Patent Application
20120004160
The present invention relates to agents, and methods for identifying compounds, which agents and compounds result in the inhibition of activation of macrophages. In addition, the invention relates to compositions and methods for their use in treating conditions that are characterized by macrophage activity including infections, allograft reactions, inflammation (such as but not limited to Chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), rheumatoid arthritis), metabolic disease, cardiovascular disease, tissue injury, allergic and autoimmune diseases, and cancer.
Inflammation is a complex response of the body to harmful events, such as pathogens, damaged cells, or irritants. Through this process the body tries to remove the harmful agents and start the healing process for the affected tissue. This process is very well controlled in most cases. However, when the control of the inflammation process is lost, inflammation can also lead to a range of diseases, such as allergies, rheumatoid arthritis, inflammatory bowel disease, and atherosclerosis. Also, inflammatory processes may contribute to metabolic diseases, cancer and allograft rejections. Amongst the cell types playing a role in the inflammatory process, macrophages have been shown to be a key player in inflammatory processes.
Despite an increased understanding of macrophages and their role in inflammatory diseases and pathological events such as infections, allograft reactions, inflammation (such as but not limited to Chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), rheumatoid arthritis), metabolic disease, cardiovascular disease, tissue injury, allergic and autoimmune diseases, and cancer, and other disorders and conditions, there is still a need for improved and specific therapies for these conditions and for the particular modulation of macrophage activation and/or their release of inflammatory factors or mediators.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
In the present invention cultivated human primary macrophages have been used to identify targets involved in inflammatory processes. The advantage of using primary cells is that their gene expression profile is similar or identical to that of macrophages in human tissue, avoiding the false positive or false negative results that may occur in utilizing passaged or established cell lines or tumour cell lines.
The present invention provides for targets identified by screening of an adenoviral expression library of shRNAs directed against mRNA sequences of drugable targets which allows the identification of drugable regulators for macrophage activation. In the knock-down approach (shRNA expression constructs), the shRNA expression constructs mimic antagonistic compounds. The invention also relates to the development of compounds that result in the modulation of macrophage activation. Preferably, the compound antagonizes the activation of macrophages, and/or inhibits the release of inflammatory mediators, particularly TNFα.
The present invention is based on the discovery that agents that inhibit the expression and/or activity of the TARGETS disclosed herein are able to result in inhibition of the activation of macrophages, as indicated by a suppression of the release of cytokines from macrophages, in particular a suppression of the release of TNFα. The present invention therefore provides TARGETS which are involved in the pathway leading to macrophage activation, methods for screening for agents capable of inhibiting macrophage activation and uses of these agents in the prevention and/or treatment of diseases associated with macrophage activation, in particular diseases associated with local or systemic inflammation including, without limitation, infections, inflammation (Chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), crohns and colitis, rheumatoid arthritis), metabolic disease, cardiovascular disease, tissue injury, allograft reactions, allergic and autoimmune diseases, and cancer.
The present invention relates to a method for identifying compounds that inhibit macrophage activation, comprising contacting the compound with the identified TARGETS or their protein domain fragments (SEQ ID. NO: 18-34) under conditions that allow said TARGETS or their protein domain fragments to bind to the compound, and measuring a compound-polypeptide property related to activation of macrophages. In one aspect the property is the release of chemokines (for example IL-8/CXCL8, IP-10/CXCL10, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5) or cytokines (for example IL-1β/IL-1F2, IL-6, and/or TNF-α/TNFSF1A) from macrophages, in particular the release of TNF-α. In an alternative aspect the property is the release of proteolytic enzymes selected from MMP-1, -2, -7, -9, and -12 and/or growth factors for example but without limitation TGF-β, VEGF, FGF, and PDGF. In a further aspect the property is the altered morphology of the macrophages or the altered expression of cell surface markers, for example but without limitation mannose receptor, sialoadhesins, macrosialin, and F480.
In particular the present invention provides TARGETS which are involved in the activation of macrophages, methods for screening for agents capable of modulating the expression and/or activity of TARGETS and uses of these agents in the prevention and/or treatment of diseases involving activated macrophages, in particular infections, inflammation, allograft reactions, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer. The invention provides uses of agents directed against these targets in the diseases discussed above. In a particular aspect the present invention provides TARGETS which are involved in rheumatoid arthritis.
Aspects of the present method include the in vitro assay of compounds using identified TARGETS, and cellular assays wherein identified TARGET inhibition is followed by observing indicators of efficacy, including release of chemokines, cytokines, proteolytic enzymes, and/or growth factors and/or altered expression of cell surface markers. A particular exemplary cytokine is TNF-α. Another aspect of the invention is a method of treatment or prevention of a condition involving activated macrophages, in a subject suffering or susceptible thereto, by administering a pharmaceutical composition comprising an agent which is able to inhibit the activation of macrophages.
The present invention relates to a method for identifying compounds that inhibit the TARGET(s), comprising contacting the compound with the identified TARGETS or their protein domain fragments (SEQ ID NO: 18-34) under conditions wherein the compounds may interact with or influence the TARGET(s), measuring the expression or release of chemokines, cytokines, proteolytic enzymes, and/or growth factors, and selecting compounds which suppress the expression or release of said chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages. In one such method regarding the release of cytokines, TNFα is measured.
The present invention relates to a method for identifying compounds that inhibit the TARGET(s), comprising contacting the compound with the identified TARGETS or their protein domain fragments (SEQ ID NO: 18-34) under conditions wherein the compounds may interact with or influence the TARGET(s), monitoring the morphological appearance of the macrophages and selecting compounds which alter the morphological appearance of said macrophages.
The present invention relates to a method for identifying compounds that inhibit the TARGET(s), comprising contacting the compound with the identified TARGETS or their protein domain fragments (SEQ ID NO: 18-34) under conditions wherein the compounds may interact with or influence the TARGET(s), monitoring the expression of cell surface markers on the surface of macrophages and selecting compounds which alter the expression of said cell surface markers.
The present invention relates to a method for identifying compounds that are able to inhibit macrophage activation, comprising contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18-34 (hereinafter “TARGETS”) and fragments thereof, under conditions that allow said polypeptide to bind to said compound, and measuring a compound-polypeptide property related to macrophage activation. In a specific embodiment, the present invention relates to a method for identifying compounds that are able to modulate the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages, comprising contacting a compound with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18-34 (hereinafter “TARGETS”) and fragments thereof, under conditions that allow said polypeptide to bind to said compound, and measuring the release of said chemokines, cytokines, proteolytic enzymes, and/or growth factors from said macrophages, in a particular aspect the release of cytokines, in particular TNFα is measured.
Aspects of the present method include the in vitro assay of compounds using a TARGET polypeptide sequence, or fragments thereof, including the amino acid sequences described by SEQ ID NO: 18-34 and cellular assays wherein TARGET inhibition is followed by observing indicators of efficacy including, for example, TARGET expression levels, TARGET enzymatic activity, activation of macrophages (e.g. by measuring release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages and/or by monitoring morphological changes and/or expression of cell surface markers of macrophages) and/or other assessments of immune or inflammatory response.
The present invention also relates to
Another aspect of the invention is a method of treatment or prevention of a disease characterized by macrophage activation, in particular infections, inflammation, allograft reactions, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury and cancer, in a subject suffering from or susceptible thereto, by administering a pharmaceutical composition comprising an effective TARGET-expression inhibiting amount of a expression-inhibitory agent or an effective TARGET activity inhibiting amount of an activity-inhibitory agent.
A further aspect of the present invention is a method for diagnosis of a disease characterized by activation of macrophages, in particular infections, inflammation, allograft reactions, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer comprising measurement of indicators of levels of TARGET expression in a subject. In particular the present invention relates to a method for the diagnosis of allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
Another aspect of this invention relates to the use of agents which inhibit a TARGET as disclosed herein in a therapeutic method, a pharmaceutical composition, and the manufacture of such composition, useful for the treatment of a disease involving the activation of macrophages. In particular, the present method relates to the use of the agents which inhibit a TARGET in the treatment of a disease characterized by activation of macrophages, suitable conditions include but are not limited to asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis.
Another aspect of this invention relates to the use of agents which inhibit a TARGET as disclosed herein in a therapeutic method, a pharmaceutical composition, and the manufacture of such composition, useful for the treatment of a disease involving inflammation, local or systemic. In particular said diseases are selected from the group consisting of allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
Other objects and advantages will become apparent from a consideration of the ensuing description taken in conjunction with the following illustrative drawings.
The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention.
The term ‘agent’ means any molecule, including polypeptides, antibodies, polynucleotides, chemical compounds and small molecules. In particular the term agent includes compounds such as test compounds or drug candidate compounds.
The term ‘agonist’ refers to a ligand that stimulates the receptor the ligand binds to in the broadest sense.
As used herein, the term ‘antagonist’ is used to describe a compound that does not provoke a biological response itself upon binding to a receptor, but blocks or dampens agonist-mediated responses.
The term ‘assay’ means any process used to measure a specific property of an agent. A ‘screening assay’ means a process used to characterize or select agents based upon their activity from a collection of agents.
The term ‘binding affinity’ is a property that describes how strongly two or more compounds associate with each other in a non-covalent relationship. Binding affinities can be characterized qualitatively, (such as ‘strong’, ‘weak’, ‘high’, or ‘low’) or quantitatively (such as measuring the KD).
The term ‘carrier’ means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition. A carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution. Examples of physiologically acceptable carriers include aqueous or solid buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.
The term ‘complex’ means the entity created when two or more compounds bind to, contact, or associate with each other.
The term ‘compound’ is used herein in the context of a ‘test compound’ or a ‘drug candidate compound’ described in connection with the assays of the present invention. As such, these compounds comprise organic or inorganic compounds, derived synthetically, recombinantly, or from natural sources.
