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WO2006069449A1 - Activite immunomodulatoire et antiproliferative independante du recepteur de la chimiokine - Google Patents

Activite immunomodulatoire et antiproliferative independante du recepteur de la chimiokine Download PDF

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Publication number
WO2006069449A1
WO2006069449A1 PCT/CA2005/001985 CA2005001985W WO2006069449A1 WO 2006069449 A1 WO2006069449 A1 WO 2006069449A1 CA 2005001985 W CA2005001985 W CA 2005001985W WO 2006069449 A1 WO2006069449 A1 WO 2006069449A1
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WIPO (PCT)
Prior art keywords
chemokine
expression
gene
immunomodulatory
cell
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PCT/CA2005/001985
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English (en)
Inventor
Robert E. Hancock
Jiang-Hong Gong
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The University Of British Columbia
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Publication of WO2006069449A1 publication Critical patent/WO2006069449A1/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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to chemokines and specifically to the discovery of a previously unknown function of chemokines using a comprehensive microarray gene analysis.
  • This discovery involves the innate immunity-boosting and antiproliferative activities of selected chemokines, and fragments and analogs thereof, via a mechanism that appears to be independent of G-protein receptors, and the use of these immunomodulatory and anti-proliferative functions in treating infectious diseases and cancer.
  • Infectious diseases are currently the third leading cause of death in North America, hi the USA and Canada, more than 300,000 deaths annually are attributed to pneumonia and 100,000 people die of septicaemia (blood stream infections). Up to 20,000 direct deaths are attributable each year to nosocomial (hospital-acquired) infections, which affect two million patients a year and result in increased health care costs of 5 billion dollars. Current sales of antibiotics are US$26 billion worldwide. However, the overuse and sometimes unwarranted use of antibiotics have resulted in the evolution of new antibiotic- resistant strains of bacteria. With the dramatic rise of antibiotic resistance, including the emergence of untreatable infections, there is a clear unmet medical need for novel types of antimicrobial therapies, and agents that impact innate immunity would be one such class of agents.
  • cancer is a major cause of death world- wide. Despite dramatic advances in cancer therapy, the metastic potential of some cancers can result in a significantly shortened survival time and reduced quality of life. In addition to cancer prevention and significant improvements in early detection for most cancers, effective novel therapeutic strategies targeting both cancer and the host immunity will provide the greatest clinical benefit, and are highly required.
  • the innate immune system is a highly effective and powerful general defense system. Elements of innate immunity are always present at low levels and are activated very rapidly when stimulated. Stimulation can include interaction of bacterial signaling molecules with pattern recognition receptors on the surface of the body's cells or other mechanisms of disease. Every day, humans are exposed to tens of thousands of potential pathogenic microorganisms through the food and water we ingest, the air we breathe and the surfaces, pets and people that we touch. The innate immune system acts to prevent these pathogens from causing disease.
  • the innate immune system differs from so-called adaptive immunity (which includes antibodies and antigen-specific B- and T-lymphocytes) because it is always present, effective immediately, and relatively non-specific for any given pathogen.
  • adaptive immunity which includes antibodies and antigen-specific B- and T-lymphocytes
  • the adaptive immune system requires amplification of specific recognition elements and thus takes days to weeks to respond. Even when adaptive immunity is pre-stimulated by vaccination, it may take three days or more to respond to a pathogen whereas innate immunity is immediately or rapidly (hours) available.
  • Innate immunity involves a variety of effector functions including phagocytic cells, complement, and the like, but is generally incompletely understood.
  • Cationic antimicrobial peptides represent good templates for a new generation of antimicrobials. They kill both Gram negative and Gram positive microorganisms rapidly and directly, do not easily select mutants, work against common clinically-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE), show a synergistic effect with conventional antibiotics, and can often activate host innate immunity without displaying immunogenicity.
  • MRSA methicillin-resistant Staphylococcus aureus
  • VRE vancomycin resistant Enterococcus
  • Chemokines are a superfamily of small, inducible, secreted, proteins involved in a variety of immune responses, acting primarily as chemoattractants of specific types of leukocytes.
  • Four classes of chemokines have been defined by their arrangement of conserved cysteine (C) residues in the mature proteins: the CXC, CC, C and CX3C families of chemokines, Chemokines have related amino acid sequences so it is not surprising that their secondary and tertiary structures are also similar.
  • the overall features, determined by structural analyses of several chemokines, include a relatively disordered N-terminus that is anchored to the rest of the molecule by disulfide bonds involving the two N-terminal domain cysteines.
  • Chemokines have the ability to attract specific cells by interacting with specific receptors on the cell surface of their target cells. These specific receptors, called chemokine receptors, belong to the G-protein-coupled receptors (GPCRs) superfamily. It is well known that all chemokine receptor mediated functions can be blocked by pertussis toxin, which specifically disrupts the G-proteins responsible for GPCR mediated signaling.
  • GPCRs G-protein-coupled receptors
  • Chemokine ligands are classified based on key signature subsequences into the CCL, CXCL, CL and CX3CL families.
  • the chemokine receptors are classified according to their respective chemokine ligands into the families CXCR, CCR, CR and CX3CR respectively.
  • chemokines have immunomodulatory and antiproliferative activities via a mechanism that appears to be independent of G-protein receptors.
  • chemokines of the invention include peptides having the amino acid sequences of SEQ ID NOS: 1-17, and analogs, derivatives, amidated variations and conservative variations thereof.
  • the invention also provides a method of inhibiting the growth of pathogenic microbes by administering to the host an effective amount of at least one chemokine of the invention.
  • the invention further provides polynucleotides that encode the peptides of the invention.
  • Exemplary polynucleotides encode the chemokines as listed in SEQ ID NOS: 1-17.
  • the invention further provides a method for identifying a chemokine receptor (GPCR) independent function of a chemokine, or analog thereof, leading to enhancement of innate immunity by contacting a target cell with a chemokine or analog thereof and detecting a change in the expression of a cytokine gene or a set of genes related to innate immunity, as compared to the expression in the absence of the chemokine.
  • GPCR chemokine receptor
  • the invention also provides a method of identifying a chemokine with chemokine (GPCR) receptor independent anti-tumor cell growth anti-proliferative activity comprising contacting a tumor cell with a chemokine and detecting the change in expression of a gene or set of genes related to cell proliferation and its regulation, wherein an increase or decrease in expression of the genes related to cell proliferation and its regulation in the presence of the chemokine as compared to the expression in the absence of the chemokine is indicative of a chemokine that is anti-proliferative, whereby the anti-proliferative activity of a chemokine is not mediated through interaction of the chemokine with its GPCR chemokine receptor.
  • the invention further provides a method of boosting innate immunity in a subject who is unable to mount an effective immune response, by administering to the subject a therapeutically effective amount of at least one chemokine of the invention.
  • the invention further provides a method of inhibiting a cell proliferation-associated disorder in a subject having or at risk of having such a disorder.
  • the method includes administering to the subject a therapeutically effective amount of at least one chemokine of the invention.
  • the invention further provides a method of identifying a pattern of polynucleotide expression for identification of a compound that selectively enhances innate immunity.
  • the invention includes detecting a pattern of polynucleotide expression for cells contacted in the presence and absence of a chemokine, wherein the pattern in the presence of the chemokine represents stimulation of innate immunity; detecting a pattern of polynucleotide expression for cells contacted in the presence of a test compound, wherein a pattern with the test compound that is similar to the pattern observed in the presence of the chemokine, is indicative of an agent that enhances innate immunity.
  • This invention also provides a method of identifying a pattern of polynucleotide expression for identification of a compound that selectively inhibits target cell proliferation.
  • the invention includes detecting a pattern of polynucleotide expression for cells contacted in the presence and absence of a chemokine, wherein the pattern in the presence of the chemokine represents inhibition of proliferation. Detecting a pattern of polynucleotide expression for cells contacted in the presence of a test compound, wherein a pattern with the test compound that is similar to the pattern observed in the presence of the chemokine, is indicative of a compound that anti-proliferation.
  • nucleic acid sequences encoding the chemokines of the invention are also provided.
  • vectors including such polynucleotides and host cells containing the vectors.
  • the invention further provides the use of these immunomodulatory and antiproliferative functions of the chemokines of the invention as therapeutic compositions in treating infections and cancer.
  • Fig. 1 is a pictoral diagram showing the 3 -dimensional structures of selective chemokines having positively charged surfaces.
  • Fig. 2 is a graphical representation showing chemokine-induced IL-8 production of THP-I cells (Fig. 2A); and the cationic chemokines MIG and MIP-3 ⁇ , but not the non-cationic chemokines MCP-I and MIP-3 ⁇ , stimulated PBMC producing substantial amounts of IL-8 (Fig 2B).
  • Fig. 3 is a graphical representation showing chemokine-induced IL-8 gene expression.
  • Fig. 4 is a graphical representation showing IL-8 production by THP-I cells induced by chemokine fragments.
  • Fig. 5 is a graphical representation showing induction of IL-8 production by full length MIG and MCP-I, and fragments thereof.
  • Fig. 6 is a pictoral representation showing a RT-PCR results that demonstrates that THP-I cells express chemokine receptor CCRl (receptor for MIP-l ⁇ /CCL3), CCR2 (receptor for MCP-1/CCL2), but do not express CCR6 (receptor for MIP-3 ⁇ ) and CXCR3 (receptor for IPlO and MIG).
  • Fig. 7 is a graphical representation showing IL-8 production after treatment of THP-I cells with pertussis toxin in the presence or absence of MIG (Fig. 7A), and with pertussis toxin (P.T.) in the presence or absence of various chemokines (Fig. 7B).
  • Fig. 8 is a graphical representation showing that the MIG-induced gene expression pattern is different than that of MCP-I -induced via chemokine receptor activation.
  • Fig. 9 is a graphical representation showing that treatment with MIG and SLC enhanced a tumor suppressor, BTG2, gene expression in THP-I cells as confirmed by Real time PCR (Fig. ' 9A), and RT-PCR for a separate experiment in that THP-I cells were treated with various chemokines at a concentration of 10 uM for 4 hours (Fig 9B).
  • Fig. 9A Real time PCR
  • Fig. 9B RT-PCR
  • PCA Principal Component Analysis
  • Fig. 11 is a graphical representation showing that treatment with MIG inhibited the growth of the human myeloid leukemia THP-I cell, while treatment with MCP-I did not influence the growth of THP-I cells.
  • Fig. 12 is a graphical representation of the induction of MIP- l ⁇ and MIP- l ⁇ protein expression in monocytes.
  • Fig. 13 is a graphical representation showing that treatment with MIG or SLC inhibited the growth of the human breast epithelial cancer cell line, MDA, while treatment with MCP-I did not influence the growth of the MDA cell line.
  • Fig. 14 is a graphical representation showing GM-CSF induction of MIG-induced MIP- l ⁇ production.
  • the present invention provides a chemokine receptor-independent immunomodulatory function of certain chemokines at concentrations that exceed those that mediate chemotaxis. These chemokines display immunomodulatory activity at ⁇ M concentrations, in addition to their previously reported antimicrobial functions.
  • This novel immunomodulatory function includes enhancement of the innate immune response by induction of gene expression and production of various cytokines and chemokines. Many of the chemokines are cationic in nature.
  • the present invention also provides a chemokine receptor-independent antiproliferative function of certain chemokines. These chemokines display antiproliferative activity at ⁇ M concentrations in addition to their previously reported antimicrobial functions. This antiproliferative function includes regulation of the expression of genes related to cell proliferation and viral replication.
  • the invention provides a number of methods, reagents, and compounds. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a peptide” includes a combination of two or more peptides, and the like.
  • “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ⁇ 20% or ⁇ 10%, more preferably ⁇ 5%, even more preferably ⁇ 1%, and still more preferably ⁇ 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
  • Innate immunity refers to the natural ability of an organism to defend itself against invasion by pathogens.
  • Pathogens or microbes as used herein may include, but are not limited to bacteria, fungi, parasites, and viruses.
  • Innate immunity is contrasted with acquired/adaptive immunity in which the organism develops a defensive mechanism based substantially on antibodies and/or immune lymphocytes that is characterized by specificity, amplifiability and self vs. non-self discrimination. With innate immunity, broad, nonspecific immunity is provided, and there is no immunologic memory of prior exposure.
  • innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the like.
  • antimicrobial means that the chemokines of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses, parasites or the like either directly or through stimulation of the host's innate immune response.
  • antiviral means that the chemokines of the present invention inhibit, prevent or destroy the growth or proliferation of viruses or of virally-infected cells either directly or through stimulation of the host's innate immune response.
  • antiitumor as used herein means that the chemokines of the present invention may be used to inhibit the growth of or destroy tumors either directly or through stimulation of the host's innate immune response.
  • antifungal means that the chemokines of the present invention may be used to inhibit the growth of or destroy fungi either directly or through stimulation of the host's innate immune response.
  • antiparasite means that the chemokines of the present invention inhibit, prevent, or destroy the growth or proliferation of any organism that lives at the expense of a host organism either directly or through stimulation of the host's innate immune response.
  • Cancer in an animal refers to the presence of cells possessing characteristics typical of cancer-causing cells, for example, uncontrolled proliferation, loss of specialized functions, immortality, significant metastatic potential, significant increase in anti-apoptotic activity, rapid growth and proliferation rate, and certain characteristic morphology and cellular markers.
  • cancer cells will be in the form of a tumor; such cells may exist locally within an animal, or circulate in the blood stream as independent cells, for example, leukemic cells.
  • detecting a cancer or “diagnosing or predicting a cancer or a cancer potential” refers to determining the presence or absence of cancer or a precancerous condition in an animal. "Detecting a cancer” also can refer to obtaining indirect evidence regarding the likelihood of the presence of precancerous or cancerous cells in the animal or assessing the predisposition of a patient to the development of a cancer. Detecting a cancer can be accomplished using the methods of this invention alone, in combination with other methods, or in light of other information regarding the state of health of the animal.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • Precancerous refers to cells or tissues having characteristics relating to changes that may lead to malignancy or cancer. Examples include adenomatous growths in lung, colon, ovary, or pancreas, tissues, or conditions, for example, dysplastic nevus syndrome, a precursor to malignant melanoma of the skin. Examples also include, abnormal neoplastic, in addition to dysplastic nevus syndromes, polyposis syndromes, prostatic dysplasia, and other such neoplasms, whether the precancerous lesions are clinically identifiable or not.
  • Inflammation or "inflammatory response” refer to an innate immune response that occurs when tissues are injured by bacteria, trauma, toxins, heat, or any other cause. The damaged tissue releases compounds including histamine, bradykinin, and serotonin. Inflammation refers to both acute responses (i.e., responses in which the inflammatory processes are active) and chronic responses (i.e., responses marked by slow progression and formation of new connective tissue). Acute and chronic inflammation can be distinguished by the cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; whereas chronic inflammation is normally characterized by a lymphohistiocytic and/or granulomatous response. Inflammation includes reactions of both the specific and non-specific defense systems.
  • a specific defense system reaction is a specific immune system reaction response to an antigen (possibly including an autoantigen).
  • a non-specific defense system reaction is an inflammatory response mediated by leukocytes incapable of immunological memory. Such cells include granulocytes, macrophages, neutrophils and eosinophils. Examples of specific types of inflammation are diffuse inflammation, focal inflammation, croupous inflammation, interstitial inflammation, obliterative inflammation, parenchymatous inflammation, reactive inflammation, specific inflammation, toxic inflammation and traumatic inflammation.
  • Sepsis is the systemic inflammatory response to infection. Sepsis is the result of the interaction between the microorganism and their products and the host factors released on response (cytokines and other mediators). This host response is an innate mechanism developed to protect the organism from harm but in sepsis the response is in excess, with negative effects, leading to organ dysfunction and frequently to death. The amplitude of the host defense depends on the amount of the invading microorganism present. The septic response then involves complex interactions among microbial signal molecules, leukocytes, humoral mediators and vascular endothelium. Inflammatory cytokines amplify and diversify the overall response.
  • Microbial toxins stimulate the production of cytokines like TNF- ⁇ and IL- l ⁇ , which in turn promote endothelial cell-leukocyte adhesion, release of proteases and arachidonic acid metabolites and activation of clotting.
  • IL-8 a neutrophil chemotaxin
  • IL-6 and IL-IO which are counter-regulatory, inhibit the generation of TNF- ⁇ , augment the action of acute phase reactants and immunoglobulins, and inhibit T-lymphocyte and macrophage function.
  • IL-6 along with other mediators can also promote intravascular coagulation.
  • Metal cancer or “metastatic cancer cell” refers to the ability of a tumor cell to form implants at a site distant from the original tumor.
  • Recurrence of cancer refers to the ability of a tumor cell to form implants near the site of the original tumor.
  • Non-metastatic cancer or “non-metastatic cancer cell” refers to a cancer or tumor cell that remains at the site of the original tumor.
  • Treating refers to any indication of success in amelioration of an injury, pathology, or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and/or psychiatric evaluation.
  • Treating includes the administration of the construct or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with the disease, condition or disorder.
  • treatment can refer to the inhibition of tumor growth, the arrest of tumor growth, or the regression of already existing tumors. It can also refer to the alleviation of cancer-related anemia.
  • Therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of cancer in the subject. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.
  • the phrase “well tolerated” refers to the absence of adverse changes in health status that occur as a result of the treatment and would affect treatment decisions.
  • each component can be administered at the same time or sequentially in any order at different points in time.
  • each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect
  • Subject refers to any mammalian patient or subject to which the compositions of the invention can be administered.
  • “Mammal” or “mammalian” refers to human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine risk factors that can be associated with the targeted or suspected disease or condition. These and other routine methods allow the clinician to select patients in need of therapy using the methods and formulations of the invention.
  • a “differentially expressed gene transcript”, as used herein, refers to a gene transcript that is found in different numbers of copies in different cell or tissue types of an organism having a tumor or cancer, for example, a lung cancer, a colon cancer, an ovarian cancer, or a pancreatic cancer, compared to the numbers of copies or state of the gene transcript found in the cells of the same tissue in a healthy organism, or in the cells of the same tissue in the same organism. Multiple copies of gene transcripts may be found in an organism having the tumor or cancer, while fewer copies of the same gene transcript are found in a healthy organism or healthy cells of the same tissue in the same organism, or vice- versa.
  • a “differentially expressed gene,” can be a target, fingerprint, or pathway gene.
  • a “fingerprint gene”, as used herein, refers to a differentially expressed gene whose expression pattern can be used as a prognostic or diagnostic marker for the evaluation of tumors and cancers, or which can be used to identify compounds useful for the treatment of tumors and cancers, for example, lung cancer, colon cancer, ovarian cancer, or pancreatic cancer.
  • the effect of a compound on the fingerprint gene expression pattern normally displayed in connection with tumors and cancers can be used to evaluate the efficacy, such as potency, of the compound as a tumor and cancer treatment, or can be used to monitor patients undergoing clinical evaluation for the treatment of tumors and cancer.
  • a “fingerprint pattern”, as used herein, refers to a pattern generated when the expression pattern of a series (which can range from two up to all the fingerprint genes that exist for a given state) of fingerprint genes is determined.
  • a fingerprint pattern also may be referred to as an "expression profile”.
  • a fingerprint pattern or expression profile can be used in the same diagnostic, prognostic, and compound identification methods as the expression of a single fingerprint gene.
  • a “target gene”, as used herein, refers to a differentially expressed gene in which modulation of the level of gene expression or of gene product activity prevents and/or ameliorates tumor and cancer, for example, lung cancer, colon cancer, ovarian cancer, or pancreatic cancer, symptoms.
  • compounds that modulate the expression of a target gene, the target gene, or the activity of a target gene product can be used in the diagnosis, treatment or prevention of tumors and cancers.
  • a particular target gene of the present invention is the SALPR ' or Relaxin-3 gene.
