US20080213319A1 - Chemorepulsion of cells - Google Patents

Chemorepulsion of cells Download PDF

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US20080213319A1
US20080213319A1 US11/867,290 US86729007A US2008213319A1 US 20080213319 A1 US20080213319 A1 US 20080213319A1 US 86729007 A US86729007 A US 86729007A US 2008213319 A1 US2008213319 A1 US 2008213319A1
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cells
chemorepellent
neutrophils
validated
cell
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Hyun Kang
Scott Sacane
Jonathan L. Moon
Erica B. Goodhew
Lopa Bhatt
Stacey L. Rose
Milton H. Werner
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Celltaxis LLC
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Celtaxsys Inc
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Assigned to CELTAXSYS, INC. reassignment CELTAXSYS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KANG, HYUN, SACANE, SCOTT, BHATT, LOPA, GOODHEW, ERICA B., MOON, JONATHAN L., ROSE, STACEY L., WERNER, MILTON H.
Publication of US20080213319A1 publication Critical patent/US20080213319A1/en
Priority to US12/698,732 priority patent/US9617330B2/en
Priority to US15/480,884 priority patent/US20180022793A1/en
Assigned to CELLTAXIS, LLC reassignment CELLTAXIS, LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CELTAXSYS, INC.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/739—Lipopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1719—Muscle proteins, e.g. myosin or actin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1732—Lectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Chemotaxis or the oriented movement of a cell with reference to a chemical agent is a complex and highly integrated process.
  • the movement may be positive (toward) or negative (away) with respect to the chemical gradient.
  • this process in turn mediates, among many things, tissue organization, organogenesis and homeostasis and ultimately orchestrates embryonic morphogenesis; contributes to tissue repair and regeneration; and drives disease progression in cancer, mental retardation, atherosclerosis, and arthritis.
  • the migrating cell is highly polarized with complex regulatory pathways that spatially and temporally integrate its component processes.
  • Chemotaxis occurs in both prokaryotes and eukaryotes. In all cases, movement toward an agent or stimulus is termed positive chemotaxis (i.e., the agent or stimulus is chemoattractive for the cell), while movement away from an agent or stimulus is termed negative chemotaxis (i.e., the agent or stimulus is chemorepulsive for the cell). Chemoattraction (CA) and chemorepulsion (CR) are therefore properties of the agent or stimulus, while chemotaxis is a property of cells.
  • positive chemotaxis i.e., the agent or stimulus is chemoattractive for the cell
  • negative chemotaxis i.e., the agent or stimulus is chemorepulsive for the cell.
  • Chemoattraction (CA) and chemorepulsion (CR) are therefore properties of the agent or stimulus, while chemotaxis is a property of cells.
  • Chemotaxis is known to occur for several types of eukaryotic cells. Within the immune system, chemotaxis is often driven by a class of biological agents, known as chemokines (or chemotactic cytokine).
  • chemokines or chemotactic cytokine
  • Chemotaxis and the related phenomenon, chemokinesis (the enhancement of random cellular movement in response to a chemical or biological agent, irrespective of agent concentration), have been examined in subpopulations of mammalian cells. Chemorepulsion, however, has rarely been described as a physiological phenomenon.
  • the chemokine SDF-1 (a.k.a. SDF-1 ⁇ , CXCL12) has been described as a chemorepellent in the context of a two-dimensional transmigration apparatus and at a concentration 10-fold above that necessary for the induction of chemoattraction in the same device.
  • the present invention provides methods of controlling the direction and/or movement of migratory cells, or cell migration.
  • chemorepellents including non-chemokines, which have not previously been identified in the art as chemorepellents.
  • a method for identifying cells which secrete a chemorepulsive agent is provided.
  • the invention is directed to the identification of unimodal fugetaxins which alter or affect the movement of primary cells involved in immune, inflammatory or cancerous phenotypes.
  • a method of modulating migration of immune cells is provided. Modulation may be toward or away from a particular location or site in an animal or subject.
  • a high throughput transmigration assay method of screening for chemorepellent agents is provided. This method generally involves contacting an agent suspected of being a chemorepellent with a cell capable of induced migration, measuring the movement of the cell, wherein movement of the cell away from the agent is indicative of chemorepulsion.
  • a method of inhibiting endothelial cell migration to a tumor site in a subject involves locally administering to an area surrounding a tumor site in need of such treatment a validated chemorepellent in an amount effective to inhibit endothelial cell migration to the tumor site in the subject.
  • the area surrounding the tumor site is not immediate to the tumor site.
  • a method of inhibiting tumor cell metastasis in a subject involves contacting or administering, either locally or systemically, to a tumor site in a subject in need of such treatment a validated chemorepellent agonist or antagonist agent in an amount effective to inhibit metastasis of tumor cells from the tumor site in the subject.
  • FIGS. 1A-C are bar graphs of the chemotactic response of neutrophils and lymphocytes to galectin-3.
  • FIGS. 2A-C are bar graphs showing the inhibition of chemotactic response of neutrophils and lymphocytes to galectin-3 using galectin-3 antibody.
  • FIG. 3 is a bar graph comparing chemotactic response of neutrophils to varying concentrations (in ⁇ g/ml) of SDF-1, IL-8, galectin-3, and LTA from Bacillus subtilis .
  • the first four bars for each agent indicate positive chemotaxis while the second four indicate negative chemotaxis.
  • FIG. 4 is a bar graph comparing positive and negative chemotactic response of neutrophils to varying concentrations (in ⁇ g/ml) of LPS, HSP-40, and HSP-65.
  • the first four bars for each agent indicate positive chemotaxis while the second four indicate negative chemotaxis.
  • FIG. 5 High throughput screening apparatus for analysis of cell migration.
  • FIG. 6 Well configuration for analysis of negative chemotaxis.
  • the present invention involves the unexpected discovery of agents which can act as chemorepellents, most of which are not related to chemokines or other proteins whose actions are mediated by GPCRs.
  • the newly-discovered chemorepellents also include agents that are of chemokine origin and yet not previously recognized as chemorepellents.
  • applicants have further identified several unimodal fugetaxins.
  • the object of the present invention is to identify and/or isolate agents which induce, elicit or trigger human migratory cells to move in a desired direction.
  • desired direction is meant in any direction whether away from or toward a site or location, whereby the movement is therapeutically relevant as contributing to the improving, lessening, ameliorating, preventing, treating or mitigating a disease state or condition.
  • the migratory cells of most interest include those involved in the process of cancer, immunity or inflammation but may include those identified to play a role in any disease state or condition.
  • a “chemorepellent” is an agent or stimulus which induces, elicits or triggers negative chemotaxis (movement away from an agent or stimulus) in a migratory eukaryotic cell.
