrate lectins are known to be specific for certain tumors.
  • Examples of such specificities are galactose for melanoma, sarcoma, colon carcinoma, and Hodgkin disease; mannose for teratocarcinoma; glucose for colon carcinoma; and L-fucose for teratoma.
  • conjugation of biocompatible polymers to PF4 can be used to enhance circulation half-life of PF4 in the blood.
  • This process can also be used to alter the biodistribution of the protein according to the type of polymer used. Specific examples are the use of polyethylene glycol (PEG), polyvinyl alcohol, and polyurethane.
  • PF4 conjugates to treat diseases involving ocular neovascularization. These diseases can be very serious, leading in some cases to blindness. PF4 is highly advantageous in that it can be used to effectively treat these conditions without toxicity.
  • the clinical utility of PF4 as a treatment for angiogenic diseases of the eye has been determined using two different animal models. The first model simulates angiogenic diseases of the cornea and the second model simulates angiogenic diseases of the retina. These models are predictive of clinical utility for treating diseases involving neovascularization of the retina or cornea or other part of the eye (collectively referred to as ocular neovascularization).
  • the assay used to determine the effect of PF4 on corneal neovascularization involves the implantation of angiogenic stimuli into a surgically-created micropocket in the cornea of animals.
  • the assay is often conducted using rats or rabbits.
  • the angiogenic stimuli induces a vigorous neovascular response which can be easily detected in this normally avascular tissue.
  • PF4 is incorporated, along with the angiogenic stimuli, into the implant pellet in order to assess the effect of PF4 on the neovascularization process.
  • This assay demonstrates the ability of PF4 to effectively inhibit corneal neovascularization.
  • retinal capillary networks The aberrant development of retinal capillary networks is a cause of significant vision loss in several disease conditions such as proliferative diabetic retinopathy, disciform macular degeneration, and retinopathy of prematurity (or retrolental fibroplasia). While the precise etiologic events or agents causing these conditions is unknown, all three are associated with retinal ischemic damage following a transient decrease of the oxygen supply in the tissue. The current understanding of these conditions suggests that the common event of transient altered oxygen levels induces tissue damage, the release of mitogenic cytokines, and vigorous vascular endothelial cell migration, proliferation, and assembly. The conditions associated with retinal neovascularization can be simulated in neonatal rats.
  • the relatively avascular retina of the full term newborn rat is similar in morphology to that of the minimally vascularized retina of a premature human baby. Under conditions of hyperbaric oxygen followed by normal air, a pathology is induced in both species' retinas, which is remarkably similar as assessed morphologically and histologically.
  • the retinal pathology associated with the adult diseases mentioned above presents a similar morphological pattern which is believed to be caused by the same type of transient ischemic event.
  • the neonatal rat model is a major step forward in the identification of broadly active antiproliferative drugs for treatment of neovascular retinopathy. Therefore, the positive results reported here establish that PF4 effectively inhibits retinal neovascularization.
  • the combined results showing the advantageous activity of PF4 in both the cornea model and the retina model establish that this protein is effective across a range of ocular diseases when those diseases involve neovascularization.
  • Kaposi's sarcoma This human clinical data was obtained from twelve patients with Kaposi's sarcoma (KS) who were treated with PF4 doses ranging from 0.1 to 2.5 mg/lesion daily for 7 days, then three times a week for 3 additional weeks. Each subject had two randomly selected lesions chosen for study, and a prospective randomized blinded administration of study drug or control was performed. Each patient had one lesion injected with rPF4 and one lesion injected with saline. No serious life-threatening adverse events occurred; two patients died of other AIDS-related events. Mild to moderate erythema was observed at the injection site in approximately 20% of the patients. Of 12 patients injected, 6 patients demonstrated some anti-KS effects, and all 3 patients at the highest dose demonstrated anti-KS effects. This study demonstrates the safety and utility of rPF4.
  • KS Kaposi's sarcoma
  • PF4 as an inhibitor of angiogenesis, has clinical utility in the management and detection of widely disseminated sites of angiogenesis and cellular proliferation. For example, progressive growth of tumors requires new blood vessel formation which, if inhibited, may not only restrict tumor growth, but stimulate regression of existing vessels, as well as enhance other responses (such as immune responses) to malignant invasion. As described below, the subject invention is particularly advantageous when the angiostatic activity of PF4 is combined in a synergistic way with toxins which destroy existing tumor cells.
  • PF4 as a targeting agent.
  • the applicants' discovery that PF4 preferentially binds to proliferating endothelial cells is the basis for the use of PF4 as a targeting agent.
  • This embodiment of the invention is highly advantageous because it enables the skilled artisan to accurately and predictably direct any one of a variety of biological activities to sites of endothelial cell proliferation located throughout the body.
  • various pharmaceutical, cytotoxic, or diagnostic agents can be covalently or non-covalently coupled to the PF4 to form a conjugate.
  • radioactive compounds e.g., 125 I, 131 I
  • agents which bind DNA such as alkylating agents or various antibiotics (daunomycin, adriamycin, chlorambucil); anti-metabolites (e.g., methotrexate); inhibitors of protein synthesis (e.g., diphtheria toxin and toxin plant proteins); and cytokines (e.g., tumor necrosis factor, interferon, and interleukin-2).
  • PF4 can be particularly advantageous as a drug delivery vehicle because of its heparin-binding characteristics. Pre-incubation with heparin before injection will prevent PF4 from binding to the vascular wall. There is evidence that the endothelium in different vascular beds is coated by unique species of heparin sulfate proteoglycans. Thus, by incubating PF4 with a mixture of unique species of heparin sulfate, PF4 may be directed predominantly to specific locations.
  • PF4 can be conjugated to a label which generates a detectable signal.
  • the conjugate is administered to an animal suspected of having a tumor, other pathological angiogenesis, or normal angiogenesis which is desired to be detected.
  • the signal generated by the label can then be detected by an appropriate detection device.
  • the detected signal can then be converted to an image of the site. This image makes it possible to localize the angiogenesis site in vivo.
  • this data can be used to develop an appropriate therapeutic strategy or to monitor disease progression.
  • Such procedures can also be used in vitro to, for example, assess the angiogenic activity of excised tissue.
  • a PF4 conjugate can be used in either an in vivo or in vitro assay to assess whether a tumor has angiogenic activity characteristic of malignant tumors.
  • tumor tissue which has been removed from an animal can be contacted with a composition comprising PF4 bound to a detectable label.
  • the tumor sample can be separated from the PF4-conjugate solution and the sample can then be analyzed to determine the extent to which PF4 bound to the tissue.
  • a high incidence of PF4 binding is associated with active cellular proliferation characteristic of a malignant tumor. This procedure also could readily be utilized by a person skilled in the art to assess the malignancy of tumors in vivo.
  • the PF4 of the subject invention can also be used in a pregnancy test.
  • the PF4 can be conjugated in a manner which reduces or eliminates angiostatic activity while maintaining the ability to bind to sites of endothelial cell proliferation.
  • pregnancy could be avoided or halted by the use of a PF4 conjugate that inhibits angiogenesis.
  • the labels that are suitable for conjugation to PF4 for angiogenesis localization include, but are not limited to, radio labels (e.g., radioisotopes) (also referred to herein as radiation emitters), fluorescent labels, and biotin labels.
  • Radioisotopes that are suitable for labeling PF4 include Iodine-131, Iodine-123, Iodine-125, Iodine-126, Iodine-133, Bromine-77, Indium-111, Indium-113m, Gallium-67, Gallium-68, Ruthenium-95, Ruthenium-97, Ruthenium- 103, Ruthenium- 105, Mercury-107, Mercury-203, Rhenium-99m, Rhenium-105, Rhenium-101, Tellurium-121m, Tellurium-99m, Tellurium-125m, Thulium-165, Thulium-167, Thulium-168, Technetium-99m, and Fluorine-18.
  • Fluorescent compounds suitable for labeling PF4 include fluorescein sodium, fluorescein isothiocyanate, and Texas red sulfonyl chloride. Those skilled in the art will, with the benefit of the current application, be able to utilize other detectable compounds that are suitable for labeling PF4.