  • a "gene” is a region on the genome that is capable of being transcribed to an RNA that either has a regulatory function, a catalytic function, and/or encodes a protein.
  • An eukaryotic gene typically has nitrons and exons, which may organize to produce different RNA splice variants that encode alternative versions of a mature protein.
  • the skilled artisan will appreciate that the present invention encompasses all SALPR- and Relaxin-3 -encoding transcripts that may be found, including splice variants, allelic variants and transcripts that occur because of alternative promoter sites or alternative polyadenylation sites.
  • a “full-length” gene or RNA therefore encompasses any naturally occurring splice variants, allelic variants, other alternative transcripts, splice variants generated by recombinant technologies which bear the same function as the naturally occurring variants, and the resulting RNA molecules.
  • a “fragment" of a gene, including an oncogene can be any portion from the gene, which may or may not represent a functional domain, for example, a catalytic domain, a DNA binding domain, etc.
  • a fragment may preferably include nucleotide sequences that encode for at least 25 contiguous amino acids, and preferably at least about 30, 40, 50, 60, 65, 70, 75 or more contiguous amino acids or any integer thereabout or therebetween.
  • Pathway genes are genes that encode proteins or polypeptides that interact with other gene products involved in, for example, tumors and cancers. Pathway genes also can exhibit target gene and/or fingerprint gene characteristics.
  • RNA expression level means a level that is detectable by standard techniques currently known in the art or those that become standard at some future time, and include for example, differential display, RT (reverse transcriptase)-coupled polymerase chain reaction (PCR), Northern Blot, and/or RNase protection analyses.
  • RT reverse transcriptase
  • PCR reverse transcriptase-coupled polymerase chain reaction
  • Northern Blot Northern Blot
  • RNase protection analyses The degree of differences in expression levels need only be large enough to be visualized or measured via standard characterization techniques.
  • Transformed cell means a cell into which (or into predecessor or an ancestor of which) a nucleic acid molecule encoding a polypeptide of the invention has been introduced, by means of, for example, recombinant DNA techniques or viruses.
  • Nucleic acid or “nucleic acid molecule” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, can encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • a polynucleotide probe is a single stranded nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a polynucleotide probe can include natural (i.e., A, G, C, or T) or modified bases (e.g., 7- deazaguanosine, inosine). Therefore, polynucleotide probes can 5-10,000, 10-5,000, 10-500, 10- 50, 10-25, 10-20, 15-25, and 15-20 bases long. Probes are typically about 10-50 bases long, and are often 15-20 bases.
  • the array includes test probes (also referred to as polynucleotide probes) more than 5 bases long, preferably more than 10 bases long, and some more than 40 bases long.
  • the probes can also be less than 50 bases long, hi some cases, these polynucleotide probes can range from about 5 to about 45 or 5 to about 50 nucleotides long, or from about 10 to about 40 nucleotides long, or from about 15 to about 40 nucleotides in length.
  • the probes can also be about 20 or 25 nucleotides in length.
  • polynucleotide probes can be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • polynucleotide probes can be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • the length of probes used as components of pools for hybridization to distal segments of a target sequence often increases as the spacing of the segments increased thereby allowing hybridization to be conducted under greater stringency to increase discrimination between matched and mismatched pools of probes.
  • the polynucleotide probes can be less than 50 nucleotides in length, generally less than 46 nucleotides, more generally less than 41 nucleotides, most generally less than 36 nucleotides, preferably less than 31 nucleotides, more preferably less than 26 nucleotides, and most preferably less than 21 nucleotides in length.
  • the probes can also be less than 16 nucleotides, less than 13 nucleotides hi length, less than 9 nucleotides in length and less than 7 nucleotides in length.
  • arrays can have polynucleotides as short as 10 nucleotides or 15 nucleotides. In addition, 20 or 25 nucleotides can be used to specifically detect and quantify nucleic acid expression levels. Where ligation discrimination methods are used, the polynucleotide arrays can contain shorter polynucleotides. Arrays containing longer polynucleotides are also suitable. High density arrays can comprise greater than about 100, 1000, 16,000, 65,000, 250,000 or even greater than about 1,000,000 different polynucleotide probes. "Target nucleic acid” refers to a nucleic acid (often derived from a biological sample), to which the polynucleotide probe is designed to specifically hybridize.
  • target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target.
  • the term target nucleic acid can refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage can be apparent from context. .
  • Subsequence refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
  • Gene refers to a unit of inheritable genetic material found in a chromosome, such as in a human chromosome. Each gene is composed of a linear chain of deoxyribonucleotides which can be referred to by the sequence of nucleotides forming the chain. Thus, “sequence” is used to indicate both the ordered listing of the nucleotides which form the chain, and the chain which has that sequence of nucleotides. The term “sequence” is used in the same way in referring to RNA . chains, linear chains made of ribonucleotides. The gene includes regulatory and control sequences, sequences which can be transcribed into an RNA molecule, and can contain sequences with unknown function.
  • RNA products products of transcription from DNA
  • messenger RNAs mRNAs
  • ribonucleotide sequences or sequence
  • the sequences which are not translated include control sequences, introns and sequences with unknowns function. It can be recognized that small differences in nucleotide sequence for the same gene can exist between different persons, or between normal cells and cancerous cells, without altering the identity of the gene.
  • the phrase "specifically (or selectively) binds" to an antibody refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • telomere binding reaction which is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions can require a binding moiety that is selected for its specificity for a particular target antigen.
  • a variety of assay formats can be used to select ligands that are specifically reactive with a particular protein.
  • solid-phase ELISA immunoassays, immunoprecipitation, Biacore and Western blot are used to identify peptides that specifically react with neuropsychiatric domain-containing proteins.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.
  • Specific binding between a monovalent peptide and its receptor means a binding affinity of at least 10 3 M "1 , and preferably 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M "1 .
  • Module includes inhibitors and activators.
  • Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of a polypeptide or polynucleotide of the invention, e.g., agonists.
  • Modulators include agents that, e.g., alter the interaction of the chemokines of the invention with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of naturally-occurring ligands, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Cell-based assays for inhibitors and activators include, e.g., applying putative modulator compounds, e.g., small molecules, drugs, chemokines and the like, to a cell expressing a particular gene or genes of interest and then determining the functional effects on the particular genes related to cell proliferation and viral replication, as described herein.
  • Cell based assays include, but are not limited to, in vivo tissue or cell samples from a mammalian subject or in vitro cell-based assays comprising a particular gene or genes of interest that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples (untreated with inhibitors) can be assigned a relative activity value of 100%. Inhibition of cell proliferation or viral replication is achieved when the activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of a particular activity is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • innate immunity the immune response is not dependent upon antigens. The innate immunity process may include the production of secretory molecules and cellular components as set forth above.
  • the pathogens are recognized by receptors encoded in the germline. These Toll-like receptors have broad specificity and are capable of recognizing many pathogens.
  • chemokines When certain immunomodulatory chemokines are present in the immune response, they aid in the host response to pathogens. This change in the immune response induces the release of chemokines, which promote the recruitment of immune cells to the site of infection.
  • chemokine as used herein includes analogs and peptides that are conservative variations of those chemokines specifically exemplified herein.
  • Chemokines are a subgroup of immune factors that mediate chemotactic and other pro- inflammatory phenomena (See, Schall, 1991, Cytokine 3:165- 183). Chemokines are small molecules of approximately 70-80 residues in length and can generally be divided into 4 subgroups, CXC, which have two N-terminal cysteines separated by a single amino acid, CC, which have two adjacent cysteines at the N terminus, and the C which have a single cysteine residue at the N terminus and CX3C which have two cysteines at the N terminus separated by 3 residues (reviewed by Horuk, R., 1994, Trends Pharmacol. Sci, 15:159- 165; Murphy, P.
  • C-terminal ⁇ -helix which in many chemokines is present as an amphipathic ⁇ -helix, is to date not well understood. While most chemokines are small, with molecular weights around 8000, the chemokines MIG/CXCL9 and SLC/CCL21 are exceptional in having 2 extra cysteines and an extended basic C-terminal tail. The function of these extended C-terminal tails is unknown to date, as truncation of the C-terminus fragment of MIG did not block binding on its chemokine receptor CXCR3 (Clark-Lewis, I. et al, J. Biol. Chem., 278: 289- 295, 2003).
  • Chemokines induce target cell migration in a bell-shaped dose dependent manner: the chemotactic activity increases along the concentration gradient and is maximal at a concentration of between 30 and 100 nM. At micromolar ( ⁇ M) concentrations, chemokines cannot induce cell migration.
  • chemokines have positively charged surfaces in their 3- dimensional structures, due to basic amino acid residues, share common structural characteristics with certain classes of cationic antimicrobial peptides and display antimicrobial activities. For instance, at ⁇ M concentrations, MIG, SLC, IP-10 and MIP-3oc are able to kill Gram negative and/or Gram positive bacteria, whereas other chemokines, such as MCP-I and MIP- l ⁇ , that are not very positively charged, have no antimicrobial activity (see Fig. 1; Yang et al, Journal Leukocyte Biology 74: 448-55, 2003). Fig. 1 and Table 1 summarize data from the literature indicating the known features of various chemokines and their published antimicrobial activity.
  • cationic is used to refer to any peptide that possesses sufficient positively charged amino acids to have a pi (isoelectric point) greater than about 9.0. Although most chemokines have a net positive charge, "cationic chemokines” refer herein to those peptides found to have antimicrobial activity and that are believed to possess positively charged surfaces in their 3-dimensional structures (Table 1, see bolded chemokines). Thus, a cationic chemokine includes all chemokines having a molecular surface electrostatic charge greater than or equal to +3.
  • amino acid residues identified herein are in the natural L-configuration.
  • abbreviations for amino acid residues are as shown in the following table.
  • the invention also includes analogs, derivatives, conservative variations, and chemokine variants of the enumerated polypeptides, provided that the analog, derivative, conservative variation, or variant has a detectable activity in which it enhances innate immunity or has anti-inflammatory activity. It is not necessary that the analog, derivative, variation, or variant have activity identical to the activity of the peptide from which the analog, derivative, conservative variation or variant is derived.
  • a peptide “variant” is any peptide that is an altered form of a referenced peptide.
  • the term “variant” includes a chemokine in which at least one amino acid of a reference chemokine is substituted in an expression library.
  • the term “reference chemokine” or “reference peptide” means any of the peptides of the invention, from which a variant, derivative, analog, or conservative variation is derived. Included within the term “derivative” are peptides in which one or more amino acids are deleted from the sequence of a chemokine enumerated herein, provided that the derivative has activity in which it enhances innate immunity or has anti-inflammatory activity.
  • amino or carboxy terminal amino acids which may not be required for enhancing innate immunity or anti-inflammatory activity of a peptide can be removed.
  • additional derivatives can be produced by adding one or a few (e.g., less than 5) amino acids to a peptide without completely inhibiting the activity of the peptide.
  • C-terminal derivatives e.g., C-terminal methyl esters, and N-terminal derivatives can be produced and are encompassed by the invention.
  • Peptides of the invention include any analog, homolog, mutant, isomer or derivative of the chemokines disclosed in the present invention, so long as the bioactivity as described herein remains.
  • a peptide encompassed by the general formulas set forth above. Additionally, an amino acid of "D" configuration may be substituted with an amino acid of "L” configuration and vice versa. Alternatively the peptide may be cyclized chemically or by the addition of two or more cysteine residues within the sequence and oxidation to form disulphide bonds.
  • isolated when used in reference to a peptide, refers to a peptide substantially free of proteins, lipids, nucleic acids, for example, with which it can be naturally associated. Those of skill in the art can make similar substitutions to achieve peptides with greater antimicrobial activity and a broader host range.
  • the invention includes the peptides depicted in SEQ ID NOS: 1-17, as well as analogs or derivatives thereof, as long as the bioactivity (e.g., antiproliferative activity) of the peptide remains.
  • Minor modifications of the primary amino acid sequence of the peptides of the invention may result in peptides that have substantially equivalent activity as compared to the specific peptides described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All of the peptides produced by these modifications are included herein as long as the biological activity of the original peptide still exists.
  • deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule that would also have utility.
  • amino or carboxy terminal amino acids that may not be required for biological activity of the particular peptide can be removed.
  • Peptides of the invention include any analog, homolog, mutant, isomer or derivative of the peptides disclosed in the present invention, so long as the bioactivity as described herein remains. All peptides were synthesized using L amino acids, however, all D forms of the peptides can be synthetically produced.
  • C-terminal derivatives can be produced, such as C-terminal methyl esters and C-terminal amidates, in order to increase the antimicrobial activity of a peptide of the invention.
  • the peptide can be synthesized such that the sequence is reversed whereby the last amino acid in the sequence becomes the first amino acid, and the penultimate amino acid becomes the second amino acid, and so on. It is well known that such reversed peptides usually have similar antimicrobial activities to the original sequence.
  • the peptides of the invention include peptide analogs and peptide mimetics. Indeed, the peptides of the invention include peptides having any of a variety of different modifications, including those described herein.
  • Peptide analogs of the invention are generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein, such as any of the following sequences disclosed in the tables.
  • the present invention clearly establishes that these peptides in their entirety and derivatives created by modifying any side chains of the constituent amino acids have the ability to inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses, parasites or the like.
  • the present invention further encompasses polypeptides up to about 50 amino acids in length that include the amino acid sequences and functional variants or peptide mimetics of the sequences described herein.
  • a peptide of the present invention is a pseudopeptide.
  • Pseudopeptides or amide bond surrogates refers to peptides containing chemical modifications of some (or all) of the peptide bonds. The introduction of amide bond surrogates not only decreases peptide degradation but also may significantly modify some of the biochemical properties of the peptides, particularly the conformational flexibility and hydrophobicity.
  • polypeptides of the present invention protein engineering can be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions, or fusion proteins.
  • modified polypeptides can show, e.g., increased/decreased biological activity or increased/decreased stability.
  • they can be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • the polypeptides of the present invention can be produced as multimers including dimers, trimers and tetramers. Multimerization can be facilitated by linkers, introduction of cysteines to permit creation of interchain disulphide bonds, or recombinantly though heterologous polypeptides such as F c regions.
  • the present invention provides polypeptides having one or more residues deleted from the amino terminus.
  • many examples of biologically functional C-terminal deletion mutants are known (see, e.g., Dobeli et al, J. Biotechnology 7: 199-216, 1988). Accordingly, the present invention provides polypeptides having one or more residues deleted from the carboxy terminus.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini as described below.
  • mutants in addition to N- and C-terminal deletion forms of the protein discussed above are included in the present invention.
  • the invention further includes variations of the polypeptides which show substantial chaperone polypeptide activity.
  • Such mutants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu and Phe; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • the polypeptide of the present invention can be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue can or cannot be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the polypeptide, such as an IgG F c fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide or a pro-protein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue can or cannot
  • polypeptides of the present invention can include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation.
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein.
  • the following groups of amino acids represent equivalent changes: (1) Ala, Pro, GIy, GIu, Asp, GIn, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) VaI, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.
  • polypeptides of the present invention can include one or more amino acid substitutions that mimic modified amino acids.
  • An example of this type of substitution includes replacing amino acids that are capable of being phosphorylated (e.g., serine, threonine, or tyrosine) with a negatively charged amino acid that resembles the negative charge of the phosphorylated amino acid (e.g., aspartic acid or glutamic acid).
  • substitution of amino acids that are capable of being modified by hydrophobic groups e.g., arginine
  • amino acids carrying bulky hydrophobic side chains such as tryptophan or phenylalanine.
  • a specific embodiment of the invention includes polypeptides that include one or more amino acid substitutions that mimic modified amino acids at positions where amino acids that are capable of being modified are normally positioned. Further included are polypeptides where any subset of modifiable amino acids is substituted. For example, a polypeptide that includes three serine residues can be substituted at any one, any two, or all three of said serines. Furthermore, any polypeptide amino acid capable of being modified can be excluded from substitution with a modification-mimicking amino acid.
  • the present invention is further directed to fragments of the polypeptides of the present invention. More specifically, the present invention embodies purified, isolated, and recombinant polypeptides comprising at least any one integer between 6 and 504 (or the length of the polypeptides amino acid residues minus 1 if the length is less than 1000) of consecutive amino acid residues. Preferably, the fragments are at least 6, preferably at least 8 to 10, more preferably 12, 15, 20, 25, 30, 35, 40, 50 or more consecutive amino acids of a polypeptide of the present invention.
  • the present invention also provides for the exclusion of any species of polypeptide fragments of the present invention specified by 5' and 3' positions or sub-genuses of polypeptides specified by size in amino acids as described above. Any number of fragments specified by 5' and 3' positions or by size in amino acids, as described above, can be excluded.
  • the peptides of the present invention include two or more modifications, including, but not limited to those described herein.
  • PEPTIDES PEPTIDE VARIANTS, AND PEPTIDE MIMETICS
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
  • Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-I, -2,3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4- isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)- phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy- biphenylphenylalan
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Protein as used herein includes peptides that are conservative variations of those peptides specifically exemplified herein.
  • Consservative variation denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include, but are not limited to, the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine.
  • the term “conservative variation” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides of the invention.
  • the biological activity of the peptides can be determined by standard methods known to those of skill in the art, such as those described in the Examples.
  • the peptides and polypeptides of the invention include all “mimetic” and “peptidomimetic” forms.
  • the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides of the invention.
  • the mimetic can be either entirely composed of synthetic, non- natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or activity.
  • a mimetic composition is within the scope of the invention if, when administered to or expressed in a cell, e.g., a polypeptide fragment of an antimicrobial and antiproliferative protein having antimicrobial and antiproliferative activity.
  • Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • peptide bonds can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'- diisopropylcarbodiimide (DIC).
  • DCC N,N'-dicyclohexylcarbodiimide
  • DIC N,N'- diisopropylcarbodiimide
  • Mimetics of acidic amino acids can be generated by substitution by, e.g., non-carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
  • Carboxyl side groups e.g., aspartyl or glutamyl
  • Carboxyl side groups can also be selectively modified by reaction with carbodiimides (R' — N — C — N — R') such as, e.g., l-cyclohexyl-3(2-morpholin-yl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide.
  • Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, guanidino-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
  • Nitrile derivative e.g., containing the CN-moiety in place of COOH
  • Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
  • Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
  • Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane.
  • N- acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
  • alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines
  • Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo- trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p- chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
  • cysteinyl residues e.g., bromo- trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid
  • chloroacetyl phosphate N- alkylmaleimides
  • 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
  • Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino- containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
  • Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
  • Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
  • mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha- amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C- terminal carboxyl groups.
  • a component of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form
  • the invention also provides polypeptides that are "substantially identical" to an exemplary polypeptide of the invention.
  • a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
  • a conservative amino acid substitution substitutes one amino acid for another of the same class ⁇ e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
  • One or more amino acids can be deleted, for example, from an antimicrobial polypeptide having antimicrobial activity of the invention, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal, or internal, amino acids that are not required for antimicrobial activity can be removed.
  • Modified peptides of the invention can be further produced by chemical modification methods, see, e.g., Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994.
  • Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers, Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems, 1995.
  • peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge, Science 269: 202, 1995; Merrifield, Methods Enzymol. 289: 3-13, 1997) and automated synthesis can be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • Peptides of the invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (See, Coligan et al, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods described in Merrifield, J. Am. Chem. Soc.
  • Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
  • Analogs polypeptide fragment of antimicrobial protein having antimicrobial activity, are generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein, such as any of the sequences including SEQ ID NOS: 1-17.
  • Polypeptide includes proteins, fusion proteins, oligopeptides and polypeptide derivatives, with the exception that peptidomimetics are considered to be small molecules herein.