  • the terms “induce”, “elicit” and “trigger,” when referring to the chemorepulsive activity of a chemorepellent, carry the same meaning. Chemorepellents are said to effect “chemorepulsion” or to have “chemorepulsive activity.”
  • a “validated chemorepellent” is an agent which exhibits a repellent index equal to or greater than 1.2 over at least a 10-fold change in concentration.
  • the “Repellent Index” is a measure of the ability of an agent to induce chemorepulsion of a cell relative to any spontaneous movement the cell might exhibit.
  • An agent's repellent index is the ratio of chemorepulsion (CR) to spontaneous migration (SM), i.e., [(CR)/(SM)].
  • Spontaneous migration is the amount of migration which occurs in the absence of an added agent.
  • agent refers generally to any compound. Such compounds in the context of the present invention may be those having suspected chemorepulsive activity or may be a known compound such as a drug or other therapeutic substance. Agents may also be agonists or antagonists of chemorepellents, validated chemorepellents or validated conditioned chemorepellents.
  • the term “stimulus” refers to any physical or chemical cue that provokes or evokes a response in a system. As such, an agent may also act as a stimulus.
  • chemorepellents identified using the methods of the present invention may upon contact with a migratory cell act to spatially or temporally modulate cell migration.
  • contact or “contacting” means the act of touching or bringing together two entities or things in such proximity as will allow an influence of at least one on the other.
  • the definition, while inclusive of physical contact is not so limited.
  • spatialally modulating cell migration refers to altering the movement of a cell or population of like cells in reference to a certain locality in a subject, especially in reference to the location of a cell or cells in a subject in relation to a the location of the same cell or cells in the subject after the movement.
  • the phrase “temporally modulating cell migration” refers to altering the movement of a cell or population of like cells over the passage of time, especially in reference to the rate a cell or cells are induced to move.
  • the agents identified and/or validated as chemorepellents according to the present invention may act to effect chemorepulsion by any pathway or mechanism.
  • the agents may bind to a surface receptor.
  • Surface receptors include but are not limited to G-protein coupled receptors; Toll-like receptors, cytokine and chemokine receptors, T-cell receptors, neuropilin receptors, tumor necrosis factor receptors, growth factor receptors, ion channels, ion pumps and porins.
  • the agents may be internalized once bound or may remain on the surface of the cell.
  • the agents may interact with a substance or biologic factor in the intercellular milieu, such as second messengers or soluble ligands, which then go on to translate the chemorepellent effects to the cell whose movement is being affected.
  • a substance or biologic factor in the intercellular milieu such as second messengers or soluble ligands
  • Chemorepulsive activity can be detected using any of the transmigration systems, including the high throughput transmigration assay described herein (see Examples). It is also possible to use a variety of other systems well known in the art (e.g. U.S. Pat. No. 5,514,555, “Assays and therapeutic methods based on lymphocyte chemoattractants”).
  • Chemorepellants may be identified from a variety of cells, including cultured homogeneous and heterogeneous cell populations which in turn may be derived from a variety of sources. Cells can be of any type or origin. Chemorepellents may be isolated from cells of healthy or diseased tissue. As used herein “diseased tissues or organs” include those tissue or organs which have or are suspected of having an impairment of health or a condition of abnormal functioning. Chemorepellents may also be isolated from or used to treat conditions associated with an immune privileged tissue.
  • An “immune-privileged” tissue is a tissue with immune-cell tolerance and includes, but is not limited to, brain tissue, central nervous system tissue, testes, eyes or placenta.
  • the chemorepellents can also be isolated from one or more biological fluids.
  • Biological fluids include, but are not limited to, synovial fluid, cerebrospinal fluid, fallopian tube fluid, seminal fluid, ocular fluid, pericardial fluid, pleural fluid, inflammatory exudates, eluates, lysates and ascitic fluid. It is also appreciated that any migratory cell as disclosed herein may also be the source of chemorepellents.
  • моду ⁇ ии cells are those cells which are capable of movement from one place to another in response to a stimulus.
  • Preferred types of cells whose migration is to be mediated by the identified and/or validated chemorepellents of the present invention include, but are not limited to immune cells including, monocytes, Natural Killer (NK) cells, dendritic cells (which could be immature or mature), subsets of dendritic cells including myeloid, plasmacytoid (also called lymphoid) or langerhans; macrophages such as histiocytes, Kupffer's cells, alveolar macrophages or peritoneal macrophages; neutrophils, eosinphils, mast cells, basophils; B cells including plasma B cells, memory B cells, B-1 cells, B-2 cells; CD45RO (naive T), CD45RA (memory T); CD4 Helper T Cells including Th1, Th2 and Tr1/Th3; CD8 Cytotoxic T Cells, Regulatory T Cell
  • Cells may readily be derived from a number of appropriate organs or tissues including, but not limited to, skin, liver, pancreas, fat, bone marrow, lymph node, thymus, kidney, colon, and brain.
  • Cells may be of “hematopoietic origin” and include, but are not limited to, pluripotent stem cells, multipotent progenitor cells and/or progenitor cells committed to specific hematopoietic lineages.
  • the progenitor cells committed to specific hematopoietic lineages may be of T cell lineage, B cell lineage, dendritic cell lineage, neutrophil lineage, Langerhans cell lineage and/or lymphoid tissue-specific macrophage cell lineage.
  • the hematopoietic cells may be derived from a tissue such as bone marrow, peripheral blood (including mobilized peripheral blood), umbilical cord blood, placental blood, fetal liver, embryonic cells (including embryonic stem cells), aortal-gonadal-mesonephros derived cells, and lymphoid soft tissue. Lymphoid soft tissue includes the thymus, spleen, liver, lymph node, skin, tonsil and Peyer's patches.
  • the “hematopoietic origin” cells may be derived from in vitro cultures of any of the foregoing cells, and in particular in vitro cultures of progenitor cells.
  • Cells of neural origin include neurons and glia, and/or cells of both central and peripheral nervous tissue.
  • Cells of epithelial origin include cells of a tissue that covers and lines the free surfaces of the body.
  • Such epithelial tissue includes cells of the skin and sensory organs, as well as the specialized cells lining the blood vessels, gastrointestinal tract, air passages, lungs, ducts of the kidneys and endocrine organs.
  • Cells of mesenchymal origin include cells that express typical fibroblast markers such as collagen, vimentin and fibronectin.
  • Cells involved in angiogenesis are cells that are involved in blood vessel formation and include cells of endothelial origin and cells of mesenchymal origin.
  • a germ cell is a cell specialized to produce haploid gametes. It is a cell further differentiated than a stem cell, which can still give rise to more differentiated germ-line cells.
  • a method is provided to identify chemorepellents which are isolated from a serum-free conditioned media.