  • PF4 is used to direct a diagnostic agent for magnetic resonance imaging of tumors to sites of cellular proliferation or neovascularization.
  • chelators for indium or gadolinium such as diethylenetriaminepentaacetic acid may be conjugated according to the protocols provided herein.
  • chlorin-e6 or related photoactivable molecules may be conjugated to PF4 as a means of photodynamic therapy for surface accessible vascular diseases such as diabetic retinopathy.
  • neutron activatable molecules such as Boron-10 can also be conjugated to PF4 for Boron neutron capture therapy.
  • chemotherapeutic drugs such as methotrexate, ricin A, or doxorubicin
  • PF4 acts as a targeting agent for tumor neovasculature sites and is able to more efficiently accumulate the conjugated drugs at those sites thereby reducing the tumor as well as inhibiting angiogenesis.
  • attachment of a DNA fragment via, for example, an acid hydrolyzable bond to PF4 can be used to facilitate efficient targeting of genes to proliferating endothelial cells.
  • the DNA fragment Upon endocytosis, the DNA fragment is captured into the endosomes and eventually released into the cell where it expresses the desired gene product.
  • PF4 can be used in angiography.
  • ophthalmologists currently carry out fluorescein angiograms by giving a single intravenous injection of fluorescein and then taking rapid photographs of the eye using blue light or cobalt light to fluoresce the blood vessels in the retina.
  • Neovascularization is detected by underlying leakage of fluorescein from the new vessels which leave a yellow perivascular stain. This is an indirect way of detecting neovascularization.
  • this procedure is subject to misinterpretation of the results because there are many other causes of leakage of fluorescein that are not caused by neovascularization.
  • PF4 in angiography is advantageous because the fluorescein does not escape from the vessels and is visible for at least one to two hours, thus providing the ophthalmologist with a "standing angiography.” In this way, the ophthalmologist has time to look directly at the eye and examine it without having to wait for films to be developed. Films can also be taken to provide a permanent record.
  • Fluorescein-labeled PF4 can also be used to augment laser therapy.
  • the subject invention embraces the specific amino acid sequences and other compositions which are specifically exemplified herein.
  • the subject invention further embraces analogs and variants of these sequences, as well as fragments of the sequences, and analogs and mutants of the fragments.
  • analog refers to compounds which are substantially the same as another compound but which may have been modified by, for example, adding side groups.
  • “Mutants” and “variants” as referred to in this application refer to amino acid sequences which are substantially the same as another sequence but which have amino acid substitutions, additions, or deletions at certain locations within the amino acid sequence or at the ends of the sequence.
  • “Fragments” refer to portions of a longer amino acid sequence. These analogs, variants, and fragments are embraced within the subject invention so long as the analog, fragment, or variant retains substantially the same relevant biological activity as the originally exemplified compound. For example, it is well within the skill of a person trained in this art to make conservative amino acid substitutions. These substitutions are discussed in more detail below. To the extent that these substitutions do not substantially alter the relevant biological activity, then the resulting compounds fall within the scope of the subject invention.
  • the term "relevant biological activity” refers to the activity of interest for a particular application of a compound. For example, several uses of PF4 are discussed below.
  • PF4 uses include inhibition of angiogenesis and endothelial cell proliferation and binding to proliferating endothelial cells.
  • variants would refer to compounds where PF4 has been modified (by a conservative amino acid substitution, for example) without, for example, substantially altering the compound's ability to inhibit angiogenesis or endothelial cell proliferation or bind to proliferating endothelial cells.
  • Conservative amino acid substitutions are only one example of the type of modifications which are within the scope of the subject matter of this invention.
  • full length recombinant PF4 inhibits growth factor-dependent human endothelial cell proliferation in vitro.
  • angiogenesis-inhibiting activity of PF4 is retained by synthetic peptides corresponding to sequences of PF4 as small as 10 amino acids in length.
  • a synthetic peptide of 13 amino acids corresponding to the carboxy terminus of PF4 (C-13) has displayed potent angiostatic activity.
  • a peptide of 41 amino acids corresponding to the carboxy terminus of PF4 (C-41) has also shown angiostatic activity.
  • the activity of the C-13 peptide is especially surprising in light of its inability to affect the anticoagulant activity of heparin.
  • the use of the C-13 peptide offers several advantages over whole PF4 such as reduced dosage (weight basis), reduced likelihood of antigenicity, and greater likelihood of effectiveness in novel dosage forms.
  • the C-13 peptide of PF4 also retains the ability to prevent Con-A induced immunosuppression in mice, an activity which is unaffected by heparin and probably independent of the ability of the peptide to inhibit angiogenesis.
  • angiogenesis is required for solid tumors to grow beyond a few cubic millimeters.
  • use of PF4, or modifications thereof, to inhibit angiogenesis presents a novel and highly advantageous means of therapy.
  • the fact that the C-13 peptide inhibits angiogenesis without affecting the anticoagulant activity of heparin demonstrates that this small peptide also has the benefit of not interfering with concurrent anticoagulant therapy.
  • small peptides are generally less antigenic than larger proteins, and, thus, the PF4 fragments of the subject invention can be used advantageously for oral and transdermal administration.
  • PF4 fragments are particularly useful in the treatment of gastrointestinal capillary proliferation and skin lesions (e.g., Kaposi's sarcoma), respectively.
  • Intralesional, as well as systemic, administration of PF4 fragments are also appropriate for treatment of these conditions.
  • Topical or aerosol administration of PF4 fragments is appropriate for skin or pulmonary lesions, respectively (e.g., Kaposi's sarcoma and lung cancer).
  • Variants of PF4 which exhibit enhanced ability to inhibit angiogenesis have been synthesized.
  • rPF4-241 One such variant, known as rPF4-241, was created by cassette mutagenesis of a synthetic PF4 gene whereby four lysine residues of the carboxy terminus of PF4 were converted to two Gln-Glu couplets in order to eliminate heparin binding activity while retaining the ⁇ -helical secondary structure.
  • PF4 conjugates One important aspect of the subject invention is the discovery of a Preferred Conjugation Site (PCS) on the PF4 molecule.
  • PCS Conjugation Site
  • PF4 is conjugated to other molecules through chemical processes which modify a free carboxyl group of an amino acid in the PF4 molecule. This modification has been found to be most preferable when the conjugation occurs near the N-terminus of the PF4 molecule.
  • a further aspect of the preferred mode of the subject invention is the discovery that conjugation involving modification of carboxyl-containing amino acid residues located at, or near, the N-terminus of the PF4 molecule can be particularly effective in the production of a biologically active conjugate.
  • Particularly attractive targets for modification are amino acids possessing side chains containing free carboxyl groups such as glutamic acid-1, glutamic acid-3, and glutamic acid-4.
  • the free carboxyl group of the aspartic acid residues can also be modified.
  • Aspartic acid residues 5 and 7 are preferred sites for modification. It was found that conjugation to PF4 at one of the first 14 amino acids of the N-terminus of PF4 advantageously results in the production of a biologically active conjugate.
  • C-activation of carboxyl residues is the underlying principle of preferred coupling methods used according to the subject invention.
  • C-activation can be achieved by attaching an electron withdrawing atom (e.g., chlorine) or group (e.g., azide) which renders the carbon atom of the carboxyl group sufficiently electrophilic to facilitate nucleophilic attack by nucleophilic reagents such as simple or complex molecules or polymers containing ⁇ -amino or hydrazide groups.
  • O-activation is an alternative method to generate an activated site at the carboxyl residue. Conversion of the carboxyl-component into a highly reactive acid anhydride can be achieved using reagents such as acid chlorides or alkyl carbonates.
  • Such O-activation can also be in the form of active esters which are generally more stable in the reaction media such as the various halogen or nitro substituted phenolates, cyanomethyl esters, substituted hydroxylamines, substituted carbodumides, and substituted azoles such as benzotriazoles.
  • a carbodiimide or bis-carbodiimide is used as an activating agent to facilitate the conjugation of desired moieties to the free carboxyl groups of PF4.
  • Various carbodumides and bis-carbodumides which can be used according to the subject invention are known to those skilled in the art. These reagents serve to activate the free carboxyl groups of amino acid residues to facilitate conjugation with a desired entity such as a label or toxin.
