Patent Application
20110177094
NOT APPLICABLE
NOT APPLICABLE
Multiple sclerosis (MS) is the most common autoimmune inflammatory disease of the central nervous system. It is characterized by demyelinating lesions in the white matter of the central nervous system that lead to neurological deficits (Sospedra M. and Martin R., Immunology of Multiple Sclerosis. Annu Rev Immunol., 23:683-747 (2005)). The pathogenesis of the disease is associated with the infiltration of immune cells, mainly activated T cells, into the brain (Sospedra M. and Martin R., Annu Rev Immunol., 23:683-747 (2005)). This infiltration is accompanied by a disruption of the blood-brain barrier (van Horssen J. et al., J Neuropathol Exp Neurol., 66:321-8 (2007)).
Intravenous immunoglobulins (IVIG) have been shown to be effective in the treatment of a number of autoimmune diseases including MS (Sospedra M. and Martin R., Immunology of Multiple Sclerosis. Annu Rev Immunol., 23:683-747 (2005)), but the exact mechanisms of action underlying the immunomodulatory activities of IVIG have not been fully explained. There are several models that try to explain the immunomodulatory efficacy of IVIG in patients suffering from autoimmune and inflammatory diseases (Kazatchkine M. D. et al., Mult Scler, 2:24-6; 33:24-26 (2000); Trebst C. and Stangel M., Curr. Pharm. Design, 12:241-2493 (2006)). These models include Fcγ-receptor-mediated immunomodulation (SOrensen P. S., Neurol Sci, 4:227-230 (2003)), modulation of idiotype/anti-idiotype networks (Samuelsson A. et al., Science, 291:484-6 (2001)), elimination of immunostimulating microbial products (Dalakas M. C., Ann Intern Med, 126:721-30 (1997)) and neutralizing antibodies against cytokines and chemokines (Bayry J. et al., Transfus Clin Biol., 10:165-9 (2003)). IVIG's potential to modify the balance between Th1 and Th2 cell immunoreactivity and to inhibit the formation of antibody/complement complexes have also been demonstrated (Andersson U. et al., Immunol Rev, 139:21-42 (1994); Bayry J. et al., Intravenous immunoglobulin in autoimmune disorders: An insight into the immunregulatory mechanisms).
The beneficial effects of IVIG in patients with MS were shown by a number of open clinical trials (Basta M. et al., Blood, 77:376-80 (1991)) and by four randomized double-blind clinical studies (SOrensen P. S. et al., Eur J Neurol, 9:557-563 (2002); Strasser-Fuchs S. et al., Mult Scler, 2:9-13 (2000); Sorensen P. S. et al., Neurology, 50:1273-1281 (1998); Lewanska M. et al., Eur J Neurol, 9:565-572 (2002)). IVIG decreased the relapse rate in MS patients and the number of gadolinium-enhancing lesions seen on brain magnetic resonance imaging (MRI) (Dudesek A. and Zettl U. K., J Neurol, 253; V/50-V/58)). Furthermore, IVIG was shown to suppress proliferation of activated peripheral T cells (Bayry J. et al., Neurol Sci, 4:217-221 (2003); Stangel M. and Gold R., Nervenarzt, (2005)). Auto-reactive peripheral T cells can cross the blood-brain barrier and are believed to be the main effector cells responsible for brain inflammation (Sospedra M. and Martin R., Annu Rev Immunol., 23:683-747 (2005); Helling N. et al., Immunol Res., 1:27-51 (2002)). Therefore, a modulation of T cell function by IVIG could explain the beneficial therapeutic effect of IVIG seen in MS patients.
Recently, we showed that IVIG is an effective alternative treatment for patients with acute exacerbations in relapsing-remitting multiple sclerosis (RRMS) (Elovaara I. et al., Intravenous Immunoglobulin is effective and well tolerated in the treatment of MS Relapse, manuscript submitted). Because peripheral auto-reactive T cells are believed to be responsible for brain inflammation in MS, we undertook to identify genes that are differentially regulated in peripheral T cells of patients with MS in acute exacerbation that are treated with IVIG. We reasoned that differences in gene expression profiles could provide important information about the potential mechanisms of action of IVIG treatment. Furthermore, changes in gene expression profiles could provide prognostic markers to predict treatment success. Such markers could also help to identify targets for developing new therapeutic agents.
Furthermore, increasing evidence has suggested a role for brain inflammation not only in MS but also in the pathogenesis of Alzheimers' disease and Parkinsons' disease (see, e.g., Wilms et al., Curr. Pharm. Des. 13:1925 (2007)). In particular microglia, the resident innate immune cells, play a major role in inflammatory processes of the brain and are known to be associated not only with MS but also with Alzheimers' disease and in Parkinsons'disease (see, e.g, Yamamoto et al., Am. J. Pathology 166:1475 (2006); Huang et al., FASEB 19:761 (2005); Kim et al., Exp. And Mol. Med. 38:333 (2006)). Thus, the present invention provides new prognostic markers to predict treatment success associated with the administration of intravenous immunoglobulin treatment as well as new therapeutic targets that may be exploited in the treatment of MS, e.g., relapsing-remitting multiple sclerosis (RRMS), Parkinsons' disease or Alzheimers disease.'
The present invention provides methods for providing a prognosis of treatment of multiple sclerosis, Parkinson's disease and Alzheimer's disease using molecular markers that are overexpressed or underexpressed in patients treated with intravenous immunoglobulins (IVIG). Also provided are methods to identify compounds that are useful for the treatment or prevention of multiple sclerosis. In some aspects, the subtype of multiple sclerosis is relapsing-remitting multiple sclerosis (RRMS).
Accordingly, in one embodiment the present invention provides method of providing a prognosis of multiple sclerosis, Parkinson's disease and Alzheimer's disease in a subject treated with intravenous immunoglobulin (IVIG) by contacting a biological sample from the subject treated with IVIG with a reagent that specifically binds to at least one marker selected from any of the nucleic acids and corresponding protein sequences shown in Table 3a, Table 3b, and Table 4, and then determining whether or not the marker is overexpressed or underexpressed in the sample, thus providing a prognosis for MS, Parkinson's disease and Alzheimer's disease in a subject treated with IVIG. In an aspect of this embodiment, the multiple sclerosis is of the relapsing-remitting multiple sclerosis (RRMS) subtype.
In various aspects of this embodiment, the reagent is an antibody, such as a monoclonal antibody. Alternatively, the reagent can be a nucleic acid, including an oligonucleotide or an RT PCR primer set. In other aspects, the sample is a blood sample, which can contain T cells. The sample can also be cerebrospinal fluid. In some aspects of this embodiment, one of the markers is a chemokine. Examples of chemokines include: CXCL3, CXCL5, CCL13, and XCL2.
Another embodiment of the invention provides a method of identifying a compound that prevents or treats multiple sclerosis, Parkinson's disease and Alzheimer's disease by contacting a compound with a sample comprising a cell that expresses a marker selected from any of the nucleic acid and corresponding protein sequences shown in Table 3a, Table 3b, Table 3c, Table 3d, and Table 4, and then determining the functional effect of the compound on the marker, thus identifying a compound that prevents or treats MS, Parkinson's disease and Alzheimer's disease. In an aspect of this embodiment, the multiple sclerosis is of the relapsing-remitting multiple sclerosis (RRMS) subtype.
In various aspects of this embodiment, the functional effect is an increase or decrease in expression of the marker. In other aspects, the functional effect is an increase or decrease in activity of the marker. Examples of compounds used in various aspects of this embodiment include: a small molecule, a siRNA, a ribozyme, an antibody, which can be a monoclonal antibody.
A further embodiment of the invention provides a method of treating or preventing multiple sclerosis, Parkinson's disease and Alzheimer's disease in a subject by administering to the subject an effective amount of an antibody which binds a chemokine, including CXCL5, CXCL3, and CCL13, in which the effective amount is sufficient to inactivate the chemokine or chemokine cell signaling, thus treating or preventing multiple sclerosis, Parkinson's disease and Alzheimer's disease. In an aspect of this embodiment, the multiple sclerosis is of the relapsing-remitting multiple sclerosis (RRMS) subtype.