  • a “protein” is a molecule having a sequence of amino acids that are linked to each other in a linear molecule by peptide bonds. Protein refers to a polypeptide that is isolated from a natural source, or produced from an isolated cDNA using recombinant DNA technology; and has a sequence of amino acids having a length of at least about 200 amino acids.
  • a “fusion protein” is a type of recombinant protein that has an amino acid sequence that results from the linkage of the amino acid sequences of two or more normally separate polypeptides.
  • a “protein fragment” is a proteolytic fragment of a larger polypeptide, which may be a protein or a fusion protein.
  • a proteolytic fragment may be prepared by in vivo or in vitro proteolytic cleavage of a larger polypeptide, and is generally too large to be prepared by chemical synthesis.
  • Proteolytic fragments have amino acid sequences having a length from about 200 to about 1,000 amino acids.
  • oligopeptide or “peptide” is a polypeptide having a short amino acid sequence (i.e., 2 to about 200 amino acids).
  • An oligopeptide is generally prepared by chemical synthesis. Although oligopeptides and protein fragments may be otherwise prepared, it is possible to use recombinant DNA technology and/or in vitro biochemical manipulations.
  • a nucleic acid encoding an amino acid sequence may be prepared and used as a template for in vitro transcription/translation reactions. In such reactions, an exogenous nucleic acid encoding a preselected polypeptide is introduced into a mixture that is essentially depleted of exogenous nucleic acids that contains all of the cellular components required for transcription and translation.
  • Radiolabeled amino acids are added before or with the exogenous DNA, and transcription and translation are allowed to proceed. Because the only nucleic acid present in the reaction mix is the exogenous nucleic acid added to the reaction, only polypeptides encoded thereby are produced, and incorporate the radiolabeled amino acid(s). In this manner, polypeptides encoded by a preselected exogenous nucleic acid are radiolabeled. Although other proteins are present in the reaction mix, the preselected polypeptide is the only one that is produced in the presence of the radiolabeled amino acids and is thus uniquely labeled.
  • polypeptide derivatives include without limitation mutant polypeptides, chemically modified polypeptides, and peptidomimetics.
  • polypeptides of this invention may generally be prepared following known techniques.
  • synthetic production of the polypeptide of the invention may be according to the solid phase synthetic method.
  • the solid phase synthesis is well understood and is a common method for preparation of polypeptides, as are a variety of modifications of that technique.
  • polypeptides of this invention may be prepared in recombinant systems using polynucleotide sequences encoding the polypeptides.
  • polypeptide derivatives include without limitation proteins that naturally undergo post- translational modifications such as, e.g., glycosylation. It is understood that a polypeptide of the invention may contain more than one of the following modifications within the same polypeptide.
  • Preferred polypeptide derivatives retain a desirable attribute, which may be biological activity; more preferably, a polypeptide derivative is enhanced with regard to one or more desirable attributes, or has one or more desirable attributes not found in the parent polypeptide. Although they are described in this section, peptidomimetics are taken as small molecules in the present disclosure.
  • a polypeptide having an amino acid sequence identical to that found in a protein prepared from a natural source is a "wild type" polypeptide.
  • Functional variants of polypeptides can be prepared by chemical synthesis, including without limitation combinatorial synthesis.
  • polypeptides larger than oligopeptides can be prepared using recombinant DNA technology by altering the nucleotide sequence of a nucleic acid encoding a polypeptide. Although some alterations in the nucleotide sequence will not alter the amino acid sequence of the polypeptide encoded thereby ("silent" mutations), many will result in a polypeptide having an altered amino acid sequence that is altered relative to the parent sequence. Such altered amino acid sequences may comprise substitutions, deletions and additions of amino acids, with the proviso that such amino acids are naturally occurring amino acids.
  • subjecting a nucleic acid that encodes a polypeptide to mutagenesis is one technique that can be used to prepare functional variants of polypeptides, particularly ones having substitutions of amino acids but no deletions or insertions thereof.
  • a variety of mutagenic techniques are known that can be used in vitro or in vivo including without limitation chemical mutagenesis and PCR-mediated mutagenesis.
  • Such mutagenesis may be randomly targeted (i.e., mutations may occur anywhere within the nucleic acid) or directed to a section of the nucleic acid that encodes a stretch of amino acids of particular interest. Using such techniques, it is possible to prepare randomized, combinatorial or focused compound libraries, pools and mixtures.
  • Polypeptides having deletions or insertions of naturally occurring amino acids may be synthetic oligopeptides that result from the chemical synthesis of amino acid sequences that are based on the amino acid sequence of a parent polypeptide but which have one or more amino acids inserted or deleted relative to the sequence of the parent polypeptide. Insertions and deletions of amino acid residues in polypeptides having longer amino acid sequences may be prepared by directed mutagenesis.
  • polypeptide includes those having one or more chemical modification relative to another polypeptide, Le., chemically modified polypeptides.
  • the polypeptide from which a chemically modified polypeptide is derived may be a wild type protein, a functional variant protein or a functional variant polypeptide, or polypeptide fragments thereof; an antibody or other polypeptide ligand according to the invention including without limitation single-chain antibodies, crystalline proteins and polypeptide derivatives thereof; or polypeptide ligands prepared according to the disclosure.
  • the chemical modification(s) confer(s) or improve(s) desirable attributes of the polypeptide but does not substantially alter or compromise the biological activity thereof.
  • Desirable attributes include but are limited to increased shelf-life; enhanced serum or other in vivo stability; resistance to proteases; and the like. Such modifications include by way of non-limiting example N-terminal acetylation, glycosylation, and biotinylation.
  • An effective approach to confer resistance to peptidases acting on the N-terminal or C- terminal residues of a polypeptide is to add chemical groups at the polypeptide termini, such that the modified polypeptide is no longer a substrate for the peptidase.
  • One such chemical modification is glycosylation of the polypeptides at either or both termini.
  • Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of polypeptides inhuman serum. Powell et al, Pharma. Res. 10: 1268-1273, 1993.
  • N-terminal alkyl group consisting of a lower alkyl of from 1 to 20 carbons, such as an acetyl group, and/or the addition of a C-terminal amide or substituted amide group.
  • N-terminal D-amino acid increases the serum stability of a polypeptide that otherwise contains L-amino acids, because exopeptidases acting on the N- terminal residue cannot utilize a D-amino acid as a substrate.
  • C- terminal D-amino acid also stabilizes a polypeptide, because serum exopeptidases acting on the C-terminal residue cannot utilize a D-amino acid as a substrate.
  • amino acid sequences of polypeptides with N-terminal and/or C- terminal D-amino acids are usually identical to the sequences of the parent L-amino acid polypeptide.
  • Substitution of unnatural amino acids for natural amino acids in a subsequence of a polypeptide can confer or enhance desirable attributes including biological activity. Such a substitution can, for example, confer resistance to proteolysis by exopeptidases acting on the N- terminus.
  • the synthesis of polypeptides with unnatural amino acids is routine and known in the art (see, for example, Coller, et al. 1993, cited above).
  • Different host cells will contain different post-translational modification mechanisms that may provide particular types of post-translational modification of a fusion protein if the amino acid sequences required for such modifications is present in the fusion protein.
  • a large number (about 100) of post-translational modifications have been described, a few of which are discussed herein.
  • One skilled in the art will be able to choose appropriate host cells, and design chimeric genes that encode protein members comprising the amino acid sequence needed for a particular type of modification.
  • Glycosylation is one type of post-translational chemical modification that occurs in many eukaryotic systems, and may influence the activity, stability, pharmacogenetics, immunogenicity and/or antigenicity of proteins.
  • Saccharomyces cerevisieae and Pichia pastoris provide for the production of glycosylated proteins, as do expression systems that utilize insect cells, although the pattern of glyscoylation may vary depending on which host cells are used to produce the fusion protein.
  • Another type of post-translation modification is the phosphorylation of a free hydroxyl group of the side chain of one or more Ser, Thr or Tyr residues, Protein kinases catalyze such reactions. Phosphorylation is often reversible due to the action of a protein phosphatase, an enzyme that catalyzes the dephosphorylation of amino acid residues.
  • bacterial proteins are synthesized with an amino terminal amino acid that is a modified form of methionine, i.e., N-formyl-methionine (fMet).
  • fMet N-formyl-methionine
  • the statement is often made that all bacterial proteins are synthesized with an fMet initiator amino acid; although this may be true for E. coli, recent studies have shown that it is not true in the case of other bacteria such as Pseudomonas aeruginosa. Newton et al, J. Biol. Chem. 274: 22143-22146, 1999. hi any event, in E.
  • E. coli mutants that lack the enzymes (such as, e.g., formylase) that catalyze such post-translational modifications will produce proteins having an amino terminal fMet residue (Guillon et al, J. Bacteriol. 174: 4294-4301, 1992).
  • acetylation of the initiator methionine residue, or the penultimate residue if the initiator methionine has been removed typically occurs co- or post-translationally.
  • the acetylation reactions are catalyzed by N-terminal acetyltransferases (NATs, a.k.a. N-alpha- acetyltransferases), whereas removal of the initiator methionine residue is catalyzed by methionine aminopeptidases (for reviews, see Bradshaw et al, Trends Biochem. ScL 23: 263- 267, 1998; and Driessen et al, CRC CHt. Rev. Biochem. 18: 281-325, 1985).
  • NATs N-terminalpha- acetyltransferases
  • Amino terminally acetylated proteins are said to be "N-acetylated,” “N alpha acetylated” or simply “acetylated.”
  • Another post-translational process that occurs in eukaryotes is the alpha-amidation of the carboxy terminus.
  • About 50% of known endocrine and neuroendocrine peptide hormones are alpha-amidated (Treston et al, Cell Growth Differ. 4: 911- 920, 1993). In most cases, carboxy alpha-amidation is required to activate these peptide hormones.
  • a polypeptide mimetic is a molecule that mimics the biological activity of a polypeptide but is no longer peptidic in chemical nature.
  • a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids).
  • the term peptidomimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi- peptides and peptoids. Examples of some peptidomimetics by the broader definition (where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below.
  • peptidomimetics Whether completely or partially non-peptide, peptidomimetics according to this invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the polypeptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems that are similar to the biological activity of the polypeptide.
  • polypeptides may exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action.
  • Peptidomimetics are often small enough to be both orally active and to have a long duration of action.
  • stability, storage and immunoreactivity for polypeptides that are not experienced with peptidomimetics There are also problems associated with stability, storage and immunoreactivity for polypeptides that are not experienced with peptidomimetics .
  • Candidate, lead and other polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities.
  • Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • the development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling.
  • the present invention provides compounds exhibiting enhanced therapeutic activity in comparison to the polypeptides described above.
  • the peptidomimetic compounds obtained by the above methods having the biological activity of the above named polypeptides and similar three-dimensional structure, are encompassed by this invention. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any of the modified polypeptides described in the previous section or from a polypeptide bearing more than one of the modifications described from the previous section. It will furthermore be apparent that the peptidomimetics of this invention can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.
  • Proteases act on peptide bonds. It therefore follows that substitution of peptide bonds by pseudopeptide bonds confers resistance to proteolysis. A number of pseudopeptide bonds have been described that in general do not affect polypeptide structure and biological activity.
  • the reduced isosteric pseudopeptide bond is a suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity (Couder et al, Int. J. Polypeptide Protein Res. 41: 181-184, 1993, incorporated herein by reference).
  • the amino acid sequences of these compounds may be identical to the sequences of their parent L-amino acid polypeptides, except that one or more of the peptide bonds are replaced by an isosteric pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • peptide bonds may also be substituted by retro- inverso pseudopeptide bonds (Dalpozzo et at, Int. J. Polypeptide Protein Res. 41: 561-566, incorporated herein by reference).
  • the amino acid sequences of the compounds may be identical to the sequences of their L-amino acid parent polypeptides, except that one or more of the peptide bonds are replaced by a retro-inverso pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • Peptoid derivatives of polypeptides represent another form of modified polypeptides that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon et ah, Proc. Natl. Acad. ScL USA 89: 9367-9371, 1992, and incorporated herein by reference).
  • Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid.
  • RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • nucleic acid sample comprising niRNA transcript(s) of the gene or genes, or nucleic acids derived from the mRNA transcript(s) is provided.
  • a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template.
  • a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample.
  • suitable samples include mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
  • a nucleic acid sample is the total mRNA isolated from a biological sample.
  • biological sample refers to a sample obtained from an organism or from components (e.g., cells) or an organism.
  • the sample can be of any biological tissue or fluid. Frequently the sample is from a patient.
  • samples include sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and fleural fluid, or cells therefrom.
  • Biological samples can also include sections of tissues such as frozen sections taken for histological purposes. Often two samples are provided for purposes of comparison.
  • the samples can be, for example, from different cell or tissue types, from different species, from different individuals in the same species or from the same original sample subjected to two different treatments (e.g., drug-treated and control).
  • the invention includes polynucleotides encoding chemokines of the invention.
  • polynucleotide refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct.
  • DNA encoding a chemokine of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
  • Polynucleotide sequences of the invention include DNA, RNA and cDNA sequences.
  • polynucleotide sequence can be deduced from the genetic code, however, the degeneracy of the code must be taken into account.
  • Polynucleotides of the invention include sequences which are degenerate as a result of the genetic code. Such polynucleotides are useful for the recombinant production of large quantities of a peptide of interest.
  • Recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams, J. Am. Chem. Soc. 105: 661, 1983; Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994; Narang, Meth. Enzymol. 68: 90, 1979; Brown Meth. Enzymol. 68: 109, 1979; Beaucage, Tetra. Lett. 22: 1859, 1981; U.S. Pat. No. 4,458,066.
  • the invention provides oligonucleotides comprising sequences of the invention, e.g., subsequences of the exemplary sequences of the invention.
  • Oligonucleotides can include, e.g., single stranded poly-deoxynucleotides or two complementary polydeoxynucleotide strands which can be chemically synthesized.
  • nucleic acids such as, e.g., subcloning, labeling probes ⁇ e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., See, for example, Sambrook, Fitsch & Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2 nd , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.
  • Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g.
  • Obtaining and manipulating nucleic acids used to practice the methods of the invention can be done by cloning from genomic samples, and, if desired, screening and re-cloning inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld, Nat. Genet.
  • MACs mammalian artificial chromosomes
  • yeast artificial chromosomes YAC
  • bacterial artificial chromosomes BAC
  • Pl artificial chromosomes see, e.g., Woon, Genomics 50: 306-316, 1998
  • Pl-derived vectors see, e.g., Kern, Biotechniques 23:120- 124, 1997
  • cosmids recombinant viruses, phages or plasmids.
  • the invention provides fusion proteins and nucleic acids encoding them.
  • a gene product or polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N- terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.).
  • metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle Wash.
  • the inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego, CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification.
  • an expression vector can include an epitope- encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams, Biochemistry 34: 1787-1797, 1995; Dobeli, Protein Expr. Purif 12: 404-414, 1998).
  • the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.
  • the nucleic acids of the invention can be operatively linked to a promoter.
  • a promoter can be one motif or an array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a "constitutive" promoter is a promoter which is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter which is under environmental or developmental regulation.
  • tissue specific promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the proteins of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of S V40), Pl-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast). See, for example, 5,707,855.
  • Vectors of the invention can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.
  • nucleic acids of the invention can be cloned, if desired, into any of a variety of vectors using routine molecular biological methods; methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039.
  • restriction enzyme sites can be "built into” a PCR primer pair.
  • the invention provides libraries of expression vectors encoding polypeptides and peptides of the invention. These nucleic acids can be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, e.g., Roberts, Nature 328: 731, 1987; Schneider, Protein Expr. Purif. 6435: 10, 1995; Sambrook, Tijssen or Ausubel.
  • the vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are stably or transiently expressed in cells ⁇ e.g., episomal expression systems).
  • Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences.
  • selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
  • the nucleic acids of the invention are administered in vivo for in situ expression of the peptides or polypeptides of the invention.
  • the nucleic acids can be administered as "naked DNA” (see, e.g., U.S. Pat. No. 5,580,859) or in the form of an expression vector, e.g., a recombinant virus.
  • the nucleic acids can be administered by any route, including peri- or intra-tumorally, as described below.
  • Vectors administered in vivo can be derived from viral genomes, including recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxyiridae, adenoviridiae, or picornnaviridiae. Chimeric vectors can also be employed which exploit advantageous merits of each of the parent vector properties (See e.g., Feng, Nature Biotechnology 15: 866-870, 1997). Such viral genomes can be modified by recombinant DNA techniques to include the nucleic acids of the invention; and can be further engineered to be replication deficient, conditionally replicating or replication competent.
  • vectors are derived from the adenoviral (e.g., replication incompetent vectors derived from the human adenovirus genome, see, e.g., U.S. Pat. Nos. 6,096,718; 6,110,458; 6,113,913; 5,631,236); adeno-associated viral and retroviral genomes.
  • Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof; see, e.g., U.S. Pat. Nos.
  • Adeno-associated virus (AAV)-based vectors can be used to adioimmun cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures; see, e.g., U.S. Pat. Nos. 6,110,456; 5,474,935; Okada, Gene Titer. 3: 957-964, 1996.
  • Expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as a polypeptide of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression can also be used, e.g., enhancers.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • Vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors ⁇ e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a. sequence encoding a polypeptide of the invention, or a vector of the invention.
  • the host cell can be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhirnurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
  • Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art.
  • the vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation.
  • Engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter can be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells can be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
  • Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
  • the expressed polypeptide or fragment can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK cell lines.
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides produced by host cells containing the vector may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may or may not also include an initial methionine amino acid residue.
  • Cell-free translation systems can also be employed to produce a polypeptide of the invention.
  • Cell-free translation systems can use mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding the polypeptide or fragment thereof, hi some aspects, the DNA construct can be linearized prior to conducting an in vitro transcription reaction. The transcribed mRNA is then incubated with an appropriate cell-free translation extract, such as a rabbit reticulocyte extract, to produce the desired polypeptide or fragment thereof.
  • an appropriate cell-free translation extract such as a rabbit reticulocyte extract
  • the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • nucleic acids encoding the polypeptides of the invention, or modified nucleic acids can be reproduced by, e.g., amplification.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids encoding polypeptides of the invention, e.g., primer pairs capable of amplifying nucleic acid sequences comprising the chemokine protein or related protein sequences, or subsequences thereof.
  • Amplification methods include, e.g., polymerase chain reaction, PCR (Per Protocols, A Guide To Methods And Applications, ed. Innis, Academic Press, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press, Lie., N. Y., ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4: 560, 1989; Landegren, Science 241: 1077, 1988; Barringer, Gene 89: 117, 1990); transcription amplification (see, e.g., Kwoh, Proc. Natl. Acad.
  • LCR ligase chain reaction
  • the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention, e.g., a sequence or related sequence, or the complement of any thereof, or a nucleic acid that encodes a polypeptide of the invention (See also SEQ ID NO: 1-17).
  • the stringent conditions are highly stringent conditions, medium stringent conditions or low stringent conditions, as known in the art and as described herein. These methods can be used to isolate nucleic acids of the invention.
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or more residues in length, or, the full length of a gene or coding sequence, e.g., cDNA. Nucleic acids shorter than full length are also included.
  • nucleic acids can be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.
  • a nucleic acid can be determined to be within the scope of the invention by its ability to hybridize under stringent conditions to a nucleic acid otherwise determined to be within the scope of the invention (such as the exemplary sequences described herein).
  • Stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but not to other sequences in significant amounts (a positive signal (e.g., identification of a nucleic acid of the invention) is about 10 times background hybridization). Stringent conditions are sequence- dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in, e.g., Sambrook, ed., 1989; Ausubel, ed. 1997; Tijssen, ed., 1993, supra).
  • stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point I for the specific sequence at a defined ionic strength pH.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30oC for short probes (e.g., 10 to 50 nucleotides) and at least about 60oC for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide as described in Sambrook (cited below).
