WO2006010070A2 - Compositions et procedes lies a des peptides se liant de facon selective avec des cellules de la leucemie - Google Patents

Compositions et procedes lies a des peptides se liant de facon selective avec des cellules de la leucemie Download PDF

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Publication number
WO2006010070A2
WO2006010070A2 PCT/US2005/024414 US2005024414W WO2006010070A2 WO 2006010070 A2 WO2006010070 A2 WO 2006010070A2 US 2005024414 W US2005024414 W US 2005024414W WO 2006010070 A2 WO2006010070 A2 WO 2006010070A2
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Prior art keywords
peptide
leukemia
agent
phage
cells
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PCT/US2005/024414
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English (en)
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WO2006010070A3 (fr
Inventor
Diana Jaalouk
Wadih Arap
Renata Pasqualini
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Board Of Regents, The University Of Texas System
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Publication of WO2006010070A2 publication Critical patent/WO2006010070A2/fr
Publication of WO2006010070A3 publication Critical patent/WO2006010070A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • the present invention concerns the fields of molecular medicine and targeted delivery of therapeutic agents. More specifically, the present invention relates to compositions and methods for identification and use of peptides that selectively target leukemia cells.
  • ⁇ 2 integrins are important receptors mediating the homing of leukocytes.
  • a peptide has been identified that blocks ⁇ 2-mediated adhesion without significant toxicity.
  • Koivunen et al. (2001) reported the screening of a phage display random peptide libraries for ligands that would inhibit the function of the ⁇ 2 integrin receptor as it related to leukocyte adhesion.
  • a peptide ligand with a compact disulfide-restrained structure (ADGACPCFLLGCCGA (SEQ ID NO: 8) termed LLG-C4) was identified as an inhibitor of leukocyte cell adhesion and migration.
  • the LLG motif supported cell adhesion when immobilized in vitro and bound only to cells expressing ⁇ 2 integrins.
  • the striking specificity of the peptide is explained by its ability to interact with the I-domain (the active site in leukocyte integrins) and with ICAM-I (a functional ligand for ⁇ 2 integrins). It is well recognized that ⁇ 2 integrins only become active after selective stimulation by inflammatory cytokines and contact with antigen-presenting cells. Selective inhibition of ⁇ 2 function would likely prevent or attenuate the progression of leukemia.
  • This peptide binds strongly to acute myelogenous leukemia (AML)-derived cell lines and to the AML cell-enriched leukocyte population isolated from blood of AML patients with no significant binding to leukocytes of healthy donors.
  • AML acute myelogenous leukemia
  • the LLG-C4 peptide significantly improved the survival of mice xenografted with human OCI-AML3 cells. Additionally, the peptide prevented the attachment and proliferation of the leukemia cells on growth-supporting mesenchymal cell layer.
  • LLG-C4 peptide has properties that are difficult to work with and is poorly soluble in aqueous environments.
  • the present invention provides additional methods and compositions for the preparation and use of targeting peptides that are selective and/or specific for leukemia cells.
  • the invention concerns particular targeting peptides selective or specific for leukemia cells, including but not limited to AYHRLRR (SEQ ID NO: 5), GFYWLRS (SEQ ID NO: 6), or SFFYLRS (SEQ ID NO: 7).
  • Other embodiments concern such targeting peptides attached to therapeutic agents.
  • leukemia or other targeting peptides may be used to selectively or specifically deliver therapeutic agents to target cells, such as leukemia cells.
  • the subject methods concern the identification, preparation, and use of targeting peptides selective or specific for a given target cell, tissue or organ, such as leukemia cells.
  • One embodiment of the invention concerns isolated peptides of 100 amino acids or less in size, comprising at least 3, 4, 5, 6, or 7 contiguous amino acids of a leukemia targeting peptide sequence.
  • the contiguous amino acids are selected from any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
  • the isolated peptide is 50 amino acids or less, more preferably 30 amino acids or less, more preferably 20 amino acids or less, more preferably 10 amino acids or less, still more preferably 7 amino acids or less in size, or even still more preferably 5 amino acids or less in size.
  • the isolated peptide may comprise at least 4, 5, 6, or 7 contiguous amino acids of a targeting peptide sequence, selected from a leukemia cell targeting peptide including, but not limited to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
  • the isolated peptide may be operatively coupled to an agent.
  • the isolated peptide is covalent coupled to an agent.
  • the agent is a drug, a chemo therapeutic agent, a radioisotope, a pro- apoptosis agent, an anti-angiogenic agent, a hormone, a cytokine, a growth factor, a cytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fab fragment of an antibody, a survival factor, an anti-apoptotic factor, a hormone antagonist, an imaging agent, a nucleic acid or an antigen.
  • the pro-apoptosis agent is gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2 (SEQ ID NO:1), (KLAKKLA) 2 (SEQ ID NO:2), (KAAKKAA) 2 (SEQ ID NO:3) or (KLGKKLG) 3 (SEQ ID NO:4).
  • the anti-angiogenic agent is angiostatin 5, pigment epithelium-derived factor, angiotensin, laminin peptides, f ⁇ bronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro- ⁇ , thrombospondin, 2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CMlOl, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon- alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, a proteasome inhibitor, a kinase inhibitor, a signal
  • the cytokine is interleukin 1 (IL-I), IL-2, IL-5, IL-10, IL-I l, IL-12, IL-18, interferon- ⁇ (IF- ⁇ ), IF- ⁇ , IF- ⁇ , tumor necrosis factor- ⁇ (TNF- ⁇ ), or GM-CSF (granulocyte macrophage colony stimulating factor).
  • IL-I interleukin 1
  • IL-2 IL-5
  • IL-10 IL-I l
  • IL-12 IL-18
  • interferon- ⁇ IF- ⁇
  • IF- ⁇ IF- ⁇
  • IF- ⁇ tumor necrosis factor- ⁇
  • GM-CSF granulocyte macrophage colony stimulating factor
  • targeting peptides attached to one or more therapeutic agents may be administered to a subject, such as an animal, mammal, cat, dog, cow, pig, horse, sheep or human subject. Such administration may be of use for the treatment of various disease states.
  • leukemia targeting peptides attached to a cytocidal, pro-apoptotic, anti-angiogenic or other therapeutic agent may be of use in methods to treat hyperproliferative diseases, such as cancer and preferably leukemia.
  • the isolated peptide may be attached to an agent that is a macromolecular complex.
  • a "macromolecular complex" refers to a collection of molecules that may be random, ordered or partially ordered in their arrangement.
  • a virus may be a papovaviruses, a simian virus 40, a bovine papilloma virus, a polyoma virus, adenovirus, vaccinia virus, adeno-associated virus (AAV), herpes virus or any of a variety of viruses known in the art.
  • AAV adeno-associated virus
  • the molecules may be identical, or may differ from each other.
  • the macromolecular complex may be a virus, a bacteriophage, a bacterium, a liposome, a microparticle, a magnetic bead, a yeast cell, a mammalian cell, a cell, or a microdevice.
  • macromolecular complexes within the scope of the present invention may include virtually any complex that may be attached, directly or indirectly, to a targeting peptide and administered to a subject.
  • the isolated peptide may be attached to a eukaryotic expression vector, more preferably a gene therapy vector.
  • the isolated peptide may be attached to a solid support, preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
  • a solid support preferably magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
  • fusion proteins comprising a leukemia targeting peptide.
  • a fusion protein may comprise at least 3, 4, 5, 6, or 7 contiguous amino acids of a sequence selected from any of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or variants and/or mimetics thereof.
  • larger contiguous sequences, up to a full-length sequence selected from any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7 may be used.
  • Certain other embodiments concern compositions comprising isolated peptides or fusion proteins in a pharmaceutically acceptable carrier. Further embodiments concern kits comprising the claimed isolated peptides or fusion proteins in one or more containers.
  • a leukemia targeting peptide for a desired organ, tissue or cell type, attaching the targeting peptide to a molecule, macromolecular complex or gene therapy vector, and providing the peptide attached to the molecule, complex or vector to a subject.
  • the targeting peptide is selected to include at least 3, 4, 5, 6, or 7 contiguous amino acids from any of selected from any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
  • the cell is a leukemia cell.
  • the molecule attached to the targeting peptide is a chemotherapeutic agent, an antigen, an imaging or a diagnostic agent.
  • nucleic acids of 300 nucleotides or less in size encoding a targeting peptide.
  • the isolated nucleic acid is 255, 250, 240, 225, 210, 200, 180, 175, 150, 126, 125, 102, 100, 75, 51, 50, 42, 40, 30, 21, 20, 12, 10 or even 9 nucleotides or less in size.
  • the isolated nucleic acid is incorporated into a eukaryotic or a prokaryotic expression vector.
  • the vector is a plasmid, a cosmid, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a virus or a bacteriophage.
  • the isolated nucleic acid is operatively linked to a leader sequence or a nucleic acid encoding a leader sequence that localizes the nucleic acid or expresses a peptide that is localized to the extracellular surface of a host cell, respectively.
  • the terms "operatively linked” or "operatively coupled” mean connected either directly or indirectly with other intervening moieties in a way that typically forms an operational or a functional connection between entities, e.g., a peptide and therapeutic agent.
  • An alternative example of operatively coupled includes a peptide coupled to a liposome, which contains a therapeutic agent.
  • the peptide is operatively coupled to the therapeutic agent due to formation of an operational connection between the peptide and therapeutic agent mediated by the intervening liposome.
  • Additional embodiments of the present invention concern methods of treating a disease state, such as leukemia, comprising selecting a targeting peptide that targets cells associated with the disease state, attaching one or more molecules effective to treat the disease state to the peptide, and administering the peptide to a subject with the disease state.
  • the targeting peptide includes at least 3, 4, 5, 6, or 7 contiguous amino acids selected from any of selected from any of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
  • targeting peptides are identified by exposing a subject or cells to a phage display library, collecting samples of one or more organs, tissues or cell types, separating the samples into isolated cells or small clumps of cells suspended in an aqueous phase, layering the aqueous phase over an organic phase, centrifuging the two phases so that the cells are pelleted at the bottom of a centrifuge tube and collecting phage from the pellet.
  • the organic phase is dibutylphtalate.
  • a phage display library displaying the antigen binding portions of antibodies from a subject is prepared, the library is screened against one or more antigens and phage that bind to the antigens are collected.
  • the antigen is a targeting peptide.
  • methods include selecting a leukemia targeting peptide by obtaining at least one sample comprising leukemia cells; exposing the sample to a peptide library; and recovering one or more peptides that bind to the leukemia cells.
  • the peptide library may be a phage display library.
  • the phage may be recovered by infecting pilus positive bacteria.
  • the phage may be recovered by amplifying phage inserts; ligating the amplified inserts to phage DNA; and producing phage from the ligated DNA.
  • phage are recovered by BRASIL (Biopanning and Rapid Analysis of Selective Interactive Ligands).
  • the method may further comprise obtaining one or more types of non-leukemic cells and exposing said cells to said peptide library and recovering one or more peptides that do not bind to said one or more types of non-leukemic cells.
  • the method may further comprise preselecting the phage library against non-leukemia cell type; removing phage that bind to the non-leukemia cell type; and selecting the remaining phage against leukemia cells, non- leukemic cells may be lymphocytes, leukocytes, stem cells, myeloid cells or the like.