  • Conditioned media include, but are not limited to, any serum-free growth medium in which a cell that secretes a chemorepellent has been grown or maintained for at least 24 hours; an ascites fluid collected from a patient with cancer; a lysate of cells in which a chemorepellent has been expressed or synthesized or other biological fluid.
  • Biological fluids include, but are not limited to, synovial fluid, cerebral spinal fluid, fallopian tube fluid, ocular fluid, pericardial fluid, pleural fluid or inflammatory exudates.
  • the chemorepellent can be isolated from a non-homogeneous solution derived from a cell culture supernatant, also known as a cell culture conditioned media.
  • a cell culture supernatant also known as a cell culture conditioned media.
  • a conditioned medium can be contained in culture flasks, plates and dishes.
  • culture vessels such as fermenters can be used. Culturing in three-dimensional porous matrices can also be used.
  • the chemorepellent present in the conditioned medium can be removed centrifugation of the cell culture to remove the cells and aspiration to recover the solution in which the cells were bathed.
  • the cultures can also be filtered to remove cells and cell debris.
  • the chemorepellent in the conditioned medium can be fractionated according to standard chromatographic procedures to facilitate isolation of the chemorepellent.
  • standard chromatographic procedures include, but are not limited to, size exclusion chromatography, FPLC, HPLC, ion-exchange chromatography, hydrophobic chromatography, immune-affinity chromatography, etc.
  • the fractions of conditioned media are then used to repel immune cells.
  • a method for identifying cells which secrete a chemorepulsive agent involves preparing a culture of mammalian cells derived from tumors or other diseased state or a culture of bacterial, viral or fungal cells and isolating a supernatant suspected of containing a chemorepellent agent from the culture, fractionating the supernatant into a plurality of fractions with chemorepulsion activity and measuring the movement of cells away from a fraction.
  • the plurality of fractions may be undiluted or concentrated.
  • the cell secretions may contain one or more putative chemorepellents and may be a mixture, aliquot or pooled fraction therefrom.
  • the agent isolated from a positive fraction can be further characterized by subjecting the fraction to protein sequencing according to standard methods or mass spectrometry coupled with chemical or electrochemical fragmentation. Any chemical information so obtained can be screened with databases for homology to existing proteins, polypeptides, carbohydrates, lipids, or combinations thereof.
  • a positive fraction can be used to generate antibodies, which recognize the chemorepellent. Such antibodies can then be used in expression cloning protocols, Western blots, and other techniques useful in the isolation of chemorepellents from a conditioned medium.
  • the chemorepellent is present in a tumor cell culture supernatant, tumor cell eluate and/or tumor cell lysate.
  • the tumor cell may be of a cancer or tumor type that is thought to escape immune recognition.
  • Such cancers can be of the following origin: biliary tract cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer, gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemia; multiple myeloma; AIDS associated leukemias and adult T-cell leukemia lymphoma; intraepithelial neoplasms, including Bowen's disease and Paget's disease; liver cancer (hepatocarcinoma); lung cancer; lymphomas, including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer, including squamous cell carcinoma; ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreas cancer; prostate cancer; rectal cancer; sarcoma
  • the chemorepellent is secreted or displayed by a microorganism.
  • microorganism it is meant a gram positive or gram negative bacterium, a fungus such as yeast or mold, viruses, whether DNA- or RNA-based.
  • high density it is meant a 96 or 384-well apparatus containing an upper chamber, a polycarbonate or polyester planar membrane and a lower chamber (see Example 9). Briefly, the cell suspension is placed in the upper chamber, on top of the planar membrane and cells induced to migrate by addition of the chemorepellent at varying concentrations to the upper well of the transmigration apparatus (see FIG. 5-6 ).
  • the transmigration apparatus is disassembled, a cell lysis and chemiluminescence agent added to the lower (i.e. migration) chamber and the liquid in the lower plate is recovered either by aspiration and delivery into a white 96- or 384-well plate suitable for luminometry or by centrifugation.
  • a cell lysis and chemiluminescence agent added to the lower (i.e. migration) chamber and the liquid in the lower plate is recovered either by aspiration and delivery into a white 96- or 384-well plate suitable for luminometry or by centrifugation.
  • the plate of lower chambers fitted with a funnel device for centrifugal transfer of all liquid solutions to a white reading plate suitable for measurement of luminescence.
  • the white reading plate is shaken orbitally for 10 minutes in the dark, loaded into an automated plate loader and read, well-by-well, in a device suitable for measurement of luminescence.
  • cells in the migration plate may be stained with a fluorescent marker, transferred by aspiration or centrifugation to a black reading plate and the fluorescence read well-by-well in a device suitable for measuring fluorescence. Migration of cells into any well is compared to the migration of cells that have not been exposed to the tested or putative chemorepellent and a repellent index is calculated.
  • the “Repellent Index” is a measure of the ability of an agent to induce chemorepulsion of a cell relative to any spontaneous movement of the cell itself. Spontaneous migration is measured without any agent added, while chemokinesis refers to migration in the presence of an agent, where the agent is at equal concentration above, and below, the membrane on which the cells are placed. It is noted that the present apparatus is unable to measure chemokinesis since the device is vertical and the cells detach from the membrane when they squeeze through the membrane pores. As such, cells can't reach a equilibrium and chemokinesis can't be measured.
  • An agent's repellent index is the ratio of chemorepulsion (CR) to spontaneous migration (SM) induced by the agent, i.e., [(CR)/(SM)].
  • a “validated chemorepellent” is an agent which exhibits a repellent index equal to or greater than 1.2 over at least a 10-fold change in concentration. For example, in calculating a RI for an agent, migratory responses including chemorepulsion (CR) and spontaneous migration (SM) may be measured over several decades or log scales of concentration. If, over any two consecutive measurements at concentrations which differ by at least 10 fold, the ratio of CR to SM at each of the two contiguous measurements is equal to or greater than 1.2, then the agent is considered a validated chemorepellent.
  • MI The “Migration Index”
  • MI is a measure of the ability of a mixture of putative chemorepellent agents, either secretions from cultured mammalian cells or collected fractions therefrom, to induce chemorepulsion of a cell relative to any spontaneous movement the mixture of putative chemorepellent agents might induce in the cell.
  • a mixture's migration index is computed as the ratio of chemorepulsion (CR) observed for the mixture of putative chemorepellent agents relative to the ability of a serum-free medium itself to induce chemorepulsion (i.e., CR is normalized to spontaneous migration (SM)) induced by the incubation medium in which the cells reside) and is represented as [(CR)/(SM)].
  • mixture when referring to studies involving the conditioned media assay and identification of chemorepellents from a system, includes solutions, cell supernatants, isolates, tissue exudates, non-homogeneous solutions, and fractions, aliquots or subportions thereof.