  • the entity to which the PF4 is conjugated will have an activated nucleophile.
  • activated nucleophiles include, but are not limited to, amines, hydrazides, and alcohols.
  • modifications include conjugations with aminoacetamidomethyl fluorescein, PEG-hydrazide, or biotinamidocaproyl hydrazide. These modifications, and the biological activity of the modified products are described in more detail in the Examples which follow.
  • modification of carboxyl groups may be preferred, as described above, for certain embodiments, modification of amino groups can also be used.
  • One specific chemical modification of PF4 according to the subject invention which results in advantageous biological properties involves modification of the free amino groups of rPF4 with fluorescein-isothiocyanate (FITC).
  • FITC fluorescein-isothiocyanate
  • the resulting adduct, FrPF4 surprisingly, retains the ability to inhibit angiogenesis as tested in the human umbilical vein endothelial cell (HUVEC) proliferation assay.
  • angiostatic activity is also found in PF4 fragments and mutants which have been modified with the bulky and hydrophobic fluorescein moiety.
  • the FITC- labeled PF4 sequences are useful for visual detection of PF4 molecules.
  • linkers used according to the subject invention can preferably comprise hydrolyzable bonds. These bonds may be acid, enzymatic, or spontaneously hydrolyzable.
  • the conjugation may occur through, for example, modification of functional groups on the protein, such as free amino, carboxyl, sulfhydryl, or guanadinium (arginine) groups of PF4. Also, conjugation to aromatic groups such as possessed by histidine and tyrosine can be done. A further option is the modification of hydroxy groups. The modification of these moieties necessary to effect the desired conjugation can be carried out by chemical procedures described herein as well as those which are well known to those skilled in this art. See, for example, Ming Lu et al. (1992) J. Immunol. Meth. 156:85-99.
  • the compounds of the subject invention can be combined with a suitable pharmaceutical carrier.
  • these compounds can be formulated in physiologically acceptable carriers, such as phosphate buffered saline, acetate buffered saline, distilled water, excipients, or the like, or may be administered neat.
  • PF4 can be used as an active agent for treatment of inappropriate angiogenesis, or PF4 can be used as a targeting agent for selectively delivering other agents to sites of inappropriate angiogenesis.
  • PF4 is used as the active ingredient
  • a physiologically-acceptable solution of PF4 is administered into the afflicted animal by any appropriate means including, but not limited to, injection into a blood vessel; direct application to a surgical site using a biocompatible gel, film, or sponge; intraperitoneal or subcutaneous injection; topical administration; and pulmonary administration.
  • compositions used in these therapies may also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, liposomes, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known those of skill in the art.
  • the compositions of the invention are in the form of a unit dose and will usually be administered to the patient one or more times a day.
  • PF4 conjugates may be administered to the patient in any pharmaceutically acceptable dosage form, including intravenous, intramuscular, intralesional, or subcutaneous injection. It should, of course, be understood that the compositions and methods of this invention may be used in combination with other therapies.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment can cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • PF4 is administered intralesionally, it can be applied such that the dosage is between about 1.0 ⁇ g/lesion and about 10 mg/lesion.
  • the dosage of PF4 can be between about 100 ⁇ g/kg of body weight and about 500 mg/kg of body weight. Similar and higher dosages can be used for the administration of PF4 conjugates and peptide fragments. For example, dosages of PF4 fragments may be twice that of PF4 or higher.
  • Ligands conjugated to PF4 can also be utilized according to the subject invention.
  • PF4 can be bound to biotin using procedures known to those skilled in the art and described herein.
  • the PF4- biotin complex can then be administered to an animal concurrently with, or followed by, an avidin complex.
  • the avidin complex can comprise avidin and a second component bound to the avidin.
  • the second component can be a detectable label such as a radiolabel.
  • the second component can be any of the various toxins, chemotherapeutic agents and other therapeutic molecules described herein or otherwise known to those skilled in the art.
  • PF4 directs the PF4-biotin conjugate to a site of angiogenesis or cellular proliferation.
  • the avidin conjugate then binds to the biotin thereby bringing the second component of the avidin conjugate to the site of angiogenesis or cellular proliferation.
  • the second component of the avidin conjugate can then either be detected in a diagnostic procedure, or exert its therapeutic effect.
  • a synergistic activity can result from the combined actions of the PF4 which inhibits angiogenesis and cellular proliferation together with the toxin effect of the toxin which destroys existing cells involved in inappropriate angiogenesis.
  • PF4 can be delivered systemically without unwanted side effects or the loss of desirable biological activity.
  • efficacy of systemically-administered PF4 has been demonstrated using several models. These models, which are described in detail in the Examples which follow, provide valuable insight into the bio-distribution patterns of systemically-administered PF4 and PF4 conjugates.
  • CAM Chicken chorioallantoic membrane assay. Fertile eggs were incubated in a stationary position for 3 days at 37°C and 70-80% relative humidity. During this time, the embryo rose to the upper surface of the egg contents. At the beginning of the 4th day, the eggs were cracked without inversion and carefully deposited into sterile plastic petri dishes such that the embryo remained on the upper surface. The shell-free eggs were incubated for an additional 72 hours at 37°C, under an atmosphere containing 2.5-3.5% CO 2 after which the growing embryos developed a recognizable CAM. Discs, made by mixing test samples with 1% (w/v) methylcellulose were dried and placed on the CAM between major veins and approximately 0.5 cm from the embryo. Following another 48 hour incubation at 37°C (2.5-3.5% CO 2 ), the samples were scored for their ability to inhibit angiogenesis.
  • Inhibition appears as an avascular zone surrounding the implant and can often include elbows formed by veins avoiding the disc and a reduced number of capillaries in the region of the implant.
  • Endothelial cell proliferation assay Human umbilical vein endothelial cells were cultured in Medium 199 (Gibco) containing 10% (v/v) fetal bovine serum (FBS),
  • rPF4 production Recombinant PF4 was produced in E. coli as an N-terminal fusion protein containing a methionine immediately preceding the PF4 sequence.
  • the insoluble fusion protein was cleaved with cyanogen bromide treatment and purified by heparin agarose affinity chromatography. The isolated protein was buffer exchanged into 20 mM sodium acetate, pH 4.0, and either frozen or lyophilized for storage.
  • Peptides were prepared by standard solid phase synthesis procedures, cleaved from the solid support and deblocked, and purified by reverse phase HPLC.
  • the lysine rich region of PF4 (residues 61-66) is also the domain associated with the binding of heparin by PF4.
  • Heparin is known to play a role in modulating angiogenesis, which can also be affected by protamine, another well characterized heparin-binding protein.
  • protamine another well characterized heparin-binding protein.
  • Protamine and platelet factor 4 are able to prevent the heparin inhibition of thrombin and Factor Xa at approximately equimolar concentrations.
  • the 41 amino acid C-terminal peptide of PF4 (C-41) prevented heparin inhibition less effectively, but the C-13 peptide was unable to prevent the inhibition of thrombin even at concentrations ten times that of an effective level of rPF4. This unexpected finding suggests that the C-13 peptide inhibits angiogenesis by some method other than heparin binding.
  • rPF4 Endothelial cell division and growth is tightly controlled and strictly dependent on the presence of growth factors.
  • rPF4 and related peptides were evaluated in vitro. rPF4 significantly inhibited endothelial cell growth in a dose-dependent fashion at a concentration as low as 10 mcg/ml. Inhibition was complete at 25 mcg/ml in the heparin-deficient medium employed here.
  • the gene for rPF4-241 was expressed as a fusion protein in E. coli with the same N-terminal amino acid sequences as with the parent rPF4 molecule.
  • the protein was cleaved from the E. coli fusion peptide by CNBr and formic acid and purified to homogeneity by DEAE-sepharose chromatography.
  • the protein was reactive with polyclonal antibodies to PF4 and was determined to possess the appropriate modifications by amino acid analysis.
  • the purified mutant protein lacked heparin-binding activity in the Factor Xa inhibition assay.