The compounds include inorganic or organic compounds such as polynucleotides, lipids or hormone analogs. Other biopolymeric organic test compounds include peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, including polypeptide ligands, enzymes, receptors, channels, antibodies or antibody conjugates.
The term ‘condition’ or ‘disease’ means the overt presentation of symptoms (i.e., illness) or the manifestation of abnormal clinical indicators (for example, biochemical indicators or diagnostic indicators). Alternatively, the term ‘disease’ refers to a genetic or environmental risk of or propensity for developing such symptoms or abnormal clinical indicators.
The term ‘contact’ or ‘contacting’ means bringing at least two moieties together, whether in an in vitro system or an in vivo system.
The term ‘derivatives of a polypeptide’ relates to those peptides, oligopeptides, polypeptides, proteins and enzymes that comprise a stretch of contiguous amino acid residues of the polypeptide and that retain a biological activity of the protein, for example, polypeptides that have amino acid mutations compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may further comprise additional naturally occurring, altered, glycosylated, acylated or non-naturally occurring amino acid residues compared to the amino acid sequence of a naturally occurring form of the polypeptide. It may also contain one or more non-amino acid substituents, or heterologous amino acid substituents, compared to the amino acid sequence of a naturally occurring form of the polypeptide, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence.
The term ‘derivatives of a polynucleotide’ relates to DNA-molecules, RNA-molecules, and oligonucleotides that comprise a stretch of nucleic acid residues of the polynucleotide, for example, polynucleotides that may have nucleic acid mutations as compared to the nucleic acid sequence of a naturally occurring form of the polynucleotide. A derivative may further comprise nucleic acids with modified backbones such as PNA, polysiloxane, and 2′-O-(2-methoxy) ethyl-phosphorothioate, non-naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection.
The term ‘endogenous’ shall mean a material that a mammal naturally produces. Endogenous in reference to the term ‘protease’, ‘kinase’, or G-Protein Coupled Receptor (‘GPCR’) shall mean that which is naturally produced by a mammal (for example, and not limitation, a human). In contrast, the term non-endogenous in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human). Both terms can be utilized to describe both in vivo and in vitro systems. For example, and without limitation, in a screening approach, the endogenous or non-endogenous TARGET may be in reference to an in vitro screening system. As a further example and not limitation, where the genome of a mammal has been manipulated to include a non-endogenous
TARGET, screening of a candidate compound by means of an in vivo system is viable.
The term ‘expressible nucleic acid’ means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.
The term ‘expression’ comprises both endogenous expression and overexpression by transduction.
The term ‘expression inhibitory agent’ means a polynucleotide designed to interfere selectively with the transcription, translation and/or expression of a specific polypeptide or protein normally expressed within a cell. More particularly, ‘expression inhibitory agent’ comprises a DNA or RNA molecule that contains a nucleotide sequence identical to or complementary to at least about 15-30, particularly at least 17, sequential nucleotides within the polyribonucleotide sequence coding for a specific polypeptide or protein. Exemplary expression inhibitory molecules include ribozymes, double stranded siRNA molecules, self-complementary single-stranded siRNA molecules (shRNA), genetic antisense constructs, and synthetic RNA antisense molecules with modified stabilized backbones.
The term ‘fragment of a polynucleotide’ relates to oligonucleotides that comprise a stretch of contiguous nucleic acid residues that exhibit substantially a similar, but not necessarily identical, activity as the complete sequence. In a particular aspect, ‘fragment’ may refer to a oligonucleotide comprising a nucleic acid sequence of at least 5 nucleic acid residues (preferably, at least 10 nucleic acid residues, at least 15 nucleic acid residues, at least 20 nucleic acid residues, at least 25 nucleic acid residues, at least 40 nucleic acid residues, at least 50 nucleic acid residues, at least 60 nucleic residues, at least 70 nucleic acid residues, at least 80 nucleic acid residues, at least 90 nucleic acid residues, at least 100 nucleic acid residues, at least 125 nucleic acid residues, at least 150 nucleic acid residues, at least 175 nucleic acid residues, at least 200 nucleic acid residues, or at least 250 nucleic acid residues) of the nucleic acid sequence of said complete sequence.
The term ‘fragment of a polypeptide’ relates to peptides, oligopeptides, polypeptides, proteins, monomers, subunits and enzymes that comprise a stretch of contiguous amino acid residues, and exhibit substantially a similar, but not necessarily identical, functional or expression activity as the complete sequence. In a particular aspect, ‘fragment’ may refer to a peptide or polypeptide comprising an amino acid sequence of at least 5 amino acid residues (preferably, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, at least 150 amino acid residues, at least 175 amino acid residues, at least 200 amino acid residues, or at least 250 amino acid residues) of the amino acid sequence of said complete sequence.
The term ‘hybridization’ means any process by which a strand of nucleic acid binds with a complementary strand through base pairing. The term ‘hybridization complex’ refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A hybridization complex may be formed in solution (for example, C0t, or R0t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (for example, paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed). The term “stringent conditions” refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, for example, formamide, temperature, and other conditions well known in the art. In particular, reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature can increase stringency. The term ‘standard hybridization conditions’ refers to salt and temperature conditions substantially equivalent to 5×SSC and 65° C. for both hybridization and wash. However, one skilled in the art will appreciate that such ‘standard hybridization conditions’ are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20NC below the predicted or determined Tm with washes of higher stringency, if desired.
The term ‘inhibit’ or ‘inhibiting’, in relationship to the term ‘response’ means that a response is decreased or prevented in the presence of a compound as opposed to in the absence of the compound.
The term ‘inhibition’ refers to the reduction, down regulation of a process or the elimination of a stimulus for a process, which results in the absence or minimization of the expression or activity of a protein or polypeptide.
The term ‘induction’ refers to the inducing, up-regulation, or stimulation of a process, which results in the expression or activity of a protein or polypeptide.
The term ‘ligand’ means a molecule, including an endogenous, naturally occurring molecule or a synthetic non-natural molecule, specific for an endogenous, naturally occurring receptor.
The term ‘pharmaceutically acceptable salts’ refers to the non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds which inhibit the expression or activity of TARGETS as disclosed herein. These salts can be prepared in situ during the final isolation and purification of compounds useful in the present invention.
The term ‘polypeptide’ relates to proteins (such as TARGETS), proteinaceous molecules, fragments of proteins, monomers, subunits or portions of polymeric proteins, peptides, oligopeptides and enzymes (such as kinases, proteases, GPCR's etc.).
The term ‘polynucleotide’ means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pairs, and more particularly 70 percent of its base pairs are in common, most particularly 90 per cent, and in a particular embodiment, 100 percent of its base pairs. The polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof. It also includes nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2′-O-(2-methoxy)ethylphosphorothioate. The polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, particularly about 100 to about 4000 bases, more particularly about 250 to about 2500 bases. One polynucleotide embodiment comprises from about 10 to about 30 bases in length. A particular embodiment of polynucleotide is the polyribonucleotide of from about 17 to about 22 nucleotides, more commonly described as small interfering RNAs (siRNAs—either double stranded siRNA or single stranded self complementary siRNA (shRNA)). Another particular embodiment are nucleic acids with modified backbones such as peptide nucleic acid (PNA), polysiloxane, and 2′-O-(2-methoxy)ethylphosphorothioate, or including non-naturally occurring nucleic acid residues, or one or more nucleic acid substituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-, amino-, propyl-, chloro-, and methanocarbanucleosides, or a reporter molecule to facilitate its detection. Polynucleotides herein are selected to be ‘substantially’ complementary to different strands of a particular target DNA sequence. This means that the polynucleotides must be sufficiently complementary to hybridize with their respective strands. Therefore, the polynucleotide sequence need not reflect the exact sequence of the target sequence. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the polynucleotide, with the remainder of the polynucleotide sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the polynucleotide, provided that the polynucleotide sequence has sufficient complementarity with the sequence of the strand to hybridize therewith under stringent conditions or to form the template for the synthesis of an extension product.
The term ‘preventing’ or ‘prevention’ refers to a reduction in risk of acquiring or developing a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop) in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset.
The term ‘prophylaxis’ is related to and encompassed in the term ‘prevention’, and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti-malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.
The term ‘solvate’ means a physical association of a compound useful in this invention with one or more solvent molecules. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. “Solvate” encompasses both solution-phase and isolable solvates. Representative solvates include hydrates, ethanolates and methanolates.
The term ‘subject’ includes humans and other mammals.
‘Therapeutically effective amount’ means that amount of a drug, compound, expression inhibitory agent, or pharmaceutical agent that will elicit the biological or medical response of a subject that is being sought by a medical doctor or other clinician.
The term ‘treating’ or ‘treatment’ of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e., arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment ‘treating’ or ‘treatment’ refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, ‘treating’ or ‘treatment’ refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, ‘treating’ or ‘treatment’ relates to slowing the progression of the disease.
The term ‘vectors’ also relates to plasmids as well as to viral vectors, such as recombinant viruses, or the nucleic acid encoding the recombinant virus.
The term ‘vertebrate cells’ means cells derived from animals having vertera structure, including fish, avian, reptilian, amphibian, marsupial, and mammalian species. Preferred cells are derived from mammalian species, and most preferred cells are human cells. Mammalian cells include feline, canine, bovine, equine, caprine, ovine, porcine murine, such as mice and rats, and rabbits.
The term ‘TARGET’ or ‘TARGETS’ means the protein(s) identified in accordance with the assays described herein and determined to be involved in the modulation of maturation of dendritic cells. The term TARGET or TARGETS includes and contemplates alternative species forms, isoforms, and variants, such as splice variants, allelic variants, alternate in frame exons, and alternative or premature termination or start sites, including known or recognized isoforms or variants thereof such as indicated in Table 1.