  • the methods and compositions may be used to identify one or more receptors for a targeting peptide.
  • compositions and methods may be used to identify naturally occurring ligands for known or newly identified receptors.
  • the methods may comprise contacting a targeting peptide to an organ, tissue or cell containing a receptor of interest, allowing the peptide to bind to the receptor, and identifying the receptor by its binding to the peptide.
  • the targeting peptide contains at least 3, 4, 5, 6, or 7 contiguous amino acids selected from SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
  • the targeting peptide may comprise a portion of an antibody against the receptor.
  • methods of the invention may utilize intact organs, tissues or cells, or may alternatively utilize homogenates or detergent extracts of the organs, tissues or cells for identifying additional targeting peptide or protein that interact, directly or indirectly with a targeting peptide.
  • cells to be contacted may be genetically engineered to express a suspected receptor for the targeting peptide.
  • the targeting peptide is modified with a reactive moiety that allows its covalent attachment to the receptor.
  • the reactive moiety is a photoreactive group that becomes covalently attached to the interacting protein or receptor when activated by light.
  • the peptide is attached to a solid support and the interacting protein or receptor is purified by affinity chromatography.
  • the solid support comprises magnetic beads, Sepharose beads, agarose beads, a nitrocellulose membrane, a nylon membrane, a column chromatography matrix, a high performance liquid chromatography (HPLC) matrix or a fast performance liquid chromatography (FPLC) matrix.
  • HPLC high performance liquid chromatography
  • FPLC fast performance liquid chromatography
  • the receptor of interest or a targeted receptor of the invention is a cell surface receptor, more preferably a member of the Plexin or GP-130 like family of receptors, and more preferably GP 130R, plexin bl, GLM-R, plexin Al, plexin A2, neuropilin-1 (NRP-I), NRP-2, or most preferably NRP-I .
  • the targeting peptide may inhibit the activity of a receptor upon binding to the receptor.
  • the receptor activity can be assayed by a variety of methods known in the art, including but not limited to catalytic activity and binding activity.
  • binding of a targeting peptide to a receptor may inhibit a transport activity of the receptor.
  • one or more ligands for a receptor of interest may be identified by the disclosed methods and compositions.
  • One or more targeting peptides that mimic all or part of a naturally occurring ligand may be identified by phage display and biopanning in vivo or in vitro.
  • a naturally occurring ligand may be identified by homology with a single targeting peptide that binds to the receptor, or a consensus motif of sequences that bind to the receptor.
  • an antibody may be prepared against one or more targeting peptides that bind to a receptor of interest. Such antibodies may be used for identification or immunoaffinity purification of native ligands.
  • inventions include methods of treating leukemia in a subject by obtaining a leukemia cell targeting peptide or peptide conjugate as described, selected or made herein, wherein the peptide i) is operatively coupled to and delivers a therapeutic agent to the leukemia cell, ii) inhibits the adhesion of the leukemia cell to a tissue or organ, or iii) is operatively coupled to and delivers a therapeutic agent to the leukemia cell and inhibits the adhesion of the leukemia cell to a tissue or organ; and administering the peptide to the subject.
  • the targeting peptides of the present invention are of use for the selective delivery of therapeutic agents, including but not limited to gene therapy vectors and fusion proteins, to specific organs, tissues or cell types.
  • therapeutic agents including but not limited to gene therapy vectors and fusion proteins
  • the skilled artisan will realize that the scope of the claimed methods of use include any disease state that can be treated by targeted delivery of a therapeutic agent to a desired organ, tissue or cell type.
  • disease states include those where the diseased cells are confined to a specific organ, tissue or cell type, other disease states may be treated by an organ, tissue or cell type- targeting approach.
  • the organ, tissue or cell type may comprise leukemia.
  • methods of targeting the delivery of an agent to a leukemia cell in a subject include obtaining a peptide described, selected or made herein operatively coupling the peptide to the agent; and administering the peptide-coupled agent to the subject.
  • the subject is preferably a human, but may include a mouse, a dog, a cat, a rat, a sheep, a horse, a cow, a goat or a pig.
  • a method of identifying a leukemia cell includes contacting a sample suspected of comprising a leukemia cell with an isolated peptide of the invention; and detecting binding of the peptide to the sample, thereby identifying sample as comprising leukemia cells.
  • a method of identifying a receptor or protein that interacts with a leukemia targeting peptide comprising the steps of obtaining a composition suspected of comprising a receptor or protein that interacts with a leukemia cell targeting peptide; contacting the composition with a peptide in accordance with the invention under conditions that permit binding of the peptide to any such receptor or protein present in the composition; and identifying a receptor or protein that binds to the peptide.
  • the composition may comprise leukemia cells.
  • the methods may further comprise isolating the receptor or protein.
  • the methods may further comprise preparing an antibody or antibody fragment that recognizes and binds to the receptor or protein.
  • the agent that one desires to have delivered to leukemia cells may be operatively coupled to the antibody or antibody fragment.
  • an antibody or antibody fragment that recognizes and binds to a receptor or protein identified by the methods of the invention.
  • the antibody or antibody fragment may further comprise an agent or macromolecular complex that one desires to have delivered to leukemia cells attached to said antibody or antibody fragment.
  • yet another aspect of the invention includes methods of selectively targeting a leukemia cell in a patient, comprising the steps of obtaining an antibody or antibody fragment in accordance with the invention; and administering the antibody or fragment to said patient to thereby target the leukemia cells.
  • the profile of phage interaction with various leukemia cells may be utilized to characterize known leukemia or to classify a leukemia cell that not been classified or its classification is unknown.
  • a set of standards or profile parameters will be established so that the phage binding profile of various unknown leukemias may then be compared to the profile of known or characterized leukemia and a designation made regarding the classification of the unknown leukemia.
  • "a” or “an” may mean one or more.
  • the words “a” or “an” in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more of an item.
  • the antigen comprises one or more targeting peptides.
  • the targeting peptides are prepared and immobilized on a solid support, serum-containing antibodies is added and antibodies that bind to the targeting peptides are collected.
  • FIG. 7 illustrates exemplary results of BRASIL on a panel of cell lines to validate binding of Molt-4 phage clones.
  • FIG. 8 illustrates exemplary results of BRASIL on AML clinical samples to validate binding of Molt-4 phage clones.
  • FIG. 9 illustrates exemplary results of BRASIL on AML clinical samples to validate binding of Molt-4 phage clones.
  • FIG. 10 illustrates exemplary results of BRASIL on AML clinical samples to validate binding of Molt-4 phage clones.
  • FIG. 11 illustrates exemplary results of BRASIL on AML clinical samples to validate binding of Molt-4 phage clones.
  • FIGs. 12A - 12B illustrates a comparison of phage binding in AML patient sample, HBMMNC, and HPBMNC.
  • FIG. 13 illustrates exemplary results of BRASIL on AML clinical samples to validate binding of Molt-4 phage clones.
  • FIG. 14 illustrates the results of studies investigating the peptide binding to Molt-4 cells.
  • FIG. 15 illustrates an exemplary internalization assay of Molt-4 phage clones in
  • FIG. 16 illustrates the validation of neuropilin-1 (NRP-I), a putative receptor for the GFYWLRS peptide insert in Molt-4 phage clone 2 as suggested from BLAST analysis. Phage binding to recombinant rat neuropilin-1 /Fc chimera (rrNRP-1/Fc) was assessed. Results shown are average of three independent assays.
  • FIG. 17. illustrates the assessment of binding of Molt-4 phage clone 2 to neuropilin-1 -related proteins such as neuropilin-2 and members of the semaphorin family.
  • FIGs. 18A-18C illustrates FACS profiles of cell surface expression of NRP-I in Molt-4 (FIG. 18A), OCI- AML3 (FIG. 18B), and K562 (FIG. 18C) leukemia cell lines.
  • Cells were stained as such: cells only, + FITC-goat anti-rabbit IgG, + control rabbit IgG and FITC- goat anti-rabbit IgG, + rabbit anti-human NRP-I and FITC-goat anti -rabbit IgG.
  • M2 indicates % of positive gated cells per sample, Mn stands for the mean fluorescence intensity of the positively gated population.
  • FIGs. 19A-19C illustrates assessment of the cytotoxic effect of C-GFYWLRS- C-GG-D(KLAKLAK)2 peptide on leukemia cells in vitro.
  • Increasing doses of either peptide alone, peptides mixed in solution, or synthetically conjugated peptides were added to cultured leukemia cells in proliferating phase and incubated for 24hr at 37 0 C.
  • the WST-I reagent was then added for 4hr and the OD was measured at 450nm. Assuming viability of untreated cells at 100%, the corresponding OD measurement was used as baseline from which % viability and proliferation of all treated samples was extrapolated based on the various OD readouts.
  • FIG. 19A-19C illustrates assessment of the cytotoxic effect of C-GFYWLRS- C-GG-D(KLAKLAK)2 peptide on leukemia cells in vitro.
  • FIG. 19A shows the results for Molt-4 cells whereby the C-GFYWLRS-C-GG- D(KLAKLAK)2 peptide resulted in maximal cytotoxicity at concentrations as low as 20 ⁇ M. At this dose, significant cytotoxicity was also obtained in treated OCI-AML3 cells (FIG. 19B). At higher doses though, either D(KLAKLAK)2 peptide alone or in mix with the ligand peptide resulted in significant loss of cell viability as well, hence reflecting non-specific toxicity at these doses in OCI- AML3 cells.
  • the CML K562 cell line (FIG.
  • BRASIL Biopanning and Rapid Analysis of Selective Interactive Ligands
  • selective binding in no way precludes binding to other cells or material, but connotes the preferential binding of leukemia cells.
  • Selective binding may include a 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fold preference for leukemia cells as compared to non-leukemia cells including normal leukocytes and mesenchymal stem cells (MSC).
  • human-derived leukemia cell lines were profiled, including those from the NCI-60 cell panel. Screening of the cell lines with a CX 7 C random phage library, for example, yielded several peptide motifs that bound leukemia cells, of which at least three clones (SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7) exhibited high frequency binding to various leukemia cell lines as compared to the control insert-less phage. Comparison of the selected motifs with available sequences in on ⁇ line protein databases suggests that a number of candidate proteins, in particular known viral proteins, share homologous sequences with these peptides.
  • peptides are being use in further studies to identify and purify protein(s) that interact, directly or indirectly, with an identified peptide, including identifying and purifying corresponding receptor(s).
  • the newly identified peptides and peptide motifs may serve as targeting moieties, drugs and/or drug leads.
  • the identified peptides can be optimized as delivery vehicles or enhancers for targeted therapy of leukemia.
  • a "targeting peptide” as used herein is a peptide comprising a contiguous sequence of amino acids, which is characterized by selective localization to an organ, tissue or cell type. Selective localization may be determined, for example, by methods disclosed below, wherein the putative targeting peptide sequence is incorporated into a protein that is displayed on the outer surface of a phage. Administration to a subject of a library of such phage that have been genetically engineered to express a multitude of such targeting peptides of different amino acid sequence is followed by collection of one or more organs, tissues or cell types that are typically derived from a subject and identification of phage found in or associated with that organ, tissue or cell type.