  • destabilizing agents such as formamide as described in Sambrook (cited below).
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65° C, with a wash in 0.2x SSC and 0.1% SDS at 65° C.
  • a positive signal e.g., identification of a nucleic acid of the invention
  • Stringent hybridization conditions that are used to identify nucleic acids within the scope of the invention include, e.g.
  • genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. Additional stringent conditions for such hybridizations (to identify nucleic acids within the scope of the invention) are those which include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C.
  • wash conditions used to identify nucleic acids within the scope of the invention include, e.g., a.
  • the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68°C for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen and Ausubel for a description of SSC buffer and equivalent conditions.
  • the invention also provides nucleic acid probes for identifying nucleic acids encoding a polypeptide which is a modulator immunomodulatory and -signaling activity.
  • the probe comprises at least 10 consecutive bases of a nucleic acid of the invention.
  • a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
  • the probes identify a nucleic acid by binding and/or hybridization.
  • the probes can be used in arrays of the invention, see discussion below.
  • the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
  • the invention provides nucleic acids having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an immunomodulatory and anti-proliferative chemokine polynucleotide or related polynucleotide of the invention.
  • the invention provides polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an immunomodulatory and anti-proliferative chemokine protein or related protein.
  • the sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Protein and/or nucleic acid sequence identities (homologies) can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of nucleic acids and proteins the BLAST and BLAST 2.2.2. or FASTA version 3.0t78 algorithms and the default parameters discussed below can be used.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • FASTA FASTA algorithm
  • Pearson & Lipman Proc. Natl. Acad. ScL U.S.A. 85: 2444, 1988. See also Pearson, Methods Enzymol. 266: 227-258, 1996.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5787, 1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. MoI. Evol. 35: 351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989.
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
  • This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences.
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12: 387-395, 1984.)
  • ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05 respectively.
  • the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sd. U.S.A. 89: 10915-10919, 1992).
  • Sequence identity refers to a measure of similarity between amino acid or nucleotide sequences, and can be measured using methods known in the art, such as those described below:
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • “Substantially identical,” in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least of at least 60%, often at least 70%, preferably at least 80%, most preferably at least 90% or at least 95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 bases or residues in length, more preferably over a region of at least about 100 bases or residues, and most preferably the sequences are substantially identical over at least about 150 bases or residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • “Homology” and “identity” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence comparison one sequence can act as a reference sequence (an exemplary sequence of an immunomodulatory and antiproliferative chemokine gene product or related gene product or polynucleotide or polypeptide) to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the numbers of contiguous residues.
  • contingous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention, e.g., an immunomodulatory and anti-proliferative chemokine polynucleotide or polypeptide, are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an immunomodulatory and anti-proliferative chemokine polynucleotide or polypeptide, that sequence is within the scope of the invention.
  • Motifs which can be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
  • the chemokine polynucleotide sequence used according to the method of the invention can be isolated from an organism or synthesized in the laboratory. Specific DNA sequences encoding the chemokine of interest can be obtained by: 1) isolation of a double-stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the cationic peptide of interest; and 3) in vitro synthesis of a double- stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • DNA sequences are frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known.
  • the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons that are more likely to be recognized by a bacterial host, thereby permitting high level expression without difficulties in translation.
  • virtually any peptide can be synthesized, including those encoding natural chemokines, variants of the same, or synthetic peptides.
  • cDNA sequences When the entire sequence of the desired peptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences.
  • the standard procedures for isolating cDNA sequences of interest is the formation of plasmid or phage containing cDNA libraries that are derived from reverse transcription of mRNA that is abundant in donor cells that have a high level of genetic expression.
  • plasmid or phage containing cDNA libraries that are derived from reverse transcription of mRNA that is abundant in donor cells that have a high level of genetic expression.
  • the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single stranded form (Jay, et al, Nuc. Acid Res., 11:2325, 1983).
  • the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
  • the words "recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media can be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
  • the terms "computer,” “computer program” and “processor” are used in their broadest general contexts and incorporate all such devices.
  • the invention further provides for nucleic acids complementary to (e.g., antisense sequences to) the nucleic acid sequences of the invention.
  • Antisense sequences are capable of modulating or inhibiting the transport, splicing or transcription of protein-encoding genes, e.g., immunomodulatory and anti-proliferative-encoding nucleic acids, differentially expressed nucleic acids, related nucleic acids and the like.
  • the modulation or inhibition can be effected through the targeting of genomic DNA or messenger RNA.
  • the transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
  • One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind gene or message, in either case preventing or inhibiting the production or function of the protein. The association can be through sequence specific hybridization.
  • Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of protein message.
  • the oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes.
  • the oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One can screen a pool of many different such oligonucleotides for those with the desired activity.
  • RNAi RNA interference
  • RNAi encompasses molecules such as short interfering RNA (siRNA), microRNAs (mRNA), small temporal RNA (stRNA).
  • siRNA short interfering RNA
  • mRNA microRNAs
  • stRNA small temporal RNA
  • the invention provides antisense oligonucleotides capable of binding immunomodulatory and antiproliferative messenger RNA which can inhibit polypeptide activity by targeting mRNA.
  • Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such oligonucleotides using the novel reagents of the invention.
  • gene walking/RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho, Methods Enzymol. 314: 168-183, 2000, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith, EMr. /. Pharm. Sci. 11: 191-198, 2000.
  • Naturally occurring nucleic acids are used as antisense oligonucleotides.
  • the antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening.
  • the antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem.
  • peptide nucleic acids containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used.
  • Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata, Toxicol Appl Pharmacol 144: 189-197, 1997; Antisense Therapeutics, ed. Agrawal, Humana Press, Totowa, NJ., 1996.
  • Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'- thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
  • Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense polypeptides sequences of the invention (see, e.g., Gold, J. of Biol. Chem. 270: 13581-13584, 1995).
  • siRNA refers to double-stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability to specifically interfere with protein expression through RNA interference (RNAi).
  • RNAi RNA interference
  • siRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more.
  • RNAi is a two-step mechanism. Elbashir et al, Genes Dev., 15: 188-200, 2001.
  • siRNAs are cleaved by an enzyme known as Dicer in 21-23 ribonucleotide (nt) fragments, called small interfering RNAs (siRNAs). Then, siRNAs associate with a ribonuclease complex (termed RISC for RNA Induced Silencing Complex) which target this complex to complementary mRNAs. RISC then cleaves the targeted mRNAs opposite the complementary siRNA, which makes the mRNA susceptible to other RNA degradation pathways.
  • siRNAs of the present invention are designed to interact with a target ribonucleotide sequence, meaning they complement a target sequence sufficiently to bind to the target sequence.
  • the present invention also includes siRNA molecules that have been chemically modified to confer increased stability against nuclease degradation, but retain the ability to bind to target nucleic acids that may be present.
  • the invention provides ribozymes capable of binding message which can inhibit polypeptide activity by targeting mRNA, e.g., inhibition of polypeptides with immunomodulatory and antiproliferative activity, e.g., signaling activity.
  • ribozymes capable of binding message which can inhibit polypeptide activity by targeting mRNA, e.g., inhibition of polypeptides with immunomodulatory and antiproliferative activity, e.g., signaling activity.
  • Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA.
  • the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence.
  • a ribozyme After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
  • a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide.
  • antisense technology where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule
  • This potential advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
  • the enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but can also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RnaseP-like RNA (in association with an RNA guide sequence).
  • hammerhead motifs are described by Rossi, Aids Research and Human Retroviruses 8: 183, 1992; hairpin motifs by Hampel, Biochemistry 28: 4929, 1989, and Hampel, Nuc. Acids Res.
  • RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.
  • the present invention provides novel methods for screening for compositions to treat, for example, tumor cell differentiation or viral infection, in a mammalian subject.
  • the expression levels of genes are determined for different cellular states of cancer or non-cancer (or viral vs. non-viral) to provide expression profiles.
  • a cancer cell expression profile of a particular cancer cell state can be a "fingerprint" of the state; while two states can have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell.
  • the evaluation of a particular treatment regime can be evaluated: does an tumor cell drug act like an tumor cell drug in this particular patient.
  • diagnosis can be done or confirmed: does this patient have the gene expression profile of cancer cell state.
  • these gene expression profiles can be used in drug candidate screening to find drugs that mimic a particular expression profile; for example, screening can be done for drugs that treat cancer as evidenced by a non-cancer expression profile. Accordingly, genes are identified and described which are differentially expressed within and among cancer or non-cancer cells in different states, from which the expression profiles are generated as further described herein. For example, determinations of differentially expressed nucleic acids are provided in the examples.
  • Gene expression profile or “gene expression profile set” refers to the set of genes of a specific tissue or cell type that are transcribed or “expressed” to form RNA molecules. Which genes are expressed in a specific cell line or tissue can depend on factors such as tissue or cell type, stage of development or the cell, tissue, or target organism and whether the cells are normal or transformed cells, such as cancerous cells. For example, a gene can be expressed at the embryonic or fetal stage in the development of a specific target organism and then become non- expressed as the target organism matures. Alternatively, a gene can be expressed in liver tissue but not in brain tissue of an adult human.
  • Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Stringent conditions are conditions under which a probe can hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • stringent conditions include a salt concentration of at least about 0.01 to 1.0 M Na+ concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide or tetraalkyl ammonium salts.
  • 5X SSPE 750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-3O 0 C. are suitable for allele-specific probe hybridizations.
  • sequence relationships between two or more nucleotide sequences or amino acid sequences include “reference sequence,” “selected from,” “comparison window,” “identical,” “percentage of sequence identity,” “substantially identical,” “complementary,” and “substantially complementary.”
  • differential expression refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among cancer cells or non-cancer cells.
  • a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation in, for example, tolerant versus immunosuppressed cells, rested, naive or activated cells, or in a cancer cell state vs. non-cancer cell state (or viral infected cell state vs. non- viral infected cell state).
  • Genes can be turned on or turned off in a particular state, relative to another state. Any comparison of two or more states can be made.
  • Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which can be detectable by standard techniques in one such state or cell type, but can be not detectable in both.
  • the determination can be quantitative in that expression is increased or decreased; that is, the expression of the gene is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript.
  • the degree to which expression differs need only be large enough to quantify using standard characterization techniques, for example, by using Affymetrix GeneChipTM expression arrays. Lockhart, Nature Biotechnology 14: 1675-1680, 1996; this reference and all references cited therein are incorporated by reference.
  • Other methods include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis and RNase protection.
  • the change or modulation in expression ⁇ i.e., upregulation or downregulation is at least about 5%, more preferably at least about 10%, more preferably, at least about 20%, more preferably, at least about 30%, or more preferably by at least about 50%, or at least about 75%, and more preferably at least about 90%.
  • genes can be evaluated. These genes include, but are not limited to, the genes disclosed herein (the accession numbers for these genes can be found in the tables). Generally, oligonucleotide sequences used in the evaluation of these genes are derived from their 3' untranslated regions.
  • Differentially expressed genes can represent "expression profile genes", which includes “target genes".
  • “Expression profile gene,” as used herein, refers to a differentially expressed gene whose expression pattern can be used in methods for identifying compounds useful in the modulation of cancer cell or non-cancer cell states or activity, or the treatment of diseases or disorders, or alternatively, the gene can be used as part of a prognostic or diagnostic evaluation of immune disorders.
  • the effect of the compound on the expression profile gene normally displayed in connection with a particular state for example, can be used to evaluate the efficacy of the compound to modulate that state, or preferably, to induce or maintain that state.
  • the gene can be used as a diagnostic or in the treatment of immune disorders as also further described below.
  • Expression profile refers to the pattern of gene expression generated from two up to all of the expression profile genes which exist for a given state. As outlined above, an expression profile is in a sense a "fingerprint” or “blueprint” of a particular cellular state; while two or more states have genes that are similarly expressed, the total expression profile of the state will be unique to that state.
  • the gene expression profile obtained for a given cancer cell or non-cancer cell state can be useful for a variety of applications, including diagnosis of a particular disease or condition and evaluation of various treatment regimes. In addition, comparisons between the expression profiles of different cancer cell or non-cancer cell states can be similarly informative.
  • An expression profile can include genes which do not appreciably change between two states, so long as at least two genes which are differentially expressed are represented.
  • the gene expression profile can also include at least one target gene, as defined below.
  • the profile can include all of the genes which represent one or more states. Specific expression profiles are described below.
  • Gene expression profiles can be defined in several ways. For example, a gene expression profile can be the relative transcript level of any number of particular set of genes. Alternatively, a gene expression profile can be defined by comparing the level of expression of a variety of genes in one state to the level of expression of the same genes in another state. For example, genes can be either upregulated, downregulated, or remain substantially at the same level in both states.
  • Target gene refers to a differentially expressed expression profile gene whose expression is unique for a particular state, such that the presence or absence of the transcript of a target gene(s) can indicate the state the cell is in.
  • a target gene can be completely unique to a particular state; the presence or absence of the gene is only seen in a particular cell state, or alternatively, cells in all other states express the gene but it is not seen in the first state.
  • target genes can be identified as relevant to a comparison of two states, that is, the state is compared to another particular state or standard to determine the uniqueness of the target gene.
  • Target genes can be used in the diagnostic, prognostic, and compound identification methods described herein.
  • a target gene for a first state can be an expression profile gene for a second state.
  • the presence or absence of a particular target gene in one state can be diagnostic of the state; the same gene in a different state can be an expression profile gene.
  • pathway genes are provided herein. "Pathway genes" are defined by the ability of their gene products to interact with expression profile genes. Pathway genes can also exhibit target gene and/or expression profile gene characteristics and can be included as modulators of expression profile genes as further described below.
  • the present invention includes the products of such expression profile, target, and pathway genes to such gene products. Furthermore, the engineering and use of cell- and animal- based models, for example, of various cancer cell or non-cancer cell states to which such profiles, genes and gene products can contribute, are also described.
  • nucleic acid arrays There are two principal categories of nucleic acid arrays.
  • One type of array detects the presence and/or levels of particular mRNA sequences that are known in advance.
  • polynucleotide probes can be selected to hybridize to particular preselected subsequences of mRNA gene sequence.
  • Such expression monitoring arrays can include a plurality of probes for each mRNA to be detected.
  • the probes are designed to be complementary to the region of the mRNA that is incorporated into the nucleic acids (i.e., the 3' end).
  • the array can also include one or more control probes.
  • Generic arrays can include all possible nucleotides of a given length; that is, polynucleotides having sequences corresponding to every permutation of a sequence.
  • the polynucleotide probes of this invention preferably include up to 4 bases (A, G, C, T) or (A, G, C, U) or derivatives of these bases, an array having all possible nucleotides of length X contains substantially 4 X different nucleic acids (e.g., 16 different nucleic acids for a 2 mer, 64 different nucleic acids for a 3 mer, 65536 different nucleic acids for an 8 mer).
  • An array comprising all possible nucleotides of length X refers to an array having substantially all possible nucleotides of length X.
  • AU possible nucleotides of length X includes more than 90%, typically more than 95%, preferably more than 98%, more preferably more than 99%, and most preferably more than 99.9% of the possible number of different nucleotides.
  • Generic arrays are particularly useful for comparative hybridization analysis between two mRNA populations or nucleic acids derived therefrom.
  • Microarray technology provides the opportunity to analyze a large number of nucleic acid sequences. This technology can also be utilized for comparative gene expression analysis, drug discovery, and characterization of molecular interactions. With respect to expression analysis, the expression pattern of a particular gene can be used to characterize the function of that gene. In addition, microarrays can be utilized to analyze both the static expression of a gene (e.g., expression in a specific tissue) as well as, dynamic expression of a particular gene (e.g., expression of one gene relative to the expression of other genes) (Duggan et al., Nature Genet. 21: 10-14, 1999).
  • microarray technology is the use of an impermeable, rigid support as compared to the porous membranes used in the traditional blotting methods (e.g., Northern and Southern analyses). Hybridization buffers do not penetrate the support resulting in greater access to the oligonucleotide probes, enhanced rates of hybridization, and improved reproducibility.
  • the microarray technology provides better image acquisition and image processing (Southern et al, Nature Genet. 21: 5-9, 1999).
  • nucleic acids e.g., RNA
  • RNA can be isolated from a biological sample.
  • Nucleic acid samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
  • proteomics offers a promising look at the biological functions of a cell.
  • Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantitating expression at the protein level, rather than at the messenger RNA level.
  • Proteomics also involves the study of non-genome encoded events including the post- translational modification of proteins, interactions between proteins, and the location of proteins within the cell.
  • Standard assays for the presence of an analyte in a solution such as those commonly used for diagnostics, for example, involve the use of an antibody which has been raised against the targeted antigen.
  • Multianalyte assays known in the art involve the use of multiple antibodies and are directed towards assaying for multiple analytes. However, these multianalyte assays have not been directed towards assaying the total or partial protein content of a cell or cell population. Furthermore, sample sizes required to adapt such standard antibody assay approaches to the analysis of even a fraction of the estimated 100,000 or more different proteins of a human cell and their various modified states are prohibitively large. Automation and/or miniaturization of antibody assays are required if large numbers of proteins are to be assayed simultaneously. Materials, surface coatings, and detection methods used for macroscopic immunoassays and affinity purification are not readily transferable to the formation or fabrication of miniaturized protein arrays.
  • DNA biochip technology is not transferable to protein-binding assays such as antibody assays because the chemistries and materials used for DNA biochips are not readily transferable to use with proteins.
  • Nucleic acids such as DNA withstand temperatures up to 100 0 C, can be dried and re- hydrated without loss of activity, and can be bound physically or chemically directly to organic adhesion layers supported by materials such as glass while maintaining their activity.
  • proteins such as antibodies are preferably kept hydrated and at ambient temperatures are sensitive to the physical and chemical properties of the support materials. Therefore, maintaining protein activity at the liquid-solid interface requires entirely different immobilization strategies than those used for nucleic acids.
  • the proper orientation of the antibody or other protein-capture agent at the interface is desirable to ensure accessibility of their active sites with interacting molecules. With miniaturization of the chip and decreased feature sizes, the ratio of accessible to non-accessible and the ratio of active to inactive antibodies or proteins become increasingly relevant and important.
  • Supports can be made of a variety of materials, such as glass, silica, plastic, nylon or nitrocellulose. Supports are preferably rigid and have a planar surface. Supports typically have from 1-10,000,000 discrete spatially addressable regions, or cells. Supports having 10-1,000,000 or 100-100,000 or 1000-100,000 cells are common. The density of cells is typically at least 1000, 10,000, 100,000 or 1,000,000 cells within a square centimeter. Typically a single probe per cell. In some supports, all cells are occupied by pooled mixtures of probes. In other supports, some cells are occupied by pooled mixtures of probes, and other cells are occupied, at least to the degree of purity obtainable by synthesis methods, by a single type of polynucleotide.
  • the strategies for probe design described in the present application can be combined with other strategies, such as those described by WO 95/11995, EP 717,113 and WO 97/29212 in the same array.
  • each different polynucleotide probe in the array is generally known.
  • the large number of different probes can occupy a relatively small area providing a high density array having a probe density of generally greater than about 60, more generally greater than about 100, and most generally greater than about 600, often greater than about 1000, more often greater than about 5,000, most often greater than about 10,000, preferably greater than about 40,000 more preferably greater than about 100,000, and most preferably greater than about 400,000 different polynucleotide probes per cm .
  • the small surface area of the array (often less than about 10 cm , preferably less than about 5 cm more preferably less than about 2 cm 2 , and most preferably less than about 1.6 cm 2 ) permits the use of small sample volumes and extremely uniform hybridization conditions.
  • Arrays of probes can be synthesized in a step-by-step manner on a support or can be attached in presynthesized form.