The term ‘disease characterized by activation of macrophages’ refers to a disease which involves, results at least in part from, or includes activation of macrophages, in particular diseases associated with local or system inflammation. The term includes, but is not limited to, exemplary diseases selected from asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis.
The term ‘disease characterized by inflammation’ refers to a disease which involves, results at least in part from or includes inflammation. The term includes, but is not limited to, exemplary diseases selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
The term ‘autoimmune disease’ refers to a disease which involves, results at least in part from or includes an immune response of the body against substances and tissues normally present in the body. The term includes, but is not limited to, exemplary diseases selected from Addison's disease, ankylosing spondylitis, coeliac disease, chronic obstructive pulmonary disease, dermatomyositis, diabetes mellitus type 1, Graves' disease, Guillain-Barré syndrome (GBS), lupus erythematosus, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, and vasculitis.
The term ‘cancer’ refers to a malignant or benign growth of cells in skin or in body tissues or organs, for example but without limitation, breast, prostate, lung, kidney, pancreas, stomach or bowel. A cancer tends to infiltrate into adjacent tissue and spread (metastasise) to distant organs, for example to bone, liver, lung or the brain. As used herein the term cancer includes both metastatic tumour cell types, such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma and types of tissue carcinoma, such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma.
The term ‘inflammatory mediators’ refers to mediators which are associated with, enhance, initiate, or facilitate an inflammatory reaction or an inflammatory response, and may be selected from the following: cytokines (e.g. TNFalpha, IL3, IL4, IL5, IL13, GM-CSF), chemokines (e.g. MDC, CCL19, CCL20, CCL21, MIP-1alpha), Prostaglandins (e.g. PGD2), Leukotrienes (e.g. LTB4, LTC4, LTD4), metalloproteases, chymase, tryptase, growth factors (e.g. VEGF).
The present invention is based on the present inventors' discovery that the TARGETS are factors in the activation of macrophages, whereby inhibition of the TARGETS results in suppression of the release of cytokines, in particular TNFα following activation of macrophages. The TARGETS are factors or protein molecules involved in the response of macrophages to triggers such that their inhibition results in a suppression of the activation of macrophages. The TARGETS may also serve a role in autoimmune and or inflammatory response in other cells, particularly in basophils and plasmacytoid dendritic cells.
The TARGETS listed in Table 1 below were identified herein as involved in the pathway that controls the activation of macrophages on stimulation, therefore, inhibitors of these TARGETS are able to inhibit the activation of macrophages and are of use in the prevention and/or treatment of diseases involving local or systemic inflammation. These TARGETS are proposed to have a general role in autoimmune and inflammatory responses via macrophages. Inhibition of these TARGETS is demonstrated herein to result in a suppression of the release of cytokines, in particular TNFα, from macrophages. Therefore these TARGETS are involved in diseases characterized by autoimmune and/or inflammatory responses.
Therefore, in one aspect, the present invention relates to a method for assaying for drug candidate compounds that inhibit the activation of macrophages comprising contacting the compound with a polypeptide comprising an amino acid sequence of SEQ ID NO: 18-34, or fragment thereof, under conditions that allow said polypeptide to bind to the compound, and detecting the formation of a complex between the polypeptide and the compound. In particular said method may be used to identify drug candidate compounds that inhibit the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages. In particular said method may be used to identify drug candidate compounds that inhibit the release of cytokines, e.g. TNFα, from macrophages. One particular means of measuring the complex formation is to determine the binding affinity of said compound to said polypeptide.
More particularly, the invention relates to a method for identifying an agent or compound that inhibits the activation of macrophages said method comprising:
In a further aspect of the present invention said method is used to identify a compound that inhibits the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages. In particular said method is used to identify a compound that inhibits the relase of cytokines from macrophages. In particular said method is used to identify a compound that inhibits the relase of TNFα from macrophages.
In a further aspect, the present invention relates to a method for assaying for drug candidate compounds that inhibit activation of macrophages comprising
In particular said method may be used to identify drug candidate compounds capable of suppressing the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages. In particular said method may be used to identify drug candidate compounds capable of suppressing the release of cytokines, for example TNFα, from macrophages. One particular means of measuring the activity or expression of the polypeptide is to determine the amount of said polypeptide using a polypeptide binding agent, such as an antibody, or to determine the activity of said polypeptide in a biological or biochemical measure, for instance the amount of phosphorylation of a target of a kinase polypeptide.
The compound-polypeptide property referred to above is related to the expression and/or activity of the TARGET, and is a measurable phenomenon chosen by the person of ordinary skill in the art. The measurable property may be, for example, the binding affinity of said compound for a peptide domain of the polypeptide TARGET, a property related to the folding or activity of the disease-related protein or the level of any one of a number of biochemical marker levels of inflammation. In a particular method, activation of macrophages may be measured by measuring release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages, in particular the release of cytokines, for example TNFα. In an alternative method, activation of macrophages is measured by monitoring morphological changes in the macrophages.
In an additional aspect, the present invention relates to a method for assaying for drug candidate compounds that inhibit activation of macrophages, comprising contacting the compound with a nucleic acid encoding a TARGET polypeptide, including a nucleic acid sequence selected from SEQ ID NO: 1-17, or fragment/portion thereof, under conditions that allow said nucleic acid to bind to or otherwise associate with the compound, and detecting the formation of a complex between the nucleic acid and the compound. In particular, said method may be used to identify drug candidate compounds able to suppress the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages. In particular, said method may be used to identify drug candidate compounds able to suppress the release of cytokines, for example TNFα, from macrophages. One particular means of measuring the complex formation is to determine the binding affinity of said compound to said nucleic acid or the presence of a complex by virtue of resistance to nucleases or by gel mobility assays. Alternatively, complex formation may be determined by inhibition of nucleic acid transcription or translation.
In a particular embodiment of the invention, the TARGET polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID No: 18-34 as listed in Table 1. In an embodiment of the invention, the nucleic acid capable of encoding the TARGET polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-17 as listed in Table 1. Table 1 provides TARGET exemplary human nucleic acid and protein sequence, including recognized variants or isoforms where more than one accession number and SEQ ID NO: is indicated. Isoforms or variants of the TARGET(S) include nucleic acid or proteins with or utilizing alternate in frame exons, alternative splicing or splice variants, and alternative or premature termination variants.
Depending on the choice of the skilled artisan, the present assay method may be designed to function as a series of measurements, each of which is designed to determine whether the drug candidate compound is indeed acting on the TARGET to thereby inhibit the activation of macrophages. For example, an assay designed to determine the binding affinity of a compound to the TARGET, or fragment thereof, may be necessary, but not sufficient, to ascertain whether the test compound would be useful for inhibiting the activation of macrophages when administered to a subject. Nonetheless, such binding information would be useful in identifying a set of test compounds for use in an assay that would measure a different property, further down the biochemical pathway, for example suppression of the release of cytokines. Such additional assay(s) may be designed to confirm that the test compound, having binding affinity for the TARGET, actually inhibits the activation of macrophages.
Suitable controls should always be in place to insure against false positive readings. In a particular embodiment of the present invention the screening method comprises the additional step of comparing the compound to a suitable control. In one embodiment, the control may be a cell or a sample that has not been in contact with the test compound. In an alternative embodiment, the control may be a cell that does not express the TARGET; for example in one aspect of such an embodiment the test cell may naturally express the TARGET and the control cell may have been contacted with an agent, e.g. an siRNA (either a double stranded siRNA or a single stranded self complementary siRNA (shRNA)), which inhibits or prevents expression of the TARGET. Alternatively, in another aspect of such an embodiment, the cell in its native state does not express the TARGET and the test cell has been engineered so as to express the TARGET, so that in this embodiment, the control could be the untransformed native cell. Whilst exemplary controls are described herein, this should not be taken as limiting; it is within the scope of a person of skill in the art to select appropriate controls for the experimental conditions being used.
The order of taking these measurements is not believed to be critical to the practice of the present invention, which may be practiced in any order. For example, one may first perform a screening assay of a set of compounds for which no information is known respecting the compounds' binding affinity for the TARGET. Alternatively, one may screen a set of compounds identified as having binding affinity for a TARGET protein domain, or a class of compounds identified as being an inhibitor of the TARGET. However, for the present assay to be meaningful to the ultimate use of the drug candidate compounds in diseases characterized by activation of macrophages, an immune response and/or inflammation (local or systemic), a measurement of the activation of macrophages is necessary. Validation studies, including controls, and measurements of binding affinity to the polypeptides of the invention are nonetheless useful in identifying a compound useful in any therapeutic or diagnostic application.
Analogous approaches based on art-recognized methods and assays may be applicable with respect to the TARGETS and compounds in any of various disease(s) characterized by activation of macrophages, autoimmune response or inflammatory diseases. An assay or assays may be designed to confirm that the test compound, having binding affinity for the TARGET, inhibits the activation of macrophages. In one such method the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages is measured. In a particular embodiment of said method the release of cytokines, e.g. TNFα, from macrophages is measured. In another such method the morphological appearance of macrophages is monitored.
The present assay method may be practiced in vitro, using one or more of the TARGET proteins, or fragments thereof, including monomers, portions or subunits of polymeric proteins, peptides, oligopeptides and enzymatically active portions thereof.