  • C-13-241 has the following sequence:
  • rPF4-241 was dried in methylcellulose discs and tested for its ability to inhibit capillary growth in the chicken chorioallantoic membrane (CAM) assay.
  • CAM chicken chorioallantoic membrane
  • mutant rPF4-241 was at least as effective in inhibiting cell growth (Figure 5). Further tests suggest that rPF4-241 was inhibitory at concentrations as low as 0.5 mcg/mL. In a test of inhibition of human umbilical vein endothelial cell proliferation by native rPF4 and mutant rPF4-241, the rPF4-241 was shown to be more effective than the native rPF4 at inhibiting the proliferation of these cells. The results of this test are shown in Figure 6.
  • mice Normal C57BL/6J female mice (6-8 weeks old) were inoculated subcutaneously with 5 ⁇ 10 5 log phase cells of a B16-F10 melanoma tumor line. This protocol led to progressive tumor growth resulting in large (300 mm 3 ) necrotic tumors after approximately 10 days, followed by death of untreated animals usually within three weeks of tumor inoculation.
  • tumor bearing animals were divided into two groups.
  • One group was injected with 50 ⁇ g rPF4 (native sequence) in 100 ⁇ l of 50 mM sodium phosphate, pH 6.5, 50 mM sodium chloride directly into the nascent tumor, daily, beginning one day after tumor inoculation.
  • a control group was treated identically with carrier buffer lacking rPF4. Tumor volume was measured at regular intervals with digital calipers by laboratory personnel uninformed of the specific treatment received by each subject animal.
  • proteins of identified structure and function may be construction by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser, E.T., F.J. Kezdy [1984] Science 223:249-255).
  • the subject invention includes mutants (or variants) of the amino acid sequences exemplified herein which do not alter the protein secondary structure, or if the structure is altered, the biological activity is retained.
  • Table 2 provides a listing of examples of several mutant sequences and their biological activity.
  • rPF4 307 [PF4 AA 1-57] - Pro Leu Tyr Gln Ile Glu Ile Gln Leu Glu Leu Glu Ser pos.
  • rPF4 308 [PF4 AA 1-57] - Pro Leu Tyr Asn Asp Ile Ile Asn Asp Leu Leu Glu Ser pos.
  • amino acids may be placed in the following classes: basic, hydrophobic, acidic, polar, and amide. Substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound.
  • Table 3 provides a listing of examples of amino acids belonging to each class.
  • non-conservative substitutions can also be made.
  • lysine may be substituted for with any of the following amino acids: Glu, Gln, Asp, Asn, Met, Ala, Leu, and Ile.
  • the critical factor is that these substitutions must not significantly detract from the biological activity of the rPF4 or the rPF4 fragment.
  • A represents all or part of the polypeptide sequence consisting of residues 1 through 57 of PF4; A may be or may not be present; wherein Xaa 10 is Lys, Gly, Glu, Gln, Asp, Asn, Met, Ala, Leu, or Ile;
  • Xaa 9 is Lys, Glu, Gln, Asp, Asn, Met, Ala, Leu, or Ile;
  • Xaa 8 is Glu, Gln, Met, Ala, Leu, Ile, Val, Pro, Phe, Trp, or Tyr;
  • Xaa 7 is Glu, Met, Ala, Leu, Ile, Val, Pro, Phe, Trp, or Tyr;
  • Xaa 6 is Lys, Gly, Glu, Gln, Asp, Asn, Met, Ala, Leu, or Ile;
  • Xaa 5 is Lys, Glu, Gln, Asp, Asn, Met, Ala, Leu, or Ile
  • Xaa 4 is Lys, Glu, Met, Ala, Leu, Ile, Val, Pro, Phe, Trp, or Tyr
  • Xaa 3 is Gln, Met, Ala, Leu, Ile, Val, Pro, Phe, Trp, or Tyr.
  • Xaa 8 is Ile, Glu, or Gln;
  • Xaa 7 is Ile or Glu
  • Xaa 6 is Leu, Gln, or Glu
  • Xaa 5 is Leu or Glu.
  • amino terminus of the proteins of the subject invention can also be modified in a variety of ways while retaining the biological activity fundamental to the subject invention. Most notably, the length of the amino terminus can be modified while retaining angiostatic or endothelial cell inhibitory activity.
  • amino terminus refers to amino acids 1-60 from the amino terminal of PF4 or its variants. As we have shown herein, up to 57 of these amino acids can be removed with a retention of angiostatic activity. The remaining peptide is the biologically active C-13 peptide. We have also shown that the C-41 peptide has biological activity. Thus, various active fragments of PF4 can be readily produced and used according to the subject invention.
  • angiostatic activity refers to a level of angiostatic activity which is characteristic of PF4. This level of angiostatic activity is, for example, at least about 75% (and preferably, greater than about 90%) of the angiostatic activity exhibited by PF4 as measured, for example, in the CAM assay. Methods for determining angiostatic activity are described herein and are well known and readily performed by those skilled in this art.
  • antiproliferative activity refers to a level of antiproliferative activity which is characteristic of PF4. This level of antiproliferative activity is at least, for example, about 75% (and preferably, greater than about 90%) of the antiproliferative activity exhibited by PF4 as measured, for example, by the HUVEC assay. Methods for determining antiproliferative activity are described herein and are well known and readily performed by those skilled in this art.
  • the term “lack of heparin binding activity” refers to a relative lack of ability to bind heparin under normal physiological conditions compared to PF4.
  • heparin binding activity is, for example, less than about 25% of PF4's heparin binding activity and, preferably, less than about 10% of PF4's heparin binding activity.
  • the ability to bind heparin can be readily determined by various assays as described herein and as is known by those skilled in this art.
  • rPF4 was batch bound to heparin-agarose at 1 mg protein/ml resin in 25 mM NaH 2 PO 4 , pH 8.0, 300 mM NaCl (Buffer F) for 24 hours at 4°C. 300 ⁇ g FITC/ml of heparin-agarose-protein complex was incubated for 18 hours in the dark at 4°C. The resin was then poured into an open column, washed with Buffer F and the protein was eluted with 25 mM NaH 2 PO 4 , pH 8.0, 2 M NaCl.
  • the eluent was dialyzed against 40 mM acetic acid, pH 3.0 in a 3,000 MWCO dialysis membrane for 18 hours in the dark at room temperature.
  • the dialyzed eluent was then loaded onto an S-Sepharose column equilibrated in 40 mM acetic acid, pH 3.0.
  • the column was washed with 40 mM acetic acid, pH 3.0, 1 M NaCl, followed by an additional wash with 40 mM acetic acid, pH 3.0, 2 M NaCl.
  • Protein was eluted with 50 mM Tris-HCl, pH 9.0, 2 M NaCl.
  • the eluent was dialyzed against 0.1% TFA:H2O in a 3,000 MWCO dialysis membrane at room temperature in the dark.
  • Example 10 Inhibition of Angiogenesis by Fluorescein-Isothiocyanate-Coniugated rPF4
  • FrPF4 produced as described in Example 9A, was tested for activity in the CAM assay as described above. Although FrPF4 lacked heparin binding activity, it retained full activity as an inhibitor of angiogenesis on the CAM. The results of these assays are shown in Table 4. Table 4. Activity of FrPF4 in the CAM assay.
  • FrPF4 and FrPF4-241, produced as described in Example 9 were tested separately to determine their ability to inhibit endothelial cell proliferation.
  • HUVE cells were tested for their sensitivity to FrPF4 as described above except that [ 3 H]-thymidine was added to the cultures 24 hours after the addition of FrPF4. The cultures were then incubated an additional 6 hours. Cells were harvested, washed, and radioactive thymidine incorporation into DNA was measured.
  • FITC-conjugated rPF4 was very effective, even at low dosages, in inhibiting DNA synthesis in human umbilical vein endothelial cells and therefore inhibiting cell proliferation. Similar results were obtained using FrPF4-241. In this case, the inhibition of HUVE cell proliferation with increasing concentrations of rPF4-241 was tested using the Endothelial Cell Proliferation Assay as described above. The results of experiments using FrPF4-241 are shown in Figure 10.