A yet further embodiment of the invention provides a method of treating or preventing multiple sclerosis, Parkinson's disease and Alzheimer's disease in a subject by administering to the subject an effective amount of an antibody which binds a chemokine receptor, including receptors for CXCL5, CXCL3, and CCL13, in which the effective amount is sufficient to inactivate the function of the chemokine receptor, thus treating or preventing multiple sclerosis, Parkinson's disease and Alzheimer's disease. In an aspect of this embodiment, the multiple sclerosis is of the relapsing-remitting multiple sclerosis (RRMS) subtype.
Another embodiment of this invention provides a method of treating or preventing multiple sclerosis, Parkinson's disease and Alzheimer's disease in a subject by administering to the subject an effective amount of an antibody which binds to a XCL2 chemokine receptor, in which the effective amount is sufficient to activate the XCL2 chemokine receptor, thus treating or preventing multiple sclerosis, Parkinson's disease and Alzheimer's disease. In an aspect of this embodiment, the multiple sclerosis is of the relapsing-remitting multiple sclerosis (RRMS) subtype.
Multiple sclerosis (MS) refers generally to an inflammatory, demyelinating disease that affects the central nervous system (CNS). During the progression of MS, the myelin surrounding the axons of neurons degenerates, resulting in subsequent axonal degeneration. The pathogenesis of MS is believed to involve an autoimmune response in which T cells attack parts of the central nervous system, triggering inflammatory responses, which results in the stimulation of other immune cells and the secretion of soluble factors such as cytokines and antibodies. The inflammatory processes triggered by T cells create leaks in the blood-brain barrier formed by endothelial cells. The leaks in the blood-brain barrier, in turn, cause a number of other damaging effects such as brain swelling, activation of macrophages, and further secretion of cytokines and other proteolytic proteins such as matrix metalloproteinases. The final outcome of these pathological processes is neuronal demyelination. See, e.g., Calabresi, P. A., American Family Physician, 70: 1935-1944 (2004), for review.
As MS progresses, gradual demyelination and transection of neuron axons in patches throughout the brain and spinal cord occur. Thus, the term multiple sclerosis refers to the multiple scars (or scleroses) found on myelin sheaths in affected individuals. This scarring causes symptoms which may vary widely depending upon the extent of scarring and which neuronal pathways are disrupted.
Among the symptoms and manifestations of MS include changes in sensation (hypoesthesia), muscle weakness, abnormal muscle spasms, difficulties in movement; difficulties with coordination and balance (ataxia); problems in speech (dysarthria) or swallowing (dysphagia), visual problems (nystagmus, optic neuritis, or diplopia), fatigue and acute or chronic pain syndromes, bladder and bowel difficulties, cognitive impairment, or emotional symptomatology (e.g., depression).
The most common initial symptoms reported are: changes in sensation in the arms, legs or face (33%), complete or partial vision loss (optic neuritis) (16%), weakness (13%), double vision (7%), unsteadiness when walking (5%), and balance problems (3%). See Navarro et al., Rev Neurol 41: 601-3 (2005); Jongen P., J Neurol Sci 245: 59-62 (2006). In some individuals, the initial MS attack is preceded by infection, trauma, or strenuous physical effort.
A number of diagnostic tests are currently in use for the diagnosis of MS. These include the clinical presentation of two separate episodes of neurologic symptoms characteristic of MS, along with the finding of consistent abnormalities on physical examination. Alternatively, magnetic resonance imaging (MRI) of the brain and spine is often used to evaluate individuals with suspected MS. MRI reveals areas of demyelination as bright lesions on T2-weighted images or FLAIR (fluid attenuated inversion recovery) sequences. Gadolinium contrast can be used to demonstrate active plaques on T1-weighted images.
The testing of cerebrospinal fluid (CSF) can provide evidence of chronic inflammation of the central nervous system, a characteristic of MS. In such a test, the CSF is tested for oligoclonal bands, which are immunoglobulins found in 85% to 95% of people with definite MS. When combined with MRI and clinical data, the presence of oligoclonal bands can help make a definite diagnosis of MS.
Because the brains MS-affected individuals often respond less actively to stimulation of the optic nerve and sensory nerves, the measurement of such brain responses can also be used as a diagnostic tool. These brain responses can be examined using visual evoked potentials (VEPs) and somatosensory evoked potentials (SEPs). Decreased activity on either test can reveal demyelination which may be otherwise asymptomatic. Along with other data, these exams can help uncover the widespread nerve involvement required for a definite diagnosis of MS.
Several subtypes, or patterns of progression, of MS have been described. In 1996, the United States National Multiple Sclerosis Society standardized the following four subtype definitions, as described below.
Relapsing-remitting MS (RRMS) refers to a subtype characterized by unpredictable attacks (relapses) followed by periods of months to years of relative quiet (remission) with no new signs of disease activity. Deficits suffered during the attacks may either resolve or may be permanent. Relapsing-remitting describes the initial course of 85% to 90% of individuals with MS.
Secondary progressive describes around 80% of those with initial relapsing-remitting MS, who then begin to have neurologic decline between their acute attacks without any definite periods of remission. This decline may include new neurologic symptoms, worsening cognitive function, or other deficits. Secondary progressive is the most common type of MS and causes the greatest amount of disability.
Primary progressive describes the approximately 10% of individuals who never have remission after their initial MS symptoms. Decline occurs continuously without clear attacks. The primary progressive subtype tends to affect people who are older at disease onset.
Progressive relapsing describes those individuals who, from the onset of their MS, have a steady neurologic decline but also suffer superimposed attacks; and is the least common of all subtypes.
While there is currently no definitive cure for MS, a number of therapies have been developed that are directed toward returning function after an attack, preventing new attacks, or preventing disability. Thus, different therapies are used for patients experiencing acute attacks; those who have the relapsing-remitting subtype; those who have the progressive subtypes; those without a diagnosis of MS who have a demyelinating event; and for managing the various consequences of MS attacks.
The phamacological agents currently in use for MS include interferons, which have been approved for use in relapsing forms of secondary progressive MS; glatiramer acetate, a synthetic medication made of four amino acids that are found in myelin, which stimulates T cells to secrete anti-inflammatory agents that reduce inflammation at lesion sites; mitoxantrone, an agent used to treat progressive, progressive-relapsing, and worsening relapsing-remitting MS; and Natalizumab, a monoclonal antibody that recognizes α4-integrin.
High doses of intravenous corticosteroids, such as methylprednisolone, are frequently administered in the treatment of RRMS and have been shown to be effective at shortening the length of relapsing-remitting symptomatic attacks. As described in greater detail herein, intravenous IgG immunoglobulins have also been used to treat MS.
Similarly to MS, other disease states are associated with brain inflammation, such as Parkinson's disease and Alzheimer's disease, as described above. For example, chemokine CCL13, described herein, activates the chemokine receptor CCR2, which is expressed in microglia and astrocytes. Both of these cell types are associated with Parkinson's disease and Alzheimer's disease. This and other markers described herein are therefore useful for drug assays, diagnostic and prognostic assays, and for therapeutic siRNA and antibody treatment for Alzheimer's disease and Parkinson's disease.
Intravenous immunoglobulins (IVIG) have been successfully used to treat a number of autoimmune diseases of the central nervous system, including multiple sclerosis (MS). However, the underlying mechanisms of action of IVIG have not been fully explained. Accordingly, we have undertaken the identification of gene expression profiles that are associated with the immunomodulatory activity of IVIG in patients with acute exacerbations in relapsing-remitting MS (RRMS). As described below, HU-133 microarrays from Affymetrix were used to study gene expression profiles of peripheral T cells in 10 RRMS patients before and after treatment with IVIG. Patients treated with intravenous methylprednisolone were included as controls. The differential expression of representative genes was confirmed by real-time polymerase chain reaction. All patients were analyzed neurologically and by brain and spinal cord magnetic resonance imaging before and after IVIG therapy.