  • a preferred method of synthesis is VLSIPSTM (see Fodor et al, Nature 364: 555-556, 1993; McGaIl et al, U.S. Ser. No. 08/445,332; U.S. Pat. No. 5,143,854; EP 476,014), which entails the use of light to direct the synthesis of polynucleotide probes in high-density, miniaturized arrays. Algorithms for design of masks to reduce the number of synthesis cycles are described by Hubbel et al, U.S. Pat. No.
  • Arrays can also be synthesized in a combinatorial fashion by delivering monomers to cells of a support by mechanically constrained flowpaths. See Winkler et al, EP 624,059. Arrays can also be synthesized by spotting monomers reagents on to a support using an ink jet printer. See id.; Pease et al, EP 728,520.
  • hybridization intensity for the respective samples is determined for each probe in the array.
  • hybridization intensity can be determined by, for example, a scanning confocal microscope in photon counting mode. Appropriate scanning devices are described by e.g., Trulson et al, U.S. Pat. No. 5,578,832; Stem et al, U.S. Pat. No. 5,631,734 and are available from Affymetrix, Inc., under the GeneChipTM label. Some types of label provide a signal that can be amplified by enzymatic methods (see Broude et al, Proc. Natl. Acad. ScI U.S.A. 91: 3072-3076, 1994).
  • arrays for expression monitoring are generally described, for example, WO 97/27317 and WO 97/10365 (these references are herein incorporated by reference).
  • arrays There are two principal categories of arrays.
  • One type of array detects the presence and/or levels of particular mRNA sequences that are known in advance.
  • polynucleotide probes can be selected to hybridize to particular preselected subsequences of mRNA gene sequence.
  • Such expression monitoring arrays can include a plurality of probes for each mRNA to be detected.
  • the probes are designed to be complementary to the region of the mRNA that is incorporated into the nucleic acids (i.e., the 3' end).
  • the array can also include one or more control probes.
  • Generic arrays can include all possible nucleotides of a given length; that is, polynucleotides having sequences corresponding to every permutation of a sequence.
  • the polynucleotide probes of this invention preferably include up to 4 bases (A, G, C, T) or (A, G, C, U) or derivatives of these bases, an array having all possible nucleotides of length X contains substantially 4 X different nucleic acids (e.g., 16 different nucleic acids for a 2 mer, 64 different nucleic acids for a 3 mer, 65536 different nucleic acids for an 8 mer).
  • An array comprising all possible nucleotides of length X refers to an array having substantially all possible nucleotides of length X.
  • AU possible nucleotides of length X includes more than 90%, typically more than 95%, preferably more than 98%, more preferably more than 99%, and most preferably more than 99.9% of the possible number of different nucleotides.
  • Generic arrays are particularly useful for comparative hybridization analysis between two mRNA populations or nucleic acids derived therefrom.
  • Either customized or generic probe arrays can contain control probes in addition to the probes described above.
  • Normalization controls are typically perfectly complementary to one or more labeled reference polynucleotides that are added to the nucleic acid sample.
  • the signals obtained from the normalization controls after hybridization provide a control for variations in hybridization conditions, label intensity, reading and analyzing efficiency and other factors that can cause the signal of a perfect hybridization to vary between arrays.
  • Signals (e.g., fluorescence intensity) read from all other probes in the array can be divided by the signal (erg., fluorescence intensity) from the control probes thereby normalizing the measurements.
  • Normalization probes can be selected to reflect the average length of the other probes present in the array, however, they can also be selected to cover a range of lengths.
  • the normalization control(s) can also be selected to reflect the (average) base composition of the other probes in the array. However one or a fewer normalization probes can be used and they can be selected such that they hybridize well (i.e. , no secondary structure) and do not match any target-specific probes.
  • Normalization probes can be localized at any position in the array or at multiple positions throughout the array to control for spatial variation in hybridization efficiently.
  • the normalization controls can be located at the corners or edges of the array as well as in the middle of the array.
  • Expression level controls can be probes that hybridize specifically with constitutively expressed genes in the biological sample. Expression level controls can be designed to control for the overall health and metabolic activity of a cell. Examination of the covariance of an expression level control with the expression level of the target nucleic acid can indicate whether measured changes or variations in expression level of a gene is due to changes in transcription rate of that gene or to general variations in health of the cell. Thus, for example, when a cell is in poor health or lacking a critical metabolite the expression levels of both an active target gene and a constitutively expressed gene are expected to decrease. The converse can also be true.
  • the change can be attributed to changes in the metabolic activity of the cell as a whole, not to differential expression of the target gene in question.
  • the expression levels of the target gene and the expression level control do not covary, the variation in the expression level of the target gene can be attributed to differences in regulation of that gene and not to overall variations in the metabolic activity of the cell.
  • Virtually any constitutively expressed gene can provide a suitable target for expression level controls.
  • expression level control probes can have sequences complementary to subsequences of constitutively expressed genes including, but not limited to the B-actin gene, the transferrin receptor gene, the GAPDH gene, and the like.
  • Mismatch controls can also be provided for the probes to the target genes, for expression level controls or for normalization controls. Mismatch controls are typically employed in customized arrays containing probes matched to known mRNA species. For example, some such arrays contain a mismatch probe corresponding to each match probe. The mismatch probe is the same as its corresponding match probe except for at least one position of mismatch.
  • a mismatched base is a base selected so that it is not complementary to the corresponding base in the target sequence to which the probe can otherwise specifically hybridize. One or more mismatches are selected such that under appropriate hybridization conditions (e.g.
  • the test or control probe can be expected to hybridize with its target sequence, but the mismatch probe cannot hybridize (or can hybridize to a significantly lesser extent).
  • Mismatch probes can contain a central mismatch.
  • a corresponding mismatch probe can have the identical sequence except for a single base mismatch (e.g., substituting a G, a C or a T for an A) at any of positions 6 through 14 (the central mismatch).
  • the probes can be provided as pairs where each pair of probes differ in one or more preselected nucleotides.
  • a priori which of the probes in the pair is the perfect match
  • the other probe of the pair can act as a mismatch control for that target sequence.
  • the perfect match and mismatch probes need not be provided as pairs, but can be provided as larger collections (e.g., 3, 4, 5, or more) of probes that differ from each other in particular preselected nucleotides.
  • mismatch probes can provide a control for nonspecific binding or cross-hybridization to a nucleic acid in the sample other than the target to which the probe is complementary. Mismatch probes thus can indicate whether a hybridization is specific or not. For example, if the complementary target is present the perfect match probes can be consistently brighter than the mismatch probes. In addition, if all central mismatches are present, the mismatch probes can be used to detect a mutation. Finally, the difference in intensity between the perfect match and the mismatch probe (1(PM)-I(MM)) can provide a good measure of the concentration of the hybridized material.
  • Sample Preparation, Amplification, and Quantitation Controls can also include sample preparation/amplification control probes. These can be probes that are complementary to subsequences of control genes selected because they do not normally occur in the nucleic acids of the particular biological sample being assayed. Suitable sample preparation/amplification control probes can include, for example, probes to bacterial genes (e.g., Bio B) where the sample in question is a biological sample from a eukaryote.
  • sample preparation/amplification control probes can include, for example, probes to bacterial genes (e.g., Bio B) where the sample in question is a biological sample from a eukaryote.
  • RNA sample can then be spiked with a known amount of the nucleic acid to which the sample preparation/amplification control probe is directed before processing. Quantification of the hybridization of the sample preparation/amplification control probe can then provide a measure of alteration in the abundance of the nucleic acids caused by processing steps (e.g., PCR, reverse transcription, or in vitro transcription).
  • processing steps e.g., PCR, reverse transcription, or in vitro transcription.
  • Quantitation controls can be similar. Typically they can be combined with the sample nucleic acid(s) in known amounts prior to hybridization. They are useful to provide a quantitation reference and permit determination of a standard curve for quantifying hybridization amounts (concentrations).
  • mRNA or nucleic acid derived therefrom are applied to an array.
  • the component strands of the nucleic acids hybridize to complementary probes, which are identified by detecting label.
  • the hybridization signal of matched probes can be compared with that of corresponding mismatched or other control probes. Binding of mismatched probe serves as a measure of background and can be subtracted from binding of matched probes. A significant difference in binding between a perfectly matched probes and a mismatched probes signifies that the nucleic acid to which the matched probes are complementary is present. Binding to the perfectly matched probes is typically at least 1.2, 1.5, 2, 5 or 10 or 20 times higher than binding to the mismatched probes.
  • nucleic acids are not labeled but are detected by template-directed extension of a probe hybridized to a nucleic acid strand with the nucleic acid strand serving as a template.
  • the probe is extended with a labeled nucleotide, and the position of the label indicates, which probes in the array have been extended.
  • By performing multiple rounds of extension using different bases bearing different labels it is possible to determine the identity of additional bases in the tag than are determined through complementarity with the probe to which the tag is hybridized.
  • the use of target-dependent extension of probes is described by U.S. Pat. No. 5,547,839.
  • probes can be extended with inosine. The inosine strand can be labeled.
  • degenerate bases such as inosine (it can pair with all other bases), can increase duplex stability between the polynucleotide probe and the denatured single stranded DNA nucleic acids.
  • degenerate bases such as inosine (it can pair with all other bases)
  • 1-6 inosines onto the end of the probes can increase the signal intensity in both hybridization and ligation reactions on a generic ligation array. This can allow for ligations at higher temperatures.
  • degenerate bases is described in WO 97/27317.
  • Ligation reactions can offer improved discriminate between fully complementary hybrids and those that differ by one or more base pairs, particularly in cases where the mismatch is near the 5' terminus of the polynucleotide probes.
  • Use of a ligation reaction in signal detection increases the stability of the hybrid duplex, improves hybridization specificity (particularly for shorter polynucleotide probes (e.g., 5 to 12-mers), and optionally, provides additional sequence information.
  • Ligation reactions used in signal detection are described in WO 97/27317.
  • ligation reactions can be used in conjunction with template-directed extension of probes, either by inosine or other bases.
  • the position of label is detected for each probe in the array using a reader, such as described by U.S. Pat. No. 5,143,854, WO 90/15070, and Trulson et al, supra.
  • the hybridization pattern can then be analyzed to determine the presence and/or relative amounts or absolute amounts of known mRNA species in samples being analyzed as described in e.g., WO 97/10365. Comparison of the expression patterns of two samples is useful for identifying mRNAs and their corresponding genes that are differentially expressed between the two samples.
  • Expression monitoring can be used to monitor the expression (transcription) levels of nucleic acids whose expression is altered in a disease state.
  • a cancer can be characterized by the overexpression of a particular marker such as the described herein.
  • Expression monitoring can be used to monitor expression of various genes in response to defined stimuli, such as a drug. This is especially useful in drug research if the end point description is a complex one, not simply asking if one particular gene is overexpressed or underexpressed. Therefore, where a disease state or the mode of action of a drug is not well characterized, the expression monitoring can allow rapid determination of the particularly relevant genes.
  • the hybridization pattern is also a measure of the presence and abundance of relative mRNAs in a sample, although it is not immediately known, which probes correspond to which mRNAs in the sample.
  • Generic arrays can also provide a powerful tool for gene discovery and for elucidating mechanisms underlying complex cellular responses to various stimuli.
  • generic arrays can be used for expression fingerprinting.
  • the mRNA from a certain cell type displays a distinct overall hybridization pattern that is different under different conditions (e.g., when harboring mutations in particular genes, in a disease state).
  • this pattern of expression an expression fingerprint
  • Both customized and generic arrays can be used in drug safety studies. For example, if one is making a new antibiotic, then it should not significantly affect the expression profile for mammalian cells.
  • the hybridization pattern can be used as a detailed measure of the effect of a drug on cells, for example, as a toxicological screen.
  • the sequence information provided by the hybridization pattern of a generic array can be used to identify genes encoding mRNAs hybridized to an array. Such methods can be performed using DNA nucleic acids of the invention as the target nucleic acids described in WO 97/27317. DNA nucleic acids can be denatured and then hybridized to the complementary regions of the probes, using standard conditions described in WO 97/27317.
  • the hybridization pattern indicates which probes are complementary to nucleic acid strands in the sample. Comparison of the hybridization pattern of two samples indicates which probes hybridize to nucleic acid strands that derive from rnRNAs that are differentially expressed between the two samples. These probes are of particular interest, because they contain complementary sequence to mRNA species subject to differential expression.
  • sequence of such probes is known and can be compared with sequences in databases to determine the identity of the full-length mRNAs subject to differential expression provided that such mRNAs have previously been sequenced.
  • sequences of probes can be used to design hybridization probes or primers for cloning the differentially expressed mRNAs.
  • the differentially expressed mRNAs are typically cloned from the sample in which the mRNA of interest was expressed at the highest level.
  • database comparisons or cloning is facilitated by provision of additional sequence information beyond that inferable from probe sequence by template dependent extension as described above.
  • the compounds tested as modulators of immunomodulatory and antiproliferative chemokine genes or chemokine-receptor dependent and independent signaling can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide, RNAi, or a ribozyme, or a lipid.
  • modulators can be genetically altered versions of immunomodulatory and antiproliferative chemokine proteins or a G-protein coupled receptor protein.
  • test compounds will be small organic molecules, peptides, lipids, and lipid analogs.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (Le., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37: 487-493, 1991 and Houghton et al, Nature 354: 84-88, 1991).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
  • WO 93/20242 random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sci. USA 90: 6909-6913, 1993), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc. 114: 6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al, J. Amer. Chem. Soc.
  • Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14: 309-314, 1996 and PCT/US96/10287), carbohydrate libraries ⁇ see, e.g., Liang et al, Science 274: 1520-1522, 1996 and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
  • antibody libraries see, e.g., Vaughn et al, Nature Biotechnology, 14: 309
  • Candidate compounds are useful as part of a strategy to identify drugs for treating infectious diseases and cancer involving innate and specific immune responses.
  • test compounds for identifying candidate or test compounds that bind to immunomodulatory and anti-proliferative chemokine gene products, or modulate the activity of immunomodulatory and anti-proliferative chemokine proteins or polypeptides or biologically active portions thereof, are also included in the invention.
  • the test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one- compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach can be used for, e.g., peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997).
  • Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. ScL U.S.A. 90: 6909, 1993; Erb et al, Proc. Natl. Acad. ScL USA 91: 11422, 1994; Zuckermann et al, J. Med. Chem.
  • test compounds are activating variants of immunomodulatory and anti-proliferative chemokine genes.
  • the ability of a test compound to modulate the activity of immunomodulatory and antiproliferative chemokine genes or related genes or a biologically active portions thereof can be determined, e.g., by monitoring the ability to form complexes in the presence of the test compound.
  • the ability of the test compound to modulate the activity of immunomodulatory and antiproliferative chemokine genes, or a biologically active portion thereof, can also be determined by monitoring the ability of proteins binding to these genes.
  • the binding assays can be cell-based or cell-free.
  • test compound In general, the ability of a test compound to bind to immunomodulatory and antiproliferative chemokine genes or their producrts, or otherwise affect induction of cytokines, is compared to a control in which the binding or induction of activity is determined in the absence of the test compound.
  • a predetermined reference value is used. Such reference values can be determined relative to controls, in which case a test sample that is different from the reference would indicate that the compound binds to the molecule of interest or modulates expression.
  • a reference value can also reflect the amount of binding or induction observed with a standard. In this case, a test compound that is similar to (e.g., equal to or less than) the reference would indicate that compound is a candidate compound.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the invention provides soluble assays using immunomodulatory and anti-proliferative chemokine gene product or protein of interest, or a cell or tissue expressing immunomodulatory and anti-proliferative chemokine gene product or protein or interest, either naturally occurring or recombinant.
  • the invention provides solid phase based in vitro assays in a high throughput format, where immunomodulatory and antiproliferative chemokine gene product or protein of interest or its ligand is attached to a solid phase substrate via covalent or non-covalent interactions. Any one of the assays described herein can be adapted for high throughput screening.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.
  • the protein of interest or a fragment thereof e.g., an extracellular domain, or a cell or membrane comprising the protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag.
  • the tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs.
  • agonists and antagonists of cell membrane receptors e.g., cell receptor-ligand interactions such as toll-like receptors, transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I, 1993.
  • toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to persons of skill in the art.
  • polyethylene glycol linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • an animal whose genome contains a polynucleotide encoding an immunomodulatory and antiproliferative chemokine gene (or related gene or differentially expressed gene) operably linked to a promoter such that the non-human or human gene is functionally expressed in the animal, or the non-human or human gene is a gain of function mutation in the macrophage of the animal is contemplated.
  • the present invention further provides methods for making a transgenic non-human animal expressing non-human or human immunomodulatory and antiproliferative chemokine genes or related genes in the animal.
  • the transgenic animal used in the methods of the invention can be, e.g., a mammal, a bird, a reptile or an amphibian.
  • Suitable mammals for uses described herein include: rodents; ruminants; ungulates; domesticated mammals; and dairy animals.
  • Preferred animals include: rodents, goats, sheep, camels, cows, pigs, horses, oxen, llamas, chickens, geese, and turkeys.
  • the non-human animal is a mouse.
  • transgenic animals are known in the art (see, e.g., Watson, et al, "The Introduction of Foreign Genes Into Mice," in Recombinant DNA, 2d Ed., W. H. Freeman & Co., New York, pp. 255-272, 1992; Gordon, Intl. Rev. Cytol. 115: 171-229, 1989; Jaenisch, Science 240: 1468-1474, 1989; Rossant, Neuron 2: 323-334, 1990).
  • An exemplary protocol for the production of a transgenic pig can be found in White and Yannoutsos, Current Topics in Complement Research: 64th Forum in Immunology, pp. 88-94; U.S. Pat. No.
  • An exemplary protocol for the production of a transgenic rat can be found in B&dev et al., Clinical and Experimental Pharmacology and Physiology, Supp. 3: S81-S87, 1996.
  • An exemplary protocol for the production of a transgenic cow can be found in Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc.
  • An exemplary protocol for the production of a transgenic sheep can be found in Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc.
  • Several exemplary methods are set forth in more detail below.
  • Transgenic animals can be produced by introducing a nucleic acid construct according to the present invention into egg cells.
  • the resulting egg cells are implanted into the uterus of a female for normal fetal development, and animals which develop and which carry the transgene are then backcrossed to create heterozygotes for the transgene.
  • Embryonal target cells at various developmental stages are used to introduce the transgenes of the invention. Different methods are used depending on the stage of development of the embryonal target cell(s).
  • Exemplary methods for introducing transgenes include, but are not limited to, microinjection of fertilized ovum or zygotes (Brinster et al., Proc. Natl Acad.
  • production of transgenic mice employs the following steps. Male and female mice, from a defined inbred genetic background, are mated. The mated female mice are previously treated with pregnant mare serum, PMS, to induce follicular growth and human chorionic gonadotropin, hCG, to induce ovulation. Following mating, the female is sacrificed and the fertilized eggs are removed from her uterine tubes. At this time, the pronuclei have not yet fused and it is possible to visualize them using light microscopy. In an alternative protocol, embryos can be harvested at varying developmental stages, e.g. blastocysts can be harvested. Embryos are recovered in a Dulbecco's modified phosphate buffered saline (DPBS) and maintained in Dulbecco's modified essential medium (DMEM) supplemented with 10% fetal bovine serum.
  • DPBS Dulbecco's modified phosphate buffered saline
  • DMEM Dulbecco
  • Foreign DNA or the recombinant construct e.g., an immunomodulatory and antiproliferative chemokine gene expression construct
  • a pronucleus is then microinjected (100-1000 molecules per egg) into a pronucleus.
  • Microinjection of an expression construct can be performed using standard micro manipulators attached to a microscope. For instance, embryos are typically held in 100 microliter drops of DPBS under oil while being microinjected. DNA solution is microinjected into the male pronucleus. Successful injection is monitored by swelling of the pronucleus. Shortly thereafter, fusion of the pronuclei (a female pronucleus and a male pronucleus) occurs and, in some cases, foreign DNA inserts into (usually) one chromosome of the fertilized egg or zygote.