The binding affinity of the compound with the TARGET or a fragment thereof can be measured by methods known in the art, such as using surface plasmon resonance biosensors (Biacore), by saturation binding analysis with a labeled compound (e.g. Scatchard and Lindmo analysis), by differential UV spectrophotometer, fluorescence polarization assay, Fluorometric Imaging Plate Reader (FLIPR®) system, Fluorescence resonance energy transfer, and Bioluminescence resonance energy transfer. The binding affinity of compounds can also be expressed in dissociation constant (Kd) or as IC50 or EC50. The IC50 represents the concentration of a compound that is required for 50% inhibition of binding of another ligand to the polypeptide. The EC50 represents the concentration required for obtaining 50% of the maximum effect in any assay that measures the TARGET function. The dissociation constant, Kd, is a measure of how well a ligand binds to the polypeptide, it is equivalent to the ligand concentration required to saturate exactly half of the binding-sites on the polypeptide. Compounds with a high affinity binding have low Kd, IC50 and EC50 values, i.e. in the range of 100 nM to 1 pM; a moderate to low affinity binding relates to a high Kd, IC50 and EC50 values, i.e. in the micromolar range.
The present assay method may also be practiced in a cellular assay. A host cell expressing the TARGET can be a cell with endogenous expression or a cell over-expressing the TARGET e.g. by transduction. When the endogenous expression of the polypeptide is not sufficient to determine a baseline that can easily be measured, one may use host cells that over-express the TARGET. Over-expression has the advantage that the level of the TARGET substrate end products is higher than the activity level by endogenous expression. Accordingly, measuring such levels using presently available techniques is easier. In one such cellular assay, the biological activity of the TARGET may be measured by measuring the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages. In a particular cellular assay, the biological activity of the TARGET may be measured by measuring the release of cytokines from macrophages.
One embodiment of the present method for identifying a compound that inhibits activation of macrophages comprises culturing a population of mammalian cells expressing a TARGET polypeptide, or a functional fragment or derivative thereof; determining a first level of release of chemokine, cytokine, proteolytic enzymes, and/or growth factors in said population of cells on activation of the population of cells (e.g. after a trigger of IgG coated beads); exposing said population of cells to a compound, or a mixture of compounds; determining a second level of chemokine, cytokine, proteolytic enzymes, and/or growth factors release in said population of cells after the same activation, during or after exposure of said population of cells to said compound, or the mixture of said compounds; and identifying the compound(s) that suppress the release of said chemokines, cytokines, proteolytic enzymes, and/or growth factors. In a specific embodiment, the cells are macrophages. In a specific embodiment the cells are human cells.
The release of chemokines, cytokines, proteolytic enzymes, and/or growth factors from macrophages can be determined by methods known in the art such as the methods as described herein.
The present inventors identified TARGET genes involved in the inhibition of activation of macrophages by using a ‘knock-down’ library. This type of library is a screen in which siRNA molecules are transduced into cells by recombinant adenoviruses, which siRNA molecules inhibit or repress the expression of a specific gene as well as expression and activity of the corresponding gene product in a cell. Each siRNA in a viral vector corresponds to a specific natural gene. By identifying a siRNA that inhibits activation of macrophages, as measured by suppression of the release of cytokines, in particular TNFα, a direct correlation can be drawn between the specific gene expression and the pathway by which macrophages are activated. The TARGET genes identified using the knock-down library (the protein expression products thereof herein referred to as “TARGET” polypeptides) are then used in the present inventive method for identifying compounds that can be used to inhibit the activation of macrophages. Indeed, shRNA compounds comprising the sequences listed in Table 2 (SEQ ID NOs: 35-75) inhibit the expression and/or activity of these TARGET genes and suppress TNFα release, confirming the role of the TARGETS in the pathway leading to the activation of macrophages after a trigger (e.g. IgG coated beads).
The present invention further relates to a method for identifying a compound that inhibits the activation of macrophages, comprising:
In one aspect, the assay method includes contacting cells expressing said polypeptide with the compound that exhibits a binding affinity in the micromolar range. In an aspect, the binding affinity exhibited is at least 10 micromolar. In an aspect, the binding affinity is at least 1 micromolar. In an aspect, the binding affinity is at least 500 nanomolar.
The assay method may be based on the particular expression or activity of the TARGET polypeptide, including but not limited to an enzyme activity. Thus, assays for the enzyme TARGETs identified as SEQ ID NO: 23 (hydrolase) or SEQ ID NO: 25 (dehydrogenase) may be based on enzymatic activity or enzyme expression. Assays for the protease TARGET identified as SEQ ID NOs: 33 may be based on protease activity or expression. Assays for the kinase TARGETs identified as SEQ ID NOs: 22, 26, 29, 30, 32 or 34 may be based on kinase activity or expression, including but not limited to phosphorylation of a kinase target. Assays for the GPCR and receptor TARGETs identified as SEQ ID NO: 20, 21, 24, 27, 28 or 31 may be based on GPCR or receptor activity or expression, including downstream mediators or activators. Assays for the ion channel TARGETs identified as SEQ ID NOs: 18 or 18 may use techniques well known to those of skill in the art including classical patch clamping, high-throughput fluorescence based or tracer based assays which measure the ability of a compound to open or close an ion channel thereby changing the concentration of fluorescent dyes or tracers across a membrane or within a cell. The measurable phenomenon, activity or property may be selected or chosen by the skilled artisan. The person of ordinary skill in the art may select from any of a number of assay formats, systems or design one using his knowledge and expertise in the art.
Table 1 lists the TARGETS identified using applicants' knock-down library in the dendritic cell maturation assay described below, including the class of polypeptides identified. TARGETS have been identified in polypeptide classes including hydrolase, dehydrogenase, kinase, protease, GPCR, receptor and ion channel for instance.
Specific methods to determine the activity of a kinase by measuring the phosphorylation of a substrate by the kinase, which measurements are performed in the presence or absence of a compound, are well known in the art.
Specific methods to determine the inhibition by a compound by measuring the cleavage of the substrate by the polypeptide, which is a protease, are well known in the art. Classically, substrates are used in which a fluorescent group is linked to a quencher through a peptide sequence that is a substrate that can be cleaved by the target protease. Cleavage of the linker separates the fluorescent group and quencher, giving rise to an increase in fluorescence.
Ion channels are membrane protein complexes and their function is to facilitate the diffusion of ions across biological membranes. Membranes, or phospholipid bilayers, build a hydrophobic, low dielectric barrier to hydrophilic and charged molecules. Ion channels provide a high conducting, hydrophilic pathway across the hydrophobic interior of the membrane. The activity of an ion channel can be measured using classical patch clamping. High-throughput fluorescence-based or tracer-based assays are also widely available to measure ion channel activity. These fluorescent-based assays screen compounds on the basis of their ability to either open or close an ion channel thereby changing the concentration of specific fluorescent dyes across a membrane. In the case of the tracer based assay, the changes in concentration of the tracer within and outside the cell are measured by radioactivity measurement or gas absorption spectrometry.
G-protein coupled receptors (GPCR) are capable of activating an effector protein, resulting in changes in second messenger levels in the cell. The activity of a GPCR can be measured by measuring the activity level of such second messengers. Two important and useful second messengers in the cell are cyclic AMP (cAMP) and Ca2+. The activity levels can be measured by methods known to persons skilled in the art, either directly by ELISA or radioactive technologies or by using substrates that generate a fluorescent or luminescent signal when contacted with Ca2+ or indirectly by reporter gene analysis. The activity level of the one or more secondary messengers may typically be determined with a reporter gene controlled by a promoter, wherein the promoter is responsive to the second messenger. Promoters known and used in the art for such purposes are the cyclic-AMP responsive promoter that is responsive for the cyclic-AMP levels in the cell, and the NF-AT responsive promoter that is sensitive to cytoplasmic Ca2−-levels in the cell. The reporter gene typically has a gene product that is easily detectable. The reporter gene can either be stably infected or transiently transfected in the host cell. Useful reporter genes are alkaline phosphatase, enhanced green fluorescent protein, destabilized green fluorescent protein, luciferase and β-galactosidase.
It should be understood that the cells expressing the polypeptides, may be cells naturally expressing the polypeptides, or the cells may be transfected to express the polypeptides, as described above. Also, the cells may be transduced to overexpress the polypeptide, or may be transfected to express a non-endogenous form of the polypeptide, which can be differentially assayed or assessed.
In one particular embodiment the methods of the present invention further comprise the step of contacting the population of cells with an agonist of the polypeptide. This is useful in methods wherein the expression of the polypeptide in a certain chosen population of cells is too low for a proper detection of its activity. By using an agonist the polypeptide may be triggered, enabling a proper read-out if the compound inhibits the polypeptide. Similar considerations apply to the measurement of the release of chemokines, cytokines, proteolytic enzymes, and/or growth factors. In a particular embodiment, the cells used in the present method are mammalian macrophages. The macrophages, in the assay contemplated, may be activated (e.g. by contacting the cells with IgG coated beads).
A method for identifying a compound that inhibits the activation of macrophages, comprising:
In one embodiment of the present invention the compound-polypeptide property related to macrophage activation is binding affinity.
In one embodiment of the present invention the compound-polypeptide property related to macrophage activation is the suppression of the release of chemokines, cytokines, proteolytic enzymes and/or growth factors, in particular cytokines, e.g. TNFα.
In one embodiment of the present invention the compound-polypeptide property related to macrophage activation is the activity of said polypeptide. In particular, in one embodiment the compound inhibits the activity of said polypeptide.
In one embodiment of the present invention the compound-polypeptide property related to macrophage activation is the expression of said polypeptide. In particular, in one embodiment the compound inhibits the expression of said polypeptide.
The present invention further relates to a method for identifying a compound that inhibits the activation of macrophages, wherein said compound exhibits at least a moderate binding affinity to an amino acid selected from the group of SEQ ID NOS: 18-34, said method comprising:
In one embodiment of the above method, the release of cytokines is measured. In one such method the cytokines that is measured is TNFα.