As shown below in the Examples, 360 genes that were differentially expressed during IVIG treatment were identified. Some encode chemokines such as CXCL3 and CXCL5 that are known to bind to CXCR2, a receptor essential for the regulation of oligodendrocyte migration in the brain. Others encode proteins that are involved in signal transduction, proliferation or apoptosis.
The studies disclosed herein indicate that among the differentially expressed genes the regulation of chemokine expression in peripheral T cells is an important new mechanism of action of IVIG in patients with acute exacerbations in MS. Thus, the genes disclosed herein may serve as diagnostic markers for predicting treatment success in IVIG therapy and provide new molecular targets for drug development.
The term “intravenous IgG” or “IVIG” treatment refers generally to a composition of IgG immunoglobulins administered intravenously to treat a number of conditions such as immune deficiencies, inflammatory diseases, and autoimmune diseases. The IgG immunoglobulins are typically pooled and prepared from serum. Whole antibodies or fragments can be used.
The term “chemokine” refers generally to a family of small cytokines which are secreted by various cells that promote chemotaxis in responsive cells. Chemokines have also gone by the nomenclature of SIS family of cytokines, SIG family of cytokines, SCY family of cytokines, Platelet factor-4 superfamily or intercrines. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine.
Some members of the chemokine family control cells of the immune system during the process of immune surveillance, such as by directing lymphocytes to the lymph nodes to allow lymphocyte surveillance invasion of pathogens through interaction with antigen-presenting cells residing in these tissues. Such chemokines are known as homeostatic chemokines and are produced and secreted without any need to stimulate their source cell(s). Some chemokines have roles in development by, e.g., promoting angiogenesis or guiding cells to tissues that provide specific signals critical for cellular maturation. Other chemokines are inflammatory and are released from a wide variety of cells in response to bacterial infection, viruses and agents that cause physical damage. The release of inflammatory chemokines is often stimulated by pro-inflammatory cytokines such as interleukin 1. Inflammatory chemokines function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils and other effector cells from the blood to sites of infection or tissue damage. Certain inflammatory chemokines activate cells to initiate an immune response or promote wound healing. They are released by many different cell types and serve to guide cells of both innate immune system and adaptive immune system.
Structurally, chemokines are small proteins, with molecular masses of between 8 and 10 kDa. Chemokines also possess conserved amino acids that are important for creating their 3-dimensional or tertiary structure, such as (in most cases) four cysteines that interact with each other in pairs to create a greek key shape that is a characteristic of this class of proteins; intramolecular disulphide bonds typically join the first to third, and the second to fourth cysteine residues, numbered as they appear in the protein sequence of the chemokine.
Members of the chemokine family are categorized into four groups depending on the spacing of their first two cysteine residues. The CC chemokines (or β-chemokines) have two adjacent cysteines near their amino terminus. There have been at least 27 distinct members of this subgroup reported for mammals, called CC chemokine ligands (CCL)-1 to -28. The first two cysteine residues in CXC chemokines (or α-chemokines) are separated by one amino acid, represented by “X”. There have been 17 different CXC chemokines described in mammals, that are subdivided into two categories, those with a specific amino acid sequence (or motif) of Glutamic acid-Leucine-Arginine (ELR) immediately before the first cysteine of the CXC motif (ELR-positive), and those without an ELR motif (ELR-negative). The third group of chemokines is known as the C chemokines (or γ chemokines), and is unlike all other chemokines in that it has only two cysteines; one N-terminal cysteine and one cysteine downstream. A fourth group has three amino acids between the two cysteines and is termed CX3C chemokine (or δ-chemokines).
Chemokine receptors are G protein-coupled receptors containing 7 transmembrane domains that are found on the surface of leukocytes. Approximately 19 different chemokine receptors have been characterized to date, which are divided into four families depending on the type of chemokine they bind; CXCR that bind CXC chemokines, CCR that bind CC chemokines, CX3CR1 that binds the sole CX3C chemokine (CX3CL1), and XCR1 that binds the two XC chemokines (XCL1 and XCL2).
“Chemokine cell signaling” refers generally to the ability of chemokine receptors to associate with G-proteins to transmit cell signals following ligand binding. Activation of G proteins, by chemokine receptors, causes the subsequent activation of phospholipase C (PLC). PLC cleaves a phosphatidylinositol (4,5)-bisphosphate (PIP2) into two second messenger molecules, inositol triphosphate (IP3) and diacylglycerol (DAG) that trigger intracellular signaling events; DAG activates another enzyme called protein kinase C (PKC), and IP3 triggers the release of calcium from intracellular stores. These events promote signaling cascades such as the MAP kinase pathway that generate responses including chemotaxis, degranulation, release of superoxide anions and changes in the avidity of cell adhesion molecules such as integrins within the cell harboring the chemokine receptor.
The term “marker” or “biomarker” refers to a molecule (typically protein, nucleic acid, carbohydrate, or lipid) that is expressed in a cell, expressed on the surface of a cell or secreted by a cell and which is useful for providing a prognosis of relapsing-remitting multiple sclerosis (RRMS) in a subject treated with IVIG. Some of the biomarkers disclosed herein are molecules that are overexpressed in individuals with relapsing-remitting multiple sclerosis (RRMS) treated with IVIG, in comparison to individuals not treated IVIG or in RRMS patients prior to treatment with IVIG, for instance, 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more. Alternatively, other biomarkers are molecules that are underexpressed in individuals with relapsing-remitting multiple sclerosis (RRMS) treated with IVIG, in comparison to individuals not treated IVIG or in RRMS patients prior to treatment with IVIG, for instance, 1-fold underexpression, 2-fold underexpression, 3-fold underexpression, or more. Further, a marker can be a molecule that is inappropriately synthesized in individuals with relapsing-remitting multiple sclerosis (RRMS) treated with IVIG, in comparison to individuals not treated IVIG or in RRMS patients prior to treatment with IVIG, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell.
It will be understood by the skilled artisan that markers may be used singly or in combination with other markers for any of the uses, e.g., prognosis of IVIG treatment of relapsing-remitting multiple sclerosis (RRMS), disclosed herein.
“Biological sample” includes biological fluid samples, such as blood and cerebrospinal fluid, sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), cerebrospinal fluid, sputum, cervicovaginal fluid, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, Mouse; rabbit; or a bird; reptile; or fish.
The terms “overexpress,” “overexpression” or “overexpressed” or “upregulated” interchangeably refer to a protein or nucleic acid (RNA) that is transcribed or translated at a detectably greater level, usually in an IVIG-treated relapsing-remitting multiple sclerosis (RRMS) patient, in comparison to a patient not undergoing IVIG treatment. The term includes overexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a control. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques). Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell. In certain instances, overexpression is 1-fold, 2-fold, 3-fold, 4-fold or more higher levels of transcription or translation in comparison to a control.
The terms “underexpress,” “underexpression” or “underexpressed” or “downregulated” interchangeably refer to a protein or nucleic acid that is transcribed or translated at a detectably lower level, usually in an IVIG-treated relapsing-remitting multiple sclerosis (RRMS) patient, in comparison to a patient not undergoing IVIG treatment. The term includes underexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a control. Underexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e., ELISA, immunohistochemical techniques). Underexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less in comparison to a control. In certain instances, underexpression is 1-fold, 2-fold, 3-fold, 4-fold or more lower levels of transcription or translation in comparison to a control.
The term “differentially expressed” or “differentially regulated” refers generally to a protein or nucleic acid that is overexpressed (upregulated) or underexpressed (downregulated) in one sample compared to at least one other sample, generally in an IVIG-treated relapsing-remitting multiple sclerosis (RRMS) patient, in comparison to a patient not undergoing IVIG treatment, in the context of the present invention.