  • a phage expressing a targeting peptide sequence is considered to be selectively localized to a tissue, organ or cell if it exhibits greater binding in that tissue, organ, or cell compared to a control tissue, organ, or cell.
  • selective localization of a targeting peptide should result in a two-fold or higher enrichment of the phage or peptide in the target organ, tissue or cell type, compared to a control organ, tissue or cell type.
  • Selective localization resulting in at least a three- fold, four ⁇ fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold or higher enrichment in the target organ compared to a control organ, tissue or cell type is more preferred.
  • a phage expressing a targeting peptide sequence that exhibits selective localization preferably shows an increased enrichment in the target organ or cell compared to a control organ or cell when phage recovered from the target organ or cell are injected into or put in contact with a second host or cell population for another round of screening. Further enrichment may be exhibited following a third round of screening.
  • phage expressing the putative target peptide preferably exhibit a two-fold, more preferably a three- fold or higher enrichment in the target organ or cell type compared to control phage that express a non-specific peptide or that have not been genetically engineered to express any putative target peptides.
  • Yet another means to determine selective localization is that localization to the target organ of phage expressing the target peptide is at least partially blocked by the co-administration of a synthetic peptide containing the target peptide sequence.
  • “Targeting peptide” and “homing peptide” are used synonymously herein.
  • Leukemia is classified by how quickly it progresses. Acute leukemia is fast- growing and can overrun the body within a few weeks or months. By contrast, chronic leukemia is slow-growing and progressively worsens over years.
  • the blood-forming (hematopoietic) cells of acute leukemia remain in an immature state, so they reproduce and accumulate very rapidly. Therefore, acute leukemia needs to be treated immediately, otherwise the disease may be fatal within a few months. Fortunately, some subtypes of acute leukemia respond very well to available therapies and they are curable. Children often develop acute forms of leukemia, which are managed differently from leukemia in adults. In chronic leukemia, the blood-forming cells eventually mature, or differentiate, but they are not "normal.” They remain in the bloodstream much longer than normal white blood cells, and they are unable to combat infection well.
  • Leukemia also is classified according to the type of white blood cell that is multiplying - that is, lymphocytes (immune system cells), granulocytes (bacteria-destroying cells), or monocytes (macrophage-forming cells). If the abnormal white blood cells are primarily granulocytes or monocytes, the leukemia is categorized as myelogenous, or myeloid, leukemia. On the other hand, if the abnormal blood cells arise from bone marrow lymphocytes, the cancer is called lymphocytic leukemia. Other cancers, known as lymphomas, develop from lymphocytes within the lymph nodes, spleen, and other organs. Such cancers do not originate in the bone marrow and have a biological behavior that is different from lymphocytic leukemia.
  • AML Acute Myelogenous Leukemia
  • CML Chronic Myelogenous Leukemia
  • ALL Acute Lymphocytic Leukemia
  • CLL Chronic Lymphocytic Leukemia
  • AML Acute Myelogenous Leukemia
  • Acute myelogenous leukemia also known as acute nonlymphocytic leukemia (ANLL) - is the most common form of adult leukemia. Initial response rates are approximately 65-75% but the overall cure rates are more on the order of 40-50%.
  • Acute leukemia, such as AML is typically categorized according to a system known as French- American-British (FAB) classification.
  • FAB divides AML into eight subtypes: undifferentiated AML (MO), myeloblasts leukemia (Ml), myeloblasts leukemia (M2), promyelocy e leukemia (M3 or M3), myelomonocytic leukemia (M4), monocytic leukemia (M5), erythroleukemia (M6), or megakaryoblastic leukemia (M7).
  • MO undifferentiated AML
  • Ml myeloblasts leukemia
  • M2 myeloblasts leukemia
  • M3 or M3 promyelocy e leukemia
  • M4 myelomonocytic leukemia
  • M5 monocytic leukemia
  • M6 erythroleukemia
  • M7 megakaryoblastic leukemia
  • patients sometimes develop isolated tumors of the myeloblasts (early granulocytes), e.g., granulocytic sarcoma
  • the invention comprises methods for the identification of one or more targeting peptides or molecular targets that could be utilized for the development of novel therapies in leukemia.
  • BRASIL Biopanning and Rapid Analysis of Selective Interactive Ligands
  • human-derived leukemia cell lines are profiled, including, but not limited to the NCI-60 cell panel.
  • Screening of the leukemia cells including Acute Myelogenous Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) cell line with CX n C, wherein in can be 4, 5, 6, 7, or more residues, random phage library that yield several peptide motifs.
  • AML Acute Myelogenous Leukemia
  • ALL Acute Lymphoblastic Leukemia
  • three clones (encoding SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7) exhibited high frequency binding to various leukemia cell lines as compared to the control insert-less phage.
  • Comparison of the selected motifs with available sequences in on-line protein databases suggests that a number of candidate proteins, in particular known viral proteins, that share homologous sequences with these peptides. Work is underway to validate the lead peptides by assessing their binding specificity in clinical samples from ALL and AML patients. Subsequently, identification and purification of the corresponding receptor(s) and investigation of the interaction mechanism of the ligand-receptor pair candidates are being conducted.
  • BRASIL has been successfully used to isolate phage in various cell systems such as activated endothelial cells and tumor cells.
  • BRASIL has also been used to isolate bone marrow homing phage using in vivolex-vivo based strategies.
  • One method includes injecting the phage libraries intravenously and recover the bone marrow after a few minutes.
  • To identify leukemia cells that can bind to peptide from patients cells are incubated with peptide encoding phage or control phage. Phage bound to the cells are recovered, and quantified. Typically, an enrichment of the peptide-displaying phage in comparison to control phage is seen in cells with active ⁇ 2 integrins.
  • Certain aspects of the invention include the identification of peptide motifs (ligands) that bind preferentially to leukemia cells from clinical samples. Other aspects include utilizing the leukemia targeted peptide to identify and purify their corresponding rece ⁇ tor(s) present on the surface of leukemia cells. Still further aspects of the invention include the study of the interaction mechanism of the ligand-receptor pair candidates in the context of leukemia. The inventor describe herein various methods and compositions for the identification of ligands, receptors, and ligand-receptor pairs useful for the development of leukemia-targeted therapy.
  • Phage display libraries expressing transgenic peptides on the surface of bacteriophage were initially developed to map epitope binding sites of immunoglobulins (Smith and Scott, 1985 and 1993). Such libraries can be generated by inserting random oligonucleotides into cDNAs encoding a phage surface protein, generating collections of phage particles displaying unique peptides in as many as 10 9 permutations (Pasqualini and Ruoslahti, 1996; Arap et al, 1998a and 1998b).
  • a "phage display library” is a collection of phage that have been genetically engineered to express a set of putative targeting peptides on their outer surface.
  • DNA sequences encoding the putative targeting peptides are inserted in frame into a gene encoding a phage capsule protein.
  • the putative targeting peptide sequences are in part random mixtures of all twenty amino acids and in part non-random.
  • the putative targeting peptides of the phage display library exhibit one or more cysteine residues at fixed locations within the targeting peptide sequence. Cysteines may be used, for example, to create a cyclic peptide.
  • BRASIL Biopanning and Rapid Analysis of Selective Interactive Ligands
  • separation of phage bound to the cells of a target organ, tissue or cell type from unbound phage is achieved using the BRASIL (Biopanning and Rapid Analysis of Soluble Interactive Ligands) technique (PCT Application PCT/USOl/28124 entitled, "Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL)" by Arap et al, filed September 7, 2001, incorporated herein by reference in its entirety).
  • BRASIL Biopanning and Rapid Analysis of Soluble Interactive Ligands
  • an organ, tissue or cell type is gently separated into cells or small clumps of cells that are suspended in an aqueous phase.
  • the aqueous phase is layered over an organic phase of appropriate density and centrifuged.
  • BRASIL may be performed in an in vivo protocol, in which organs, tissues or cell types are exposed to a phage display library by intravenous administration, or by an ex vivo protocol, where the cells are exposed to the phage library in the aqueous phase before centrifugation.
  • primary phage libraries are amplified before injection into a human subject.
  • a phage library is prepared by ligating targeting peptide-encoding sequences into a phage vector, such as fUSE5.
  • the vector is transformed into pilus negative host E. coli such as strain MC 1061.
  • the bacteria are grown overnight and then aliquots are frozen to provide stock for library production.
  • Use of pilus negative bacteria avoids the bias in libraries that arises from differential infection of pilus positive bacteria by different targeting peptide sequences.
  • To freeze, bacteria are pelleted from two thirds of a primary library culture (5 liters) at 4000 x g for 10 min. Bacteria are resuspended and washed twice with 500 ml of 10% glycerol in water, then frozen in an ethanol/dry ice bath and stored at -80°C.
  • phage display libraries Various methods of phage display and methods for producing diverse populations of peptides are well known in the art. For example, U.S. Patents 5,223,409; 5,622,699 and 6,068,829, each of which is incorporated herein by reference in its entirety, disclose methods for preparing a phage library.
  • the phage display technique involves genetically manipulating bacteriophage so that small peptides can be expressed on their surface (Smith and Scott, 1985 and 1993).
  • phage particles In addition to peptides, larger protein domains such as single-chain antibodies can also be displayed on the surface of phage particles (Arap et al., 1998a).
  • Targeting peptides selective for a given organ, tissue or cell type can be isolated by "biopanning" (Pasqualini and Ruoslahti, 1996; Pasqualini, 1999).
  • biopanning Pasqualini and Ruoslahti, 1996; Pasqualini, 1999.
  • a library of phage containing putative targeting peptides is administered to an animal or human and samples of organs, tissues or cell types containing phage are collected.
  • the phage may be propagated in vitro between rounds of biopanning in pilus-positive bacteria.
  • the bacteria are not lysed by the phage but rather secrete multiple copies of phage that display a particular insert.
  • Phage that bind to a target molecule can be eluted from the target organ, tissue or cell type and then amplified by growing them in host bacteria. If desired, the amplified phage can be administered to a host and samples of organs, tissues or cell types again collected. Multiple rounds of biopanning can be performed until a population of selective binders is obtained.
  • the amino acid sequence of the peptides is determined by sequencing the DNA corresponding to the targeting peptide insert in the phage genome. The identified targeting peptide can then be produced as a synthetic peptide by standard protein chemistry techniques (Arap et al., 1998a, Smith and Scott, 1985).
  • a candidate target is identified as the receptor of a targeting peptide, it can be isolated, purified and cloned by using standard biochemical methods (Pasqualini, 1999; Rajotte and Ruoslahti, 1999).
  • a subtraction protocol is used may be used to further reduce background phage binding.
  • the purpose of subtraction is to remove phage from the library that bind to cells other than the cell of interest, or that bind to inactivated cells.
  • the phage library may be prescreened against a subject who does not possess the targeted cell, tissue or organ. For example, placenta-binding peptides may be identified after prescreening a library against a male or non-pregnant female subject. After subtraction the library may be screened against the cell, tissue or organ of interest. In another alternative embodiment, an unstimulated, quiescent cell type, tissue or organ may be screened against the library and binding phage removed.