In one such method, the compound exhibits a binding affinity to an amino acid selected from the group of SEQ ID NOS: 18-34 of at least 10 micromolar.
The present invention further relates to a method for identifying a compound that inhibits the activation of macrophages, said method comprising:
The present invention further relates to a method for identifying a compound that inhibits the activation of macrophages said method comprising:
In a particular aspect of the present invention the methods described above include the additional step of comparing the compound to be tested to a control, where the control is a population of cells that have not been contacted with the test compound.
In a particular aspect of the present invention the methods described above include the additional step of comparing the compound to be tested to a control, where the control is a population of cells that do not express said polypeptide.
For high-throughput purposes, libraries of compounds may be used such as antibody fragment libraries, peptide phage display libraries, peptide libraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries (BioMol), synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich, BioFocus DPI) or natural compound libraries (Specs, TimTec).
Preferred drug candidate compounds are low molecular weight compounds. Low molecular weight compounds, i.e. with a molecular weight of 500 Dalton or less, are likely to have good absorption and permeation in biological systems and are consequently more likely to be successful drug candidates than compounds with a molecular weight above 500 Dalton (Lipinski et al. (1997)). Peptides comprise another preferred class of drug candidate compounds. Peptides may be excellent drug candidates and there are multiple examples of commercially valuable peptides such as fertility hormones and platelet aggregation inhibitors. Natural compounds are another preferred class of drug candidate compound. Such compounds are found in and extracted from natural sources, and which may thereafter be synthesized. The lipids are another preferred class of drug candidate compound.
Another preferred class of drug candidate compounds is an antibody. The present invention also provides antibodies directed against the TARGETS. These antibodies may be endogenously produced to bind to the the TARGETS within the cell, or added to the tissue to bind to the TARGET polypeptide present outside the cell. These antibodies may be monoclonal antibodies or polyclonal antibodies. The present invention includes chimeric, single chain, and humanized antibodies, as well as FAb fragments and the products of a FAb expression library, and Fv fragments and the products of an Fv expression library.
In certain embodiments, polyclonal antibodies may be used in the practice of the invention. The skilled artisan knows methods of preparing polyclonal antibodies. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. Antibodies may also be generated against the intact TARGET protein or polypeptide, or against a fragment, derivatives including conjugates, or other epitope of the TARGET protein or polypeptide, such as the TARGET embedded in a cellular membrane, or a library of antibody variable regions, such as a phage display library.
It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). One skilled in the art without undue experimentation may select the immunization protocol.
In some embodiments, the antibodies may be monoclonal antibodies. Monoclonal antibodies may be prepared using methods known in the art. The monoclonal antibodies of the present invention may be “humanized” to prevent the host from mounting an immune response to the antibodies. A “humanized antibody” is one in which the complementarity determining regions (CDRs) and/or other portions of the light and/or heavy variable domain framework are derived from a non-human immunoglobulin, but the remaining portions of the molecule are derived from one or more human immunoglobulins. Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa. The humanization of antibodies may be accomplished by methods known in the art (see, e.g. Mark and Padlan, (1994) “Chapter 4. Humanization of Monoclonal Antibodies”, The Handbook of Experimental Pharmacology Vol. 113, Springer-Verlag, New York). Transgenic animals may be used to express humanized antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries (Hoogenboom and Winter, (1991) J. Mol. Biol. 227:381-8; Marks et al. (1991). J. Mol. Biol. 222:581-97). The techniques of Cole, et al. and Boerner, et al. are also available for the preparation of human monoclonal antibodies (Cole, et al. (1985) Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77; Boerner, et al (1991). J. Immunol., 147(1):86-95).
Techniques known in the art for the production of single chain antibodies can be adapted to produce single chain antibodies to the TARGETS. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively; the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens and preferably for a cell-surface protein or receptor or receptor subunit. In the present case, one of the binding specificities is for one domain of the TARGET; the other one is for another domain of the the TARGET.
Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, (1983) Nature 305:537-9). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. Affinity chromatography steps usually accomplish the purification of the correct molecule. Similar procedures are disclosed in Trauneeker, et al. (1991) EMBO J. 10:3655-9.
According to another preferred embodiment, the assay method uses a drug candidate compound identified as having a binding affinity for the TARGET, and/or has already been identified as having down-regulating activity such as antagonist activity for the TARGET.
In vivo animal models of inflammation or inflammatory diseases may be utilized by the skilled artisan to further or additionally screen, assess, and/or verify the agents or compounds identified in the present invention, including further assessing TARGET modulation in vivo. Such animal models include, but are not limited to, ulcerative colitis models, arthritis models, allergic asthma models, airway inflammation models, and acute inflammation models.
The present invention further relates to a method for inhibiting the activation of macrophages comprising contacting said cells with an expression inhibitory agent comprising a polynucleotide sequence that complements at least about 15 to about 30, particularly at least 17 to about 30, most particularly at least 17 to about 25 contiguous nucleotides of a nucleotide sequence encoding a polypeptide TARGET or portion thereof including the nucleotide sequences selected from the group consisting of SEQ ID NO: 1-17.
Another aspect of the present invention relates to a method for inhibiting the activation of macrophages, comprising contacting said cell with an expression-inhibiting agent that inhibits the translation in the cell of a polyribonucleotide encoding the TARGET. A particular embodiment relates to a composition comprising a polynucleotide including at least one antisense strand that functions to pair the agent with the the TARGET mRNA, and thereby down-regulate or block the expression of the TARGET. The inhibitory agent preferably comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence encoding a portion of a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 18-34. In a preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-17. In another preferred embodiment the expression-inhibiting agent is complementary to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 35-75.
An embodiment of the present invention relates to a method wherein the expression-inhibiting agent is selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for SEQ ID NO: 18-34, a small interfering RNA (siRNA, preferably shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for SEQ ID NO: 18-34, such that the siRNA, preferably shRNA, interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide. Preferably the expression-inhibiting agent is an antisense RNA, ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA, complementary to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-17. In another preferred embodiment, the nucleotide sequence is complementary to a polynucleotide selected from the group consisting of SEQ ID NO: 35-75.
The down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level. Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding the TARGET or the corresponding messenger RNA. In addition, antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding the TARGET by inhibiting splicing of its primary transcript. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for the TARGETS. Preferably, the antisense sequence is at least about 17 nucleotides in length. The preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of oligo and genetic antisense is known in the art.
One embodiment of expression-inhibitory agent is a nucleic acid that is antisense to a nucleic acid selected from the group consisting of SEQ ID NO: 1-17. For example, an antisense nucleic acid (e.g. DNA) may be introduced into cells in vitro, or administered to a subject in vivo, as gene therapy to inhibit cellular expression of a nucleic acid selected from the group constisting of SEQ ID NO: 1-17. Antisense oligonucleotides preferably comprise a sequence containing from about 15 to about 100 nucleotides and more preferably the antisense oligonucleotides comprise from about 17 to about 30, most particularly at least 17 to about 25. Antisense nucleic acids may be prepared from about 10 to about 30 contiguous nucleotides complementary to a nucleic acid sequence selected from the sequences of SEQ ID NO: 1-17.
The skilled artisan can readily utilize any of several strategies to facilitate and simplify the selection process for antisense nucleic acids and oligonucleotides effective in inhibition of TARGET expression. Predictions of the binding energy or calculation of thermodynamic indices between an olionucleotide and a complementary sequence in an mRNA molecule may be utilized (Chiang et al. (1991) J. Biol. Chem. 266:18162-18171; Stull et al. (1992) Nucl. Acids Res. 20:3501-3508). Antisense oligonucleotides may be selected on the basis of secondary structure (Wickstrom et al (1991) in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wickstrom, ed., Wiley-Liss, Inc., New York, pp. 7-24; Lima et al. (1992) Biochem. 31:12055-12061). Schmidt and Thompson (U.S. Pat. No. 6416,951) describe a method for identifying a functional antisense agent comprising hybridizing an RNA with an oligonucleotide and measuring in real time the kinetics of hybridization by hybridizing in the presence of an intercalation dye or incorporating a label and measuring the spectroscopic properties of the dye or the label's signal in the presence of unlabelled oligonucleotide. In addition, any of a variety of computer programs may be utilized which predict suitable antisense oligonucleotide sequences or antisense targets utilizing various criteria recognized by the skilled artisan, including for example the absence of self-complementarity, the absence hairpin loops, the absence of stable homodimer and duplex formation (stability being assessed by predicted energy in kcal/mol). Examples of such computer programs are readily available and known to the skilled artisan and include the OLIGO 4 or OLIGO 6 program (Molecular Biology Insights, Inc., Cascade, Colo.) and the Oligo Tech program (Oligo Therapeutics Inc., Wilsonville, Oreg.). In addition, antisense oligonucleotides suitable in the present invention may be identified by screening an oligonucleotide library, or a library of nucleic acid molecules, under hybridization conditions and selecting for those which hybridize to the target RNA or nucleic acid (see for example U.S. Pat. No. 6,500,615). Mishra and Toulme have also developed a selection procedure based on selective amplification of oligonucleotides that bind target (Mishra et al (1994) Life Sciences 317:977-982). Oligonucleotides may also be selected by their ability to mediate cleavage of target RNA by RNAse H, by selection and characterization of the cleavage fragments (Ho et al (1996) Nucl Acids Res 24:1901-1907; Ho et al (1998) Nature Biotechnology 16:59-630). Generation and targeting of oligonucleotides to GGGA motifs of RNA molecules has also been described (U.S. Pat. No. 6,277,981).
The antisense nucleic acids are preferably oligonucleotides and may consist entirely of deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both. The antisense nucleic acids can be synthetic oligonucleotides. The oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage. Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases. In addition, when a phosphorothioate antisense oligonucleotide hybridizes to its target site, the RNA-DNA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule. Oligonucleotides may also contain one or more substituted sugar moieties. Particular oligonucleotides comprise one of the following at the 2′ position: OH, SH, SCH3, F, OCN, heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide.