  • a chemorepellent Once a chemorepellent is identified, it may be validated using the techniques described above. It is further contemplated that in some situations, using the chemorepellent itself as a therapeutic composition may not be optimal. For example, if a very large protein is identified as a chemorepellent, it would not be feasible to develop that entity as a therapeutic. It would however be advantageous to identify regions, epitopes or fragments of that protein which elicit the migratory response and then to design agonists or antagonists of the chemorepellent agent.
  • agonists and antagonists may then be employed in pharmaceutical applications or as pharmaceutical compositions which may be administered to a subject to ameliorate, prevent, treat or cure disease indications or conditions associated with, caused by or which have as a component of their etiology the aberrant control or regulation of cell migration.
  • ameliorate means to make a situation better or more tolerable.
  • the present invention embraces the screening of tissues, cells and biological samples for the secretion, expression or presentation of agents which act as chemorepellents.
  • Chemorepellent agents identified may be polypeptides, nucleic acid based, may be modified with any posttranslational moieties including but not limited to carbohydrates, lipids and the like. Agonists and antagonists do not have to be of the same type of molecule as the identified or validated chemorepellent.
  • an antagonist of a polypeptide may be a small molecule, antibody, aptamer, adnectin, or any agent which antagonizes the chemorepellent activity of the agent identified.
  • Chemorepellent agents may be hybrid molecules such as glycoproteins, peptidoglycans, or protein-nucleic acid conjugates. They may be modified by conjugation of delivery moieties, stabilizing moieties or any other moiety which improves or facilitates the pharmaceutical or diagnostic properties of the composition.
  • the present invention also makes it possible to isolate proteins, lipids or carbohydrates that bind the chemorepellent, including cell surface receptors.
  • the proteins, carbohydrates and lipids that bind the chemorepellent can be used, for example, in screening assays to detect the presence, or absence, of the repellent in cell- or medium-derived mixtures.
  • the binding proteins, carbohydrates or lipids can also be used to block the effect of the isolated chemorepellent.
  • the invention therefore embraces any binding agent, which can, for example, be an antibody or fragment thereof which has the ability to selectively bind the chemorepellent.
  • tissue injury-induced inflammation ischemia-reperfusion injury
  • crystal-induced inflammation complement-induced inflammation and oxidative stress
  • hemodialysis immune complex-induced inflammation
  • cytokine-induced inflammation rheumatoid arthritis
  • antineutrophil cytoplasmic antibodies and vascullitis autoimmunity against neutrophil components
  • genetic disorders of neutrophil regulations implant related inflammation
  • cystic fibrosis
  • the invention provides a method for inhibiting migration of immune cells to a site of inflammation.
  • “Inflammation” refers to a localized protective response elicited by a foreign (non-self) antigen and/or by injury or destruction of tissue(s), which serves to destroy the foreign antigen, the injurious agent and/or the injured tissue.
  • An “inflammatory response” is that response by the human body to an inflammatory insult.
  • an “inflammatory insult” is any event which triggers inflammation. Inflammation occurs when tissues are injured by viruses, bacteria, trauma, chemicals, heat, cold or any other harmful stimulus.
  • immune cells such as T cells, B cells or macrophages interface with cells and soluble products that mediate the inflammatory response (e.g. neutrophils, basophils, eosinophils, kinin and coagulation systems and complement cascades).
  • Inflammation may also be caused by a self-antigen and the subject in need of treatment has an autoimmune disease.
  • Autoimmune disease results when a subject's immune system attacks its own organs or tissues, producing a clinical condition associated with the destruction of that tissue, exemplified by diseases such as rheumatoid arthritis, uveitis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, Crohn's disease, Guillain-Barre syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, systemic lupus erythrematosus, etc.
  • an “immune response” means a physiological response in humans and higher animals to defend the body against the introduction of foreign material. Inhibition of immune cell migration to a select site or area of inflammation in any of the foregoing conditions is beneficial, for it inhibits the escalation of the inflammatory response, protecting the specific site involved from ‘self damage’.
  • a method of inhibiting endothelial cell migration to a tumor site in a subject involves locally administering to or contacting an area surrounding a tumor site in need of such treatment a validated chemorepellent in an amount effective to inhibit endothelial cell migration into the tumor site in the subject.
  • Validated chemorepellent antagonists may also be administered to or be contacted with the tumor site or location.
  • the area surrounding the tumor site is not immediate to the tumor site.
  • a method of inhibiting tumor cell metastasis in a subject involves contacting or administering, either locally or systemically, to a tumor site in a subject in need of such treatment a validated chemorepellent, mimic, agonist or antagonist agent in an amount effective to inhibit metastasis of tumor cells from the tumor site in the subject.
  • a method of enhancing an immune response in a subject having a condition that involves a selected site involves administering, either locally or systemically or contacting a selected site in a subject in need of such a treatment a chemorepellent antagonist in an amount effective to inhibit immune cell-specific chemorepulsion in the subject.
  • a method of contraception involves administering an antagonist of a validated chemopellent in an amount effective to inhibit migration of germ cells in the subject.
  • a method of treating infertililty and premature labor involves administering a validated chemorepellent in an amount effective to inhibit immune cells from migrating close to a germ cell in the subject.
  • the administration is local to a germ cell-containing site of the subject.
  • any of the agents, compounds or pharmaceutical compositions of the present invention may be used for the treatment of conditions characterized by a need to modulate the direction of cell migration to or from specific sites, locations, tissues, organs, organ transplants or grafts in a subject or patient.
  • compositions of the present invention may also direct, trigger, induce or elicit the movement of cells away from a non-biologic such as a transplant, implant, area or site of microbial infiltration, or device not of biologic origin (material surface) such as those used in orthopedic applications like pins, screws and the like or those used for example in cardiovascular applications such as pacemakers, valves, stents, and the like or dental appliances.
  • a non-biologic such as a transplant, implant, area or site of microbial infiltration, or device not of biologic origin (material surface) such as those used in orthopedic applications like pins, screws and the like or those used for example in cardiovascular applications such as pacemakers, valves, stents, and the like or dental appliances.
  • the validated chemorepellent or a mimic or agonist coats the material surface with an amount effective to repel immune cells.
  • the material could be coated with a cell-growth potentiating agent, an anti-infective agent and/or an anti-inflammatory agent.
  • compositions and methods of the present invention will be useful in any situation where it is desired to prevent, reduce or eliminate the aggregation of cells, cellular secretions or infiltrates which may interfere with normal body function or healing.
  • it will be possible to modulate the chemorepulsion of cells by employing antagonists of such chemorepellents, thereby allowing the movement of cells to progress where they otherwise would have been repelled.