  • Recombinant ES cells which are prepared as set forth below, can be injected into blastocysts using similar techniques.
  • recombinant DNA molecules of the invention can be introduced into mouse embryonic stem (ES) cells. Resulting recombinant ES cells are then microinjected into mouse blastocysts using techniques similar to those set forth in the previous subsection.
  • ES mouse embryonic stem
  • ES cells are obtained from pre-implantation embryos and cultured in vitro (Evans et al, Nature 292: 154-156, 1981; Bradley et al, Nature 309: 255-258, 1984; Gossler et al, Proc. Natl Acad. ScL USA 83: 9065-9069, 1986; Robertson et al, Nature 322: 445-448, 1986).
  • Any ES cell line that is capable of integrating into and becoming part of the germ line of a developing embryo, so as to create germ line transmission of the targeting construct, is suitable for use herein.
  • a mouse strain that can be used for production of ES cells is the 129 J strain.
  • a preferred ES cell line is murine cell line D3 (American Type Culture Collection catalog no.
  • the ES cells can be cultured and prepared for DNA insertion using methods known in the art and described in Robertson, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. IRL Press, Washington, D.C., 1987, in Bradley et al, Current Topics in Devel. Biol 20: 357-371, 1986 and in Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986, the contents of which are incorporated herein by reference.
  • the expression construct can be introduced into the ES cells by methods known in the art, e.g., those described in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., ed., Cold Spring Harbor laboratory Press: 1989, the contents of which are incorporated herein by reference. Suitable methods include, but are not limited to, electroporation, microinjection, and calcium phosphate treatment methods.
  • the expression construct (e.g., an immunomodulatory and anti-proliferative chemokine gene) to be introduced into the ES cell is preferably linear. Linearization can be accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the gene (e.g., an immunomodulatory and anti-proliferative chemokine gene).
  • the ES cells are screened for the presence of the construct.
  • the cells can be screened using a variety of methods.
  • a marker gene is employed in the construct, the cells of the animal can be tested for the presence of the marker gene.
  • the marker gene is an antibiotic resistance gene, the cells can be cultured in the presence of an otherwise lethal concentration of antibiotic (e.g. G418 to select for neo). Those cells that survive have presumably integrated the transgene construct.
  • the marker gene is a gene that encodes an enzyme whose activity can be detected (e.g., . ⁇ -galactosidase)
  • the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
  • ES cell genomic DNA can be examined directly.
  • the DNA can be extracted from the ES cells using standard methods and the DNA can then be probed on a Southern blot with a probe or probes designed to hybridize specifically to the transgene.
  • the genomic DNA can also be amplified by PCR with probes specifically designed to amplify DNA fragments of a particular size and sequence of the transgene such that, only those cells containing the targeting construct will generate DNA fragments of the proper size.
  • the zygote harboring a recombinant nucleic acid molecule of the invention is implanted into a pseudo-pregnant female mouse that was obtained by previous mating with a vasectomized male.
  • a recombinant nucleic acid molecule of the invention e.g. immunomodulatory and anti-proliferative chemokine gene
  • recipient females are anesthetized, paralumbar incisions are made to expose the oviducts, and the embryos are transformed into the ampullary region of the oviducts.
  • the body wall is sutured and the skin closed with wound clips.
  • the embryo develops for the full gestation period, and the surrogate mother delivers the potentially transgenic mice. Finally, the newborn mice are tested for the presence of the foreign or recombinant DNA.
  • mice Of the eggs injected, on average 10% develop properly and produce mice. Of the mice born, on average one in four (25%) are transgenic for an overall efficiency of 2.5%. Once these mice are bred they transmit the foreign gene in a normal (Mendelian) fashion linked to a mouse chromosome.
  • Transgenic animals can be identified after birth by standard protocols. DNA from tail tissue can be screened for the presence of the transgene construct, e.g., using southern blots and/or PCR. Offspring that appear to be mosaics are then crossed to each other if they are believed to carry the transgene in order to generate homozygous animals. If it is unclear whether the offspring will have germ line transmission, they can be crossed with a parental or other strain and the offspring screened for heterozygosity. The heterozygotes are identified by southern blots and/or PCR amplification of the DNA. The heterozygotes can then be crossed with each other to generate homozygous transgenic offspring.
  • Homozygotes can be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice. Probes to screen the southern blots can be designed based on the sequence of the human or non-human immunomodulatory and antiproliferative chemokine gene, or the marker gene, or both.
  • western blots can be used to assess the level of expression of the gene introduced in various tissues of these offspring by probing the western blot with an antibody against the protein encoded by the gene introduced ⁇ e.g., the human or non-human immunomodulatory and antiproliferative chemokine protein), or an antibody against the marker gene product, where this gene is expressed.
  • an antibody against the protein encoded by the gene introduced e.g., the human or non-human immunomodulatory and antiproliferative chemokine protein
  • an antibody against the marker gene product where this gene is expressed.
  • In situ analysis such as fixing the cells and labeling with an antibody, and/or FACS (fluorescence activated cell sorting) analysis of various cells, e.g. erythrocytes, from the offspring can be performed using suitable antibodies to look for the presence or absence of the transgene product.
  • FACS fluorescence activated cell sorting
  • flow cytometry can be performed using antibodies specific for human immunomodulatory and antiproliferative chemokine gene, that are directly conjugated or used in conjunction with a secondary antibody that is fluorophore- conjugated and recognizes the antibody for the immunomodulatory and antiproliferative chemokine.
  • human erythrocytes can be used as a positive control and normal mouse erythrocytes can be used as a negative control for the presence of the immunomodulatory and anti-proliferative chemokine gene.
  • E Mice Containing Multiple Transgenes or an Additional Mutation
  • Transgenic mice expressing, for example, an immunomodulatory and antiproliferative chemokine gene as described herein, can be crossed with mice that a) harbor additional transgene(s), or b) contain mutations in other genes. Mice that are heterozygous or homozygous for each of the mutations can be generated and maintained using standard crossbreeding procedures.
  • the invention further pertains to cells derived from transgenic animals. Because certain modifications can occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • GPCR's G-protein coupled, 7 transmembrane domain receptors
  • CCRl which binds RANTES and MIP- l ⁇
  • CCR4 which binds RANTES, MIP-Ia, and MCP-I (Power et al, 1995, J. Biol. Chem. 270:19495-19500)
  • CCR5 which binds RANTES, MIP-Ia, and MlP-l ⁇
  • Receptor binding initiates a cascade of intracellular events mediated by receptor-associated heterotrimeric G-proteins.
  • sequence and structural homologies evident among chemokines and their receptors allows some overlap in receptor-ligand interactions.
  • the invention provides an isolated antimicrobial chemokine.
  • isolated antimicrobial chemokine examples include: IP-IO:
  • ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPA SPIVKKIIEKMLNSDKSN (SEQ ID NO: 7)
  • VLEVYYTSLRCRCVQESSVFIPRRFIDRIQILPRGNGCPRKEHVWKKNKSIVCVDP QAEWIQRMMEVLRKRSSSTLPVPVFKRKIP SEQ ID NO: 12
  • ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRAICSDPN NKRVKNAVKYLQSLERS (SEQ ID NO: 17)
  • the present invention demonstrates that highly positively charged chemokines at ⁇ M concentrations display novel activities that appear not to be mediated by chemokine (GPCR) receptors, but by molecular mechanisms (i.e., gene profile) that are different than chemokine (GPCR) receptor mediated mechanisms. 15. METHODS OF USE
  • the present invention demonstrates that highly positively charged chemokines display immunomodulatory activity at ⁇ M concentrations.
  • This novel immunomodulatory function includes, but is not limited to, the enhancement of the innate immune response by induction of gene expression and production of various cytokines and chemokines.
  • the present invention provides a use for chemokines to stimulate or enhance innate immunity by acting directly on host cells, hi this aspect, a method of identification of a polynucleotide or polynucleotides that are regulated by an immunomodulatory chemokine is provided.
  • the expression of the polynucleotide in the presence and absence of the chemokine is observed, and a change in expression is indicative of a polynucleotide or pattern of polynucleotides that is regulated by the immunomodulatory chemokine.
  • the invention provides a polynucleotide identified by the method.
  • immunomodulatory chemokines or compounds will be useful in methods of screening for compounds that can enhance innate immunity by affecting the expression of the polynucleotide. Such an effect on expression may be either up regulation or down regulation of expression.
  • the present invention also presents a method of identifying other enhancers of innate immunity. Additionally, the present invention provides compounds that are used or identified in the above methods.
  • the present invention provides a use of antiproliferative chemokines to inhibit tumor cell growth or virus replication by acting directly on host cells.
  • the expression of the polynucleotide in the presence or absence of the anti-proliferative chemokine is observed, and a change in expression is indicative of a polynucleotide or pattern of polynucleotides that is regulated by the anti-proliferative chemokine.
  • the invention provides specific polynucleotides identified by the method. Also provided is a method of identification of a polynucleotide or polynucleotides that are regulated by an anti-proliferative chemokine.
  • the anti-proliferative chemokines or the compounds will be useful in methods of screening for compounds that can inhibit tumor cell proliferation by affecting the expression of the polynucleotide. Such an effect on expression may be either up regulation or down regulation of expression.
  • the present invention also presents a method of identifying other anti-proliferative compounds. Additionally, the present invention provides compounds that are used or identified in the above methods.
  • "Host cells” or "Recipient cells” encompassed by the invention are any cells in which the chemokines of the invention can be used to express the polypeptides of the invention. The term also includes any progeny of a recipient or host cell.
  • Preferred recipient or host cells of the invention include E. coli, S. aureus and P. aeruginosa, although other Gram-negative and Gram- positive bacterial, fungal and mammalian cells (normal and cancer cells) and organisms known in the art can be utilized as long as the expression vectors contain an origin of replication to permit expression in the host.
  • Candidate compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. The chemokines, analogs and fragments thereof of this invention, or the newly discovered compounds may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the like to produce structural analogs.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, peptidiomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, polypeptides, polynucleotides, chemical compounds, derivatives, structural analogs or combinations thereof.
  • Incubating components of a screening assay includes conditions that allow contact between the test chemokine or compound and the targets of interest. Contacting includes in solution and in solid phase, or in a cell.
  • the test compound may optionally be a combinatorial library for screening a plurality of compounds.
  • Compounds identified in the method of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a compound.
  • a chemokine is utilized to detect and locate a polynucleotide that is essential in the process of innate immunity or cell proliferation. Once identified, a pattern of polynucleotide expression may be obtained by observing the expression in the presence and absence of the chemokine. The pattern obtained in the presence of the chemokine is then useful in identifying additional compounds that can influence expression of the polynucleotide and therefore enhance innate immunity or reduce proliferation. It is well known to one of skill in the art that non-peptidic chemicals and peptidomimetics can mimic the ability of peptides to bind to receptors and enzyme binding sites, and thus can be used to block or stimulate biological reactions.
  • an additional compound of interest provides a pattern of polynucleotide expression similar to that of the expression in the presence of a chemokine
  • that compound is also useful in the modulation of an innate immune response.
  • the chemokines of the invention which are enhancers of innate immunity, are useful as tools in the identification of additional compounds that enhance innate immunity.
  • the invention provides a method of using a chemokine to detect and locate a polynucleotide that is essential in the process of reducing proliferation. Once identified, a pattern of polynucleotide expression may be obtained by observing the expression in the presence and absence of the chemokine.
  • the pattern obtained in the presence of the chemokine is then useful in identifying additional compounds that can influence expression of the polynucleotide and therefore anti-proliferation. It is well known to one of skill in the art that non- peptidic chemicals and peptidomimetics can mimic the ability of peptides to bind to receptors and enzyme binding sites, and thus can be used to block or stimulate biological reactions. Where an additional compound of interest provides a pattern of polynucleotide expression similar to that of the expression in the presence of a chemokine, that compound is also useful in treating cancer and viral infection, hi this manner, the chemokines of the invention, which are antiproliferative, are useful as tools in the identification of additional compounds that are antiproliferative.
  • the invention provides a method of identifying a chemokine that enhances innate immunity activity or inhibits proliferation by stimulating target cell in a manner that cannot be blocked by an antagonist or inhibitor of classical chemokine receptor function.
  • a polypeptide e.g., chemokine
  • a chemokine receptor signaling inhibitor such as, for example, pertussis toxin.
  • cytokine e.g., IL-8
  • chemokine e.g., IL-8
  • Increased expression of cytokine is indicative of a chemokine that enhances innate immunity
  • the chemokine does not stimulate a septic reaction as revealed by the minimal production of the proinflammatory cytokine TNF- ⁇ .
  • chemokines of the invention have a widespread ability to downregulate particular signaling pathways such as, for example, NFKB pathways, to up-regulate many genes such as, for example, within p53 pathways and activate cells that do not carry the corresponding chemokine receptors.
  • chemokine-receptor independent activity include MIG-induced IL-8 production and the inhibition of the growth of THP-I cells.
  • the immunomodulatory activity of the chemokines of the invention can be very potent.
  • Example 2 shows that non-antimicrobial chemokines having a molecular surface that is not strongly positively charged and not having antimicrobial activity, such as MCP-I and MIP- l ⁇ , do not induce IL-8 production.
  • ⁇ M concentrations of three human chemokines, MIG, IP-IO, MIP-3 ⁇ (all of which have potent antimicrobial activity), stimulated THP-I cells to produce EL- 8, a chemokine responsible for neutrophil recruitment.
  • the IL-8 induction was dependent on the dose of the chemokines, with a minimal effective concentration of 1 to 2 ⁇ M, a dose that is above the maximum at which chemotactic activity can be observed.
  • MIG was the most potent, and induced a large amount of IL-8.
  • IP-10 and MD?-3 ⁇ induction of EL-8 production was much less than that of MIG.
  • this novel immunomodulatory function was correlated to the antimicrobial activity of these chemokines.
  • Chemokines, such as MIG that are potent antimicrobial agents against both Gram positive and Gram negative bacteria, exhibited very strong immunomodulatory activity.
  • MD?-3 ⁇ and IP-10 that have antimicrobial activity that is reduced relative to MIG, correspondingly displayed relatively lower immunomodulatory activity than that of MIG.
  • the chemokines of the invention have been further observed to induce gene expression.
  • Example 3 demonstrates that the immunomodulatory chemokines MIG and SLC could significantly enhance IL-8 gene expression by 6- to 25-fold in THP-I cells.
  • MD?-3 ⁇ and IP-10 had a substantially lesser effect on IL-8 gene expression.
  • the non-antimicrobial chemokines MCP-I and MIP- l ⁇ did not induce IL-8 gene expression.
  • Example 4 demonstrates that IL-8 induction by these fragments correlated with that of their parent chemokines (full length MIG and MCP-I), but with somewhat lower potency. Both the N-terminal (1-73) and C-terminal (73-103) fragments of MIG led to induction of IL-8 release from THP-I cells. However, to get a similar level of response as that of full length MIG, the concentration of the fragments had to be 20 to 100 fold higher.
  • IL-8 production induced by both MIG fragments and MIG itself could be enhanced by co-stimulation with GM-CSF, a factor widely present in the environment where innate immunity occurs.
  • the relatively more efficient stimulation by full length MIG was still maintained, and the concentration at which stimulation occurred was still 10-fold higher for the fragments than for the full length MIG.
  • the IL-8 induction remained much lower than that induced by full length MIG, indicating that the specific structure of MIG is responsible for its potent immunomodulatory activity. Fragments of the non-antimicrobial chemokine MCP-I did not induce IL-8 production at any concentration, similar to the observation for full length MCP-I
  • Chemokines MIG and IP-IO both act through a common counterpart receptor, CXCR3, which is specifically expressed by activated T lymphocytes, but not by monocytic cells. Chemokine MIP-3 ⁇ interacts with it's counterpart receptor, CCR6. It has been known that the human myeloid cell line THP-I does not express CXCR3, and demonstrates no chemotactic response to any of the 3 CXCR3 ligands (MIG, IP-IO and I-TAC). Example 5 demonstrates that THP-I cells do not express the CXCR3 and CCR6 genes, as detected by PCR using CXCR3- or CCR6 specific primers.
  • Chemokines and their receptors interact in a redundant manner in that one chemokine can bind to several different receptors.
  • the effect of pertussis toxin was observed in Example 6. It is well known that all chemokine receptors belong to the G-Protein coupled receptor (GPCR) superfamily, and thus their signaling will be disrupted by the G-protein inhibitor pertussis toxin. The data demonstrated that treatment with various doses of pertussis toxin did not inhibit MIG, DP-IO or MD?-3 ⁇ induced IL-8 production.
  • chemokines induced a greater amount of IL-8 production in the presence of pertussis toxin (most likely because pertussis toxin itself induced a small amount of IL-8 release).
  • Examples 5 and 6 clearly demonstrate that the immunomodulatory activity of the chemokines of the invention is not mediated by any chemokine GPCR, but rather by other mechanisms.
  • Examples 7 and 8 microarray analysis was used, probing expression of 21,000 human genes, to explore the alterations in gene expression of THP-I cells that were induced by the selective chemokines.
  • the gene expression profile induced by the immunomodulatory chemokine MIG was then compared to that of the non-antimicrobial chemokine MCP-I.
  • Treatment with both chemokines was performed under identical conditions for 4 hours using 10 ⁇ M of each of the chemokines, an optimal dose based on their induction of DL-8 production, to stimulate the THP-I cells.
  • Four separate biological repeats were performed with 2 to 4 technical repeats for each biological sample.
  • ArrayPipe Hokamp K, Roche FM, Acab M, Rousseau ME, Kuo B, Goode D, Aeschliman D, Bryan J, Babiuk L, Hancock REW, and Brinkman FSL. 2004.
  • ArrayPipe a flexible processing pipeline for microarray data.
  • Nucleic Acids Research 32: W471-474 and Genespring (Silicon Genetics), and filtered by statistical significance with a p value ⁇ 0.05.
  • Completely different subsets of genes were observed for the two chemokines, and results for MIG were consistent with an immunomodulatory activity including induction of chemokines and differentiation genes.
  • MIG and SLC when applied to a tumor target, down-regulated the expression of a set of oncogenes (based on principal component analysis showing 145 genes with substantially reduced expression). Analogously, 18 genes related to cell proliferation, cycling and cell growth of target cells were down regulated, hi contrast, markers for mature leukocytes were up-regulated.
  • a set of tumor suppressor genes including ALEX3 protein (ALEX3), BTG family member 2 (BTG2), Tissue inhibitor of metalloprotein 3 (TIMP3), cyclin-dependent kinase inhibitor IA (p21, Cipl) were up-regulated, hi addition, expression of genes related to transcription or regulating transcription including PHTFl, ZNF148, NFAT5, MTFl, HCNGP, RNF4, SCAl, PAFAHlBl, SRF are modulated by MIG.
  • chemokines such as MIG and SLC are antiproliferative and induce tumor cells to undergo terminal differentiation. The conclusion was confirmed by showing that MIG and SLC directly inhibited THP-I cells (human myeloid leukemia cell line) and MDA cells (human breast cancer cell line) growth.
  • CDKs cyclin-dependent kinases
  • HSV-I HSV-I
  • antiproliferative chemokines such as MIG and SLC down-regulate the expression of genes related to the transcriptional promoter CCAAT box and TATA box families, down-regulate expression of transcription factor E2F, and up-regulate several tumor suppressor genes including,cyclin kinase regulatory proteins p21 and pi 6, (and thus consequently down-regulate CDK activity such as cyclin D2/CCND2), it is feasible that these chemokines possess antiviral activity.