  • FrPF4 was produced as described in Example 9. B-16 Melanoma tumors were grown in C57BL6/J mice as described previously. Treatment was begun 24 hours following implantation of tumor cells (Day 1) and consisted of 25 ⁇ g/day of FrPF4 in 100 ⁇ l of sodium acetate buffer, pH 4.0. Control mice were injected with 25 ⁇ g/day of FITC labeled cytochrome-C in the same buffer. A statistically significant suppression of tumor growth by FrPF4 was observed by Day 11 ( Figure 11).
  • Example 13 Inhibition of Liver Metastases with Systemic Administration of PF4 PF4 administered as multiple intraperitoneal injections has been found to inhibit the growth of liver metastases (murine M5076 reticulum cell sarcoma). This finding further establishes systemic antitumor activity of rPF4 and shows that the effect is neither tumor type nor organ site specific.
  • Implantation of angiogenic or inflammatory stimuli into a surgically created micropocket in the cornea induces a vigorous neovascular response which can be easily detected in this normally avascular tissue.
  • Encapsulation of rPF4 in the implant pellet effectively suppresses the angiogenic response to purified basic fibroblast growth factor in both rats and rabbits. See Table 6. Evaluation of the dose response relation of rPF4 suppression to bFGF stimulation showed rPF4 to be remarkably active in this assay system, with 20 ng of rPF4 capable of completely blocking the effect of 100 ng bFGF (Table 6) in both species. rPF4 alone induced neither an angiogenic nor an inflammatory response.
  • rPF4 The safety and efficacy of rPF4 when used to treat humans having lesions associated with Kaposi's sarcoma have been evaluated.
  • rPF4 was administered as a multiple dose (16 total) intralesional injection. Three lesions were monitored on each patient: one untreated, one treated with vehicle only, and one treated with rPF4 and the vehicle.
  • the vehicle used for this study was a standard pharmaceutical carrier consisting of 10 mM sodium acetate and 150 mM sodium chloride at a p H of about 5.0.
  • the two treated lesions were assigned and treated in a double blind fashion with neither the patient nor the physician aware of the specific agent in the coded syringes.
  • the study utilized sets of three patients at each of four dose levels. The initial set of patients was treated with the lowest dose of rPF4.
  • Kaposi's sarcoma lesions were assessed in detail for signs of disease progression, stabilization, or regression. Lesion sizes (in two dimensions) were measured and documented on a regular schedule throughout the study. Lesion palpability and color were documented and assigned numerical scores according to a standard scale as well. Lesion biopsies were taken prior to and at the completion of therapy to evaluate subclinical evidence of biological effects of treatment.
  • Brain tumors can be treated with PF4 conjugates alone or in combination with radiation and/or chemotherapy, as follows:
  • Formulations and dose will depend on the method of administration.
  • a preferred administration method is the direct injection or instillation of a PF4 conjugate by a subcutaneous reservoir/catheter system that is limited to about 0.05 to
  • a single dose may contain from 0.25 to 25 mg of PF4 (0.003 to 0.4 mg/kg) and may be administered as frequently as daily, wherein the patient receives one week of daily treatment per month, or three times a week, wherein treatment is administered continuously until significant patient improvement, or treatment may be administered as infrequently as one time per month.
  • Tumors of the brain can also be treated by adopting methods widely used in the treatment of gynecological and oral cancers, and are now applied with increasing frequency to brain and other cancers.
  • Such methods termed “brachy therapy,” involve the delivery of locally high doses of radiotherapy (Cancer, Principles and Practice of Oncology, Third Edition; DeVita, V.T., Jr., S. Hellman, S.A. Rosenber, eds.).
  • PF4 conjugates can be used in place of a radioactive agent.
  • Implantable reservoirs for intracranial infusion of therapeutic agents are widely available and include the Omaya reservoir and similar devices. Implantation of the reservoir can be done at the time of initial surgery or later in a minor surgical procedure under local anaesthesia, by stereotactic methods.
  • brachytherapy which is described above.
  • Brain tumors can be treated as described above on an out- or an in-patient basis, as the patient's health permits.
  • Example 18 Treatment of Kaposi's sarcoma, Psoriasis, Basal Cell Carcinoma, and Other Skin Disorders
  • Disorders of the skin can be treated intralesionally, wherein formulations and dose will depend on the method of administration and on the size and severity of the lesion to be treated.
  • Preferred methods include intradermal and subcutaneous injection. Patients may be capable of self-administration.
  • Preferred dosages are 0.05 to 5 mg PF4 per dose (0.7 to 70 mg/kg) contained within a volume of 0.1 to 1 ml.
  • Systemic treatment can be used for conditions such as small cell lung carcinoma, head and neck cancer, sarcoma, breast cancer, and colon cancer.
  • PF4 conjugates can be administered by direct intravenous injection, or preferably by intravenous infusion lasting from 0.5 to 4 hours per single treatment.
  • Patients can be treated as in- or out-patients. Patients may also be treated using implantable subcutaneous portals, reservoirs, or pumps. Multiple intravenous or subcutaneous doses are possible, and in the case of implantable methods for treatment, formulations designed for sustained release will be especially useful.
  • Patients can be treated at dosages of 0.3 to 12 g of PF4 per period; preferably with 4 to 180 mg/kg in a volume of 60 ml to 2.5 liters per day.
  • a dosage is defined as a single dose administered as a bolus injection or intravenous infusion; or the compound can be administered to the patient as an intravenous infusion over a period of a day; alternatively, the compound can be administered in several bolus injections interrupted by periods of time such that the dose is delivered over the course of a 24 hour period.
  • the most preferred method of treatment is to administer the compound to the patient in one injection or infusion per day.
  • Patients may be treated daily on alternative weeks for six weeks, or possibly for life. They may also be treated three times per week continuously, or they may be treated daily for life.
  • Regional treatment is useful for treatment of cancers in specific organs in the patient including, but not limited to, primary liver cancer, brain and kidney cancer, and liver metastases from colon/rectal cancer.
  • Treatment can be accomplished by intraarterial infusion.
  • a catheter can be surgically or angiographically implanted to direct treatment to the affected organ.
  • a subcutaneous portal connected to the catheter can be used for chronic treatment, or an implantable, refillable pump may also be employed.
  • Patients can receive 0.05 to 1 g PF4 (1 to 20 mg/kg) in a volume of 10 to 400 ml per single dose.
  • the schedule for treatment is the same as that described above for systemic treatment.
  • mice/sex In addition to human clinical trials, the lack of rPF4 toxicity has been demonstrated in reliable animal models. Specifically, a range-finding study investigated the acute toxicity associated with a single intravascular administration of a number of different test articles in mice. The general design allowed for two mice/sex to be administered a test article and be observed for 48-72 hours in order to determine the potential for acute toxicity. Based on the results of each dose level, the subsequent group of 2 mice/sex would receive either a higher or lower dose level of that test article. Mortality, clinical observations, body weight changes, and gross necroscopy findings were used to assess toxicity.
  • rPF4 was administered to 2 male and 2 female mice each at 100 mg/kg (Group 1) and 200 mg/kg (Group 2) and to 1 male and 2 female mice at 300 mg/kg (Group 3).
  • Clinical observations revealed bruising at the site of injection in 1 male and 2 female mice. Body weight examinations revealed that there were no remarkable differences from dosing to termination. At necropsy, bruised tail was noted in one
  • Group 1 male and two Group 3 females bilateral enlarged preputial glands were noted in one Group 1 male, there were two foci on the left lateral lobe of the liver in one Group 2 male, the mandibular lymph node was enlarged in one Group 1 female, and there was a cyst on the ovary of one Group 2 female. Based on the incidence of the findings and a clear lack of dose response, it is believed that these findings are incidental to treatment (except for the bruising on the tail due to the physical trauma of the injection). Therefore, a single intravenous administration of 300 mg/kg rPF4 does not appear to have any adverse effects in mice.
  • protamine sulfate was administered to 2 mice/sex at dose levels of 50 (Group 4), 25 (Group 5), and 40 (Group 6) mg/kg.