“Therapeutic treatment” refers to drug therapy, hormonal therapy, immunotherapy, and biologic (targeted) therapy.
By “therapeutically effective amount or dose” or “sufficient amount or dose” herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1987-2005, Wiley Interscience)).
A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
“RNAi molecule” or an “siRNA” refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when the siRNA expressed in the same cell as the gene or target gene. “siRNA” thus refers to the double stranded RNA formed by the complementary strands. The complementary portions of the siRNA that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, an siRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded siRNA. The sequence of the siRNA can correspond to the full length target gene, or a subsequence thereof. Typically, the siRNA is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded siRNA is 15-50 nucleotides in length, and the double stranded siRNA is about 15-50 base pairs in length, preferable about preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
An “antisense” polynucleotide is a polynucleotide that is substantially complementary to a target polynucleotide and has the ability to specifically hybridize to the target polynucleotide.
Ribozymes are enzymatic RNA molecules capable of catalyzing specific cleavage of RNA. The composition of ribozyme molecules preferably includes one or more sequences complementary to a target mRNA, and the well known catalytic sequence responsible for mRNA cleavage or a functionally equivalent sequence (see, e.g., U.S. Pat. No. 5,093,246, which is incorporated herein by reference in its entirety). Ribozyme molecules designed to catalytically cleave target mRNA transcripts can also be used to prevent translation of subject target mRNAs.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
A particular nucleic acid sequence also implicitly encompasses “splice variants” and nucleic acid sequences encoding truncated forms of a protein. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant or truncated form of that nucleic acid. “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. Nucleic acids can be truncated at the 5′ end or at the 3′ end. Polypeptides can be truncated at the N-terminal end or the C-terminal end. Truncated versions of nucleic acid or polypeptide sequences can be naturally occurring or recombinantly created.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M). See, e.g., Creighton, Proteins (1984).
A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al., supra.
For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding. Antibodies can be polyclonal or monoclonal, derived from serum, a hybridoma or recombinantly cloned, and can also be chimeric, primatized, or humanized.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).
An antibody immunologically reactive with a particular biomarker protein of the present invention can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science, 246:1275-1281 (1989); Ward et al., Nature, 341:544-546 (1989); and Vaughan et al., Nature Biotech., 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen.
Methods of preparing polyclonal antibodies are known to the skilled artisan (e.g., Harlow & Lane, 1988, Antibodies: A Laboratory Manual. (Cold Spring Harbor Press)). Polyclonal antibodies can be raised in a mammal, e.g., 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. The immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. 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 which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
The antibodies can, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler & Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. Generally, either peripheral blood lymphocytes (“PBLs”) are used if cells of human origin are desired, or spleen cells or lymph node cells are used if nonhuman mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (1986)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
Human antibodies can be produced using various techniques known in the art, including phage display libraries (Hoogenboom & Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., BioTechnology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-13 (1994); Fishwild et al., Nature Biotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg & Huszar, Inter. Rev. Immunol., 13:65-93 (1995).
In one embodiment, the antibody is conjugated to an “effector” moiety. The effector moiety can be any number of molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety. In one aspect the antibody modulates the activity of the protein.
The nucleic acids of the differentially expressed genes of this invention or their encoded polypeptides refer to all forms of nucleic acids (e.g., gene, pre-mRNA, mRNA) or proteins, their polymorphic variants, alleles, mutants, and interspecies homologs that (as applicable to nucleic acid or protein): (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein; (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising a referenced amino acid sequence, immunogenic fragments thereof, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence, and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a reference nucleic acid sequence. A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. The nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules. Truncated and alternatively spliced forms of these antigens are included in the definition.
The phrase “specifically (or selectively) binds” when referring to a protein, nucleic acid, antibody, or small molecule compound refers to a binding reaction that is determinative of the presence of the protein or nucleic acid, such as the differentially expressed genes of the present invention, often in a heterogeneous population of proteins or nucleic acids and other biologics. In the case of antibodies, under designated immunoassay conditions, a specified antibody may bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
The phrase “functional effects” in the context of assays for testing compounds that modulate a marker protein includes the determination of a parameter that is indirectly or directly under the influence of a biomarker of the invention, e.g., a chemical or phenotypic effect such as altered chemokine cell signaling. A functional effect therefore includes ligand binding activity, transcriptional activation or repression, the ability of cells to proliferate, the ability to migrate, among others. “Functional effects” include in vitro, in vivo, and ex vivo activities.
By “determining the functional effect” is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a biomarker of the invention, e.g., measuring physical and chemical or phenotypic effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g., shape), chromatographic; or solubility properties for the protein; ligand binding assays, e.g., binding to antibodies; measuring inducible markers or transcriptional activation of the marker; measuring changes in enzymatic activity; the ability to increase or decrease cellular proliferation, apoptosis, cell cycle arrest, measuring changes in cell surface markers. The functional effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels for other genes expressed in chemokine-responsive cells, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, etc.
“Inhibitors,” “activators,” and “modulators” of the markers are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS). Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS). “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate activity of biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS), e.g., agonists. Inhibitors, activators, or modulators also include genetically modified versions of biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS), e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, RNAi molecules, small organic molecules and the like. Such assays for inhibitors and activators include, e.g., expressing biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS) in vitro, in cells, or cell extracts, applying putative modulator compounds, and then determining the functional effects on activity, as described above.
Samples or assays comprising biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS) that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition of biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS) is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation of biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS) is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.
The term “test compound” or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, peptide, circular peptide, lipid, fatty acid, siRNA, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly modulate biomarkers responsive to IVIG treatment of relapsing-remitting multiple sclerosis (RRMS). The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
A “small organic molecule” refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.
The present invention provides methods of providing a prognosis of IVIG treatment of multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS), Alzheimer's disease, or Parkinson's disease by detecting the expression of markers overexpressed or underexpressed in patients treated with IVIG. Providing a prognosis involves determining the level of one or more IVIG responsive biomarker polynucleotides or the corresponding polypeptides in a patient or patient sample and then comparing the level to a baseline or range. Typically, the baseline value is representative of levels of the polynucleotide or nucleic acid in a relapsing-remitting multiple sclerosis (RRMS) patient prior to IVIG treatment, as measured using a biological sample such as a sample of a bodily fluid (e.g., blood or cerebrospinal fluid). Variation of levels of a polynucleotide or corresponding polypeptides of the invention from the baseline range (either up or down) indicates that the patient is benefiting from IVIG treatment of relapsing-remitting multiple sclerosis (RRMS).
As used herein, the term “providing a prognosis” refers to providing a prediction of the probable course and outcome of treatment of a patient suffering from multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS), Alzheimer's disease, or Parkinson's disease with IVIG. The methods can also be used to devise a suitable alternative or additional therapy for multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS) treatment, Alzheimer's disease, or Parkinson's disease, e.g., by indicating the failure of IVIG treatment to alleviate multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS), Alzheimer's disease, or Parkinson's disease. The prognosis can be used to adjust dose or frequency of IVIG administration as well.
Antibody reagents can be used in assays to detect expression levels of the biomarkers of the invention in patient samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, “Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries, 1997; and Gosling, “Immunoassays: A Practical Approach,” Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used. See, e.g., Self et al., Curr. Opin. Biotechnol., 7:60-65 (1996). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence. See, e.g., Schmalzing et al., Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. Sci., 699:463-80 (1997). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. See, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 (1997). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, Calif.; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)).
Specific immunological binding of antibodies can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine-125 (125I) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for the nucleic acid is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p-nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals; St. Louis, Mo.).
A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 125I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, Calif.) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
Alternatively, nucleic acid binding molecules such as probes, oligonucleotides, oligonucleotide arrays, and primers can be used in assays to detect differential RNA expression in patient samples, e.g., RT-PCR. In one embodiment, RT-PCR is used according to standard methods known in the art. In another embodiment, PCR assays such as Taqman® assays available from, e.g., Applied Biosystems, can be used to detect nucleic acids and variants thereof. In other embodiments, qPCR and nucleic acid microarrays can be used to detect nucleic acids. Reagents that bind to selected biomarkers can be prepared according to methods known to those of skill in the art or purchased commercially.