  • the cell line, tissue or organ is then activated, for example by administration of a hormone, growth factor, cytokine or chemokine and the activated cell type, tissue or organ screened against the subtracted phage library.
  • Other subtraction protocols are known and may be used in the practice of the present invention, for example as disclosed in U.S. Patents 5,840,841, 5,705,610, 5,670,312 and 5,492,807, which are incorporated herein by reference in their entirety.
  • Phage libraries displaying linear, cyclic, or double cyclic peptides may be used within the scope of the present invention. However, phage libraries displaying 3 to 10 random residues in a cyclic insert (CX 3- i 0 C) are preferred, since single cyclic peptides tend to have a higher affinity for the target organ than linear peptides. Libraries displaying double-cyclic peptides (such as CX 3 C X 3 CX 3 C; Rojotte et al, 1998) have been successfully used.
  • phage from a single organ are collected, amplified and injected into a new host, where tissue from the same organ is collected for phage rescue and a new round of biopanning.
  • This protocol is feasible in animal subjects.
  • the limited availability and expense of processing samples from humans requires an improvement in the protocol.
  • the polyorgan targeting protocol may be repeated for as many rounds of biopanning as desired. In this manner, it is possible to significantly reduce the number of subjects required for isolation of targeting peptides for multiple organs, while still achieving substantial enrichment of the organ-homing phage.
  • phage are recovered from human organs, tissues or cell types after injection of a phage display library into a human subject.
  • phage may be recovered by exposing a sample of the organ, tissue or cell type to a pilus positive bacterium, such as E. coli K91.
  • phage may be recovered by amplifying the phage inserts, ligating the inserts to phage DNA and producing new phage from the ligated DNA.
  • the insertion of the RGD-4C peptide into a surface protein of an adenovirus has produced an adenoviral vector that may be used for tumor targeted gene therapy (Arap et al, 1998b).
  • the present invention describes methods and compositions for the selective targeting of leukemia cells.
  • a "receptor” for a targeting peptide includes but is not limited to any molecule or macromolecular complex that binds to a targeting peptide.
  • Non-limiting examples of receptors include peptides, proteins, glycoproteins, lipoproteins, epitopes, lipids, carbohydrates, multi-molecular structures, and a specific conformation of one or more molecules.
  • a "receptor” is a naturally occurring molecule or complex of molecules that is present on the surface of cells within a target organ, tissue or cell type. More preferrably, a "receptor” is a naturally occurring molecule or complex of molecules that is present on the surface of leukemia cells.
  • phage display biopanning in the mouse model system require substantial improvements for use with humans.
  • Techniques for biopanning in human subjects are disclosed in PCT Patent Application PCT/US01/28044, filed September 7, 2001, the entire text of which is incorporated herein by reference.
  • a "subject" refers generally to a mammal. In certain preferred embodiments, the subject is a mouse or rabbit. In more preferred embodiments, the subject is a human. In general, humans suitable for use with phage display are either brain dead or terminal wean patients.
  • the amount of phage library (preferably primary library) required for administration must be significantly increased, preferably to 10 1 TU or higher, preferably administered intravenously in approximately 200 ml of Ringer lactate solution over about a 10 minute period.
  • the amount of phage required for use in humans has required substantial improvement of the mouse protocol, increasing the amount of phage available for injection by five orders of magnitude.
  • the transformed bacterial pellets recovered from up to 500 to 1000 transformations are amplified up to 10 times in the bacterial host, recovering the phage from each round of amplification and adding LB Tet medium to the bacterial pellet for collection of additional phage.
  • the phage inserts remain stable under these conditions and phage may be pooled to form the large phage display library required for humans.
  • Samples of various organs and tissues are collected starting approximately 15 minutes after injection of the phage library. Samples are processed as described below and phage collected from each organ, tissue or cell type of interest for DNA sequencing to determine the amino acid sequences of targeting peptides.
  • biopanning technique involves polyorgan targeting.
  • the present invention concerns novel compositions comprising at least one protein or peptide.
  • a protein or peptide generally refers, but is not limited to, a protein of greater than about 200 amino acids, up to a full length sequence translated from a gene; a polypeptide of greater than about 100 amino acids; and/or a peptide of from about 3 to about 100 amino acids.
  • proteins proteins
  • polypeptide and “peptide are used interchangeably herein.
  • the size of at least one protein or peptide may comprise, but is not limited to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
  • amino acid residue refers to any naturally occurring amino acid, any amino acid derivative or any amino acid mimic known in the art.
  • residues of the protein or peptide are sequential, without any non-amino acid interrupting the sequence of amino acid residues.
  • sequence may comprise one or more non-amino acid moieties.
  • sequence of residues of the protein or peptide may be interrupted by one or more non-amino acid moieties.
  • protein or peptide encompasses amino acid sequences comprising at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid, including, but not limited to, 2 Aminoadipic acid (Aad), N Ethylasparagine (EtAsn), 3 Aminoadipic acid (Baad), Hydroxylysine (HyI), ⁇ alanine, ⁇ Amino propionic acid (BaIa), allo Hydroxylysine (AHyI), 2 Aminobutyric acid (Abu), 3 Hydroxyproline (3Hyp), 4 Aminobutyric acid (4Abu), 4 Hydroxyproline (4Hyp), 6 Aminocaproic acid (Acp), Isodesmosine (Ide), 2 Aminoheptanoic acid (Ahe), allo Isoleucine (AIIe), 2 Aminoisobutyric acid (Aib), N Methylglycine (M
  • Proteins or peptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. Coding regions for known genes may be amplified and/or expressed using the techniques disclosed herein or as would be know to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
  • peptide mimetics are molecules that mimic elements of protein secondary structure (see, for example, Johnson et al, 1993, incorporated herein by reference).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • Fusion proteins Other embodiments of the present invention concern fusion proteins. These molecules generally have all or a substantial portion of a targeting peptide, linked at the N- or C-terminus, to all or a portion of a second polypeptide or protein. For example, fusions may employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification.
  • fusion proteins include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
  • the fusion proteins of the instant invention comprise a targeting peptide linked to a therapeutic protein or peptide.
  • proteins or peptides that may be incorporated into a fusion protein include cytostatic proteins, cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents, hormones, cytokines, growth factors, peptide drugs, antibodies, Fab fragments antibodies, antigens, receptor proteins, enzymes, lectins, MHC proteins, cell adhesion proteins and binding proteins.
  • Such proteins can be produced, for example, by chemical attachment using bifunctional cross-linking reagents, by de novo synthesis of the complete fusion protein, or by attachment of a DNA sequence encoding the targeting peptide to a DNA sequence encoding the second peptide or protein, followed by expression of the intact fusion protein.
  • a protein or peptide may be isolated or purified.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the homogenization and crude fractionation of the cells, tissue or organ to polypeptide and non-polypeptide fractions.
  • the protein or peptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing.
  • An example of receptor protein purification by affinity chromatography is disclosed in U.S.
  • a particularly efficient method of purifying peptides is fast performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).
  • FPLC fast performance liquid chromatography
  • HPLC high performance liquid chromatography
  • a purified protein or peptide is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • An isolated or purified protein or peptide therefore, also refers to a protein or peptide free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the protein or peptide in the composition.
  • Various methods for quantifying the degree of purification of the protein or peptide are known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of protein or peptide within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity therein, assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification, and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "- fold" purification than the same technique utilizing some other chromatography systems. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • Affinity chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind. This is a receptor-ligand type of interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (e.g., altered pH, ionic strength, and temperature).
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • the targeting peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques.
  • Various automatic synthesizers are commercially available and can be used in accordance with known protocols (see, for example, Stewart and Young, 1984; Tarn et al, 1983; Merrifield, 1986; or Barany and Merrifield, 1979, each incorporated herein by reference).
  • Short peptide sequences usually from about 6 up to about 35 to 50 amino acids, can be readily synthesized by such methods.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell, and cultivated under conditions suitable for expression.
  • the appropriate targeting peptide or receptor, or portions thereof may be coupled, bonded, bound, conjugated, or chemically-linked to one or more agents via linkers, polylinkers, or derivatized amino acids. This may be performed such that a bispecific or multivalent composition or vaccine is produced. It is further envisioned that the methods used in the preparation of these compositions are familiar to those of skill in the art and should be suitable for administration to humans, i.e., pharmaceutically acceptable.
  • Preferred agents are the carriers are keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).
  • antibody is used to refer to any antibody like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. Techniques for preparing and using various antibody based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g., Harlow and Lane, 1988; incorporated herein by reference). F. Cytokines and chemokines
  • cytokine is a generic term for proteins released by one cell population that act on another cell as intercellular mediators.
  • cytokines examples include lymphokines, monokines, growth factors and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor- .
  • growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
  • parathyroid hormone thyroxine
  • insulin proinsulin
  • relaxin prorelaxin
  • glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH)
  • FSH f
  • TGFs transforming growth factors
  • CSFs colony stimulating factors
  • M-CSF macrophage-CSF
  • GM-CSF granulocyte- macrophage-CSF
  • G-CSF granulocyte-CSF
  • ILs interleukins
  • Chemokines generally act as chemoattractants to recruit immune effector cells to the site of chemokine expression. It may be advantageous to express a particular chemokine gene in combination with, for example, a cytokine gene, to enhance the recruitment of other immune system components to the site of treatment. Chemokines include, but are not limited to, RANTES, MCAF, MIPl -alpha, MIPl -Beta, and IP-IO. The skilled artisan will recognize that certain cytokines are also known to have chemoattractant effects and could also be classified under the term chemokines. G. Imaging agents and radioisotopes
  • the claimed peptides or proteins of the present invention may be attached to imaging agents of use for imaging and diagnosis of various diseased organs, tissues or cell types.
  • imaging agents are known in the art, as are methods for their attachment to proteins or peptides (see, e.g., U.S. Patents 5,021,236 and
  • Certain attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a
  • DTPA attached to the protein or peptide (U.S. Patent 4,472,509).
  • Proteins or peptides also may be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • Non-limiting examples of paramagnetic ions of potential use as imaging agents include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III), with gadolinium being particularly preferred.
  • Ions useful in other contexts, such as X ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
  • Radioisotopes of potential use as imaging or therapeutic agents include astatine 2 ", l4 carbon, 5 l chromium, 36 chlorine, "cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 1 ", 59 iron, 32 phosphorus, rhenium 186 , rhenium 188 , 75 selenium, 35 sulphur, technicium” 1 " and yttrium 90 .
  • 125 I is often being preferred for use in certain embodiments, and technecium 99 “ 1 and indium 1 " are also often preferred due to their low energy and suitability for long range detection.
  • Radioactively labeled proteins or peptides of the present invention may be produced according to well known methods in the art. For instance, they can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • a chemical oxidizing agent such as sodium hypochlorite
  • an enzymatic oxidizing agent such as lactoperoxidase.
  • Proteins or peptides according to the invention may be labeled with technetium 99 '" by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the peptide to this column or by direct labeling techniques, e.g., by incubating pertechnate, a reducing agent such as SNCl 2 , a buffer solution such as sodium potassium phthalate solution, and the peptide.