  • Animals administered 50 mg/kg protamine sulfate displayed lethargy and rapid breathing, and one male and one female died shortly after administration. Based on the mortality, a dose level of 25 mg/kg was administered to the next group of 2 mice/sex. Both males displayed a ruffled haircoat postdose on day 1, but neither female appeared to be affected. Based on these results, the remaining two mice/sex were administered 40 mg/kg protamine sulfate intravenously. All four animals displayed rapid breathing and lethargy postdose, and one of the males also appeared to be sensitive to the touch.
  • Biodistribution characteristics are considered by those skilled in this art to be particularly relevant in establishing that a compound will have clinical utility—especially for systemic use.
  • an angiogenesis-inhibiting compound For an angiogenesis-inhibiting compound to have clinical efficacy, it must be possible to administer the compound at concentrations which will have the desired angiostatic effect at a particular physiological location without being toxic or causing any other deleterious effect.
  • the biodistribution characteristics observed for PF4 further establish its clinical utility for the treatment of angiogenic diseases. Specifically, these results confirm the efficacy of systemic administration of PF4 to treat angiogenic diseases.
  • rPF4 The biodistribution of rPF4 was assessed at three concentrations in both male and female rats. Perfusion of animals at 30 minutes and 24 hours following intravenous injection of 125 -I-rPF4 and analysis of organ distribution of radioactivity demonstrated significant retention of counts within organ tissues at the 30 minute time point ( Figures 1 and 2) which decayed substantially over the subsequent 24 hour period. At the higher doses of rPF4, the relative distribution shifted from the liver, spleen, and kidney to include the lungs and other organs. Little radioactivity was found in the urine at the early time point, suggesting that the kidney-associated protein was not being eliminated by rapid filtration. At the 24 hour time point, however, substantial counts were detected in urine, suggesting that clearance in the urine may be linked to protein degradation.
  • rPF4 found in the lungs (5-100 ⁇ g/g tissue) at the highest doses injected in these studies correlates with inhibitory activity of this agent on endothelial cells in vitro. In addition, this dose correlates with the level of rPF4 that effectively suppresses metastatic tumor growth in the lungs when the protein is administered systemically. In mouse tumor studies, a single injection of rPF4 is sufficient to suppress the growth of lesions in the lung, suggesting that the tissue residence of rPF4 is of greater importance in mediating the antitumor effects of rPF4 than is plasma level of the drug. Similar results are obtained when rPF4 is administered in a multidose subcutaneous regimen with the first injection delayed until 24 hours following tumor seeding, demonstrating that the effects of rPF4 can be induced by alternative systemic approaches.
  • rPF4 conjugates will be useful as an intravenous infusion, or through other systemic administration approach, as a treatment for disseminated angiogenic diseases such as cancer, ocular neovascularization, and Kaposi's sarcoma.
  • PF4 preferentially binds to sites of active angiogenesis. We have discovered that this unique binding property is retained even if PF4 is conjugated to another molecule.
  • PF4 can be transported systemically without loss of activity, and, advantageously, the PF4 will bind to, and accumulate at, sites of neovascularization.
  • the advantageous binding and distribution properties of PF4 have been observed in a number of contexts as described throughout this specification.
  • One compelling demonstration of the unique and advantageous properties of PF4 has been observed with systemically-administered rPF4.
  • rPF4 in hamster cheeks was used to evaluate the vascular transportation and binding characteristics of systemically-administered rPF4.
  • PF4 it was conjugated to a fluorescent label.
  • the rPF4 is conjugated to FITC as described above in Example 9B.
  • the conjugation of rPF4 to FITC is done so that the location of the rPF4 can be readily visualized using cameras which detect the fluorescence emitted by the FITC-labeled rPF4.
  • This experimental procedure is essentially as described by Rosengren et al. ([1989] Microvascular Research 38:242-254).
  • the fluorescently-labeled PF4 is clearly seen to accumulate at the location of angiogenesis and move through, but not accumulate in, the surrounding vasculature.
  • the results of this assay clearly demonstrate that PF4 preferentially binds to sites of angiogenesis.
  • PF4 is highly advantageous for its ability to move through existing vasculature and ultimately accumulate at sites of active angiogenesis.
  • the fluorescence associated with PF4 was monitored for hours and was found to be stably associated with the proliferating endothelial cells for periods of time clearly sufficient to facilitate accurate imaging of sites of neovascularization.
  • the binding of PF4 to these sites was qualitatively and quantitatively higher than the presence of PF4 in vessels and tissues not involved in neovascularization.
  • PF4 can, itself, be used as an effective angiogenesis-inhibiting compound which advantageously accumulates at sites of angiogenesis. Furthermore, because of PF4's preferential binding to sites of endothelial cell proliferation, a characteristic which is not lost by systemic administration routes or destroyed by conjugation to other entities, PF4 can be used to target second entities having desired properties to sites of angiogenesis. These second entities may have, for example, therapeutic or diagnostic utility.
  • PF4 for treatment of certain conditions, it is sometimes advantageous to direct biological activity to a specific location.
  • the monoclonal antibody which can be produced using techniques that are well-known in the art, will selectively seek out the target site. As the antibody moves to the desired location, it brings with it the PF4. Thus, the PF4 activity can be concentrated at a specific location.
  • the PF4 may also be targeted to specific locations via analogous conjugation with binding proteins (e.g., thrombospondin or fibroblast growth factor), cell receptor molecules (e.g., CD4, lymphocyte function associated antigen-1 [LFA-1], and von Willebrand Factor [vWF]) or the complementary ligands, and non-proteinaceous biological effector molecules (e.g., ICAM-1, tumor associated-antigens, and prostaglandins).
  • binding proteins e.g., thrombospondin or fibroblast growth factor
  • cell receptor molecules e.g., CD4, lymphocyte function associated antigen-1 [LFA-1], and von Willebrand Factor [vWF]
  • non-proteinaceous biological effector molecules e.g., ICAM-1, tumor associated-antigens, and prostaglandins.
  • the monoclonal antibody or other moiety, can be associated with
  • PF4 at one or both pairs of lysine residues located near the carboxy terminus of PF4. It is preferable to modify less than all of the lysine residues.
  • a monoclonal antibody (or other moiety) at these residues, the angiostatic activity is retained while heparin binding is eliminated.
  • other amino acid residues may be substituted for the lysine residues before conjugation with appropriate moieties at these and other positions. Therefore, the compounds described here can be represented as follows:
  • A represents all or part of the polypeptide sequence consisting of residues 1 through 57 of PF4; A may or may not be present on said hybrid polypeptide;
  • F, G, H, and I are selected from the group consisting of monoclonal antibodies, polyclonal antibodies, fluorescein-isothiocyanate, fluorophores, toxins, cell receptor molecules, non-proteinaceous biological effector molecules, polyamino acids, polysaccharides, and chelators; at least one of the moieties designated F, G, H, and I must be present on said hybrid polypeptide.
  • the vertical lines represent chemical bonding interactions as do the spaces between the amino acids on the horizontal line. The existence of specifically illustrated moieties associated at the Xaa positions does not exclude the possibility of conjugation occurring at other residues.
  • PF4 can be chemically or genetically crosslinked to the toxin ricin A or the diphtheria toxin.
  • a fusion protein comprised of PF4 and ricin A can be produced recombinantly in a prokaryotic or eukaryotic host.
  • the resulting purified toxin will have the high specificity for endothelial cells or cells in close proximity to endothelial cells, e.g., tumor cells.
  • PF4 and ricin can be linked with cross linkers.
  • DSS can cross link PF4 and ricin A while retaining both PF4 and ricin A activities.
  • PF4 can also be covalently linked with a cross linker to photoactivatable molecules, for example, hematoporphyrin derivative (HPD).
  • HPD hematoporphyrin derivative
  • Water soluble carbodumides e.g., EDC
  • HPD hematoporphyrin derivative
  • EDC Water soluble carbodumides
  • PF4 can be crosslinked to a large carrier protein, e.g., human serum albumin (HSA) or immunoglobulin, by disuccinimidyl suberate (DSS) through free primary amino groups (i.e., lysine E-amino groups or N-terminal ⁇ -amino groups; see Montesano et al. [1982] Biochem. Biophys. Res. Comm. 109:7-13).