Analysis of nucleic acids can be achieved using routine techniques such as Southern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), or any other methods based on hybridization to a nucleic acid sequence that is complementary to a portion of the marker coding sequence (e.g., slot blot hybridization) are also within the scope of the present invention. Applicable PCR amplification techniques are described in, e.g., Ausubel et al. and Innis et al., supra. General nucleic acid hybridization methods are described in Anderson, “Nucleic Acid Hybridization,” BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of nucleic acid sequences (e.g., genomic DNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, “Microarrays Methods and Applications: Nuts & Bolts,” DNA Press, 2003; and Baldi et al., “DNA Microarrays and Gene Expression From Experiments to Data Analysis and Modeling,” Cambridge University Press, 2002.
Analysis of nucleic acid markers and their variants can be performed using techniques known in the art including, without limitation, microarrays, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis. A non-limiting example of a PCR-based analysis includes a Taqman® allelic discrimination assay available from Applied Biosystems. Non-limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al., Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al., Methods Mol. Cell Biol., 3:39-42 (1992)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al., Nat. Biotechnol., 16:381-384 (1998)), and sequencing by hybridization. Chee et al., Science, 274:610-614 (1996); Drmanac et al., Science, 260:1649-1652 (1993); Drmanac et al., Nat. Biotechnol., 16:54-58 (1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Other methods for detecting nucleic acid variants include, e.g., the INVADER® assay from Third Wave Technologies, Inc., restriction fragment length polymorphism (RFLP) analysis, allele-specific oligonucleotide hybridization, a heteroduplex mobility assay, single strand conformational polymorphism (SSCP) analysis, single-nucleotide primer extension (SNUPE) and pyrosequencing.
A detectable moiety can be used in the assays described herein. A wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.
Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different markers. Such formats include microarrays and certain capillary devices. See, e.g., Ng et al., J. Cell Mol. Med., 6:329-340 (2002); U.S. Pat. No. 6,019,944. In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more markers for detection.
Analysis can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate a prognosis in a timely fashion.
Alternatively, the antibodies or nucleic acid probes of the invention can be applied to sections of patient biopsies immobilized on microscope slides. The resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art.
In another format, the various markers of the invention also provide reagents for in vivo imaging such as, for instance, the imaging of labeled regents that detect the nucleic acids or encoded proteins of the biomarkers of the invention. For in vivo imaging purposes, reagents that detect the presence of proteins encoded by IVIG-responsive relapsing-remitting multiple sclerosis (RRMS) biomarkers, such as antibodies, may be labeled using an appropriate marker, such as a fluorescent marker.
IVIG compositions comprising whole antibodies have been described for the treatment of certain autoimmune conditions. (See, e.g., U.S. Patent Publication US 2002/0114802, US 2003/0099635, and US 2002/0098182.) The IVIG compositions disclosed in these references include polyclonal antibodies.
Immunoglobulin preparations according to the present invention can be prepared from any suitable starting materials. For example, immunoglobulin preparations can be prepared from donor serum or monoclonal or recombinant immunoglobulins. In a typical example, blood is collected from healthy donors. Usually, the blood is collected from the same species of animal as the subject to which the immunoglobulin preparation will be administered (typically referred to as “homologous” immunoglobulins). The immunoglobulins are isolated from the blood by suitable procedures, such as, for example, Cohn fractionation, ultracentrifugation, electrophoretic preparation, ion exchange chromatography, affinity chromatography, immunoaffinity chromatography, polyethylene glycol fractionation, or the like. (See, e.g., Cohn et al., J. Am. Chem. Soc. 68:459-75 (1946); Oncley et al., J. Am. Chem. Soc. 71:541-50 (1949); Barundern et al., Vox Sang. 7:157-74 (1962); Koblet et al., Vox Sang. 13:93-102 (1967); U.S. Pat. Nos. 5,122,373 and 5,177,194; the disclosures of which are incorporated by reference herein.)
In certain embodiments, immunoglobulin is prepared from gamma globulin-containing products produced by the alcohol fractionation and/or ion exchange and affinity chromatography methods well known to those skilled in the art. Purified Cohn Fraction II is commonly used. The starting Cohn Fraction II paste is typically about 95 percent IgG and is comprised of the four IgG subtypes. The different subtypes are present in Fraction II in approximately the same ratio as they are found in the pooled human plasma from which they are obtained. The Fraction II is further purified before formulation into an administrable product. For example, the Fraction II paste can be dissolved in a cold purified aqueous alcohol solution and impurities removed via precipitation and filtration. Following the final filtration, the immunoglobulin suspension can be dialyzed or diafiltered (e.g., using ultrafiltration membranes having a nominal molecular weight limit of less than or equal to 100,000 daltons) to remove the alcohol. The solution can be concentrated or diluted to obtain the desired protein concentration and can be further purified by techniques well known to those skilled in the art.
Preparative steps can be used to enrich a particular isotype or subtype of immunoglobulin. For example, protein A, protein G or protein H sepharose chromatography can be used to enrich a mixture of immunoglobulins for IgG, or for specific IgG subtypes. (See generally Harlow and Lane, Using Antibodies, Cold Spring Harbor Laboratory Press (1999); Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); U.S. Pat. No. 5,180,810.)
Commercial sources of immunoglobulins can also be used. Such sources include but are not limited to: Gammagard S/D® (Baxter Healthcare); BayRho-D® products (Bayer Biological); Gamimune N®, 5% (Bayer Biological); Gamimune N®, 5% Solvent/Detergent Treated (Bayer Biological); Gamimune N®, 10% (Bayer Biological); Sandoglobulin I.V.® (Novartis); Polygam S/D® (American Red Cross); Venoglobulin-S® 5% Solution Solvent Detergent Treated (Alpha Therapeutic); Venoglobulin-S® 10% Solution Solvent Detergent/Treated (Alpha Therapeutic); and VZIG® (American Red Cross). The commercial source of immunoglobulin preparation for use in the methods of the present invention is not critical.
An alternative approach is to use fragments of antibodies, such as Fc fragments of immunoglobulins. An Fc preparation comprises Fc fragments of immunoglobulins. The term “Fc fragment” refers to a portion of an immunoglobulin heavy chain constant region containing at least one heavy chain constant region domain (e.g., CH2, CH3 and/or CH4) or an antigenic portion thereof, but excluding the variable regions of the immunoglobulin. (As used herein, a variable region refers to region of the immunoglobulin that binds to an antigen, but excludes the CH1 and CL domains.) The Fc preparation can contain entire Fc fragments and/or portions thereof (e.g., one or more heavy chain constant region domains or portions thereof containing an epitope(s) bound by the rheumatoid factors). An Fc fragment optionally can include an immunoglobulin hinge region, a heavy chain CH1 domain, and/or a heavy chain CH1 domain joined to a light chain CL domain.
An Fc preparation includes Fc fragments of at least one Fc isotype and can contain a mixture of immunoglobulin Fc fragments of different isotypes (e.g., IgA, IgD, IgE, IgG and/or IgM). The Fc preparation also can contain predominantly (at least 60%, at least 75%, at least 90%, at least 95%, or at least 99%) Fc fragments from one immunoglobulin isotype, and can contain minor amounts of the other subtypes. For example, an Fc preparation can contain at least at least about 75%, at least about 90%, at least about 95%, or at least about 99% IgG Fc fragments. In addition, the Fc preparation can comprise a single IgG subtype or a mixture two or more of IgG Fc subtypes. Suitable IgG subtypes include IgG1, IgG2, IgG3, and IgG4. In a specific embodiment, the Fc preparation comprises IgG1 Fc fragments.