  • anti-proliferative chemokines such as MIG
  • MIG anti-viral growth factor
  • anti-proliferative chemokines has the potential for targeting both the etiological agents (i.e., the virus) and the host pathogenic mechanisms.
  • the observed gene profile of MIG shows that MIG not only inhibits proliferation, but also down-regulates the apoptosis signal pathway of the host cell, a common injury process seen in many viral pathologies, such as Hepatitis B and C induced liver cell apoptosis, and HIV-induced T lymphocytes apopotosis.
  • Virus replication can also be inhibited by other mechanisms. For instance, it has been reported that HIV-I replication can be inhibited by inhibiting the p38 pathway. Interruption of HIV infection by p38 inhibitors underscores the value of exploring antiviral drugs that target host cellular proteins. In the present microarray study it has been shown that p38 pathway was down- regulated by treatment with MIG (Table 2).
  • MIG can potentially inhibit viral associated disease by boosting the innate immunity of the host.
  • Example 9 gene expression differences obtained using microarray results were confirmed for randomly selected genes using limited cycle RT-PCR.
  • the invention provides methods of direct polynucleotide regulation by chemokines, and the use of the chemokines to stimulate elements of innate immunity.
  • the invention provides a method of identification of a pattern of polynucleotide expression for identification of a test compound that enhances innate immunity. An initial detection of a pattern of polynucleotide expression for cells contacted in the presence and absence of a chemokine is made. The pattern resulting from polynucleotide expression in the presence of the chemokine represents stimulation of innate immunity.
  • a pattern of polynucleotide expression is then detected in the presence of a test compound, where a resulting pattern with the test compound that is similar to the pattern observed in the presence of the chemokine is indicative of a compound that enhances innate immunity.
  • the invention provides compounds that are identified in the above methods.
  • the compound is a peptide, peptidomimetic, chemical compound, or a nucleic acid molecule.
  • Example 12 In contrast to certain cationic peptides that are toxic to the target cells, no cytotoxicity was observed in Example 12 by any of the chemokines tested at the concentrations which demonstrated immunomodulatory activities. This conclusion was made based on various biological assays including the lactate dehydrogenase (LDH) release assay and the tetrazolium dye (MTT) test.
  • LDH lactate dehydrogenase
  • MTT tetrazolium dye
  • the methods of the invention may be used in combination, to identify an agent with multiple characteristics, i.e., a chemokine with anti-inflammatory/anti-sepsis activity, and the ability to enhance innate immunity, in part by inducing cytokines/chemokines in vivo.
  • an agent with multiple characteristics i.e., a chemokine with anti-inflammatory/anti-sepsis activity, and the ability to enhance innate immunity, in part by inducing cytokines/chemokines in vivo.
  • the compounds and modulators identified by the methods of the present invention can be used in a variety of methods of treatment.
  • the present invention provides compositions and methods for treating an infectious disease, a Gram positive bacterial infection, G-coupled signaling defects, immunomodulatory and antiproliferative gene mutation or gene expression defect or immunomodulatory and antiproliferative gene product defect.
  • Exemplary infectious disease include but are not limited to, Gram positive bacterial skin infections, for example, S. pyogenes or S. aureus.
  • Gram positive cocci S. pyogenes or S. aureus are leading agents of human impetigo, cellulites, and wound infection.
  • infectious disease include but are not limited to, viral or bacterial diseases.
  • the polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases can be treated.
  • the immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the polypeptide or polynucleotide of the present invention can also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellu
  • parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
  • These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • the method of inhibiting the growth of bacteria can further include the addition of antibiotics for combination or synergistic therapy.
  • antibiotics for combination or synergistic therapy.
  • the appropriate antibiotic administered will typically depend on the susceptibility of the bacteria such as whether the bacteria is Gram negative or Gram positive, and will be easily discernable by one of skill in the art.
  • antibiotics useful for synergistic therapy with the peptides of the invention include aminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin), cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g., ciprofloxacin), carbapenems (e.g., imipenem), tetracyclines and macrolides (e.g., erythromycin and clarithromycin).
  • aminoglycosides e.g., tobramycin
  • penicillins e.g., piperacillin
  • cephalosporins e.g., ceftazidime
  • fluoroquinolones e.g., ciprofloxacin
  • carbapenems e.g., imipenem
  • tetracyclines and macrolides e.g., erythromycin and clarithromycin
  • the peptides and/or analogs or derivatives or the invention thereof can be administered to any host, including a human or non-human animal, in an amount effective to inhibit not only growth of a bacterium, but also a virus, parasite or fungus. These peptides are useful as antimicrobial agents, antiviral agents, and antifungal agents.
  • the peptides and/or analogs or derivatives thereof may be administered to any host, including a human or non-human animal, in an amount effective to inhibit not only growth of a bacterium, but also a virus or fungus. These peptides are useful as antimicrobial agents, antiviral agents, and antifungal agents.
  • bactenecin has been shown to be cytotoxic to rat embryonic neurons, fetal rat astrocytes and human glioblastoma cells. Radermacher et al., J. Neuro. Res. 36: 657, 1993.
  • the peptides of the present invention can be used to inhibit the growth of a eukaryotic cell by contacting the eukaryotic cell with an inhibiting effective amount of a peptide of the invention.
  • the immunomodulatory and antiproliferative chemokines of the invention e.g., chemokine peptides
  • the antibiotic is selected from the group consisting of aminoglycosides, penicillins, cephalosporins, carbapenems, monobactams, quinolones, tetracyclines, and glycopeptides.
  • typical antibiotics include aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, t-obramycin, streptomycin), macrolides ( azithromycin, clarithromycin, erythromycin, erythromycin estolate/ethylsuccinate/ gluceptate/lactobionate/stearate), beta-lactams such as penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin and piperacillin), or cephalosporins (e.g., cephalothin, cefazolin, cefaclor, cefamandole, cefoxitin, cefuroxime, cefonicid, cefmetazole, ce
  • antibiotics include quinolones (e.g., fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin), tetracyclines (e.g., doxycycline, minocycline, tetracycline), and glycopeptides (e.g., vancomycin, teicoplanin), for example.
  • quinolones e.g., fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin
  • tetracyclines e.g., doxycycline, minocycline, tetracycline
  • glycopeptides e.g., vancomycin, teicoplanin
  • antibiotics include chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, linezolid, synercid, polymyxin B, colisitin, colimycin, methotrexate, daptomycin, phosphonomycin and mupirocin.
  • cellular proliferative and/or differentiative disorders include, but not limited to, cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias.
  • a metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of liver, prostate, colon, lung, and breast origin.
  • “Cancer,” “hyperproliferative” and “neoplastic” refer to cells having the capacity for autonomous growth, Le., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, Le., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • Cancer or “neoplasms” include malignancies of the various organ systems, such as affecting liver, lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • Carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including liver carcinoma, hepatocellular carcinoma (HCC), respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • the term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • hematopoietic neoplastic disorders includes diseases involving hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia.
  • myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), reviewed in Vaickus, Crit Rev. in Oncol/Hemotol. 11: 267-97, 1991; lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B -lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • WM Waldenstrom's macroglobulinemia
  • malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease.
  • treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient ⁇ ex vivo therapy).
  • the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
  • the immunomodulatory and antiproliferative chemokines, or analogs, derivatives, amidated variations and conservative variations thereof can be administered to any host, including a human or non-human animal, in an amount effective to stimulate innate immunity resulting in the inhibition of growth of a bacterium, but also a virus, parasite or fungus. These chemokines are useful as replacements for antimicrobial agents, antiviral agents, and antifungal agents, by stimulating anti-infective immunity.
  • the chemokines, or analogs, derivatives, amidated variations and conservative variations thereof can be administered to any host, including a human or non-human animal, in an amount.
  • the immunomodulatory chemokines and antiproliferative chemokines, or analogs, derivatives, amidated variations and conservative variations thereof can be used as therapeutic compositions either alone or in combination with other therapies, for example, with other drugs, or with radiation or chemotherapies.
  • the invention provides pharmaceutical compositions comprising one or a combination of the immunomodulatory chemokines and anti-proliferative chemokines, or analogs, derivatives, amidated variations and conservative variations thereof of the invention, for example, formulated together with a pharmaceutically acceptable carrier.
  • Some compositions include a combination of multiple (e.g., two or more) analogs, derivatives, amidated variations and conservative variations thereof of the invention.
  • Some compositions include a combination of an immunomodulatory chemokine and antiproliferative chemokine of the invention together with other drugs or agents (i.e., antimicrobial drugs, antimicrobial agents, antiviral agents, or antifungal agents).
  • the immunomodulatory and antiproliferative chemokines and/or analogs or derivatives thereof may be administered to any host, including a human or non-human animal, in an amount effective to stimulate innate immunity resulting in the inhibition of growth of a bacterium, but also a virus, parasite or fungus. These chemokines are useful as replacements for antimicrobial agents, antiviral agents, and antifungal agents, by stimulating anti-infective immunity.
  • the chemokines and/or analogs or derivatives thereof may be administered to any host, including a human or non-human animal, in an amount effective to inhibit not only growth of a bacterium, but also a virus or fungus.
  • pharmaceutically acceptable carrier and “pharmaceutically acceptable excipient” include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for parenteral administration.
  • the carrier can be suitable for intravenous, intraperitoneal or intramuscular administration.
  • the carrier is suitable for oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. 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 compatible with the active compound, use thereof in the pharmaceutical compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • “Pharmaceutically acceptable salts and esters” means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.
  • Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene- sulfonic acids such as methanesulfonic acid and benzenesulfonic acid).
  • Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., Cl-6 alkyl esters.
  • a pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified.
  • Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters.
  • certain compounds named in this invention may be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., as a result of bacteria, fungi, viruses, parasites or the like) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • a disease or condition i.e., as a result of bacteria, fungi, viruses, parasites or the like
  • compositions or medicants are administered to a patient suspected of, or already suffering from such a disease or condition in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease or condition (e.g., biochemical and/or histologic), including its complications and intermediate pathological phenotypes in development of the disease or condition.
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.
  • agents are usually administered in several dosages until a sufficient response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to wane.
  • the pharmaceutical composition of the present invention should be sterile and fluid to the extent that the composition is deliverable by syringe, hi addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • an agent which delays absorption for example, aluminum monostearate or gelatin.
  • the active compound when suitably protected, as described above, the compound can be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • compositions of the invention also can be administered in combination therapy, i.e., combined with other agents.
  • the combination therapy can include a composition of the present invention with at least one agent or other conventional therapy.
  • Concomitant administration of a known antimicrobial drag with a pharmaceutical composition of the present invention means administration of the drug and the immunomodulatory chemokine and antiproliferative chemokine composition at such time that both the known drag and the composition of the present invention will have a therapeutic effect.
  • Such concomitant administration can involve concurrent ⁇ i.e., at the same time), prior, or subsequent administration of the antimicrobial drag with respect to the administration of a compound of the present invention.
  • a person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drags and compositions of the present invention
  • a composition of the present invention can be administered by a variety of methods known in the art.
  • the route and/or mode of administration vary depending upon the desired results.
  • the phrases "parenteral administration” and “administered parenterally” mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the chemokine of the invention can be administered parenterally by injection or by gradual infusion over time.
  • the chemokine can also be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems Further methods for delivery of the chemokine include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation. To administer a chemokine of the invention by certain routes of administration, it can be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • the method of the invention also includes delivery systems such as microencapsulation of chemokines into liposomes or a diluent.
  • Microencapsulation also allows co-entrapment of antimicrobial molecules along with the antigens, so that these molecules, such as antibiotics, may be delivered to a site in need of such treatment in conjunction with the chemokines of the invention.
  • Liposomes in the blood stream are generally taken up by the liver and spleen.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes. Strejan et ah, J. Neuroimmunol. 7: 27, 1984.
  • the method of the invention is particularly useful for delivering antimicrobial chemokines to such organs.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Robinson, 1978, Sustained and Controlled Release Drug Delivery Systems. Other methods of administration will be known to those skilled in the art.
  • the chemokines of the invention can be administered parenterally by injection or by gradual infusion over time.
  • the chemokines can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Further methods for delivery of the chemokines include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation.
  • the method of the invention also includes delivery systems such as microencapsulation of chemokines into liposomes.
  • Microencapsulation also allows co- entrapment of antimicrobial molecules along with the antigens, so that these molecules, such as antibiotics, may be delivered to a site in need of such treatment in conjunction with the chemokines of the invention.
  • Liposomes in the blood stream are generally taken up by the liver and spleen.
  • the method of the invention is particularly useful for delivering antimicrobial chemokines to such organs.
  • Other methods of administration will be known to those skilled in the art.
  • Preparations for parenteral administration of a chemokine of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions typically must be sterile, substantially isotonic, and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • 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), and suitable mixtures thereof.
  • 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.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Therapeutic compositions can also be administered with medical devices known in the art.
  • a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in, e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in, e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No.
  • chemokines of the present invention When administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (or 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical nucleic acid, peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, etc.
  • the amount of nucleic acid, peptide or polypeptide adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use i.e., the "dosing regimen” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like, hi calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like.
  • Dosage regimens of the pharmaceutical compositions of the present invention are adjusted to provide the optimum desired response ⁇ e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • a physician or veterinarian can start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).
  • an effective dose of each of the chemokines disclosed herein as potential therapeutics for use in treating microbial diseases and conditions is from about 1 ⁇ g to 500 mg/kg body weight, per single administration, which can readily be determined by one skilled in the art. As discussed above, the dosage depends upon the age, sex, health, and weight of the recipient, kind of concurrent therapy, if any, and frequency of treatment. Other effective dosage range upper limits are 100 mg/kg body weight, 50 mg/kg body weight, 25 mg/kg body weight, and 10 mg/kg body weight.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • the therapeutic compounds of the invention can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, See, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes can comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (See, e.g., Ranade, /. Clin. Pharmacol. 29: 685, 1989).
  • Exemplary targeting moieties include folate or biotin (See, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al, Biochem. Biophys. Res. Commun. 153: 1038, 1988); antibodies (Bloeman et al, FEBS Lett. 357: 140, 1995; Owais et al, Antimicrob. Agents Chemother. 39: 180, 1995); surfactant protein A receptor (Briscoe et al, Am. J. Physiol 1233: 134, 1995), different species of which can comprise the formulations of the inventions, as well as components of the invented molecules; pl20 (Schreier et al, J.
  • the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety.
  • the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection.
  • the composition should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the chemokines of the invention can be utilized as broad spectrum antimicrobial agents directed toward various specific applications. Such applications include use of the chemokines as preservatives in processed foods (organisms including Salmonella, Yersinia, Shigella), either alone or in combination with antibacterial food additives such as lysozymes; as a topical agent (Pseudomonas, Streptococcus) and to kill odor producing microbes (Micrococci).
  • the relative effectiveness of the chemokines of the invention for the applications described can be readily determined by one of skill in the art by determining the sensitivity of any organism to one of the chemokines.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.
  • Topical application can result in transdermal or intradermal delivery.
  • Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins.
  • Co- administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin patch or using transferosomes.
  • Paul et ah Eur. J. Immunol. 25: 3521-24, 1995; Cevc et ah, Biochem. Biophys. Acta 1368: 201-15, 1998.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. 21. KITS
  • kits comprising the compositions, e.g., nucleic acids, expression cassettes, vectors, cells, polypeptides (e.g., chemokine polypeptides) of the invention, and the like.
  • the kits also can contain instructional material teaching the methodologies and uses of the invention, as described herein.
  • the human monocytic cell line THP-I was purchased from the American Type Culture Collection (ATCC). The cells were cultured in the RPMI 1640 medium with 10% fetal calf serum (FCS) and other supplements, however, without any antibiotic. In this study only THP-I cells that had been cultured for less than 5 passages were used.
  • monocytes were purified from healthy donor's peripheral blood according to a well-established protocol. Briefly, 50 to 100 ml of heparinized blood was diluted 1:1 with RPMI medium and loaded onto a Ficoll gradient. After centrifugation at 1450 rpm for 20 minutes, the cells at the interface were collected. T- lymphocytes were removed by co-incubation with neurominidase-treated sheep red blood cells for 1 h, followed by Ficoll separation as above. The recovered cells were allowed to adhere onto a plastic surface at 37°C for 1 h. Contaminated B-lymphocytes were then removed by washing with phosphate-buffered saline (PBS). The remaining adherent cells were 85% monocytes that were immediately used for study.
  • PBS phosphate-buffered saline
  • Chemokines were purchased from Biomedical Research Centre of the University of British Columbia (UBC). The chemokines were chemically synthesized, folded, HPLC-purified, and evaluated and confirmed by mass spectrometry. The chemotactic activities of these chemokines were confirmed by standard assays.
  • THP-I cells were added to a 48-well plate (1.25 x 10 5 cells per well). The cells were stimulated for 48 hours with various chemokines at different concentrations as indicated in the Figures. The supernatant of the cells were collected. The amount of IL-8 in the supernatants was then determined by ELISA (Biosource) compared to an IL-8 standard. Fig. 2A shows the results. Chemokines, such as MCP-I and MIP- l ⁇ with molecular surfaces that are not positively charged and that are not antimicrobial, did not induce IL-8 production. In contrast, MIG, IP-10, MIP-3 ⁇ , three randomly picked human cationic chemokines having antimicrobial activity, all stimulated THP-I cells to produce IL-8.
  • the IL-8 induction was dependent on the dose of chemokines, at a minimal concentration of 1 to 2 ⁇ M. Amongst the three chemokines, MIG was the most potent, and induced a large amount of IL-8. IP-IO and MIP-3 ⁇ induced IL-8 production was much less than that of MIG.
  • PBMC peripheral blood mononuclear cells
  • IL-8 gene expression was studied by semi-quantitative (limited cycle) PCR using specific primers for the human IL-8 gene.
  • Fig. 3 shows a representative result of at least 9 experiments. The result is expressed as the level relative to that of the control (cells treated with buffer only).
  • Non-antimicrobial chemokines MCP-I and MIP-Ia did not induce IL-8 gene expression.
  • the human immunomodulatory (antimicrobial) peptide LL-37 did not stimulate EL-8 production, indicating that this property of MIG-I and SLC differs from the classical cationic host defence peptides.
  • N-MIG e.g., MIG (1-76
  • C-MIG e.g., MIG (73-103)
  • fragments of MCP-I C-MCP-I, e.g., MCPl (53-76)
  • full length MIG e.g., MCPl (53-76)
  • full length MCP-I e.g., MCPl (53-76)
  • CXCR3 receptor for MIG and IP-10
  • CCR2 receptor for MCP-I
  • CCR6 receptor for MIP-3a
  • CCRl receptor for MIP-Ia
  • CCR2 CCAACGAGAGCGGTGAAGAAGTC (SEQ ID NO: 11)
  • CCR6 CCTGGGGAATATTCTGGTGGTGA (SEQ ID NO: 13) CATCGCTGCCTTGGGTGTTGTAT (SEQ ID NO: 14)
  • RNA was prepared from THP-I cells. RT-PCR was done using the method described in the Example 3. No CXCR3 (receptor for immunomodulatory chemokine MIG and IP-IO) or CCR6 (receptor for immunomodulatory chemokine MIP3alpha) gene expression was detected by PCR using 0.2 ⁇ g of total cDNA for a total of 35 cycles. As a positive control, amplification using, as a template, the cDNA for human CXCR3 and CCR6 demonstrated strong amplification.
  • THP-I cells did express the CCRl (the receptor for the non- immunomodulatory chemokine MCP-I), CCR2 (the receptor for the non-immunomodulatory chemokine MIP-loc) receptors (Fig. 6).
  • chemokine activity can be blocked by pertussis toxin which disrupts GPCR signalling.
  • THP-I cells were pre- incubated for 1 hour without or with various dose of pertussis toxin as indicated.