  • the present invention provides cardiovascular stents coated, impregnated or infused with one or more validated chemorepellents.
  • a coated stent can result in high cellular uptake of the drug and thus low viability of vascular smooth muscle cells (VSMC), thus attaining better effects in preventing restenosis compared to cardiovascular stents of the prior art.
  • VSMC vascular smooth muscle cells
  • a method of inhibiting migration of immune cells to a selected site in a subject involves administering, either locally or systemically, to a selected site in a subject in need of such a treatment a chemorepulsive agent in an amount effective to elicit, trigger, enhance, induce chemorepulsion of immune cells from the site.
  • chemorepulsive agents include chemorepellents, validated chemorepellents, mimics of chemorepellents or validated chemorepellents, and agonists of chemorepellents, and the like.
  • an agonist of the chemorepellent may be administered for increased effect.
  • Such agonists may be preferred over the chemorepellent because of certain pharmacokinetic or pharmacodynamic properties.
  • the chemorepellent effective dose may be calculated based on binding constants. It is expected that the agents of the present invention would exhibit binding constants of between 1-10 nM, preferably between 3-7 nM and more preferably between 4-5 nM for a target. Binding constants may further be approximately 5 nM for the target.
  • the chemorepellents and pharmaceutical compositions comprising chemorepellents may be co-administered with a second agent (e.g., another chemorepellent or with any drug or agent which is not itself a chemorepellent).
  • the co-administered agents may act cooperatively, additively, or synergistically.
  • Co-administered agents, compounds, chemorepellents or therapeutics need not be administered at exactly the same time.
  • a chemorepellent agent is administered substantially simultaneously. By “substantially simultaneously,” it is meant that the chemorepellent agent is administered before, at the same time, and/or after the administration of the second agent.
  • Chemorepellent agents, their antagonists or agonists, and pharmaceutical compositions thereof may be co-administered with any second agent including anti-inflammatory agents, anti-cancer agents, anti-infective agents, immune therapeutics (immunosuppresants) or any therapeutic compound.
  • An anti-infective agent is an agent which reduces the activity of or kills a microorganism and includes: Aztreonam; Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; Pirazmonam Sodium; Propionic Acid; Pyrithione Sodium; Sanguinarium Chloride; Tigemonam Dicholine; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocill
  • Anti-cancer agents include Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Cris
  • Immunosuppressants include Azathioprine; Azathioprine Sodium; Cyclosporine; Daltroban; Gusperimus Trihydrochloride; Sirolimus; Tacrolimus.
  • Anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium;
  • a subject is an animal.
  • Animals include, but are not limited to, all primates (human and non-human), cows, horses, pigs, sheep goats, dogs, cats, and any other animal which may benefit in any way from receiving as a treatment or therapy the compounds, agents or compositions of the present invention.
  • a subject may also be a patient in need of treatment or prophylaxis.
  • the present invention provides a method for the control of the movement of such cells by contacting these cells with unimodal fugetactic ligands unimodal fugetaxins, their agonists or antagonists.
  • unimodal fugetaxin is galectin-3.
  • a “fugetaxin” is an agent which elicits fugetaxis in a cell or group of cells. “Fugetaxis” is defined as the active movement of a cell away from a chemokinetic agent. A “chemokinetic agent” being any agent or stimulus which elicits, triggers or causes the movement of cells in any direction. In this sense, a fugetaxin is analogous to (i.e., the same as) a chemorepellent.
  • a “unimodal fugetaxin” is a compound which (1) elicits a fugetactic response which is comparable to or greater than the chemotactic (chemoattractant) response elicited by the compound at any given dose; and (2) where the fugetactic response elicited by the compound exceeds the chemotactic response elicited by the compound at a fugetactic effective dose.
  • a “fugetactic effective dose” is that dose of a fugetaxin which causes, induces or triggers the movement of a cell away from the fugetaxin.
  • unimodal fugetaxins and methods of the present invention may be developed to treat such as inflammation and diseases or conditions associated with autoimmune responses and various cancers.
  • the compounds and methods of the present invention may also be used to treat various cancers by controlling leukocytes and acting as novel types of chemorepellent molecules (i.e., unimodal fugetaxins) in various cell types.
  • chemorepellent molecules i.e., unimodal fugetaxins
  • the fugetactic response elicited by the unimodal fugetaxin occurs with rapid onset.
  • rapid-onset is defined as the production of a measurable fugetactic or chemotactic response within or at approximately one hour after treatment. For certain cell types, rapid-onset may occur at 2, 3, or 4 hours but always by five hours post treatment.
  • the fugetactic effective dose is at least 10 uM.
  • the migratory cell is a neutrophil. It is contemplated that the fugetactic effective dose may occur at least within the ranges of 0.01 uM-0.1 uM, 0.1 uM-15 uM, 1 uM-15 uM, 2 uM-10 uM, 5 uM-8 uM or preferably less than 0.1 uM.
  • a method of altering the migration of immune, inflammatory or cancerous cells involves administering or contacting a subject in need of such treatment with a unimodal fugetaxin at a fugetactic effective dose to alter the migration of immune, inflammatory or cancer cells in the subject.
  • Administration may be by any route, local, systemic or oral.
  • the migration of immune, inflammatory or cancerous cells may be altered by providing to a subject or contacting a subject in need of such treatment with an antagonist of a unimodal fugetaxin.
  • an antagonist of a unimodal fugetaxin In this embodiment, the activity of the unimodal fugetaxin would be attenuated and thereby inhibit the fugetaxis of the immune, inflammatory or cancer cell.
  • the unimodal fugetaxin antagonist is a galectin-3 antagonist.
  • unimodal fugetaxin antagonists may comprise small molecules, antisense compounds of either RNA or DNA, siRNA, miRNA, aptamers, antibodies, peptides or peptide fragments, any of which act to interfere, abrogate, attenuate, alter or otherwise inhibit the activity of the unimodal fugetaxin on an immune, inflammatory or cancer cell.
  • the method further comprises co-administering with a unimodal fugetaxin, a fugetactic agent, a non-fugetactic agent or other therapeutic compound to the subject.
  • the fugetactic agent may be stromal derived factor-1 (sdf-1); the non-fugetactic agent may include anti-inflammatory agents and/or an immunosuppressants and the other therapeutic compound may include any drug or moiety previously identified to have therapeutic benefit.
  • the subject has an autoimmune disease. In one embodiment the subject has an inflammatory disease or condition. In one embodiment the subject has a hyperproliferative condition. In one embodiment the hyperproliferative condition comprises cancer.
  • Fetal Calf Serum (FCS) in Iscove's Modified Dulbecco's Medium is prepared and stored on ice (Assay Medium).