  • Intermediary functional groups that are often used to bind radioisotopes that exist as metallic ions to peptides are diethylenetriaminepenta-acetic acid (DTPA) and ethylene diaminetetra-acetic acid (EDTA).
  • fluorescent labels including rhodamine, fluorescein isothiocyanate and renographin.
  • the claimed proteins or peptides may be linked to a secondary binding ligand or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromo genie substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose oxidase.
  • Preferred secondary binding ligands are biotin and avidin or streptavidin compounds. The use of such labels is well known to those of skill in the art in light and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 ; each incorporated herein by reference.
  • Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.
  • Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.
  • Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.
  • the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specific groups.
  • reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied.
  • a majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.
  • ligands can be covalently bound to liposomal surfaces through the cross- linking of amine residues.
  • Liposomes in particular, multilamellar vesicles (MLV) or unilamellar vesicles such as microemulsified liposomes (MEL) and large unilamellar liposomes (LUVET), each containing phosphatidylethanolamine (PE), have been prepared by established procedures.
  • MLV multilamellar vesicles
  • MEL microemulsified liposomes
  • LVET large unilamellar liposomes
  • PE in the liposome provides an active functional residue, a primary amine, on the liposomal surface for cross-linking purposes.
  • Ligands such as epidermal growth factor (EGF) have been successfully linked with PE-liposomes. Ligands are bound covalently to discrete sites on the liposome surfaces. The number and surface density of these sites are dictated by the liposome formulation and the liposome type. The liposomal surfaces may also have sites for non-covalent association.
  • cross-linking reagents have been studied for effectiveness and biocompatibility.
  • Cross-linking reagents include glutaraldehyde (GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether (EGDE), and a water soluble carbodiimide, preferably l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
  • GID glutaraldehyde
  • OXR bifunctional oxirane
  • EGDE ethylene glycol diglycidyl ether
  • EDC water soluble carbodiimide
  • linkage of the amine residues of the recognizing substance and liposomes is established.
  • heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described (U.S. Patent 5,889,155, specifically incorporated herein by reference in its entirety).
  • the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols.
  • the cross-linking reagent can be modified to cross- link various functional groups.
  • Nucleic acids according to the present invention may encode a targeting peptide, a receptor protein, a fusion protein, or other protein or peptide.
  • the nucleic acid may be derived from genomic DNA, complementary DNA (cDNA) or synthetic DNA. Where incorporation into an expression vector is desired, the nucleic acid may also comprise a natural intron or an intron derived from another gene. Such engineered molecules are sometime referred to as "mini-genes.”
  • nucleic acid as used herein includes single-stranded and double-stranded molecules, as well as DNA, RNA, chemically modified nucleic acids and nucleic acid analogs. It is contemplated that a nucleic acid within the scope of the present invention may be of almost any size, determined in part by the length of the encoded protein or peptide. It is contemplated that targeting peptides, fusion proteins and receptors may be encoded by any nucleic acid sequence that encodes the appropriate amino acid sequence. The design and production of nucleic acids encoding a desired amino acid sequence is well known to those of skill in the art, using standardized codon tables. In preferred embodiments, the codons selected for encoding each amino acid may be modified to optimize expression of the nucleic acid in the host cell of interest. Codon preferences for various species of host cell are well known in the art.
  • the present invention encompasses complementary nucleic acids that hybridize under high stringency conditions with such coding nucleic acid sequences.
  • High stringency conditions for nucleic acid hybridization are well known in the art.
  • conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50 0 C to about 7O 0 C.
  • the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleotide content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethyl ammonium chloride or other solvent(s) in a hybridization mixture.
  • expression vectors are employed to express the targeting peptide or fusion protein, which can then be purified and used.
  • the expression vectors are used in gene therapy. Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are known.
  • expression construct or "expression vector” are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid coding sequence is capable of being transcribed.
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter.
  • a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell it is preferable to position the nucleic acid coding region adjacent and under the control of a promoter that transcriptionally active in human cells.
  • a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest.
  • CMV cytomegalovirus
  • the use of other viral or mammalian cellular or bacterial phage promoters that are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a cDNA insert one will typically include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed, such as human growth hormone and SV40 polyadenylation signals.
  • a terminator also contemplated as an element of the expression construct. These elements can serve to enhance message levels and to minimize read through from the construct into other sequences.
  • the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • Preferred gene therapy vectors are generally viral vectors.
  • a preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
  • DNA viruses used as gene vectors include the papovaviruses (e.g., simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
  • papovaviruses e.g., simian virus 40, bovine papilloma virus, and polyoma
  • adenoviruses Rosgeway, 1988; Baichwal and Sugden, 1986.
  • adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
  • helper cell line which is transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977.). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El , the E3, or both regions (Graham and Prevec, 1991.).
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or human embryonic mesenchymal or epithelial cells.
  • helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells. As discussed, the preferred helper cell line is 293. Racher et al (1995) disclose improved methods for culturing 293 cells and propagating adenovirus.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Animal studies have suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford- Perricaudet et al, 1990; Rich et al, 1993).
  • LTR long terminal repeat
  • a nucleic acid encoding a protein of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • Retroviral vectors are capable of infecting a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).
  • viral vectors may be employed as expression constructs.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988), adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and van der Eb, 1973.; Chen and Okayama, 1987.; Rippe et al, 1990; DEAE dextran (Gopal, et al, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
  • the expression construct may be entrapped in a liposome.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al. (1980) demonstrates the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
  • Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.
  • compositions - expression vectors, virus stocks, proteins, antibodies and drugs - it may be necessary to prepare pharmaceutical compositions - expression vectors, virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of impurities that could be harmful to humans or animals.
  • Aqueous compositions of the present invention may comprise an effective amount of a protein, peptide, antibody, fusion protein, recombinant phage and/or expression vector, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically or pharmacologically acceptable refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the proteins or peptides of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
  • compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention are via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial or intravenous injection. Such compositions normally would be administered as pharmaceutically acceptable compositions, described supra.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • therapeutic agents may be attached to a targeting peptide or fusion protein for selective delivery to, for example, leukemic cells or derivatives thereof.
  • Agents or factors suitable for use may include any chemical compound that induces apoptosis, cell death, cell stasis and/or anti-angiogenesis.
  • Apoptosis or programmed cell death, is an essential process for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis (Kerr et al, 1972).
  • the Bcl-2 family of proteins and ICE-like proteases have been demonstrated to be important regulators and effectors of apoptosis in other systems.
  • the BcI 2 protein discovered in association with follicular lymphoma, plays a prominent role in controlling apoptosis and enhancing cell survival in response to diverse apoptotic stimuli (Bakhshi et al, 1985; Cleary and Sklar, 1985; Cleary et al, 1986; Tsujimoto et al, 1985; Tsujimoto and Croce, 1986).
  • the evolutionarily conserved BcI 2 protein now is recognized to be a member of a family of related proteins, which can be categorized as death agonists or death antagonists.
  • BcI 2 acts to suppress cell death triggered by a variety of stimuli. Also, it now is apparent that there is a family of BcI 2 cell death regulatory proteins that share in common structural and sequence homologies. These different family members have been shown to either possess similar functions to BcI 2 ⁇ e.g., BcIXL, BcIW, BcIS, McI-I, Al, BfI-I) or counteract BcI 2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
  • Non-limiting examples of pro-apoptosis agents contemplated within the scope of the present invention include gramicidin, magainin, mellitin, defensin, cecropin, (KLAKLAK) 2 (SEQ ID NO: 1), (KLAKKLA) 2 (SEQ ID NO: 2), (KAAKKAA) 2 (SEQ ID NO: 3) or (KLGKKLG) 3 (SEQ ID NO: 4).
  • the present invention may concern administration of targeting peptides attached to anti-angiogenic agents, such as angiotensin, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin 12, platelet factor 4, IP-10, Gro- ⁇ , thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CMlOl, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM- 14
  • Chemotherapeutic (cytotoxic) agents of potential use include, but are not limited to, 5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin
  • CDDP cyclophosphamide
  • dactinomycin daunorubicin
  • doxorubicin estrogen receptor binding agents
  • etoposide VP 16
  • farnesyl-protein transferase inhibitors gemcitabine, ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol, temazolomide (an aqueous form of DTIC), transplatinum, vinblastine and methotrexate, vincristine, or any analog or derivative variant of the foregoing.
  • chemotherapeutic agents fall into the categories of alkylating agents, antimetabolites, antitumor antibiotics, corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormone agents, miscellaneous agents, and any analog or derivative variant thereof.
  • Chemotherapeutic agents and methods of administration, dosages, etc. are well known to those of skill in the art (see for example, the “Physicians Desk Reference”, Goodman & Gilman's “The Pharmacological Basis of Therapeutics” and in “Remington's Pharmaceutical Sciences” 15th ed., pp 1035-1038 and 1570-1580, each incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Examples of specific chemotherapeutic agents and dose regimes are also described herein.
  • Alkylating agents are drugs that directly interact with genomic DNA to prevent cells from proliferating. This category of chemotherapeutic drugs represents agents that affect all phases of the cell cycle, that is, they are not phase-specific.
  • An alkylating agent may include, but is not limited to, a nitrogen mustard, an ethylenimene, a methylmelamine, an alkyl sulfonate, a nitrosourea or a triazines. They include but are not limited to: busulfan, chlorambucil, cisplatin, cyclophosphamide (Cytoxan), dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.
  • Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents, they specifically influence the cell cycle during S phase. Antimetabolites can be differentiated into various categories, such as folic acid analogs, pyrimidine analogs and purine analogs and related inhibitory compounds. Antimetabolites include but are not limited to, 5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, and methotrexate.
  • 5-FU 5-fluorouracil
  • Ara-C cytarabine
  • fludarabine gemcitabine
  • gemcitabine gemcitabine
  • methotrexate methotrexate
  • Natural products generally refer to compounds originally isolated from a natural source, and identified as having a pharmacological activity. Such compounds, analogs and derivatives thereof may be, isolated from a natural source, chemically synthesized or recombinantly produced by any technique known to those of skill in the art. Natural products include such categories as mitotic inhibitors, antitumor antibiotics, enzymes and biological response modifiers.
  • Mitotic inhibitors include plant alkaloids and other natural agents that can inhibit either protein synthesis required for cell division or mitosis. They operate during a specific phase during the cell cycle. Mitotic inhibitors include, for example, docetaxel, etoposide (VP 16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
  • Mitotic inhibitors include, for example, docetaxel, etoposide (VP 16), teniposide, paclitaxel, taxol, vinblastine, vincristine, and vinorelbine.
  • Taxoids are a class of related compounds isolated from the bark of the ash tree, Taxus brevifolia. Taxoids include but are not limited to compounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin (at a site distinct from that used by the vinca alkaloids) and promotes the assembly of microtubules.
  • Vinca alkaloids are a type of plant alkaloid identified to have pharmaceutical activity. They include such compounds as vinblastine (VLB) and vincristine.
  • antibiotics have both antimicrobial and cytotoxic activity. These drugs also interfere with DNA by chemically inhibiting enzymes and mitosis or altering cellular membranes. These agents are not phase specific so they work in all phases of the cell cycle.
  • cytotoxic antibiotics include, but are not limited to, bleomycin, dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin (mithramycin) and idarubicin.