  • HSA human serum albumin
  • DSS disuccinimidyl suberate
  • An alternative means of extending the half-life of PF4 can be achieved by covalent linkage of PF4 to one or more units of a polymer such as a polyamino acid, a polysaccharide, or a biocompatible polymer.
  • a polymer such as a polyamino acid, a polysaccharide, or a biocompatible polymer.
  • the polyamino acid may be, for example, polyglutamate
  • the polysaccharide may be, for example, dextran
  • the biocompatible polymer may be, for example, polyethylene glycol (PEG).
  • PEG is a water-soluble, non-immunogenic, linear polymer which is available in many well-defined molecular weight ranges (200-20,000 daltons).
  • the dramatic increase in molecular weight of PEG protein conjugates significantly reduces glomerular filtering of the protein by the kidney.
  • PEG polymers may sterically hinder attack by proteolytic enzymes and immunoglobulin molecules, again adding to the serum life of the protein.
  • Activated esters of PEG are readily coupled to lysine amino groups on a protein.
  • pH controlled acylation Katre, N.V., M.J. Knauf, W.J. Laird [1987] Proc. Natl. Acad. Sci. USA 84:1487-1491
  • synthesis of rPF4-PEG conjugates with currently available reagents is straightforward.
  • PEG can be covalently attached to rPF4 by free amino groups using a modification of the procedure outlined by Katre et al. (supra).
  • PEG-glutaryl- N-hydroxysuccinimide MW-5000, Polysciences, Inc.
  • the reaction is allowed to proceed at 37°C for 48 hours.
  • rPF4 with varying degrees of PEG addition.
  • the extent of reaction can be conveniently monitored by SDS-PAGE. Products of the reaction are clearly visible as a series of bands, each differing by 5000 molecular weight and representing species with one or more PEG modifications.
  • each salt fraction can be further separated by other chromatographic techniques (gel filtration or hydrophobic interaction chromatography) to separate molecules with a defined PEG:rPF4 ratio.
  • the conjugation process can be carried out in the presence of heparin.
  • the presence of heparin prevents modification of the heparin binding site of PF4 and results in a conjugate which will still bind heparin.
  • the disulfides of rPF4 can be reduced by dithiothreitol
  • DTT heparin binding activity
  • the sulfhydryls of rPF4 were specifically and irreversibly modified by prereduction with DTT followed by treatment with fluorescein maleimide (FM).
  • the reduced and purified rPF4 (5 mg in sodium carbonate buffer, pH 8.5 [SCB]) was treated with 10 mg of FM for 3 hours at room temperature. Residual FM was removed by gel filtration in SCB and then dialyzed against the same buffer. The FM- rPF4 partially retained heparin binding activity.
  • FM-rPF4 When tested in the CAM and endothelial cell proliferation assays, FM-rPF4 exhibited inhibitory activity indicating that neither free sulfhydryls nor correct disulfide bonds are required for the angiostatic activity of PF4.
  • This FM-modified rPF4 can be used as an alternative endothelial cell labeling or inhibiting compound. These results show that the cysteine residues of PF4 are appropriate targets for conjugating or cross linking PF4 to other molecules for diagnostic or therapeutic applications.
  • Conjugates of PF4 can be prepared using ester chemistry to facilitate modification of a free carboxyl group of PF4. As would be appreciated by one skilled in the art, this methodology can be used to conjugate a variety of different entities to PF4. Specifically, this methodology can be used to conjugate the free carboxy groups on PF4 to molecules having alkyl groups. Specific examples would be the conjugation of methyl and ethyl groups to the carboxy groups of PF4.
  • the product was purified using a heparin-agarose column.
  • the conjugate produced by this method was modified at the PCS and retained the useful biological properties of PF4.
  • HUVEC proliferation assay showed inhibition with IC 50 similar to that of unmodified rPF4 ( Figure 12).
  • mice were treated on days 1-12 with daily intralesional injections of 50 ⁇ g of the test modified PF4 (Gly-PF4), or 50 ⁇ g of rPF4 (positive control), or 50 ⁇ g of RP347 (negative peptide control) in 50 ⁇ l of 10 mM acetate buffer at pH 5. 50 ⁇ l of the 10 mM acetate buffer at pH 5 was used as the buffer control.
  • Tumor size was calculated as the product of the length, width, and depth of a tumor. Six mice were included per experimental group.
  • PF4 can be conjugated to a fluorescent molecule such as fluorescein, or a derivative thereof.
  • AMF Aminoacetamidomethyl fluorescein
  • AMF is a derivative of fluorescein having an activated nucleophilic site which makes this molecule particularly appropriate for conjugation to a free carboxyl group of PF4 using a carbodiimide procedure.
  • Such a procedure can be performed as follows: to rPF4 (4 mg, 0.5 ⁇ moles) in 2 ml of 100 mM potassium monophosphate and 500 mM sodium chloride solution at pH 5.5 was added solid 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 100 mg, 520 ⁇ moles). The pH was the adjusted to pH 8.0 using 1 N sodium hydroxide.
  • EDC 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride
  • HUVE cells were obtained from the American Type Culture
  • HUVE cells were allowed to attach for ⁇ 4 hours before the addition of the rPF4 or rPF4-AMF (100 ⁇ l of final volume), incubated with the protein for 24 hours, then treated with [ 3 H]-thymidine at 1 ⁇ Ci per well. The plates were incubated for an additional 6 hours, then frozen at -70°C. The plates were subjected to two freeze/thaw cycles before harvest to assure complete removal of cells and matrix. Well contents were aspirated onto a fiber filter, washed with distilled water, and fixed with methyl alcohol. Each sample represents the average of three wells.
  • the inhibitory effect of recombinant PF4 on HUVE cells is correlated with the inhibition of DNA synthesis.
  • the inhibitory effect of the modified PF4-AMF in this assay was found to be as effective as that for rPF4.
  • the PF4 conjugates of the subject invention have highly advantageous biological properties and can be detected in vivo.
  • the fluorescent conjugate produced according to this procedure is particularly useful for the detection of areas of endothelial cell proliferation.
  • PF4 The half-life of PF4 in the biological system can be extended by conjugation of PF4 to polymers such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • a PEG-hydrazide reagent with activated nucleophilic site can be used.
  • Such a procedure can be performed as follows: to rPF4 (1 mg, 0.128 ⁇ moles) and PEG-hydrazide (5 mg, 1 ⁇ mole) in 250 ⁇ l of 100 mM potassium monophosphate and 500 mM sodium chloride solution at pH 5.5 was added solid l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 2.5 mg, 13 ⁇ moles). The reaction mixtures were stirred overnight at 4°C.
  • B16/F10 murine melanoma cells which had been derived in vivo and maintained in vitro, were used in the lung metastasis model. These cells were originally obtained from the Division of Cancer Treatment Tumor Repository (DCT, Frederick, MD). Cells were cultured in monolayers in an atmosphere of 5% CO 2 at 37°C, and maintained in Eagles MEM containing Earle's salts supplemented with 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acids, 1.0% MEM vitamin solution, 2 mM L-glutamine, 50 U/ml penicillin G, 50 ⁇ g/ml streptomycin, and 10% heat inactivated fetal bovine serum.
  • DCT Division of Cancer Treatment Tumor Repository
  • mice Female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were used when 8-14 weeks old.
  • PEG-rPF4 was prepared as described in Example 32.
  • Lungs were fixed in 10% buffered formalin overnight, weighed, and the number of tumor colorles at the surface were enumerated with the aid of a dissecting microscope.
  • the injection schedule systematically alternated between animals in the control group and animals in the experimental groups in order to minimize variances that may be attributed to the duration of the injection protocol.
  • the administration of rPF4 or PEG-rPF4 can substantially reduce the number and total weight of lung metastases.
  • mice Five hundred thousand B16/F10 murine melanoma cells were implanted cutaneously on day 0 into shaved and depilated flanks of syngeneic C57BL/6J female mice. The mice were treated on days 1-12 with daily intralesional injections of 50 ⁇ g of PEG-rPF4, or 50 ⁇ g of rPF4 (positive control), or 50 ⁇ g of PEG Control (negative control) in 50 ⁇ l of 10 mM acetate buffer at pH 5. Fifty microliters of the 10 mM acetate buffer at pH 5 was used as the buffer control.