  • Migratory cells at a concentration of 2 ⁇ 10 7 cells/ml are suspended in Assay Medium.
  • T-cells isolated from donor blood or buffy coats are allowed to rest overnight in Assay Medium at 37° C. in 5% CO 2 .
  • Neutrophils, monocytes and B cells are utilized as soon as they are purified from donor blood or other source of human origin.
  • Four serial (10 ⁇ ) dilutions of the ligand are then prepared in Assay Medium (normally 10 ⁇ g/ml to 0.01 ⁇ g/ml).
  • the assay plates Neuroprobe ChemoTx (part number 106-5; 5 ⁇ m pore size), are used for leukocytes or granulocytes.
  • part number 106-5 For cultured cell lines (Jurkat, Molt4, HL-60 and SupT-1 etc.), part number 106-8 (8 ⁇ m pore size) is used.
  • part number 106-3 For neutrophils, part number 106-3 (3 ⁇ m pore size) is used.
  • the plate is then covered with the supplied lid and incubated 1-3 hours in a humidified incubator at 37° C. in 5% CO 2 . Following the incubation period, the liquid from the top of the plate is removed with a tissue or similar blotting material (e.g. a Kimwipe). The membrane is carefully removed from the top of the plate and discarded. Using a multichannel pipettor set at 30 ⁇ l, the contents of the wells are transferred to a white microplate (Perkin-Elmer CulturPlate 96 #6005689).
  • a tissue or similar blotting material e.g. a Kimwipe
  • each well of the microplate is rinsed with 25 ⁇ l Phosphate Buffered Saline (Mediatech # 21-040-CV) and transferred to the same wells in the white microplate.
  • 50 ⁇ l CellTiter-Glo Luminescent Cell Viability Assay (Promega # G7572) is added to each well and mixed.
  • the plate is read using the Perkin Elmer Victor 3 V plate reader using the Large Aperture Luminescence setting at 8 mm height above the plate with a dwell time of 1 s to quantify the number of migrated cells.
  • HSF hybridoma serum free
  • HSF+PS hybridoma serum free
  • Adherent cells are grown to approximately 70 ⁇ 90% confluence in 8-75 cm 2 flasks. The cells are rinsed two times with approximately 10 ml of HSF+PS and covered with 30 ml HSF+PS.
  • the flasks are incubated at 37° C., 5% CO 2 , and the culture supernatant of the flask harvested at varying time periods (one hour after placing back in the incubator (Day 0), 24 h, 48 h, 72 h, etc up to 7 days).
  • the culture fluid should be centrifuged at 500 ⁇ g for 10 minutes and then filtered to a new tube using 0.2 ⁇ m Costar syringe filters (Catalog #8110). Storage of multiple aliquots of 250- ⁇ l each can be in microcentrifuge tubes. The remaining supernatant can be stored in a falcon tube. All samples are stored at ⁇ 80° C.
  • a positive control which can be stromal derived factor-1 (SDF-1) (0.1, 1, and 10 ⁇ g/ml) and a HSF+PS negative control should also be run in the assay.
  • SDF-1 stromal derived factor-1
  • Galectin-3 is a unimodal fugetaxin that can be titrated to control cellular behavior.
  • Each of galectin-1, 2, 3, 5, 7 and 8 was tested in a 96 trans-well migration assay for their effects on neutrophils, CD4+ and CD8+ T lymphocytes.
  • the full-length galectin protein molecules were purchased from different vendors (Sigma, R&D and BioVision).
  • the assay was performed according to the methods described herein but with the following modifications to eliminate noise and increase the signal to noise ratio.
  • FIG. 1 shows the results of the study of Galectin-3.
  • RU are luminescence units from a CellTiter-Glo (Promega, Madison, Wis.) assay of cells that have migrated through a polycarbonate membrane with a 5 ⁇ m pore size to the lower portion of a transwell (Neuro Probe, Gaithersburg, Md.).
  • Human primary neutrophils (A), CD4 + lymphocytes (B), or CD8 + lymphocytes (C) were added to the upper chamber.
  • fugetaxis chemorepulsion
  • the upper chamber also contained galectin-3 in the indicated concentrations while the lower chamber contained media.
  • CT chemotaxis
  • CT left side of each graph
  • the media control contained only media in both chambers.
  • the data represent the measurements taken at 1 hour. It is shown in the figure that galectin-3 is fugetactic (chemorepulsive) in the assay at a dose of 10 ⁇ g/ml. It was determined and can be seen in the figure that micromolar concentrations of this molecule can induce the movement of several cell types (Neutophils, CD4+ and CD8+ T lymphocytes) in a predominantly unidirectional manner. In all three cell types, 2 to 3 fold more cells are repelled by 10 ⁇ g/ml galectin-3 than are attracted by it over the same time period. To applicant's knowledge, this effect has never been documented.
  • neutrophils were investigated for their response at 1 hour to other ligands.
  • neutrophils acted identically in their chemotactic (chemoattractant) and fugetactic (chemorepellent) responses to stromal derived factor-1(sdf-1) and lipoteichoic acid (LTA).
  • Neutrophils were also over twice as chemotactic (chemoattractant) as fugetactic (chemorepellent) in response to interleukin-8 (IL8).
  • neutrophils were almost three times as fugetactic (chemorepellent) as chemotactic (chemoattractant) in response to galectin-3 and were almost twice as fugetactic (chemorepellent) as chemotactic (chemoattractant) in response to heat shock protein 65 (HSP65). It is also shown in FIG. 4 that at 10 uM, neutrophils acted identically in their chemotactic (chemoattractant) and fugetactic (chemorepellent) responses to lipopolysacharide (LPS).
  • LPS lipopolysacharide
  • RU are luminescence units from a CellTiter-Glo (Promega, Madison, Wis.) assay of cells that have migrated through a polycarbonate membrane with a 5 ⁇ m pore size to the lower portion of a transwell (Neuro Probe, Gaithersburg, Md.). Human primary neutrophils (A), CD4 + lymphocytes (B), or CD8 + lymphocytes (C) were added to the upper chamber. The media control contained only media in both chambers.
  • both the top and bottom chambers of the well contained a monoclonal antibody to galectin-3 (BD Biosciences, San Jose, Calif.) at 1, 10, or 100 ⁇ g/ml.
  • galectin-3 BD Biosciences, San Jose, Calif.
  • the upper chamber contained only media while the lower chamber contained 1 ⁇ g/ml galectin-3.
  • the upper chamber contained 10 ⁇ g/ml galectin-3 while the lower contained only media.
  • antibody was present in both upper and lower chambers at the indicated concentrations.
  • the protocol was the same as described above, and the system was challenged with antibody to galectin-3.
  • the Figure shows that the number of migrating cells decreased with increasing concentrations of anti galectin-3 antibody.