  • Miscellaneous cytotoxic agents that do not fall into the previous categories include, but are not limited to, platinum coordination complexes, anthracenediones, substituted ureas, methyl hydrazine derivatives, amsacrine, L-asparaginase, and tretinoin.
  • Platinum coordination complexes include such compounds as carboplatin and cisplatin (cis- DDP).
  • An exemplary anthracenedione is mitoxantrone.
  • An exemplary substituted urea is hydroxyurea.
  • An exemplary methyl hydrazine derivative is procarbazine (N- methylhydrazine, MIH).
  • BRASIL Prior to performing binding assays in clinical samples from leukemia patients, BRASIL was used to profile several human-derived leukemia cell lines from the NCI-60 cell panel (dtpws4.ncifcrf.gov) including MOLT-4, CCRF-CEM, SR, RPMI-8226, K-562, and HL-60.
  • the cells were incubated with either the CX 5 C or CX 7 C random phage libraries, or with the insert-less control phage (Fd-tet). Phage bound to the cells was then recovered, and quantified in 4 consecutive rounds of panning. In comparison to control phage binding to cells, enrichment in particular for phage displaying peptides that bind specifically to the targeted cell type was obtained.
  • PCR master mix was prepared in that 20 ⁇ l reaction per sample included l ⁇ l of 1 :1000 dilution in PBS of each phage clone, l ⁇ l Fuse-5 reverse primer (8 pmol/ ⁇ l), l ⁇ l Fuse-5 forward primer (8 pmol/ ⁇ l), l ⁇ l dNTP mix (2.5mM, Promega), 2 ⁇ l Taq polymerase buffer (10X, Promega), 0.5 ⁇ l Taq DNA polymerase (5U/ ⁇ l, Promega), and 13.5 ⁇ l dH 2 O.
  • PCR was performed in polypropylene V-bottom microplates (MJ Research Inc.) using 94°C annealing temperature (T) for 3 min, then 30 cycles of 94°C 10 sec, 60°C 30 sec, 72°C 30 sec. Dilutions (1 : 10) of the PCR products were prepared and stored at 4°C until sent for sequencing while the rest were stored at -2O 0 C. Seven samples were sequenced at DNA CORE facility at M.D. Anderson Cancer Center, Houston, Tx. Validation of Selected Molt-4 Phage Clones by Assessing their Binding to a
  • BRASIL Panel of Leukemia Cell Lines Using BRASIL. Cells grown in suspension were washed once with PBS and cell count was determined using trypan blue vital stain. Cells were suspended at 1x10 6 cell per l ⁇ l in 1 % BSA/RPMI. Cell suspension was then transferred in 200 ⁇ l aliquots into micro-centrifuge tubes. Each phage clone (1 x 10 9 TU) were added to 200 ⁇ l cell sample and incubated for 5 hr on ice with occasional slight vortexing. Meanwhile, K91 Kana bacteria were inoculated in TB medium containing supplements and 100 ⁇ g/ml kanamycin. Bacteria were used when 1 :10 dilution reached 0.180 to 0.200 OD (600 nm).
  • the oil (45 ml dibutyl phtalate + 5 ml cyclohexane in a 50 ml Falcon) was stored at RT and protected from light. Two hundred ⁇ l of oil were added to a 400 ⁇ l microcentrifuge tube (VWR Scientific Products, cat. No. 20170-326). The phage-cell mix (similar volume) on top of the oil was transferred and spun at 10,000 rpm for 10 min at 4°C. If the pellet remained in the interface, centrifugation was repeated at room temperature. The tubes were placed at - 80 0 C for approximately 10 min (until top layer was frozen or until bacteria were ready).
  • the bottom of the BRASIL tube was cut close to the pellet and then put into a 1.5 ml eppendorf tube. Any residual oil was then removed. Two-hundred ⁇ l of the K91-Kana growth medium were added and the cell pellet suspended using a pipette tip. Cell- bound phage and bacteria were incubated for 1- 1.5 hrs at room temperature. Infected bacteria (200 ⁇ l) was transferred to 10 ml of pre-warmed LB liquid medium supplemented with 20 ⁇ g/ml Tetracyclin and 100 ⁇ g/ml Kanamycin and incubated at room temperature for approximately 30 minutes. Dilutions (1:1, 1:10, 1:100) of each sample were plated in triplicate on LB+Tet+Kana plates and incubated at 37°C overmight for colony counts the next day.
  • MSCs from patients and from healthy marrow will be expanded in culture and BRASIL will be done on them to evaluate if any of the Molt-4 phage clones shows differential binding to MSCs from AML patient as compared to MSCs from healthy marrow.
  • Phage binding assays performed so far on all 9 AML patient samples were performed using all the mononuclear cells in the sample and not MSCs in particular. If any difference is observed upon assaying MSCs only, additional AML samples may be requested from the Leukemia Cell Bank to perform BRASIL on MSCs exclusively.
  • A. Material and Methods The selection of the peptides for which the receptor will be pursued is based on three criteria: (i) the binding avidity of the peptide, (ii) databank searches for identification of the ligand for a given peptide, and (iii) various biochemical and genetic approaches for identification and/or validation of a ligand/receptor pair.
  • BLAST is an approach that is used to elucidate the identity of the molecules to which the peptide inserts of the selected phage bind.
  • BLAST is an approach that is used to elucidate the identity of the molecules to which the peptide inserts of the selected phage bind.
  • peptide motifs were identified with selective affinities for many organs and tumors. The receptors to which the organ and tumor specific phage bind to have been characterized in the context of different vascular beds.
  • Single phage clones are then added to their respective wells using the same TU number and are incubated at RT for 2hr to allow for binding. Unbound phage are removed by several wash steps, followed by K91 infection, transfer to LB media, and plating. Colonies are counted the next day and results are interpreted in terms of fold difference in phage binding to a particular recombinant protein relative to insertless phage which is used as baseline.
  • Synthetic peptides will be used that correspond to the sequence displayed by the phage to perform inhibitory studies. This assay determines whether phage binding is entirely mediated by the peptide displayed by the phage. It is contemplated that the synthetic peptides will inhibit the binding of the corresponding phage in a dose-dependent manner. A control peptide containing unrelated amino acids will be tested at identical concentrations.
  • GST-fusion proteins can be designed for receptor identification and/or validation by biochemical approaches.
  • Peptide-coding DNA sequences will be amplified by colony PCR using forward and reverse primers that contained BamHI and EcoRI sites, respectively.
  • the amplified sequences will be cloned into the BamHI-EcoRI sites of the GST vector, pGEX-2TK (Amersham/Pharmacia), and the presence of the inserted sequences will be verified by sequence analysis.
  • Positive clones will be transformed into a bacterial expression host strain, BL21(DE3)pLysS (Stratagene) and expression of the GST-fusion proteins will be induced with 200 ⁇ M IPTG.
  • the GST-fusion proteins will be affinity purified from bacterial lysates by affinity chromatography to immobilized glutathione.
  • the GST-fusion proteins will batch-bound to glutathione Sepharose 4B beads, and the resin rinsed to remove non-specific proteins.
  • GST-fusion proteins will be eluted by incubating the resin with an excess of reduced glutathione, followed by extensive dialysis of the eluted protein against phosphate buffered saline, pH 7.4 (PBS) to remove the glutathione.
  • PBS phosphate buffered saline
  • leukemia cell lines can be found to express a particular receptor (this can be evaluated using phage binding assays), it will be possible to use positive cell lines as the source of protein.
  • the options are to microsequence the protein or to prepare antibodies to it. If the sequence is new, it will be used to design oligonucleotide probes for the isolation of cDNA clones.
  • Antibodies may also be used for the isolation of clones from bacterial expression libraries. A possible, but as yet untested, alternative way of isolating clones for the receptor molecules is to screen a bacterially expressed library with the phage carrying the appropriate insert.
  • Mass Spectrometry Mass spectrometric peptide mapping can be used to identify novel target receptors. Polyclonal and monoclonal antibodies raised against the candidate receptors will also be used to purify target proteins. These proteins will be resolved by SDS- PAGE, will be cut out from the SDS gels, and digested in-gel with trypsin. After extraction of the peptides, MALDI-TOF mass spectrometry analysis will be performed to produce a list of peptide masses. This list of peptide masses, in combination with protease specificity, produces a relatively specific "signature" that can be used to search sequence databases. If the protein sequence is present in a database, the protein can be identified with high confidence by this method. The lower detection limit for this approach is currently 1 pmol, at least 10-20- fold better than N-terminal Edman sequencing methods.
  • Biacore and Protein Arrays Prospective ligand-receptor pairs will be validated using a Biacore system.
  • the surface plasmon resonance (SPR) measurements will monitor the specificity of interaction, in real time, between two species without the need for labels.
  • Receptor identification and validation by genetic approaches Yeast two hybrid cloning can be used for receptor identification.
  • DNA encoding the peptide motif with homing properties is fused to a binding domain to screen a cDNA library fused to a transactivation domain. It is contemplated that clones will be found that are capable of binding to the peptide used as the bait; those clones may code for the relevant receptor.
  • the encoded protein may also be an irrelevant binder of the peptide, but even in that case it may be useful because antibodies against it may recognize the relevant receptor.
  • Antibodies will be developed to an identified receptor and used to identify cDNA clones, as well as to study the expression of the receptor.
  • two New Zealand White female rabbits will be immunized with 100-500 ⁇ g of purified recombinant receptor protein in Freund's complete adjuvant. Rabbits will be boosted every 3-4 weeks with lOO ⁇ g of purified recombinant protein in Freund's incomplete adjuvant.
  • a sample of the rabbit sera along with a sample of the pre-immune sera, will be analyzed for reactivity against the purified recombinant receptor protein by a standard ELISA assay.
  • the anti-receptor serum titers will be adequately high against the receptor, the final bleed will be obtained from the rabbits. IgGs will be isolated from the sera by standard affinity purification techniques using Protein G resin.
  • Immunohistochemical staining Immunohistochemistry will be performed as described previously (Zurita, 2004). Briefly, 4 ⁇ m sections will undergo antigen retrieval as well as blocking for endogenous biotin. After protein blocking, tissue sections will be incubated with the anti-receptor antibodies followed by secondary detection with the LSAB kit (DAKO). The staining will be scored and evaluated statistically. Additionally, co- localization analyses will be performed using fluorescence with known leukemia cell markers.
  • Tissue sections are rinsed, blocked with 5% normal serum for 60 minutes and then incubated with primary antibodies overnight. The following day, sections are rinsed with PBS and incubated with secondary antibodies for 4 hr. Samples are rinsed, fixed with 4% paraformaldehyde, rinsed and then mounted with VECTASHIELD (Vector Laboratories, CA). Slides are observed using a laser scanning confocal microscope (Carl Zeiss Inc, Germany) to detail the cellular context of putative target receptors. Assessment of the expression patterns of identified receptors in human leukemia by phage overlay assays.