In addition, antisense oligonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation. Furthermore, chemical groups can be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its target site. Modifications may include 2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates, modified bases, as well as other modifications known to those of skill in the art.
Another type of expression-inhibitory agent that can reduce the level of the TARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains. The substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its target sequence. The catalytic portion cleaves the target RNA at a specific site. The substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence. The ribozyme recognizes and then binds a target mRNA through complementary base pairing. Once it is bound to the correct target site, the ribozyme acts enzymatically to cut the target mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its target sequence, it is released and can repeatedly bind and cleave at other mRNAs.
Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9). A ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21:3249-55).
Ribozymes may be chemically synthesized by combining an oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the target mRNA after transcription. The oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers. The amplification product is cloned into a eukaryotic expression vector.
Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21:2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15).
A particularly preferred inhibitory agent is a small interfering RNA (siRNA, preferably shRNA). siRNA, preferably shRNA, mediate the post-transcriptional process of gene silencing by double stranded RNA (dsRNA) that is homologous in sequence to the silenced RNA. siRNA according to the present invention comprises a sense strand of 15-30, particularly 17-30, most particularly 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence of a sequence selected from the group consisting of SEQ ID NO: 1-17, and an antisense strand of 17-23 nucleotides complementary to the sense strand. Exemplary sequences are described as sequences complementary to SEQ ID NO: 35-75. The most preferred siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the target polynucleotide sequence. Preferably the siRNA further comprises a loop region linking the sense and the antisense strand.
A self-complementing single stranded siRNA molecule polynucleotide according to the present invention comprises a sense portion and an antisense portion connected by a loop region linker. Preferably, the loop region sequence is 4-30 nucleotides long, more preferably 5-15 nucleotides long and most preferably 12 nucleotides long. In a most particular embodiment the linker sequence is UUGCUAUA or GUUUGCUAUAAC (SEQ ID NO: 76). Self-complementary single stranded siRNAs form hairpin loops and are more stable than ordinary dsRNA. In addition, they are more easily produced from vectors.
Analogous to antisense RNA, the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified internucleoside linkages, modified nucleic acid bases, modified sugars and/or chemical linkage the siRNA to one or more moieties or conjugates. The nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the TARGET sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO 2004/094636, and US 2003/0198627, are hereby incorporated by reference).
The present invention also relates to compositions, and methods using said compositions, comprising a DNA expression vector capable of expressing a polynucleotide capable of inhibiting the maturation of dendritic cells, and described hereinabove as an expression inhibition agent.
A particular aspect of these compositions and methods relates to the down-regulation or blocking of the expression of the TARGET by the induced expression of a polynucleotide encoding an intracellular binding protein that is capable of selectively interacting with the TARGET. An intracellular binding protein includes any protein capable of selectively interacting, or binding, with the polypeptide in the cell in which it is expressed and neutralizing the function of the polypeptide. Preferably, the intracellular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody having binding affinity to an epitope of a TARGET selected from the group consisting of SEQ ID NO: 18-34. More preferably, the intracellular binding protein is a single chain antibody.
A particular embodiment of this composition comprises the expression-inhibiting agent selected from the group consisting of antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for a TARGET selected from the group consisting of SEQ ID NO: 18-34, and a small interfering RNA (siRNA) that is sufficiently homologous to a portion of the polyribonucleotide coding for a TARGET selected from the group consisting of SEQ ID NO: 18-34, such that the siRNA interferes with the translation of the TARGET polyribonucleotide to the TARGET polypeptide.
The polynucleotide expressing the expression-inhibiting agent, or a polynucleotide expressing the TARGET polypeptide in cells, is particularly included within a vector. The polynucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a cell utilizing, preferably, recombinant vector constructs, which will express the antisense nucleic acid once the vector is introduced into the cell. A variety of viral-based systems are available, including adenoviral, retroviral, adeno-associated viral, lentiviral, herpes simplex viral or a sendaiviral vector systems, and all may be used to introduce and express polynucleotide sequence for the expression-inhibiting agents or the polynucleotide expressing the TARGET polypeptide in the target cells.
Particularly, the viral vectors used in the methods of the present invention are replication defective. Such replication defective vectors will usually pack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques may be performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents. Preferably, the replication defective virus retains the sequences of its genome, which are necessary for encapsidating, the viral particles.
In a preferred embodiment, the viral element is derived from an adenovirus. Preferably, the vehicle includes an adenoviral vector packaged into an adenoviral capsid, or a functional part, derivative, and/or analogue thereof. Adenovirus biology is also comparatively well known on the molecular level. Many tools for adenoviral vectors have been and continue to be developed, thus making an adenoviral capsid a preferred vehicle for incorporating in a library of the invention. An adenovirus is capable of infecting a wide variety of cells. However, different adenoviral serotypes have different preferences for cells. To combine and widen the target cell population that an adenoviral capsid of the invention can enter in a preferred embodiment, the vehicle includes adenoviral fiber proteins from at least two adenoviruses. Preferred adenoviral fiber protein sequences are serotype 17, 45 and 51. Techniques or construction and expression of these chimeric vectors are disclosed in US 2003/0180258 and US 2004/0071660, hereby incorporated by reference.
In a preferred embodiment, the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof. An adenoviral late protein, for instance an adenoviral fiber protein, may be favorably used to target the vehicle to a certain cell or to induce enhanced delivery of the vehicle to the cell. Preferably, the nucleic acid derived from an adenovirus encodes for essentially all adenoviral late proteins, enabling the formation of entire adenoviral capsids or functional parts, analogues, and/or derivatives thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and/or analogue thereof. Preferably, the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and/or analogue thereof, which facilitates, at least in part, replication of an adenoviral derived nucleic acid in a cell. The adenoviral vectors used in the examples of this application are exemplary of the vectors useful in the present method of treatment invention.
Certain embodiments of the present invention use retroviral vector systems. Retroviruses are integrating viruses that infect dividing cells, and their construction is known in the art. Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV (“murine Moloney leukemia virus”) MSV (“murine Moloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus. Lentiviral vector systems may also be used in the practice of the present invention.
In other embodiments of the present invention, adeno-associated viruses (“AAV”) are utilized. The AAV viruses are DNA viruses of relatively small size that integrate, in a stable and site-specific manner, into the genome of the infected cells. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
In the vector construction, the polynucleotide agents of the present invention may be linked to one or more regulatory regions. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Regulatory regions include promoters, and may include enhancers, suppressors, etc.
Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters. The promoters may be prokaryotic or eukaryotic depending on the host. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are lac, lacZ, T3, T7, lambda Pr, P1, and trp promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue-specific promoters, including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals, e.g. chymase gene control region which is active in mast cells (Liao et al., (1997), Journal of Biological Chemistry, 27: 2969-2976), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature 318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95), beta-globin gene control region which is active in myeloid cells (Mogram, et al. (1985) Nature 315:338-40; Kollias, et al. (1986) Cell 46:89-94), the CMV promoter and the Visna LTR (Sidiropoulos et al., (2001), Gene Therapy, 8:223-231)
Other promoters which may be used in the practice of the invention include promoters which are preferentially activated in dividing cells, promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, E1a, and MLP promoters. Further promoters which may be of use in the practice of the invention include promoters which are active and/or expressed in macrophages or other cell types contributing to inflammation such as dendritic cells, monocytes, neutrophils, mast cells, endothelial cells, epithelial cells, muscle cells, etc.
Additional vector systems include the non-viral systems that facilitate introduction of polynucleotide agents into a patient. For example, a DNA vector encoding a desired sequence can be introduced in vivo by lipofection. Synthetic cationic lipids designed to limit the difficulties encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7); see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer, et al. (1993) Science 259:1745-8). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, (1989) Nature 337:387-8). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, for example, pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides, e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to liposomes chemically. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, for example, a cationic oligopeptide (e.g., International Patent Publication WO 95/21931), peptides derived from DNA binding proteins (e.g. International Patent Publication WO 96/25508), or a cationic polymer (e.g. International Patent Publication WO 95/21931).
It is also possible to introduce a DNA vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Naked DNA vectors for therapeutic purposes can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wilson, et al. (1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem. 263:14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA 88:2726-30). Receptor-mediated DNA delivery approaches can also be used (Curiel, et al. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J. Biol. Chem. 262:4429-32).
The present invention also provides biologically compatible compositions which can act to inhibit the maturation of dendritic cells wherein said compositions comprise an effective amount of one or more compounds identified as TARGET inhibitors, and/or the expression-inhibiting agents as described hereinabove.
A biologically compatible composition is a composition, that may be solid, liquid, gel, or other form, in which the compound, polynucleotide, vector, and antibody of the invention is maintained in an active form, e.g., in a form able to effect a biological activity. For example, a compound of the invention would have inverse agonist or antagonist activity on the TARGET; a nucleic acid would be able to replicate, translate a message, or hybridize to a complementary mRNA of the TARGET; a vector would be able to transfect a target cell and express the antisense, antibody, ribozyme or siRNA as described hereinabove; an antibody would bind a the TARGET polypeptide domain.
A particular biologically compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. Usually the concentration of salt ions will be similar to physiological levels. Biologically compatible solutions may include stabilizing agents and preservatives. In a more preferred embodiment, the biocompatible composition is a pharmaceutically acceptable composition. Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques. The composition may be administered parenterally in dosage unit formulations containing standard, well-known non-toxic physiologically acceptable carriers, adjuvants and vehicles as desired.