  • Tumor size was calculated as the product of the length, width, and depth of a tumor. Six mice were included per experimental group.
  • the objective of this study was to obtain tissue distribution and blood clearance information for 125 I-PEG-rPF4 following a single intravenous administration in Sprague-Dawley rats and to compare data similarly obtained with 125 I-rPF4.
  • the rats were assigned to four groups (A through D) of three rats per group.
  • Two groups (A and B) were treated with a low dose (70 ⁇ g/kg), and the other two other groups (C and D) received a medium (5 mg/kg) dose.
  • One low dose group and one high dose group received rPF4, and one low dose group and one high dose group received PEG-rPF4.
  • Each animal was surgically implanted with a femoral arterial catheter one day prior to dose administration. On the day of dosing, each animal received a bolus intravenous dose of 125 I-PEG-rPF4 or 125 I-rPF4 via the tail vein.
  • Blood samples were collected from each rat in dose groups A and C post-treatment at 1.0, 2.5, 5.0, 7.5, 10, 20, and 30 minutes, and for B and D at 30 minutes, 1, 2, 4, 6, 12, and 24 hours. Approximately 200 ⁇ l of blood was collected at each sampling time via the indwelling arterial catheter. Duplicate 40 ⁇ l aliquots of the whole blood collected in EDTA tubes were analyzed for 125 I activity. The data were compiled and graphed based on percent PF4 in whole blood calculated from its specific radioactivity/ml of blood from 1 minute. A fifth group of rats received a medium dose (50 mg/kg) of rPF4 and was evaluated in a similar manner.
  • the pharmacokinetic profile of both radiolabeled proteins were observed to be biphase in nature with a rapid initial distribution phase followed by a slower elimination phase.
  • the PEG-rPF4 showed an advantageous clearance rate from the bloodstream compared to rPF4. Specifically, the clearance of PEG-rPF4 occurs more slowly than rPF4. Clearance rates of the 70 ⁇ g/kg and the 5 mg/kg PEG-rPF4 doses at the rapid ⁇ -phase were comparable to the 50 mg/kg rPF4 and were much better than the 70 ⁇ g/kg and the 5 mg/kg rPF4 doses.
  • PEG-rPF4 retained higher protein levels in the plasma throughout the study and was completely cleared to background levels in 24 hours.
  • the number of functional molecules attached to rPF4 can be increased by conjugation of the rPF4 to a biocompatible polymer having these multiple functional molecules.
  • These functional molecules can be any of the therapeutically or diagnostically useful entities which are described herein or would be utilized by one skilled in the art having the benefit of this disclosure.
  • the molecules may be multiples of the same molecule or may be different molecules.
  • the biocompatible molecule can be conjugated to a chelator which, in turn, is bound to a functional molecule such as a metal atom or other radiation emitter.
  • biocompatible polymers useful according to this aspect of the invention include polyethylene glycol and polyethylene vinyl alcohol.
  • the size of the polymer can be from about 500 to about 20,000 daltons, but preferably the polymer will be from about 2,000 to about 5,000 daltons.
  • the polymer can be a block copolymer wherein one part of the block copolymer can, for example, impart solubility, enhance plasma circulation, alter biodistribution, and/or reduce immunogenicity.
  • the second part of the block copolymer can be a polymer with repeating units which are also biocompatible and contain residues which are reactive, or can be treated in such a way as to be readily attached with molecules having desired properties. These residues can be, for example, amino acids such as lysine, ornithine, glutamic acid, or aspartic acid. The number of such monomer units can range from 1 to about 100.
  • Such polymers or block copolymers can be conjugated to rPF4 by a person skilled in the art utilizing the teachings which are provided herein.
  • the conjugation can be through the N-terminal of a polyamino acid to the preferred conjugation site of rPF4.
  • An increase in the number of functional molecules attached to rPF4 according to this aspect of the subject invention has useful applications for magnetic resonance imaging, boron neutron capture therapy, photodynamic therapy, and chemotherapy.
  • This embodiment of the subject invention is particularly advantageous for applications requiring an increase of signal to noise ratio for diagnostic applications.
  • This technology can also be used by those skilled in the art to increase the amount of a therapeutic reagent or toxin which is delivered to a target site.
  • Example 37 Biotinamidocaproylhydrazo Conjugates of PF4 Inhibit Angiogenesis
  • a variety of useful compounds can be converted to hydrazides and then conjugated to PF4. This procedure can be used to conjugate biotin to the free carboxyl groups of PF4. This can be accomplished using a biotin derivative which is specifically suited for this procedure by virtue of having an activated nucleophilic site.

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Abstract

L'invention concerne l'utilisation du facteur plaquettaire 4 (PF4) modifié pour inhiber l'angiogenèse. Le PF4 modifié s'utilise dans le traitement des affections angiogéniques et pour inhiber la prolifération des cellules endothéliales. L'invention concerne en outre des modifications du PF4 qui augmentent la demi-vie et facilitent le ciblage de l'activité biologique du PF4 au niveau de sites spécifiques. De plus, le PF4 s'utilise pour cibler les activités d'autres molécules au niveau des sites d'angiogenèse et de prolifération des cellules endothéliales.
PCT/US1994/012737 1993-11-05 1994-11-04 Nouvelles compositions a base de pf4 modifie et procedes d'utilisation WO1995012414A1 (fr)

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WO1996032133A2 (fr) * 1995-04-13 1996-10-17 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugue pour le traitement de maladies inflammatoires, infectieuses et/ou dermiques
WO2000004926A2 (fr) * 1998-07-22 2000-02-03 Osprey Pharmaceuticals Limited Traitement de degats tissulaires secondaires, etats inflammatoires et autres troubles, et compositions a cet effet
WO2000018439A2 (fr) * 1998-09-29 2000-04-06 Schering Aktiengesellschaft Utilisation de marqueurs de neoangiogenese pour diagnostiquer et traiter des tumeurs
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WO1996032133A2 (fr) * 1995-04-13 1996-10-17 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Conjugue pour le traitement de maladies inflammatoires, infectieuses et/ou dermiques
WO1996032133A3 (fr) * 1995-04-13 1997-02-13 Deutsches Krebsforsch Conjugue pour le traitement de maladies inflammatoires, infectieuses et/ou dermiques
US6706713B1 (en) 1995-04-13 2004-03-16 Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts Conjugate for treating inflammatory infectious and/or skin diseases
WO2000004926A2 (fr) * 1998-07-22 2000-02-03 Osprey Pharmaceuticals Limited Traitement de degats tissulaires secondaires, etats inflammatoires et autres troubles, et compositions a cet effet
WO2000004926A3 (fr) * 1998-07-22 2000-11-02 Osprey Pharmaceuticals Ltd Traitement de degats tissulaires secondaires, etats inflammatoires et autres troubles, et compositions a cet effet
EP1346731A1 (fr) * 1998-07-22 2003-09-24 Osprey Pharmaceuticals Limited Conjugués pour le traitement des maladies inflammatoires et des lésions tissulaires associeés
AU2007201755B2 (en) * 1998-07-22 2007-09-20 Osprey Pharmaceuticals Usa, Inc. Conjugates for treating inflammatory disorders and associated tissue damage
AU2007201759B2 (en) * 1998-07-22 2008-05-29 Osprey Pharmaceuticals Usa, Inc. Conjugates for treating inflammatory disorders and associated tissue damage
JP2010075194A (ja) * 1998-07-22 2010-04-08 Osprey Pharmaceuticals Usa Inc 二次的組織損傷ならびにその他の炎症症状および疾患を治療する方法および組成物
WO2000018439A2 (fr) * 1998-09-29 2000-04-06 Schering Aktiengesellschaft Utilisation de marqueurs de neoangiogenese pour diagnostiquer et traiter des tumeurs
WO2000018439A3 (fr) * 1998-09-29 2000-09-14 Schering Ag Utilisation de marqueurs de neoangiogenese pour diagnostiquer et traiter des tumeurs

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