  • Phage overlay assays will be carried out on human leukemia tissue samples to study the ligand-receptor binding of the identified peptide ligands. Phage clones isolated from the biopanning on leukemia cells will be evaluated for binding to tissue samples derived from leukemia patients. A well-established phage overlay protocol will be used (Arap et al, 2002; Pasqualini and Ruoslahti, 1996). Briefly, selected phage clones or the negative control fd-tet phage (1-5 x 109 TU/tissue section) will be incubated for two hours at room temperature on 4 ⁇ m tissue sections. If necessary, the tissue sections will be treated for antigen retrieval prior to incubation.
  • the specifically bound phage will be detected with an anti-bacteriophage antibody (1 :500 dilution, Sigma) incubated for one hour at room temperature followed by a peroxidase-conjugated anti-rabbit secondary antibody. Tissue sections will then be washed, developed with DAB, counterstained with hematoxylin, dehydrated and mounted. Additionally, data on ligand-receptor interaction will be obtained using phage/antibody inhibition assays. To evaluate whether the receptor binding phage can inhibit the anti-receptor antibody (either a commercial or a generated polyclonal source) staining, receptor binding phage and negative control phage will be overlaid on serial human tissue sections prior to adding the anti-receptor antibody or negative control antibody.
  • the synthetic peptides are characterized as to their ability to bind to and interfere with receptor function. In certain aspects it is important that the synthetic peptide exhibits capacity to deliver a toxic entity such as a pro-apoptotic moiety into target cells.
  • a toxic entity such as a pro-apoptotic moiety into target cells.
  • in vitro assays can be performed on leukemia cells with the targeting synthetic peptides. These include cell viability and cytotoxicity assays to determine the proportion of live and dead cells in the tested populations, cell proliferation assays to assess the growth rate and density of a tested cell population based on monitoring changes in total nucleic acid content (L3224, V231 1 1, and C7026, Molecular Probes).
  • the analysis can be done using a flow cytometer, a fluorescence microplate reader, and/or a fluorescence microscope.
  • colorimetric-based assays could be used such as the MTT cell proliferation assay (ATCC) or the WST-I cell proliferation and cell viability assay (Roche) for which the sample measurements can be acquired and analyzed using an absorbance microplate reader.
  • the WST-I assay is primarily based on the cleavage of the WST-I tetrazolium salt by mitochondrial dehydrogenases in viable cells, not only it allows for the measurement of cell proliferation in response to growth factors or mitogens, but it also permits the assessment of growth inhibition by physiological mediators or inhibitory antibodies.
  • the WST-I cleavage product is water-soluble so it is a ready-to-use solution.
  • the APO-BrdU TUNEL Assay (Molecular Probes) can be used coupled to flow cytometry and/or fluorescence imaging.
  • fluorescent conjugates to annexin V, a phospholipid - binding protein can be used whereas late stage in apoptosis can be evaluated based on cytoskeletal collapse and cell membrane permeability to propidium iodide using flow cytometry and/or fluorescence microscopy.
  • Trans-endothelial migration assays can be performed in 6.5mm- diameter Transwell Plates (Beckton Dickinson). A monolayer of human vascular endothelial cells is grown on gelatin - coated filters (3 micron-pore size for lymphocytic leukemia cell lines and 8 micron-pore size for monocytic leukemia cell lines). A cocktail of chemotactic agents is added to the lower chamber to mediate migration of cells into the lower chamber. Migrating cells are then collected, stained with 0.1% crystal violet, and counted microscopically.
  • Phage internalization into leukemia cells Phage internalization assays were performed on K-562 cells (FIG. 15) to determine if the sequence insert in either one of the 3 Molt-4 phage clones is capable of receptor-mediated internalization. This would be indicative of whether the corresponding peptide motif has the potential to deliver drugs or apoptotic moieties into leukemia cells.
  • K562 cells mixed with either phage clone or Fd-tet control phage at 2 x 10 5 cells per 1 x 10 9 TU were incubated at 37°C overnight to permit for any potential receptor-mediated internalization to occur.
  • the cells were adhered to glass chamber slides pre-coated with poly-D lysine. Subsequently, the samples were fixed and permeabilized with pre-cooled methanol, blocked with 5% normal goat serum and stained with either an anti-fd phage antibody (SIGMA) or non ⁇ immune rabbit IgG (DAKO). FITC-conjugated goat anti-rabbit antibody (Jackson Labs) was used for secondary staining after which the samples were mounted with VECTASHIELD/DAPI. As shown in FIG.
  • the C-AYHRLRR-C motif insert in clone 1 mediates very strong internalization into K562 cells.
  • the C-GFYWLRS-C insert in clone 2 is also capable of internalizing.
  • the C- SFFYLRS-C sequence insert in clone 3 showed the strongest binding to leukemia cells in cell lines and patient samples, it did not exhibit any cytoplasmic staining, hence suggesting that it is incapable of internalizing.
  • Proteins mimicked by the peptide motifs and putative receptors approach Preliminary analysis of MOLT-4 binding peptides done by comparison of the selected motif sequences with available sequences in on-line protein databases suggests that a number of candidate proteins in particular known viral proteins share homologous sequences with these peptides (Table 3). Peptide motifs have been identified that home to tumors in mice (Pasqualini and Ruoslahti, 1996; Pasqulaini et al, 1997 and 2000).
  • a peptide may mimic a ligand of a receptor via a motif sufficient for receptor recognition.
  • peptide motifs corresponding to Molt-4 phage clone inserts were matched for similarity to known human proteins by searching in available online NCBI databases (ncbi.nlm.nih.gov/BLAST/). A minimum of tripeptide homology was used in the matching searches. Examples of candidate proteins (ligands) potentially mimicked by the leukemia- binding peptides and their corresponding putative receptors are listed in Table 3. Some of the proteins mimicked by the peptide motifs either play a role in cell differentiation and adhesion, or have growth factor, or signaling properties with known bio- functional relevance to cancer and leukemia.
  • the AYHRLRR motif insert in Molt-4 clone 1 mimics galectin -9, a lectin that binds to beta-galactoside and a potent eosinophil chemo-attractant derived from antigen-stimulated T-cells that also plays a role in myeloid differentiation and in cell adhesion.
  • Gal-9 over-expression was restricted to hematological, colorectal, and ovarian malignancies (Lahm et al, 2001).
  • T-cell lymphoma invasion and metastasis 1 protein a guanine nucleotide exchange factor that plays a role in Racl activation and src- induced transformation
  • TIAMl can directly bind to c-myc and can interfere with apoptosis of cells including leukemia cells (Van Leeuwen et al, 2002).
  • LPAl receptor G-protein coupled lysophosphatidic acid receptor
  • LTBP-2 latent transforming growth factor-beta-binding protein
  • PML tumor-suppressive promyelocytic leukemia protein
  • PML is delocalized from nuclear bodies in acute promyelocytic leukemia (APL) and is degraded in cells infected by several viruses.
  • SUMO-I can also modify E-26 transforming specific ETS-related gene product (ETV6), a transcription repressor that is rearranged in leukemias and congenital fibrosarcoma. It is known that some of the protein modifications mediated by SUMO-I could lead to abnormal cellular localization resulting in neoplastic transformation (Chakraborty et al, 2001).
  • GLM-R GP130-like monocyte receptor
  • Plexin Bl GP130-like monocyte receptor
  • GLM-R GP130-like monocyte receptor
  • LDM-R GP130-like monocyte receptor
  • Plexin Bl a novel family of trans-membrane receptors for semaphorins that play a role in axonal guidance, in immune regulation, and in tumori genesis.
  • Plexin- Al and Plexin- A2 form complexes with neuropilin- 1 (NRP-I) as well as with neuropilin-2 (NRP-2) and act as receptors for semaphorins 3A and 3F respectively.
  • NPP-I neuropilin- 1
  • NPP-2 neuropilin-2
  • semaphorins 3A and 3F semaphorins 3A and 3F respectively.
  • both neuropilins were found to function as co-receptors for VEGFi 65 and that as homo- or heterodimers, they can modulate its binding to VEGFR2 (KDR) and VEGFRl (Fltl).
  • VEGF family members such as the heparin binding from of placental growth factor (both), VEFG-B (only binds NRP-I), and VEGF-C (only binds NRP-2) (Zachary and Gliki, 2001; Neufeld et al, 2002a and 2002b; Nakarmura and Goshima, 2002; Miao et al, 2000; Kawakami et al, 2002; Bachelder et al, 2003).
  • placental growth factor both
  • VEFG-B only binds NRP-I
  • VEGF-C only binds NRP-2
  • NRP-I related proteins such as neuropillin-2 (NRP-2) and members of the semaphorin family were assessed.
  • NRP-2 neuropillin-2
  • clone 2 showed a 6.05 ⁇ 3.69 (average ⁇ SEM) - fold and 8.94 ⁇ 2.70 - fold stronger binding to recombinant NRP-2 and to recombinant semaphorin 3A (Sema3A) respectively.
  • the binding was specific since clone 2 showed no significant binding to Sema ⁇ A and to other unrelated recombinant proteins.
  • NRP-I and NRP-2 can form heterodimers and Sema3A is a natural ligand to NRP-I, the results indicate that clone 2 likely mimics a dimerization domain in NRP-I itself.
  • the effect of the pure cyclic form of clone2 peptide on proliferation and viability of leukemia cells in vitro was assessed.
  • a C-GFYWLRS-C-GG-KLAKLAKKLAKLAK-NH2 conjugated form of the peptide was also used to assess its anti-leukemia effect via delivery of the pro-apoptotic moiety.
  • the uncoupled peptide added in doses ranging from 20 ⁇ M up to 100 ⁇ M had no significant effect on viability of leukemia cells cultured at 8 x 10 4 cells/100 ⁇ l/ well in 96- well plates for 24hr.
  • the peptide slightly enhanced cell proliferation in some of the cell lines tested.
  • FIG. 19 represents the results from cytotoxicity assays performed on Molt-4, OCI- AML3, and K562 cell lines.
  • the large-scale screening approach allowed identification of leukemia binding ligands and extraction of useful biological information.
  • the C-GFYWLRS-C a leukemia- binding motif that utilizes neuropilin-1 as receptor, was shown to likely mimic a dimerization domain.
  • a pro-apoptotic peptide was targeted and internalized through this functional ligand-receptor pair.
  • Treatment of leukemia cells in vitro with a pro-apoptotic peptide guided by the C-GFYWLRS-C motif resulted in dose-dependent apoptosis in all 9 leukemia cell lines assessed. Together, these data illustrate the ability of phage-based screening systems for identification of relevant targets in the context of leukemia.
  • Inhibition assays will be performed to confirm the specificity of peptide binding. Additionally, alternative biochemical and genetic based approaches will be used to confirm that neuropilin-1 is the receptor for the GFYWLRS motif and to better map the interaction domain. For that purpose, GST-fusion constructs of Molt-4 clone 2 will be produced. Phage clones isolated from bio-panning on Molt-4 cells will be further evaluated for binding to human tissues in an overlay assay. Paraffin-embedded tissue sections from spleen and lymph node of human leukemia will be overlaid with leukemia-binding CGFYWLRSC-displaying phage. Phage will be detected by using an anti-M13 phage antibody.
  • Neuropilin-1 expression in similar tissue sections will be evaluated by conventional immunostaining with an anti-NRP-1 antibody.
  • Neuropilin-1 will be studied in regard to its elevated in leukemia patients through pathological and functional analyses to confirm that it is a potential target for intervention in leukemia.