A particular embodiment of the present composition invention is a pharmaceutical composition comprising a therapeutically effective amount of an expression-inhibiting agent as described hereinabove, in admixture with a pharmaceutically acceptable carrier. Another particular embodiment is a pharmaceutical composition for the treatment or prevention of a disease characterized by dendritic cell activity including infections, allograft reactions, inflammation, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer, or a susceptibility to said disease, comprising an effective amount of the TARGET antagonist or inverse agonist, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier. A further particular embodiment is a pharmaceutical composition for the treatment or prevention of a disease involving inflammation, or a susceptibility to the condition, comprising an effective amount of the TARGET antagonist or inverse agonist, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier. A further particular embodiment is a pharmaceutical composition for the treatment or prevention of an autoimmune disease, or a susceptibility to said disease, comprising an effective amount of the TARGET antagonist or inverse agonist, its pharmaceutically acceptable salts, hydrates, solvates, or prodrugs thereof in admixture with a pharmaceutically acceptable carrier.
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Pharmaceutical compositions for oral use can be prepared by combining active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethyl-cellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Preferred sterile injectable preparations can be a solution or suspension in a non-toxic parenterally acceptable solvent or diluent. Examples of pharmaceutically acceptable carriers are saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium; calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
The agents or compositions of the invention may be combined for administration with or embedded in polymeric carrier(s), biodegradable or biomimetic matrices or in a scaffold. The carrier, matrix or scaffold may be of any material that will allow composition to be incorporated and expressed and will be compatible with the addition of cells or in the presence of cells. Particularly, the carrier matrix or scaffold is predominantly non-immunogenic and is biodegradable. Examples of biodegradable materials include, but are not limited to, polyglycolic acid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suture material, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin, alginate, cotton, or other naturally-occurring biodegradable materials. It may be preferable to sterilize the matrix or scaffold material prior to administration or implantation, e.g., by treatment with ethylene oxide or by gamma irradiation or irradiation with an electron beam. In addition, a number of other materials may be used to form the scaffold or framework structure, including but not limited to: nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox (TPX), polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and a variety of polyhydroxyalkanoates, and combinations thereof. Matrices suitable include a polymeric mesh or sponge and a polymeric hydrogel. In the particular embodiment, the matrix is biodegradable over a time period of less than a year, more particularly less than six months, most particularly over two to ten weeks. The polymer composition, as well as method of manufacture, can be used to determine the rate of degradation. For example, mixing increasing amounts of polylactic acid with polyglycolic acid decreases the degradation time. Meshes of polyglycolic acid that can be used can be obtained commercially, for instance, from surgical supply companies (e.g., Ethicon, N.J.). In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
The composition medium can also be a hydrogel, which is prepared from any biocompatible or non-cytotoxic homo- or hetero-polymer, such as a hydrophilic polyacrylic acid polymer that can act as a drug absorbing sponge. Certain of them, such as, in particular, those obtained from ethylene and/or propylene oxide are commercially available. A hydrogel can be deposited directly onto the surface of the tissue to be treated, for example during surgical intervention.
Embodiments of pharmaceutical compositions of the present invention comprise a replication defective recombinant viral vector encoding the polynucleotide inhibitory agent of the present invention and a transfection enhancer, such as poloxamer. An example of a poloxamer is Poloxamer 407, which is commercially available (BASF, Parsippany, N.J.) and is a non-toxic, biocompatible polyol. A poloxamer impregnated with recombinant viruses may be deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentially the same advantages as hydrogel while having a lower viscosity.
The active expression-inhibiting agents may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.
Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™. (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37° C., resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S—S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
As defined above, therapeutically effective dose means that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
The pharmaceutical compositions according to this invention may be administered to a subject by a variety of methods. They may be added directly to target tissues, complexed with cationic lipids, packaged within liposomes, or delivered to target cells by other methods known in the art. Localized administration to the desired tissues may be done by direct injection, transdermal absorption, catheter, infusion pump or stent. The DNA, DNA/vehicle complexes, or the recombinant virus particles are locally administered to the site of treatment. Alternative routes of delivery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pill form), topical, systemic, ocular, intraperitoneal and/or intrathecal delivery. Examples of ribozyme delivery and administration are provided in Sullivan et al. WO 94/02595.
Antibodies according to the invention may be delivered as a bolus only, infused over time or both administered as a bolus and infused over time. Those skilled in the art may employ different formulations for polynucleotides than for proteins. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
As discussed hereinabove, recombinant viruses may be used to introduce DNA encoding polynucleotide agents useful in the present invention. Recombinant viruses according to the invention are generally formulated and administered in the form of doses of between about 104 and about 1014 pfu. In the case of AAVs and adenoviruses, doses of from about 106 to about 1011 pfu are preferably used. The term pfu (“plaque-forming unit”) corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate cell culture and measuring the number of plaques formed. The techniques for determining the pfu titre of a viral solution are well documented in the prior art.
In one aspect the present invention provides methods of preventing and/or treating diseases characterized by activation of macrophages including infections, allograft reactions, inflammation, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer, said methods comprising administering to a subject a therapeutically effective amount of an agent as disclosed herein. In a particular embodiment, the agent is selected from an expression-inhibiting agent and an antibody. In a particular embodiment the disorder is selected from asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis.
In a further aspect the present invention provides a method of preventing and/or treating a disease characterized by inflammation, said method comprising administering to a subject a therapeutically effective amount of an agent as disclosed herein. In a particular embodiment, the agent is selected from an expression-inhibiting agent and an antibody. In a particular embodiment, the disease is selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
In a further aspect the present invention provides a method of preventing and/or treating an autoimmune disease, said method comprising administering to a subject a therapeutically effective amount of an agent as disclosed herein. In a particular embodiment, the agent is selected from an expression-inhibiting agent and an antibody. In a particular embodiment, the disease is selected from Addison's disease, ankylosing spondylitis, coeliac disease, chronic obstructive pulmonary disease, dermatomyositis, diabetes mellitus type 1, Graves' disease, Guillain-Barré syndrome (GBS), lupus erythematosus, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, and vasculitis.
The invention also relates to the use of an agent as described above for the preparation of a medicament for treating or preventing a disease characterized by activation of macrophages including infections, allograft reactions, inflammation, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer. In a particular embodiment, the disease is characterised by inflammation. In a particular embodiment the disease is an autoimmune disease. In a particular embodiment of the present invention the disease is selected from asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis. In a particular embodiment the disease is selected from allergic airways disease (e.g. asthma, rhinitis), transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
The present invention also provides a method of treating and/or preventing a disease involving activation of macrophages said method comprising administering, to a subject suffering from, or susceptible to, a disease characterized by dendritic cell activity including infections, allograft reactions, inflammation, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer., a pharmaceutical composition or compound as described herein, particularly a therapeutically effective amount of an agent which inhibits the expression or activity of a TARGET as identified herein. In one embodiment, the disease is characterized by inflammation. In a particular embodiment the disorder is selected from asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis. In a particular embodiment the disease is selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
The invention also relates to an agent or a pharmaceutical composition as described above for use in the treatment and/or prevention of a disease involving activation of macrophages. In a particular embodiment, the disease is characterised by inflammation. In a particular embodiment the disorder is selected from asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis. In a particular embodiment the disease is selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
Administration of the agent or pharmaceutical composition of the present invention to the subject patient includes both self-administration and administration by another person. The patient may be in need of treatment for an existing disease or medical condition, or may desire prophylactic treatment to prevent or reduce the risk for diseases and medical conditions characterized by activation of macrophages. The agent of the present invention may be delivered to the subject patient orally, transdermally, via inhalation, injection, nasally, rectally or via a sustained release formulation.
Still another aspect of the invention relates to a method for diagnosing a pathological condition involving involving activation of macrophages, comprising determining the amount of apolypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18-34 in a biological sample, and comparing the amount with the amount of the polypeptide in a healthy subject, wherein an increase of the amount of polypeptide compared to the healthy subject is indicative of the presence of the pathological condition. In a particular embodiment the disorder is selected from asthma, allergic diseases (for example, allergy, allergic rhinitis, atopic dermatitis, urticaria, angioedema, food allergy, allergic conjunctivitis, anaphylaxis resulting from an allergic reaction), Chronic Obstructive Pulmonary Disease (COPD), Type I hypersensitivity reactions, multiple sclerosis, rheumatoid arthritis, parasitic infections, eczema, and atherosclerosis. In one embodiment, the disease is characterized by inflammation. In a particular embodiment the disorder is selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
Still another aspect of the invention relates to a method for diagnosing a pathological condition involving activation of macrophages, comprising determining the activity of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 18-34 in a biological sample, and comparing the activity with the activity of the polypeptide in a healthy subject, wherein an increase of the activity of polypeptide compared to the healthy subject is indicative of the presence of the pathological condition. In a particular embodiment the disorder is selected from infections, allograft reactions, inflammation, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer. In one embodiment, the disease is characterized by inflammation. In a further embodiment the disease is selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
Still another aspect of the invention relates to a method for diagnosing a pathological condition involving involving maturation of dendritic cells, comprising determining the nucleic acid sequence of at least one of the genes of SEQ ID NO: 1-17 within the genomic DNA of a subject; comparing the sequence with the nucleic acid sequence obtained from a database and/or a healthy subject; and identifying any difference(s) related to the onset or prevalence of the pathological conditions disclosed herein. In a particular embodiment the disorder is selected from infections, allograft reactions, inflammation, allergic and autoimmune diseases, metabolic disease, cardiovascular disease, tissue injury, and cancer. In one embodiment, the disease is characterized by inflammation. In a further embodiment the disease is selected from allergic airways disease (e.g. asthma, rhinitis), autoimmune diseases, transplant rejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenile idiopathic arthritis, colitis, and inflammatory bowel diseases.