  • the data clearly show that the fugetactic (chemorepellent) effect of galectin-3 can be abrogated in a directly competitive manner and supports the mechanism proposed in Example 3.
  • galectin-3 antibodies can act as unimodal fugetaxin antagonists.
  • Example 3 Using the methods described in Example 3 above, other compounds and/or ligands were tested for their ability to act as unimodal fugetaxins.
  • Table 1 lists those compounds and their effect on human neutrophils, CD4+ lymphocytes and CD8+ lymphocytes. A “+” in the Table indicates a positive hit as a unimodal fugetaxin, as that term is defined herein, for that particular cell type. No data indicates that the agent did not satisfy the definition of a unimodal fugetaxin.
  • Eo-HL60 which corresponds to a human myeloid leukemia cell line known as HL-60 clone 15. These cells, which have been treated with compounds known to cause their differentiation from a myeloid cell type to an eosinophil-like cell type, were utilized as surrogates for eosinophils due to their relative scarcity.
  • HL60 cells (clone 15) were obtained from the American Type Culture Collection (ATCC, Manasas Va.). To maintain and expand the cells in an undifferentiated phenotype, the cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 100 I.U./ml penicillin, and 100 ug/mL streptomycin in a humidified incubator at 37° C. with 5% CO 2 .
  • Eo-HL60 eosinophil-like phenotype
  • Eo-HL60 chemorepulsion Poly(I:C) Eo-HL60 chemorepulsion FLS-1
  • Eo-HL60 chemorepulsion Imiquimod Eo-HL60 chemorepulsion
  • Paclitaxel Eo-HL60 chemorepulsion Lipid A Diphos Eo-HL60 chemorepulsion P. gingavalis LPS
  • Eo-HL60 chemorepulsion Lipomannan M. smegmatis Eo-HL60 chemorepulsion LTA Staph Eo-HL60 chemorepulsion Standard LTA from S.
  • PMNs Primary Human Neutrophils
  • IMDM Iscove's Modified Dubelco's Medium
  • the PMN are again centrifuged for 10 minutes at 500 ⁇ g and the supernatant removed.
  • the number of cells is quantitated using a hemacytometer (Reichert). It is noted that if the original blood volume was less than 30 mL then cells were resuspended in 10 mL to achieve an accurate hemocytometer count.
  • Residual red blood cells contaminating the PMN preparation are then removed by positive selection using glycophorin A microbeads (Miltenyi Biotec, Auburn, Calif.) per the manufacturer's instructions.
  • the resulting PMN are then diluted to 50 ml with IMDM. After centrifugation at 500 ⁇ g for 10 minutes, the supernatant is removed and the PMN resuspended in 15 ml IMDM supplemented with 0.5% heat inactivated fetal calf serum (FCS) (ATCC, Manasas, Va.).
  • FCS heat inactivated fetal calf serum
  • PBMCs Peripheral Blood Mononucleocytes
  • PBMC peripheral blood mononucleocyte
  • the tubes are then filled to 50 ml with IMDM and centrifuged for 10 minutes at 500 ⁇ g. All but approximately 5 ml of the supernatant is then removed and the pellet resuspended with 25 ml IMDM and the centrifugation repeated. After removal of the supernatant, the cells are resuspended and combined to one 50 ml tube (per subject) and brought to a total volume of 50 ml. The cells are then counted using a hemacytometer (Reichert). After centrifugation under the same conditions, the supernatant is removed and the cells processed using the appropriate Miltenyi Biotec (Auburn, Calif.) negative selection kit to enrich for the desired cell type according to manufacturers instructions.
  • the resulting purified cells are then diluted to 50 ml with IMDM. After centrifugation at 500 ⁇ g for 10 minutes, the supernatant is removed and the cells resuspended in 15 ml IMDM supplemented with 0.5% heat inactivated fetal calf serum (FCS) (ATCC, Manasas, Va.). After another centrifugation for 10 minutes at 500 ⁇ g, the supernatant is again removed and the cells resuspended in 7 ml 0.5% FCS in IMDM (assay medium) and transferred to a 15 ml conical centrifuge tube. The cells are then quantitated using a hemacytometer.
  • FCS heat inactivated fetal calf serum
  • the concentration of cells is adjusted to 2 ⁇ 10 7 per ml by centrifugation and removal of the appropriate volume of assay medium.
  • the tube cap is replaced with one from a 25 cm 2 tissue culture flask (Corning) and the cells stored overnight in a humidified 37° C. incubator with 5% CO 2 .
  • the concentration of the cells is adjusted as for monocytes just prior to transmigration assay of Example 9.
  • this procedure may be used to isolate and/or purify monocytes.
  • monocyte purification method a further step is included for platelet removal via the use of CD61 microbeads also purchased from Miltenyi Biotec (Auburn, Calif.).
  • ChemoTx plates (Neuroprobe). These plates contain a planar polycarbonate or polyester membrane that has been etched and perforated so that cells can move along the etched tracks and fall through perforation holes, enabling the cells to be collected and the number of migrated cells quantified ( FIGS. 5-6 ). Several dilutions (normally 4 in 10-fold increments) are prepared of the agent of interest. For neutrophils, plates with 3 ⁇ m pore membranes are used, while for all other primary cell types, 5 ⁇ m pore membranes are used. Membranes may, or may not, be coated with polyvinylpyrrolidine (PVP) as a wetting agent.
  • PVP polyvinylpyrrolidine
  • CR spontaneous migration controls and chemorepulsion
  • 31 ⁇ l of assay medium is pipetted into the lower well of the transmigration apparatus ( FIGS. 5-6 ).
  • 29 ⁇ l of assay medium is pipetted into upper chamber of the transmigration apparatus ( FIG. 5 ).
  • the upper well assay medium contains the agent or candidate chemorepellent.
  • the upper well assay medium contains only IMDM to which FCS has been added to a final concentration of 0.5% (v/v).
  • Cells are aliquoted into 200 uL fractions and each kept at 37° C. until ready to plate. This subdivision and preservation of cells at the appropriate temperature allows better consistency across the timecourse of the assay.
  • the liquid on top of the membrane is removed by gentle wiping with a KimWipe (Kimberly-Clark), followed by removal of the membrane from the migration (lower) plate.
  • a multichannel pipettor 5 ⁇ l of CellTiter-Glo (Promega, city state) is added to each well of the migration plate to quantitate the number of cells present in the well.
  • the contents of each well are then transferred to a 96 well white reading plate suitable for measurement of luminescence (e.g. OptiPlate (Perkin Elmer)) in which 25 ⁇ l of phosphate buffered saline (PBS) (Cellgro) has been added.