  • work is underway to assess the cytotoxicity of C- GFYWLRS-C-GG-D(KLAKLAK)2 on imatinib (Gleevec) - resistant, Philadelphia chromosome positive, chronic myelogenous leukemia (CML) cell lines, namely KBM5-R and KBM7-R 41.
  • CML chronic myelogenous leukemia
  • the choice of the leukemia model will be based on several criteria: (i) the efficiency of engraftment of the leukemia cell line into Severe Combined Immune Deficient (SCID) mice, (ii) the data generated from in vitro cytotoxicity assays considering the most sensitive cell line, and (iii) the aggressiveness of the leukemia model.
  • SCID Severe Combined Immune Deficient
  • Several leukemia cell lines may initially be evaluated in pilot in vivo studies to determine optimal experimental conditions such as required number of cells to be injected for successful disease dissemination as well as optimal dose and number of injections of the control and test peptides.
  • cultured leukemia cells will be maintained in RPMI-1640 medium (GIBCO) supplemented with 25 mmol/L HEPES buffer, 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 2 mmol/L 1-glutamine. Cells will be suspended in PBS. Prior to IV injection of the leukemia cells, mice will be first irradiated using 200-250 Rad (cesium source) in order to enhance engraftment. A receptor targeting peptide conjugated to an apoptotic moiety will be evaluated for its anti-leukemia efficacy in combination with chemotherapy (AraC, Gleevec).
  • Peptide-based therapy and/or chemotherapy will be administered IP into tumor-bearing mice.
  • the number of treatment days per cycle as well as the number of successive cycles if necessary is as yet to be determined.
  • Experiments to monitor engraftment and disease progression will include measuring human CD45+ cells in blood samples collected from tail veins of control and test mice using flow cytometry. RBCs will be cleared by lysis. Additionally, the expression of human HLAs in various organs (bone marrow, spleen, liver, lungs, kidneys, and brain) will be evaluated by performing PCR on DNA extracted from these samples and by doing immunohistochemistry on human CD45+ expression in fixed tissue sections. During and following completion of therapeutic treatment, the animals will be monitored daily for survival and for any potential non-specific toxicity.
  • compositions, methods and apparatus disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it are apparent to those of skill in the art that variations maybe applied to the compositions, methods and apparatus and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it are apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Tur-Kaspa et al MoI. Cell Biol, 6:716-718, 1986. Van Leeuwen et al, J. Biol Chem., 278:400-406, 2002. Vehvilainen et al, J. Biol. Chem., 278:24705-24713, 2003. ⁇ ong et al, Gene, 10:87-94, 1980.

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Abstract

L'invention concerne des procédés et compositions supplémentaires pour la préparation et l'utilisation de peptides de ciblage sélectifs et/ou spécifiques à la leucémie. Dans certains modes de réalisation, l'invention concerne des peptides de ciblage particuliers sélectifs ou spécifiques à des cellules de la leucémie, notamment mais pas exclusivement les SEQ ID NO: 5, SEQ ID NO: 6, ou SEQ ID NO: 7. D'autres modes de réalisation de cette invention concernent de tels peptides de ciblage fixés à des agents thérapeutiques. Dans d'autres modes de réalisation encore, des peptides de ciblage de la leucémie ou autre peuvent être utilisés pour administrer de façon sélective ou spécifique des agents thérapeutiques à des cellules cibles telles que des cellules de la leucémie. Dans d'autres modes de réalisation enfin, les procédés concernent l'identification, la préparation et l'utilisation de peptides de ciblage sélectifs ou spécifiques à une cellule, un tissu ou un organe cibles donnés tels que des cellules de la leucémie.
PCT/US2005/024414 2004-07-10 2005-07-11 Compositions et procedes lies a des peptides se liant de facon selective avec des cellules de la leucemie WO2006010070A2 (fr)

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* Cited by examiner, † Cited by third party
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EP2002036A2 (fr) * 2006-03-09 2008-12-17 The Board of Regents of The University of Texas System Compositions et procédés concernant le profilage d'une pluralité de lignées cellulaires en fonction de leur liaison à des peptides
WO2009107971A3 (fr) * 2008-02-25 2009-11-26 경북대학교 산학협력단 Polypeptide spécifiquement couplé à la phosphatidylsérine et utilisation de celui-ci
WO2011031955A3 (fr) * 2009-09-11 2011-06-23 Mallinckrodt Inc. Surveillance optique de la leucémie
WO2011084571A2 (fr) 2009-12-16 2011-07-14 Mallinckrodt Inc. Dérivés azides pour photothérapie
US8313729B2 (en) 2007-03-01 2012-11-20 Medibeacon, LLC Integrated photoactive small molecules and uses thereof
US8350032B2 (en) 2007-07-31 2013-01-08 Medibeacon Development, Llc Integrated photoactive agents for real-time monitoring of hemostasis
WO2013038392A1 (fr) * 2011-09-18 2013-03-21 Ariel-University Research And Development Company, Ltd. Peptides capables de se lier aux cellules leucémiques b, conjugués, et compositions les contenant et leurs utilisations
US9068187B1 (en) 2010-02-09 2015-06-30 David Gordon Bermudes Protease inhibitor: protease sensitivity expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria
US9486513B1 (en) 2010-02-09 2016-11-08 David Gordon Bermudes Immunization and/or treatment of parasites and infectious agents by live bacteria
US9593339B1 (en) 2013-02-14 2017-03-14 David Gordon Bermudes Bacteria carrying bacteriophage and protease inhibitors for the treatment of disorders and methods of treatment
US9657085B1 (en) 2009-02-09 2017-05-23 David Gordon Bermudes Protease inhibitor: protease sensitive expression system and method improving the therapeutic activity and specificity of proteins and phage and phagemids delivered by bacteria
US9737592B1 (en) 2014-02-14 2017-08-22 David Gordon Bermudes Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment
WO2018085413A1 (fr) * 2016-11-01 2018-05-11 Northwestern University Tri couche par couche de disulfure de rhénium par ultracentrifugation en gradient de densité isopycnique haute densité
US10087451B2 (en) 2006-09-22 2018-10-02 Aviex Technologies Llc Live bacterial vectors for prophylaxis or treatment
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria

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US20030054375A1 (en) * 2000-01-31 2003-03-20 Human Genome Sciences, Inc. Nucleic acids, proteins, and antibodies

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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2002036A2 (fr) * 2006-03-09 2008-12-17 The Board of Regents of The University of Texas System Compositions et procédés concernant le profilage d'une pluralité de lignées cellulaires en fonction de leur liaison à des peptides
EP2002036A4 (fr) * 2006-03-09 2010-01-27 Univ Texas Compositions et procédés concernant le profilage d'une pluralité de lignées cellulaires en fonction de leur liaison à des peptides
US10087451B2 (en) 2006-09-22 2018-10-02 Aviex Technologies Llc Live bacterial vectors for prophylaxis or treatment
US8313729B2 (en) 2007-03-01 2012-11-20 Medibeacon, LLC Integrated photoactive small molecules and uses thereof
US8350032B2 (en) 2007-07-31 2013-01-08 Medibeacon Development, Llc Integrated photoactive agents for real-time monitoring of hemostasis
US8133971B2 (en) 2008-02-25 2012-03-13 Kyungpook National University Industry Academic Cooperation Foundation Polypeptide specifically bound to phosphatidylserine and the use thereof
KR100968839B1 (ko) 2008-02-25 2010-07-09 경북대학교 산학협력단 포스파티딜세린과 특이적으로 결합하는 폴리펩티드 및 이의용도
WO2009107971A3 (fr) * 2008-02-25 2009-11-26 경북대학교 산학협력단 Polypeptide spécifiquement couplé à la phosphatidylsérine et utilisation de celui-ci
US9657085B1 (en) 2009-02-09 2017-05-23 David Gordon Bermudes Protease inhibitor: protease sensitive expression system and method improving the therapeutic activity and specificity of proteins and phage and phagemids delivered by bacteria
US11485773B1 (en) 2009-02-09 2022-11-01 David Gordon Bermudes Protease inhibitor:protease sensitive expression system and method improving the therapeutic activity and specificity of proteins and phage and phagemids delivered by bacteria
US10590185B1 (en) 2009-02-09 2020-03-17 David Gordon Bermudes Protease inhibitor: protease sensitive expression system and method improving the therapeutic activity and specificity of proteins and phage and phagemids delivered by bacteria
WO2011031955A3 (fr) * 2009-09-11 2011-06-23 Mallinckrodt Inc. Surveillance optique de la leucémie
WO2011084571A2 (fr) 2009-12-16 2011-07-14 Mallinckrodt Inc. Dérivés azides pour photothérapie
US10364435B1 (en) 2010-02-09 2019-07-30 David Gordon Bermudes Immunization and/or treatment of parasites and infectious agents by live bacteria
US9878023B1 (en) 2010-02-09 2018-01-30 David Gordon Bermudes Protease inhibitor: protease sensitive expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria
US9486513B1 (en) 2010-02-09 2016-11-08 David Gordon Bermudes Immunization and/or treatment of parasites and infectious agents by live bacteria
US10954521B1 (en) 2010-02-09 2021-03-23 David Gordon Bermudes Immunization and/or treatment of parasites and infectious agents by live bacteria
US9068187B1 (en) 2010-02-09 2015-06-30 David Gordon Bermudes Protease inhibitor: protease sensitivity expression system composition and methods improving the therapeutic activity and specificity of proteins delivered by bacteria
US11219671B1 (en) 2010-02-09 2022-01-11 David Gordon Bermudes Protease inhibitor:protease sensitive expression system, composition and methods for improving the therapeutic activity and specificity of proteins delivered by bacteria
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
WO2013038392A1 (fr) * 2011-09-18 2013-03-21 Ariel-University Research And Development Company, Ltd. Peptides capables de se lier aux cellules leucémiques b, conjugués, et compositions les contenant et leurs utilisations
US9593339B1 (en) 2013-02-14 2017-03-14 David Gordon Bermudes Bacteria carrying bacteriophage and protease inhibitors for the treatment of disorders and methods of treatment
US11827890B1 (en) 2013-02-14 2023-11-28 David Gordon Bermudes Bacteria carrying bacteriophage and protease inhibitors for the treatment of disorders and methods of treatment
US10501746B1 (en) 2013-02-14 2019-12-10 David Gordon Bermudes Bacteria carrying bacteriophage and protease inhibitors for the treatment of disorders and methods of treatment
US9737592B1 (en) 2014-02-14 2017-08-22 David Gordon Bermudes Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment
US10828350B1 (en) 2014-02-14 2020-11-10 David Gordon Bermudes Topical and orally administered protease inhibitors and bacterial vectors for the treatment of disorders and methods of treatment
US10702803B2 (en) 2016-11-01 2020-07-07 Northwestern University Layer-by-layer sorting of rhenium disulfide via high-density isopycnic density gradient ultracentrifugation
WO2018085413A1 (fr) * 2016-11-01 2018-05-11 Northwestern University Tri couche par couche de disulfure de rhénium par ultracentrifugation en gradient de densité isopycnique haute densité
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria

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