The polypeptides or the polynucleotides of the present invention employed in the methods described herein may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. To perform the methods it is feasible to immobilize either the polypeptide of the present invention or the compound to facilitate separation of complexes from uncomplexed forms of the polypeptide, as well as to accommodate automation of the assay. Interaction (e.g., binding of) of the polypeptide of the present invention with a compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix. For example, the polypeptide of the present invention can be “His” tagged, and subsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusions with the polypeptides of the present invention can be adsorbed to IgG, which are then combined with the cell lysates (e.g., (35)S-labelled) and the candidate compound, and the mixture incubated under conditions favorable for complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the plates are washed to remove any unbound label, and the matrix is immobilized. The amount of radioactivity can be determined directly, or in the supernatant after dissociation of the complexes. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of the protein binding to the protein of the present invention quantitated from the gel using standard electrophoretic techniques.
Other techniques for immobilizing protein on matrices can also be used in the method of identifying compounds. For example, either the polypeptide of the present invention or the compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated protein molecules of the present invention can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with the polypeptides of the present invention but which do not interfere with binding of the polypeptide to the compound can be derivatized to the wells of the plate, and the polypeptide of the present invention can be trapped in the wells by antibody conjugation. As described above, preparations of a labeled candidate compound are incubated in the wells of the plate presenting the polypeptide of the present invention, and the amount of complex trapped in the well can be quantitated.
The polynucleotides encoding the TARGET polypeptides are identified as SEQ ID NO: 18-34. The present inventors show herein that transfection of mammalian cells with Ad-siRNAs targeting these genes inhibits the activation of macropages after trigger with IgG coated beads.
The invention is further illustrated in the following figures and examples.
Macrophages contribute to inflammation by releasing high levels of TNFα. One of the inducers of TNFα release by macrophages during an inflammatory reactions are immune complexes. Such immune complexes can be mimicked by beads coated with human immunoglobulins (IgG).
The IgG complex-induced TNFα release assay that has been developed for the screening of the SilenceSelect® collection has following distinctive features:
On day 0, mononuclear cells were isolated from buffy coats of healthy donors (Sanquin #N0231000) by centrifugation through Ficoll (Amersham 17-1440-02 #305224). The mononuclear cell layer was harvested, washed three times with phosphate-buffered saline (PBS; Gibco 10010015) containing 2 mM EDTA (Sigma #E-7889) and resuspended in MACS buffer (PBS; 2 mM EDTA, 30% BSA (Sigma #A-9576)). Red blood cells were lysed with RBC lysing fluid (Sigma #R-7757). The cells were washed twice in MACS buffer and incubated with magnetic MACS® CD14 Microbeads (Miltenyi BioTec #502-01) in MACS buffer according to the manufacturer's protocol. Labeled cells were isolated on columns in a magnetic field on a VarioMACS. (Miltenyi type 130-090-282). The purity of the isolated cells was assessed by flow cytometry on a FACSCalibur flow cytometer (BD Biosciences) Pharmingen, San Diego, Calif.) using PE-conjugated anti-CD14 monoclonal antibodies (BD Biosciences Pharmingen). The purity of the CD14+ cells was >90%. Cell numbers were counted on a coulter counter (Beckman Coulter, type Z2). On the day of cell isolation, the cells were seeded in 96-well plates using a Multidrop 96 (Labsystems, type 832) at a density of 20,000 cells/well in media (RPMI 1640 with 25 mM HEPES (Invitrogen #42401-018), 10% heat-inactivated FBS (ICN Biomedicals #29-167-54), 100 U/mL penicillin plus 100 μg/mL Streptomycin (Invitrogen #15140-122), 2 mM L-Glutamine (Invitrogen #25030-024) containing 100 ng/mL M-CSF (Peprotech #300-25). Plates were incubated in a humidified incubator at 5% CO2 and 37° C.
On day 4 of the culture, cells were transduced by adding virus using a multichannel or TECAN
Robot at 2500 virus particles per cell to the wells. Plates were incubated in a humidified incubator at 5% CO2 and 37° C. On day 5 medium virus was washed off by removal of 110 μL of culture supernatant and addition of 150 μL fresh medium containing M-CSF. Plates were again incubated in a humidified incubator at 5% CO2 and 37° C.
On day 10 of the culture the medium was replaced with fresh macrophage medium containing M-CSF. Subsequently, IgG-coated beads were added to the plates at a final concentration of 1% beads. These beads were generated by incubating human IgG (Sigma 12511) with OptiBind® polystyrene microparticles (Seradyn 8100-2197).
Plates were incubated in a humidified incubator at 5% CO2 and 37° C. After 24 hr of incubation with the beads, 80 μL of supernatant per well was transferred into a well of a Greiner 384-well flat bottom plate. These plates were sealed and stored at −20° C. until further use.
20 μL of supernatant was used in a TNFα readout assay (Meso Scale Discovery, (MSD), Gaithersburg, Md.). The measurements were performed according to manufacturer's instructions. 20 μL of sample solution were transferred by Tecan384 head on a Tecan freedom station to wells of a 384 well TNFα MSD readout plate(Meso Scale Discovery Catalog #K211BHB-6). A calibrator curve using calibrator from MSD was created using a multichannel pipette. The calibrator curve typically started at 10 ng/mL followed by 6 steps of a 3-fold dilution with a final volume of 10 μL. Plates were sealed and incubated for 2 hours on a shaking platform (set at ˜100 rounds per minute) at room temperature. Detection antibody (1.0 μg/mL) was added to the wells (10 μL for the calibrator and 20 μL for the samples) using a repetitive multichannel. Plates were sealed and incubated overnight at room temperature on a shaking platform (set at ˜100 rounds per minute). The plates were washed 3 times with PBS+0.05% Tween-20 using a Tecan washer (TECAN type, PW384). 35 μl of 2× Read Buffer T was added to each well using a multidrop. After addition of Read Buffer, the plates were read on an MSD SI6000 reader.
The TNFα release assay may be used to screen an arrayed collection of 11,330 different recombinant adenoviruses mediating the expression of shRNAs in macrophages. These shRNAs cause a reduction in expression levels of genes that contain homologous sequences by a mechanism known as RNA interference (RNAi). As positive controls, shRNAs for SYK and IKBKB were taken along. In
Hitcalling was performed using the raw data from the TNFα measurements. The data were log transformed to obtain a normal distribution of the data. For every plate the average and standard deviation is calculated for the all the samples and was used to convert each data point into a “cutoff value” that indicates the difference between the sample and the average of all samples in terms of standard deviation of all samples. All shRNAs that, in biological duplicate, induced TNFα levels equal to or lower than the average of the samples on the plate minus 1.3× the standard deviation were considered hits. In total, 465 shRNAs induced TNFα levels equal or lower than this cut-off value.
Hits were rescreened in the same assay combination with a set of negative controls. Per 96-well plate 24 hits were rescreened together with 24 wells containing negative controls for hitcalling. The positive controls are taken along in order to QC the individual plate. An exemplary lay-out of a rescreen plate is shown in
For the measurements of additional cytokines, such as IL-10, the supernatants were transferred using the TECAN Freedom with a TEMO-384 head. The cytokines were measured using 384 well plates purchased at Meso Scale Discovery and the methodology used is the same as described in Example 5. Hits were prioritized based on effects on IL-10 release.
Prior to additional validation assays, virus stocks were repropagated. The Ad-shRNA hits are repropagated using PerC6 cells (Crucell, Leiden, The Netherlands) at a 96-well plate level, followed by retesting in the TNFα release assay. First, tubes containing the crude lysates of the identified hit Ad-shRNA's samples are picked from the SilenceSelect® collection and rearranged in 96 well plates together with negative/positive controls as exemplified in
To exclude toxic hits, a cell viability assay was performed on the hits. The CellTiter-Blue® Cell
Viability assay from Promega was used as viability assay. The CellTiter-Blue® Cell Viability Assay provides a homogeneous, fluorometric method for estimating the number of viable cells present in multiwell plates. It uses the indicator dye resazurin to measure the metabolic capacity of cells, an indicator of cell viability. Viable cells retain the ability to reduce resazurin into resorufin, which is highly fluorescent and can be measured by using the Envision 2102, containing a 492 nm excitation and a 615 nm emission filter. A calibration curve was established by seeding different cell densities in a 96 well plate. A linear correlation between number of cells and fluorescence was obtained (see
In an shRNA screen there is the possibility that the observed effects are due to off-target effects: the shRNA knock down construct has an effect on expression of a different mRNA than the intended mRNA. To exclude possible off-target effects by the knockdown constructs the following “on-target” analysis was followed. For each target, the original Ad-shRNA plus 5 extra Ad-shRNA constructs against the same target were tested in the primary screening assay. If at least two independent shRNA sequences (including original confirmed drug targetAd-shRNA) give the same effect in one donor, the probability this is due to off target shRNA effects is negligible and the effect is declared “on-target”. For the on-target assay, the methods similar to the screen and rescreen were used as described in Examples 5 and 6. The TNFα release assay was performed as per Rescreen (see Example 6) except that the negative control viruses (Luc_v13) were repropagated alongside the on-target viruses. In total, 40 targets were taken along in the on-target assay, and for 15 targets multiple shRNA came up as a hit. These shRNAs are listed in Table 1. In addition, IL-10 release was measured in the same supernatant as described in Examples 5 and 7. Table 3 indicates the effects of the shRNAs on the IL-10 release.
From the foregoing description, various modifications and changes in the compositions and methods of this invention will occur to those skilled in the art. All such modifications coming within the scope of the appended claims are intended to be included therein.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP10/52030 | 2/18/2010 | WO | 00 | 8/19/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61208271 | Feb 2009 | US |