An Fc preparation is substantially free of F(ab′)2 fragments (i.e., heavy and light chain variable and first constant regions and a portion of the hinge region, which can be produced by pepsin digestion of the antibody molecule), Fab′ fragments (i.e., Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2 fragment), or Fab fragments (i.e., which can be generated by treating the antibody molecule with papain and a reducing agent). In this context, “substantially free” means the Fc preparation contains less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% F(ab′)2, Fab′ or Fab fragments. In another embodiment, the Fc preparation contains Fc fragments which are essentially free of F(ab′)2, Fab′ or Fab fragments. The Fc preparations are typically substantially free of whole (i.e., full length) immunoglobulins. In this context, “substantially free” means less than about 25%, or less than about 10%, or less than about 5%, or less than about 2%, less than about 1% or are free of full length immunoglobulins.
Immunoglobulins can be cleaved at any suitable time during preparation to separate the Fc fragments from the Fab, F(ab′) and/or F(ab′)2 fragments, as applicable. A suitable enzyme for cleavage is, for example, papain, pepsin or plasmin. (See, e.g., Harlow and Lane, Using Antibodies, Cold Spring Harbor Laboratory Press (1999); Plan and Makula, Vox Sanguinis 28:157-75 (1975).) After cleavage, the Fc portions can be separated from the Fab F(ab′) and/or F(ab′)2 fragments by, for example, affinity chromatography, ion exchange chromatography, gel filtration, or the like. In a specific example, immunoglobulins are digested with papain to separate the Fc fragment from the Fab fragments. The digestion mixture is then subjected to cationic exchange chromatography to separate the Fc fragments from the Fab fragments.
Immunoglobulin or Fc fragments can also be prepared from hybridomas or other culture system which express monoclonal antibody. (See, e.g., Kohler and Milstein, Nature 256:495-97 (1975); Hagiwara and Yuasa, Hum. Antibodies Hybridomas 4:15-19 (1993); Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985).) Human monoclonal antibodies can be obtained, for example, from human hybridomas (see, e.g., Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-30 (1983)) or by transforming human B cells with EBV virus in vitro (see, e.g., Cole et al., supra). Monoclonal antibodies produced from hybridomas can be purified and the Fc fragments separated from the Fab, F(ab′) and/or F(ab)2 fragments as described herein or as known to the skilled artisan.
Immunoglobulin or Fc fragments also can be produced recombinantly, such as from eukaryotic cell culture systems. For example, an Fc fragment of an immunoglobulin can be recombinantly produced by Chinese hamster ovary (CHO) cells transfected with a vector containing a DNA sequence encoding the Fc fragment. Methods for creating such recombinant mammalian cells are described in, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Manual, 3rd ed. (Cold Spring Harbor Laboratory Press (New York) 2001) and Ausubel et al., Short Protocols in Molecular Biology, 4th ed. (John Wiley & Sons, Inc. (New York) 1999) and are known to the skilled artisan. Recombinant Fc can also be produced in other mammalian cell lines, such as baby hamster kidney (BHK) cells. Methods of culturing recombinant cells to produce recombinant proteins are also known to the art.
A variety of other expression systems can be utilized to express recombinant immunoglobulins or Fc fragments. These include, but are not limited to, insect cell systems and microorganisms such as yeast or bacteria which have been transfected or transformed with an expression cassette encoding the desired Fc fragment. In certain embodiments, the microorganism optionally can be engineered to reproduce glycosylation patterns of mammalian or human Fc fragments.
In certain embodiments, further preparative steps can be used in order to render an immunoglobulin or Fc preparation safe for use in the methods according to the present invention. Such steps can include, for example, treatment with solvent/detergent, pasteurization and sterilization. Additional preparative steps may be used in order to ensure the safety of an Fc preparation. Such preparative steps can include, for example, enzymatic hydrolysis, chemical modification via reduction and alkylation, sulfonation, treatment with β-propiolactone, treatment at low pH, or the like. Descriptions of suitable methods can also be found in, for example, U.S. Pat. Nos. 4,608,254; 4,687,664; 4,640,834; 4,814,277; 5,864,016; 5,639,730 and 5,770,199; Romer et al., Vox Sang. 42:62-73 (1982); Romer et al., Vox Sang. 42:74-80 (1990); and Rutter, J. Neurosurg. Psychiat. 57 (Suppl.):2-5 (1994) (the disclosures of which are incorporated by reference herein).
An effective amount of an immunoglobulin or Fc preparation is administered to the subject generally by intravenous means. The term “effective amount” refers to an amount of an immunoglobulin or Fc preparation that results in an improvement or remediation of RRMS in the subject. An effective amount to be administered to the subject can be determined by a physician with consideration of individual differences in age, weight, disease severity and response to the therapy. In certain embodiments, an immunoglobulin or Fc preparation can be administered to a subject at about 5 mg/kilogram to about 500 mg/kilogram each day. In additional embodiments, an mmunoglobulin or Fc preparation can be administered in amounts of at least about 10 mg/kilogram, at last 15 mg/kilogram, at least 20 mg/kilogram, at least 25 mg/kilogram, at least 30 mg/kilogram or at least 50 mg/kilogram. In additional embodiments, an mmunoglobulin or Fc preparation can be administered to a subject at doses up to about 100 mg/kilogram, to about 150 mg/kilogram, to about 200 mg/kilogram, to about 250 mg/kilogram, to about 300 mg/kilogram, to about 400 mg/kilogram each day. In other embodiments, the doses of the mmunoglobulin or Fc preparation can be greater or less. Immunoglobulin or Fc preparations can be administered in one or more doses per day.
In accordance with the present invention, the time needed to complete a course of the treatment can be determined by a physician and may range from as short as one day to more than a month. In certain embodiments, a course of treatment can be from 1 to 6 months.
The invention provides compositions, kits and integrated systems for practicing the assays described herein using antibodies specific for the polypeptides or nucleic acids specific for the polynucleotides of the invention.
Kits for carrying out the diagnostic assays of the invention typically include a probe that comprises an antibody or nucleic acid sequence that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe. The kits may include several antibodies or polynucleotide sequences encoding polypeptides of the invention, e.g., a cocktail of antibodies that recognize the proteins encoded by the biomarkers of the invention.
A variety of methods may be used to identify compounds that prevent or treat multiple sclerosis, including relapsing-remitting multiple sclerosis (RRMS), Alzheimer's disease, or Parkinson's disease. Typically, an assay that provides a readily measured parameter is adapted to be performed in the wells of multi-well plates in order to facilitate the screening of members of a library of test compounds as described herein. Thus, in one embodiment, an appropriate number of cells, e.g., T cells, can be plated into the cells of a multi-well plate, and the effect of a test compound on the expression of an IVIG-responsive relapsing-remitting multiple sclerosis (RRMS) biomarker can be determined.
The compounds to be tested can be any small chemical compound, or a macromolecule, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a test compound in this aspect of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
In one preferred embodiment, high throughput screening methods are used which involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. In this instance, such compounds are screened for their ability to reduce or increase the expression of the relapsing-remitting multiple sclerosis (RRMS) biomarkers of the invention.
A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton et al., Nature, 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., PNAS USA, 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc., 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc., 116:2661 (1994)), oligocarbamates (Cho et al., Science, 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).
In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or 100,000 or more different compounds is possible using the integrated systems of the invention.
Because the biomarkers of the present invention are overexpressed or underexpressed in response to IVIG treatment of multiple sclerosis, Alzheimer's disease, or Parkinson's disease, the biomarker proteins or their cellular receptors, may serve as targets for multiple sclerosis therapy using antibodies. In the case of, for instance, of chemokines, such as CXCL5, CXCL3, and CCL13, whose expression is decreased upon treatment of RRMS with IVIG, antibodies that bind to and inactivate these chemokines or their receptors can be used in the treatment of multiple sclerosis, Alzheimer's disease, or Parkinson's disease. Alternatively, in the case of chemokines, such as XCL2, whose expression is increased upon IVIG treatment, antibodies may be generated which bind to and activate XCL2 receptors, thus mimicking the effect of XCL2 binding.