  • chemokines MIG final concentration 10 ⁇ M
  • Fig. 7B THP-I cells were treated with various chemokines (at 10 ⁇ M) in the presence or absence of pertussis toxin (100 ng/ml) for 48 hrs, as indicated. Results show that pertussis toxin did not inhibit IL-8 production in response to the chemokines D?-10, MIG, or MIP-3 ⁇ . Instead IL-8 production increased in the presence of pertussis toxin itself.
  • the result (Fig. 7) clearly demonstrates that the immunomodulatory activity of chemokines is not mediated by any chemokine GPCR, but rather via some other mechanism.
  • THP-I cells were treated under the identical conditions for 4 hours with MIG, MCP-I, or with water only as a control. Ten ⁇ M of each chemokine, an optimal dose based on their induction of IL- 8 production, was used. Four biological repeats were performed to ensure the robustness of the data.
  • Total RNA of treated THP-I cells was isolated using the Qiagen MiniprepTM kit (Qiagen). The quantity and integrity of each RNA sample was checked out by UV spectrophotometery assessing the ratio of 260/280 nm, by 1% agarose gel and using an Agilent Bioanalyzer. The mRNA was freshly reverse-transcripted into cDNAs, followed by amplification using an Ambion Amplification kit.
  • Fig. 8 is a Venn diagram that illustrates the differences in gene expression profiles induced by MIG and by MCP-I in one experiment.
  • MIG significantly changed expression of 277 genes
  • MCP-I changed the expression of 122 genes.
  • laminin a member of a family of extracellular matrix glycoproteins that is the major noncollagenous constituent of basement membranes, that has been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signalling, outgrowth and metastasis.
  • the result clearly demonstrated that immunomodulatory and antiproliferative activities of MIG are through completely different molecular mechanisms and gene profiles from that of MCP-I, a chemoattractant activity mediated through chemokine receptor.
  • Table 2 shows significantly increased or decreased gene expression in TK-P- 1 cells stimulated with chemokine MIG or MCP-I.
  • Index NC means no change relative to control.
  • MIG strongly up-regulated the expression of a set of chemokines that contribute to the recruitment of immune cells including monocytes (MCP-I), neutrophils (IL-8), dendritic cells (MIP-Ia and MlP-l ⁇ ) and activated T lymphocytes (IP-IO).
  • MCP-I monocytes
  • IL-8 neutrophils
  • MIP-Ia and MlP-l ⁇ dendritic cells
  • IP-IO activated T lymphocytes
  • ThI type immune response cytokines such as TNF- o ⁇ ;
  • interferon- ⁇ that is involved in controlling viral infection;
  • significantly enhanced leukocyte surface markers such as CD84, adhesion molecules including CD31 and integrin, and factors related to antigen processing and presentation.
  • PCA principal component analysis
  • MCP-I up-regulated genes related to cytoskeleton function, such as actin and tubulin, and genes involved in the remodelling of proteoglycan (heparin sulphate 3-O-sulfotransferase, proteoglycan 3). MCP-I also highly up-regulated a set of genes related to G-protein-dependent reactions such as GTPase-activating protein, cATPase type 8B, and guanylate cyclase activator IB.
  • a 4 h treatment with MIG led to significant down-regulation of a set of 145 oncogenes and related genes, such as myb, myc, and mammaglobin 2. This trend was observed in all experiments. This was consistent with the observation of increased expression of genes associated with tumour cell differentiation, such as lipoprotein lipase and phorbol ester-induced protein that has been previously reported to be associated with leukemia cell HL-60 terminal differentiation, as well as neuronal or hormone receptors, such as adrenomedullin receptor, putative opioid receptor, and thyroid hormone receptor.
  • tumour cell differentiation such as lipoprotein lipase and phorbol ester-induced protein that has been previously reported to be associated with leukemia cell HL-60 terminal differentiation
  • neuronal or hormone receptors such as adrenomedullin receptor, putative opioid receptor, and thyroid hormone receptor.
  • Up-regulated genes by MIG also included p53 -regulated tumour suppressor factors including Cyclin-dependent kinase inhibitor IA (p21, Cipl), cyclin E2, and Tp53 target gene 1, and other tumour suppressor, such as BTG2, arrestin and ALEX3 protein, etc.
  • the transcription factor E2F was downregulated.
  • Dramatically down-regulated genes also included certain growth factor receptors (such as PDGF and TGF) from the protein kinase C family. It is well known that protein kinase C is involved in tumor cell proliferation, and plays an important role in a variety of cancers.
  • PCA Principle component analysis
  • FIG. 10 is a graphical representation showing a PCA of the microarray results of MIG- and MCP-I induced gene responses, indicating that MIG stimulation resulted in down regulation of oncogens (145 genes, 10A), cell proliferation (18 gens, 10B), transcription gene promoter CCAAT box (9 genes, C) and TATA box (23 gens, D), transcription protein E2F (8 genes, E), and up-regulation of leukocytes marker (4 gens, F), MHC (10 genes, G), anti-microbial mediators (4 genes, H). This indicates that these chemokines induce tumor cells to undergo terminal differentiation. This gene analysis indicates that certain chemokines, such as MIG, is antiproliferative.
  • IKBKB IKB kinase B
  • AP-2 transcription factor AP-2
  • tumor suppressor genes genes within the cell cycling pathway were significantly changed, amongst them, many functionally annotated as tumor suppressor genes, or genes encoding molecules that inhibit cell transcription and cell cycling, such as cyclin- dependent kinase inhibitor IA (p21, Cipl), cyclin E2, and Tp53 target gene 1, and other tumor suppressors, such as BTG2, arrestin, ALEX3, and the like. Also found were signaling molecules involved in the TGF- ⁇ pathway, such as transforming growth factor beta-activated kinase binding protein 1 (TABl) that was up-regulated by MIG.
  • TGF- ⁇ pathway such as transforming growth factor beta-activated kinase binding protein 1 (TABl) that was up-regulated by MIG.
  • TABl transforming growth factor beta-activated kinase binding protein 1
  • MCP-I in contrast to MIG, did not induce any of the above-mentioned signaling factors.
  • the only signaling protein on the MCP-I -induced gene list was Agouti signaling protein that may be involved in lipid metabolism. It is well known that chemokine receptor signaling is mediated through the PIP3 pathway that, however, was not changed by MIG, probably because the concentrations used were much higher than those that signal chemotactic responses. MIG activation of target cells apparently occurs via entirely different signal pathways than those of chemokine receptor-mediated pathways. This result further suggests that cationic chemokhies, such as MIG at ⁇ M concentrations, display immunomodulatory and antiproliferative activity that is chemokine receptor-independent.
  • Table 3 shows the results of confirmation Studies for THP-I cells induced by MIG. Data obtained from one of the microarray studies done on THP-I stimulated with MIG was validated by limited cycle RT-PCR on the same RNA sample, and/or in 3 separate biological repeats (BRl - BR3).
  • a lipase lipoprotein lipase;
  • b ND not detectable;
  • NC no change; Blank means not done.
  • MIG MCP-I
  • CCL3/MIP-l ⁇ CCL4/MIP- ⁇
  • CCL2/MCP-1 CCL25/TECK
  • IL-12 chemokines in addition to CXCL8/IL-8
  • IFITl Table 1
  • IFNK interferon kappa
  • PBEFl pre-B-cell colony enhancing factor 1
  • FCAR IgA Fc receptor
  • NOTCHl Notch homolog 1, translocation-associated
  • MAPKIl mitogen-activated protein kinase 11, ⁇ 38beta
  • MAPKl 3 mitogen-activated protein kinase 13, p38delta, PTX3
  • THP-I cells were incubated with water (control) or with different chemokines at final concentrations of 10 ⁇ M. After 4 hours of incubation, RNAs was isolated as described before. RT-PCR was performed to study gene expression. Results are shown in Table 5. The results demonstrate that the expression of the IL-8, MCP-I, MIP-Ia, and MlP-l ⁇ genes, were up- regulated by the cationic chemokines MIG and SLC, but to a lesser degree by MIP-3 ⁇ .
  • Bolded data refer to the cationic immunomodulatory chemokines.
  • Human monocytes were isolated from the peripheral blood of 3 donors, although the cells from donor 2 and donor 3 were pooled together to obtain a larger cell number required for study gene expression. Monocytes were incubated with water (the control) or with different chemokines at a final concentration of 10 ⁇ M. After 4 hours incubation, the RNAs were isolated as described before. RT-PCR was performed to study changes in gene expression. Results are summarized in Table 6. Gene expression of EL-8, MEP- l ⁇ and MIP- l ⁇ were significantly up- regulated by the cationic chemokines MIG, SLC and by MD?-3 ⁇ . In addition, expression of TNF- ⁇ and MCP-I was induced by MIG and MD?-3 ⁇ , but not by SLC.
  • THP-I cells cationic chemokines not only have immunomodulatory effects on THP-I cells, but also on human normal monocytes.
  • MIP- l ⁇ and MIP- l ⁇ protein expression in monocytes was also induced at concentrations as low as 0.3 ⁇ M. (See Figure 12A and 12B).
  • THP-I cells were treated with 10 ⁇ M of various chemokines or fragments thereof for 48 hours. Cytotoxicity was determined by LDH assay and MTT assay using commercial kits according the provided protocols. No cytotoxicity was observed for any of the chemokines or their fragments with either method (Table 7). TABLE 7
  • Table 6 shows the lack of cytoxicity of chemokines and their fragments at a concentration of 10 ⁇ M for 36 hours on THP-I cells. Results were consistently seen in 2 to 3 experiments. NC means no significant change relative to the buffer control that was normalized to a value of 0% LDH release or 100% MTT activity. Conversely the positive control Triton X-100 yielded values of 100% and 0% respectively.
  • Fig. 9A shows the Real time PCR results that confirmed that MIG treatment resulted in around a 7 fold increase in BTG2 gene expression in all 3 separate experiments; in contrast, MCP-I treatment under the exact same conditions did not change BTG2 gene expression, hi Fig.
  • THP-I cells were seeded into a 96 well plate and were co-incubated with 10 ⁇ M concentrations of either MIG or MCP-I, or with an equal volume of solvent for 8 days. At day 4, fresh medium with the same concentration of chemokines was added. The cell number was determined at days 4, 6 and 8. MIG treatment inhibited THP-I cell growth by 75% when compared to the no-chemokine control or MCP-1-treated cells (Fig. 11). No significant apoptosis of THP-I cells was observed after treatment with any of the given chemokines. EXAMPLE 15
  • MDA human breast cancer cell line
  • the novel immunomodulatory and antiproliferative functions are apparently correlated to the antimicrobial activity of these chemokines, although obviously separable as the immunomodulatory activity represents a stimulatory effect on mammalian (eukaryotic) cells while the antimicrobial effects represents and inhibitory activity on bacterial (prokaryotic) cells.
  • Chemokines, such as MIG that are relatively potent antimicrobials, exhibit very strong immunomodulatory activity.
  • Those chemokines, such as MCP-I and MIP- l ⁇ which have no antimicrobial activity, and have molecular surfaces that are not as positively charged, do not show immunomodulatory activity.
  • This novel immunomodulatory function apparently acts via mechanisms not related to the known classic G-protein coupled chemokine receptors, and is likely contained within structural features other than receptor binding regions (N-terminal region) of chemokines. This conclusion is made based on the following observations:
  • the immunomodulatory and antiproliferative chemokines displayed immunomodulatory and antiproliferative activities on target cells in a chemokine receptor- independent manner; these chemokines strongly activated target cells that do not carry the corresponding chemokine receptors. Also the activity could not be blocked by pertussis toxin, which blocks the activity of all chemokine receptors;
  • the immunomodulatory and antiproliferative chemokines stimulated target cells, giving rise to a gene expression profile that was completely different from that stimulated by other chemokines that are more weakly cationic and do not display immunomodulatory activities; 3.
  • the immunomodulatory and antiproliferative chemokines disregulated particular signalling pathways (such as the protein kinase C and NFKB pathways), which were entirely different from the classical chemokine receptor mediated pathway.
  • chemokine receptors mediate intracellular changes that are largely involved in the synthesis and rearrangement of cytoskeleton and proteoglycan, and other G-protein mediated reactions.
  • the cationic immunomodulatory chemokines described herein can modulate multiple types of cells, including monocytic cell lines and human monocytes.
  • chemokines can induce a dramatic change in the expression of particular molecules. For instance MIG induced target cells to produce large amounts of chemokines, cytokines and inflammatory mediators, and also induced the expression of surface molecules that are critical for innate immune response (such as CD14) and adoptive immunity (MHC antigen). Other immunomodulatory chemokines, such as IP-IO and MBP-3 ⁇ , stimulated target cells more mildly and induced chemokine production.
  • MIG down-regulated the expression of a set of oncogenes.
  • genes related to cell proliferation, cycling and cell growth of target cells were down regulated.
  • Genes encoding markers for mature leukocytes and MHC antigens were up-regulated.
  • Expression of genes related to transcription or regulating transcription including PHTFl, ZNF148, NFAT5, MTFl, HCNGP, RNF4, SCAl, PAFAHlBl, SRF are modulated by MIG.
  • This gene analysis indicates that certain chemokines, such as MIG and SLC are antiproliferative and induce tumor cells to undergo terminal differentiation. The conclusion was confirmed by showing that MIG and SLC directly inhibited THP-I cells (human myeloid leukemia cell line) and MDA cells (human breast cancer cell line) growth (Examples 14 & 15).
  • chemokines may inhibit tumor cell proliferation and induce cell differentiation.
  • This invention further provides genetic fingerprints for the immunomodulatory chemokines.
  • a specific pattern of altered gene expression was found to be unique for a specific chemokine.
  • MIP-Ia, MIP-I ⁇ , and IL-8 were each significantly up-regulated by MIG, and this profile has been observed in a sustained fashion throughout all biological repeats of both the THP-I monocytic cell line and human peripheral blood monocytes, in contrast to other genes that were induced by a broader set of cationic chemokines.
  • This invention also shows that not only the full length chemokine, but also the segments thereof can activate target cells. For instance both the N-terminal and C-terminal fragments of MIG activated target cells to release IL- 8. No cytotoxicity was found for any tested chemokines at the concentrations having immunomodulatory activity. The data indicates that these selective chemokines are able to enhance innate immunity, and directly inhibit tumor proliferation, and could be used as immunomodulators to treat or to prevent infection and cancer.
  • GM-CSF granulocyte-macrophage colony stimulating factor

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Abstract

L'invention concerne une classe de chimiokines présentant une activité antimicrobienne et antiproliférative. L'invention concerne également des méthodes d'identification de chimiokines et des composés présentant une activité immunomodulatoire et antiproliférative; des méthodes de renforcement de l'immunité naturelle chez un sujet; et des méthodes d'inhibition d'infections et de cancers utilisant les chimiokines de l'invention.
PCT/CA2005/001985 2004-12-29 2005-12-29 Activite immunomodulatoire et antiproliferative independante du recepteur de la chimiokine WO2006069449A1 (fr)

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Cited By (7)

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WO2008073957A2 (fr) * 2006-12-12 2008-06-19 Emory University Composés et procédés de modulation de silençage d'un polynucléotide d'intérêt
WO2008134020A1 (fr) * 2007-04-26 2008-11-06 University Of Vermont And State Agricultural College Procédés et compositions de ccl18 et ccl3 pour la détection et le traitement du cancer
CN103626906A (zh) * 2013-10-25 2014-03-12 江苏大学 一种可选择性识别四环素的亲水性聚合微球的合成方法
EP2926815A1 (fr) * 2014-04-03 2015-10-07 Institut Curie Dérivés de céphalosporines pour traiter le cancer
CN108939051A (zh) * 2012-10-02 2018-12-07 科瑞恩生物科技（股份）责任有限公司 先兆子痫胎盘间充质干细胞条件培养基在肿瘤治疗的应用
WO2019057808A1 (fr) * 2017-09-21 2019-03-28 University Of Copenhagen Peptides potentialisant la chimiotaxie et leurs utilisations
EP3693041A1 (fr) * 2011-06-13 2020-08-12 TLA Targeted Immunotherapies AB Traitement du cancer

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WO2002059377A2 (fr) * 2001-01-24 2002-08-01 Protein Design Labs Procedes de diagnostic du cancer du sein, compositions et procedes de criblage de modulateurs du cancer du sein
WO2003106488A2 (fr) * 2002-06-12 2003-12-24 Applied Research Systems Ars Holding N.V. Nouveaux antagonistes de chimiokines cxc a liaison cxcr3
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WO1995021921A1 (fr) * 1994-02-08 1995-08-17 Schering Corporation Genes de thymokine de mammiferes
WO2000069884A2 (fr) * 1999-05-14 2000-11-23 Genesis Research & Development Corporation Limited Compositions isolees a partir de cellules cutanees, et leurs procedes d'utilisation
WO2002004015A1 (fr) * 2000-07-12 2002-01-17 Gryphon Therapeutics, Inc. Chemokines synthetiques bioactives a polymeres modifies et procedes de fabrication et d'utilisation
WO2002059377A2 (fr) * 2001-01-24 2002-08-01 Protein Design Labs Procedes de diagnostic du cancer du sein, compositions et procedes de criblage de modulateurs du cancer du sein
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WO2003106488A2 (fr) * 2002-06-12 2003-12-24 Applied Research Systems Ars Holding N.V. Nouveaux antagonistes de chimiokines cxc a liaison cxcr3

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008073957A3 (fr) * 2006-12-12 2008-07-31 Univ Emory Composés et procédés de modulation de silençage d'un polynucléotide d'intérêt
WO2008073957A2 (fr) * 2006-12-12 2008-06-19 Emory University Composés et procédés de modulation de silençage d'un polynucléotide d'intérêt
WO2008134020A1 (fr) * 2007-04-26 2008-11-06 University Of Vermont And State Agricultural College Procédés et compositions de ccl18 et ccl3 pour la détection et le traitement du cancer
US8445442B2 (en) 2007-04-26 2013-05-21 University Of Vermont And State Agricultural College CCL18 and CCL3 methods and compositions for detecting and treating cancer
EP3693041A1 (fr) * 2011-06-13 2020-08-12 TLA Targeted Immunotherapies AB Traitement du cancer
CN108939051A (zh) * 2012-10-02 2018-12-07 科瑞恩生物科技（股份）责任有限公司 先兆子痫胎盘间充质干细胞条件培养基在肿瘤治疗的应用
CN108939051B (zh) * 2012-10-02 2022-04-15 科瑞恩生物科技（股份）责任有限公司 先兆子痫胎盘间充质干细胞条件培养基在肿瘤治疗的应用
CN103626906A (zh) * 2013-10-25 2014-03-12 江苏大学 一种可选择性识别四环素的亲水性聚合微球的合成方法
WO2015150516A1 (fr) * 2014-04-03 2015-10-08 Institut Curie Nouveaux dérivés de céphalosporine pour le traitement du cancer
JP2019069990A (ja) * 2014-04-03 2019-05-09 アンスティテュ・キュリＩｎｓｔｉｔｕｔ Ｃｕｒｉｅ 癌を処置するためのセファロスポリンの新規誘導体
CN106255500B (zh) * 2014-04-03 2019-08-02 法国居里学院 用于治疗癌症的新的头孢菌素衍生物
CN106255500A (zh) * 2014-04-03 2016-12-21 法国居里学院 用于治疗癌症的新的头孢菌素衍生物
US10821114B2 (en) 2014-04-03 2020-11-03 Institut Curie Derivatives of cephalosporin for treating cancer
EP2926815A1 (fr) * 2014-04-03 2015-10-07 Institut Curie Dérivés de céphalosporines pour traiter le cancer
WO2019057808A1 (fr) * 2017-09-21 2019-03-28 University Of Copenhagen Peptides potentialisant la chimiotaxie et leurs utilisations

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