  • PBS phosphate buffered saline
  • the white reading plate is then incubated with orbital shaking in the dark for 10 minutes before reading luminescence in a microplate reader (e.g. Victor3 (Perkin Elmer)).
  • a microplate reader e.g. Victor3 (Perkin Elmer)
  • 50 plates can be read in an automated plate loading and plate reading mode using a suitable microplate reader.
  • the luminescence output correlates directly with the number of migrated cells.
  • the extent of cell migration is computed relative to spontaneous migration for the specific cell type and donor in any given experiment.
  • a conditioned medium was employed to identify chemorepellents.
  • the conditioned medium was prepared by incubation of cells of mammalian origin in a serum-containing growth medium until the cell culture reached a critical density. Critical density is reached for cells grown in suspension when the cell number exceeds 120 ⁇ 10 6 . For adherent cells, critical density is reached when the cells reach 70-90% confluency.
  • HSF ⁇ PS Hybridoma Serum Free Medium
  • ATCC 30-2300 Penicillin-Streptomycin
  • HSF ⁇ PS Penicillin-Streptomycin
  • the culture fluid was centrifuged at 500 ⁇ g for 10 minutes and then filtered to a new tube using 0.2 ⁇ m syringe filters.
  • the supernatants can be filtered using sterile 0.2 ⁇ m filter units and the supernatant stored in the included bottles. Storage of multiple aliquots of 250 ⁇ l each can be in microcentrifuge tubes. The remaining supernatant can be stored in a conical tube or left in the sterile filter unit receiver flask. All samples were stored at ⁇ 80° C.
  • the supernatants may be subsequently fractionated according to standard chromatographic procedures to facilitate isolation of the chemorepellent. Fractionation may be carried out by size exclusion chromatography, FPLC, HPLC, ion-exchange chromatography, hydrophobic chromatography, immune-affinity chromatography, and the like.
  • the results of migration index calculations determined the fractions to be pooled and carried to the next step, thereby effecting enrichment of the amount of any putative chemorepellent agents in any subsequent pooled fraction.
  • the pooled fractions having the highest migration indices, are analyzed by one or two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), the separated components visualized by silver or fluorescent stain and the visualized components eluted by techniques known in the art.
  • the eluted gel spots are subjected to chemical or enzymatic fragmentation and identification using mass spectrometry, amino-acid sequencing or other techniques known to anyone skilled in the art.
  • the pooled fractions with the highest migration indices could be directly analyzed by liquid chromatography (LC) mass spectrometry (MS).
  • the results include agents such as carbohydrate binding proteins (Table 3), serpins (Table 4), bacterial cell wall components (Table 5), heat shock proteins (Table 6), natural products (Table 7), Toll-like receptor ligands (Table 8) viral factors (Table 9), semaphorins (Table 10), elastase inhibitors (Table 11), antibiotics (Table 12), muscle cell proteins (Table 13), plant cell wall components (Table 14) and chemokines (Table 15).
  • agents such as carbohydrate binding proteins (Table 3), serpins (Table 4), bacterial cell wall components (Table 5), heat shock proteins (Table 6), natural products (Table 7), Toll-like receptor ligands (Table 8) viral factors (Table 9), semaphorins (Table 10), elastase inhibitors (Table 11), antibiotics (Table 12), muscle cell proteins (Table 13), plant cell wall components (Table 14) and chemokines (Table 15).
  • the repellent index for each agent was calculated from the independent assessment of three batches of donor blood from three different individuals, with each measurement from donor blood conducted in triplicate at four different concentrations of ligand. The ranges are given in the tables and covered a 1000-fold change in concentration.
  • the repellent indices reported in Tables 3-15 represents the mean value of three donor blood samples at the concentration of ligand at which the highest activity was observed.
  • the observed chemorepulsion is likely independent of physiological differences across the donor groups and likewise the human population, suggesting strongly that induction of chemorepulsion by a validated chemorepellent is a true physiological phenomenon.
  • Concentrations expressed in ⁇ M unless otherwise noted. It is noted that for heat killed Listeria , the concentration can only be expressed as number of cells per milliliter.
  • Table 16 the repellent indices and standard deviations measured at four concentrations of selected members of the classes of compounds of Tables 3-15 are given.
  • the standard deviations given in Table 16 represent that calculated based on one experiment (one donor) conducted in triplicate.
  • TLR Ligands Concentration Accession Repellent Range Ligand Cell Type # Index ( ⁇ M) 1 Pam3CSK4 Neutrophils Synthetic 4.9 0.00125-1.25 bacterial lipoprotein Heat Neutrophils 7.7 10 6 -10 9 Cells/ml killed Listeria monocytogenes Poly(I:C) Neutrophils Synthetic 1.4 0.01-10 ⁇ g/ml analog of dsRNA Ecoli K12 Neutrophils 8.2 0.01-10 ⁇ g/ml LPS S.typhimurium B cells M11332 1.7 0.01-10 ⁇ g/ml Flagellin Neutrophils 5.5 0.01-10 ⁇ g/ml FLS-1 Neutrophils Synthetic 6.8 0.01-10 ⁇ g/ml lipoprotein ssRNA40/ Neutrophils ssRNA 1.3 0.01-10 ⁇ g/ml LyoVec complexed with cationic lipid ODN2006 Neutrophils Synthetic 1.4 0.01-10 ⁇ g/ml
  • MI migration index
  • Mixtures whose migration index is equal to or greater than 1.2, are considered to contain at least one “validated conditioned chemorepellent.”

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US20100093677A1 (en) * 2008-10-10 2010-04-15 Erica Brook Goodhew Method of inducing negative chemotaxis
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US8575217B2 (en) 2009-03-16 2013-11-05 Genmedica Therapeutics Sl Anti-inflammatory and antioxidant conjugates useful for treating metabolic disorders
US20100239552A1 (en) * 2009-03-16 2010-09-23 Genmedica Therapeutics Sl Combination Therapies for Treating Metabolic Disorders
US20100234452A1 (en) * 2009-03-16 2010-09-16 Genmedica Therapeutics Sl Anti-Inflammatory and Antioxidant Conjugates Useful for Treating Metabolic Disorders
US8466197B2 (en) 2010-12-14 2013-06-18 Genmedica Therapeutics Sl Thiocarbonates as anti-inflammatory and antioxidant compounds useful for treating metabolic disorders
US11389478B2 (en) * 2011-12-08 2022-07-19 Eliaz Thereapeutics, Inc. Galectin-3 plasmapheresis therapy
US11389477B2 (en) * 2011-12-08 2022-07-19 Eliaz Thereapeutics, Inc. Galectin-3 plasmapheresis therapy
US11389476B2 (en) * 2011-12-08 2022-07-19 Eliaz Thereapeutics, Inc. Galectin-3 plasmapheresis therapy
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