The antibodies described above may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material which when combined with the antibody does not interfere with function of the antibody and is non-reactive with the subject's immune systems. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences, 20th ed., 2003).
Antibody formulations may be administered via any route capable of delivering the antibodies to an individual suffering from multiple sclerosis. Potentially effective routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intradermal, and the like. One preferred route of administration is by intravenous injection. A preferred formulation for intravenous injection comprises the antibodies in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. The antibody preparation may be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water containing, for example, benzyl alcohol preservative, or in sterile water prior to injection.
Treatment will generally involve the repeated administration of antibody preparations via an acceptable route of administration such as intravenous injection (IV), at an effective dose. Dosages will depend upon various factors generally appreciated by those of skill in the art, including without limitation the type, stage, the severity, grade, or stage of multiple sclerosis, the binding affinity and half life of the antibody used, the degree of biomarker or receptor expression in the patient, the desired steady-state antibody concentration level, frequency of treatment, and the influence of any other agents used in combination with the treatment method of the invention. Typical daily doses may range from about 0.1 to 100 mg/kg. Doses in the range of 10-500 mg mAb per week may be effective and well tolerated, although even higher weekly doses may be appropriate and/or well tolerated. The principal determining factor in defining the appropriate dose is the amount of a particular antibody necessary to be therapeutically effective in a particular context. Repeated administrations may be required in order to achieve longer lasting remission in RRMS. Initial loading doses may be higher. The initial loading dose may be administered as an infusion. Periodic maintenance doses may be administered similarly, provided the initial dose is well tolerated.
A variety of nucleic acids, such as antisense nucleic acids, siRNAs or ribozymes, may be used to inhibit the function of the markers of this invention. Ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target mRNAs, particularly through the use of hammerhead ribozymes. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. Preferably, the target mRNA has the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art.
Gene targeting ribozymes necessarily contain a hybridizing region complementary to two regions, each of at least 5 and preferably each 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length of a target mRNA. In addition, ribozymes possess highly specific endoribonuclease activity, which autocatalytically cleaves the target sense mRNA.
With regard to antisense, siRNA or ribozyme oligonucleotides, phosphorothioate oligonucleotides can be used. Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. Phosphorothioate is used to modify the phosphodiester linkage. An N3′-P5′ phosphoramidate linkage has been described as stabilizing oligonucleotides to nucleases and increasing the binding to RNA. Peptide nucleic acid (PNA) linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimization as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. Finally, modification of the sugar may also be considered. 2′-O-propyl and 2′-methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo.
Inhibitory oligonucleotides can be delivered to a cell by direct transfection or transfection and expression via an expression vector. Appropriate expression vectors include mammalian expression vectors and viral vectors, into which has been cloned an inhibitory oligonucleotide with the appropriate regulatory sequences including a promoter to result in expression of the antisense RNA in a host cell. Suitable promoters can be constitutive or development-specific promoters. Transfection delivery can be achieved by liposomal transfection reagents, known in the art (e.g., Xtreme transfection reagent, Roche, Alameda, Calif.; Lipofectamine formulations, Invitrogen, Carlsbad, Calif.). Delivery mediated by cationic liposomes, by retroviral vectors and direct delivery are efficient. Another possible delivery mode is targeting using antibody to cell surface markers for the target cells.
For transfection, a composition comprising one or more nucleic acid molecules (within or without vectors) can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described, for example, in Gilmore, et al., Curr Drug Delivery (2006) 3:147-5 and Patil, et al., AAPS Journal (2005) 7:E61-E77, each of which are incorporated herein by reference. Delivery of siRNA molecules is also described in several U.S. Patent Publications, including for example, 2006/0019912; 2006/0014289; 2005/0239687; 2005/0222064; and 2004/0204377, the disclosures of each of which are hereby incorporated herein by reference. Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, by electroporation, or by incorporation into other vehicles, including biodegradable polymers, hydrogels, cyclodextrins (see, for example Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and US Patent Application Publication No. 2002/130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). In another embodiment, the nucleic acid molecules of the invention can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
Examples of liposomal transfection reagents of use with this invention include, for example: CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL); and (5) siPORT (Ambion); HiPerfect (Qiagen); X-treme GENE (Roche); RNAicarrier (Epoch Biolabs) and TransPass (New England Biolabs).
In some embodiments, antisense, siRNA, or ribozyme sequences are delivered into the cell via a mammalian expression vector. For example, mammalian expression vectors suitable for siRNA expression are commercially available, for example, from Ambion (e.g., pSilencer vectors), Austin, Tex.; Promega (e.g., GeneClip, siSTRIKE, SiLentGene), Madison, Wis.; Invitrogen, Carlsbad, Calif.; InvivoGen, San Diego, Calif.; and Imgenex, San Diego, Calif. Typically, expression vectors for transcribing siRNA molecules will have a U6 promoter.
In some embodiments, antisense, siRNA, or ribozyme sequences are delivered into cells via a viral expression vector. Viral vectors suitable for delivering such molecules to cells include adenoviral vectors, adeno-associated vectors, and retroviral vectors (including lentiviral vectors). For example, viral vectors developed for delivering and expressing siRNA oligonucleotides are commercially available from, for example, GeneDetect, Bradenton, Fla.; Ambion, Austin, Tex.; Invitrogen, Carlsbad, Calif.; Open BioSystems, Huntsville, Ala.; and Imgenex, San Diego, Calif.
The following examples are offered to illustrate, but not to limit the claimed invention.
10 consecutive patients with acute MS relapse as rated on McDonald's criteria (McDonald W. I. et al., Ann Neurol, 50:121-27 (2001)) were included. The diagnosis of definite MS was based on McDonald's criteria (Kurtzke J. F., Neurology, 33:1444-1452 (1983)). The EDSS (Dastidar P. et al., Med Biol Eng Comput, 37:104-7 (1999)) and volumetric brain MRI were evaluated at baseline (at relapse immediately before treatment) and 3 weeks after completion of IVIG therapy (Elovaara I. et al., Intravenous Immunoglobulin is effective and well tolerated in the treatment of MS Relapse, Manuscript submitted). The primary outcome measure of the study was a change in the EDSS score from baseline to week 3 after the start of IVIG therapy on day 21. Secondary outcome measures were changes in the volumes of T1-, T2-, Flair- and gadolinium (Gd)-enhanced lesions, the number of Gd-enhanced lesions, and brain volumes (Elovaara I. et al., Intravenous Immunoglobulin is effective and well tolerated in the treatment of MS Relapse, Manuscript submitted; Dastidar P. et al., Med Biol Eng Comput, 37:104-7 (1999)). Patients' characteristics are listed in Table 1. Before entry into the study each patient signed a form of consent. The study was approved by the Ethics Committee of Tampere University, Tampere, Finland.
Patients who received treatment with immunosuppressants in the preceding nine months or patients who received corticosteroids in the preceding 8 weeks were excluded. All patients received 0.4 g/kg/day Endobulin (Baxter AG, Vienna, Austria) for 5 days. Clinical evaluation of the patients was done before treatment with IVIG, 1 day after completion of therapy on day 6 as well as 3 weeks after the beginning of therapy on day 21. Clinical evaluation included neurological examination, determination of the EDSS score, arm index and ambulation index. A control group of five patients received standard treatment of IVMP 100 mg/day for 3 days.
40 ± 10.6
5 ± 3.2
Brain MRI examinations were done using a 1.5 Tesla MRI unit (Philips Gyroscan ACS NT Intera, Best, Netherlands) as described (Kurtzke J. F., Neurology, 33:1444-1452 (1983)). The MRI protocol included sagittal T1 localizer, axial fluid attenuated inversion recovery (FLAIR), T1 magnetization transfer contrast (MTC), T1 spin echo (SE), T2 turbo spin echo (TSE) (3 mm thick and 0 mm gap) and gadolinium-enhanced T1 MTC sequences. T1 axial SE (3 mm thick and 0 mm gap) and axial FLAIR (5 mm thick and 1 mm gap) sequences were used for volumetric analyses of plaques. Computerized semiautomatic segmentation and volumetric analyses were done using Anatomatic software operating in a Windows environment. The inter- and intra-observer variability of the volumetric results has been reported elsewhere (Dastidar P. et al., Med Biol Eng Comput, 37:104-7 (1999); Heinonen T. et al., J Med Eng Technol, 22:173-8 (1998)). The volumetric accuracy of the Anatomatic program was analyzed as described (Dastidar P. et al., Med Biol Eng Comput, 37:104-7 (1999)). Good head repositioning was controlled using the same head coil, the same anatomic locations and the same pack of images in different MRI sequences. Whole spinal cords were scanned separating into upper and lower parts. The same scanner was used for all MRI examinations.
Blood samples were obtained using Vacutainer CPTTM Cell Preparation Tubes (Becton Dickinson, Franklin Lakes, N.J.). Peripheral blood mononuclear cells (PBMC) were separated from peripheral blood within 60 min after blood sampling using density gradient (Lymphoprep, Nycomed, Roskilde, DK) centrifugation according to the manufacturer's protocol. The cells were separated into T cells and non-T cells using a mixture of non-stimulating anti-CD4+ and anti-CD8+ magnetic Dynabeads (Dynal Biotech, Oslo, N) at 4° C. Cell pellets obtained from 5×106 cells were thoroughly mixed with 1 ml TRIzol (Invitrogen, Carlsbad, Calif.). Aliquots were frozen and stored at −80° C. until further processing. Total RNA was isolated according to the manufacturer's protocol. RNA pellets were dissolved in nuclease-free water (Invitrogen, Carlsbad, Calif.) and stored at −80° C.
The HU-133A Genechip (Affymetrix, Santa Clara, Calif.) containing approximately 33,000 human genes was used. 5 μg of total RNA were transcribed, labelled and hybridized in vitro on the array according to the manufacturer's protocol (see Affymetrix.com). The quality of the RNA was checked before in vitro processing using a Bioanalyzer (Agilent Technologies, Palo Alto, Calif.).
Statistical analysis of gene expression data was done at the Microarray Facility Tübingen, Eberhard-Karls-University Tübingen, Germany. The Affymetrix CHP files were imported into Genespring 7.1 for statistical data analysis. The signals of each array were divided by the median of all signals of the arrays from time point zero. Subsequently, a “per-gene” normalization was done by dividing all signals of a gene by the median signal of this gene. Thus the signals of each gene start at time point zero around 1 and display values greater than 1 upon increase and vice versa. The signals were log-transformed, and fold change and p-values (Welch's t-test) (Han T. et al., BMC bioinformatics, 7:9 (2006)) were calculated for each gene in pair-wise comparisons. Probe sets with a fold change of more than 2 and a p-value of less than 0.05 were identified in volcano plots and called statistically significant.
The gene expression data obtained by microarray analysis for four representative genes were confirmed by quantitative real-time polymerase chain reaction (PCR). For this purpose, 1 μg of total T cell RNA was used for reverse transcription into cDNA according to the manufacturer's protocol (MBI Fermentas, Burlington, Canada). For each sample to be analyzed, 100 mg cDNA were dissolved in 5 μl nuclease-free water (Invitrogen, Carlsbad, Calif.) and quantitatively analyzed using different TaqMan Assays-on-Demand and the ABPrism 7000 (both from Applied Biosystems, Foster City, Calif.). Data were analyzed using the ̂̂CT-method, which is commonly used for relative quantification (Livak K. J. and Schmittgen T. D., Methods, 25:402-40 (2001)). For normalization of expression data human glyceraldhyde-3 phosphate dehydrogenase was included as a housekeeping gene. For verification of normalization, a second housekeeping gene, β-2 microglobulin, was used as a control (data not shown).
Analysis of the clinical outcome of the study showed that a 5-day course of IVIG therapy resulted in a significant reduction of the EDSS score in all 10 patients (
PBMCs obtained from peripheral blood were separated into T cells and non-T cells using a mixture of non-stimulating anti-CD4+ and anti-CD8+ magnetic Dynabeads at 4° C. This procedure was chosen to prevent stimulation of T cells during cell separation. To ensure that potential differences in gene expression profiles are not due to differences in the cellular composition of the different samples, we compared the expression of genes that encode CD3, CD4, CD8 and CD14 between samples obtained at different time points for each patient. Our results show that the cellular composition of the samples obtained from each patient on different days is similar (
Statistic analysis of gene expression data included all results obtained from microarray analysis done at three different time points (before treatment, 1 day and 21 days after beginning of treatment) and included all 10 patients treated with IVIG. The analysis revealed that 360 genes in peripheral T cells were significantly changed in expression during the course of IVIG treatment. The expression of 91 of these genes changed between day 0 and day 6, the expression of 147 genes changed between day 0 and day 21, and the expression of 122 genes changed between day 6 and day 21.
Statistical analysis of the control-patient group treated with IVMP showed differential expression of 583 genes, with the majority (218 genes) being changed between day 0 and day 6.
Tables 3a-3d present the 20 most significant changes in gene expression observed in patients treated with IVIG and IVMP.
Genes mostly affected in expression by IVIG treatment include genes that encode proteins that regulate cell cycle (transcriptional regulating factor 1, TRERF1; cyclin-dependent kinase inhibitor 1C, CDKN1C; breast cancer 1, BRCA 1; SH3-domain binding protein 4, SH3BP4); but also proteins that regulate inflammation [chemokine (C-X-C motif) ligand 5, CXCL5], cell adhesion (FAT tumor suppressor homolog 2, FAT2) or cell differentiation (early growth response, EGR2). Other genes included in the list encode proteins that are involved in electron transport, phosphorylation, glycosylation, skeletal development or proteins that have not yet been defined in function.
Other genes of interest that were differentially regulated upon IVIG treatment encoded proteins involved in immune regulation such as interleukin 11 (IL11), chemokine (C motif) ligand 2 (XCL2), prostaglandin E receptor 4 (PTGER4), caspase 2 (CASP2), killer cell immunoglobin-like receptor, two domains, short cytoplasmic tail 1 (KIR2DS1), mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), chemokine (C-X-C motif) ligand 5 (CXCL5), chemokine (C-X-C motif) ligand 3 (CXCL3), C-type lectin domain family 4, member E (CLEC4E), chemokine (C-C motif) ligand 13 (CCL13) and alpha-fetoprotein (AFP) (see Table 4).
When gene expression data obtained from patients treated with IVIG were compared with gene expression data obtained from patients treated with IVMP, 17 genes were identified that significantly changed in expression in both groups of patients (Table 5). Most of the proteins that are encoded by these 17 genes regulate cell cycle (HABP4, STAT1, CDKN1, SH3BP4 and ORC1L). These results indicate that cell cycle regulation might be a mechanism of therapeutic effectiveness that both drugs have in common. The other genes that were found to be differentially regulated were only found in one of the two treatment groups and, therefore, reflect mechanisms of action that are specific for only one of the two drugs.
Data obtained with microarray analysis were confirmed by quantitative real-time PCR. For this purpose, 4 genes were selected that encoded proteins known to regulate immune regulation (see Table 4): PTGER4, CXCL5, IL11 and CASP2. Results of real-time PCR are shown in
The present study was designed to identify genes that are differentially expressed in peripheral T cells of patients with RRMS in acute exacerbation after treatment with IVIG. Peripheral T cells (CD4+ and CD8+ T cells) have been shown to be involved in the disease pathogenesis, in particular in the process of demyelination and axonal damage (Stinissen P. et al., Mult Scler., 4:203-11 (1998)). This is supported by a recent study in which a number of genes in peripheral blood cells of MS patients were shown to be differentially expressed compared with those in healthy twins (Särkijärvi S. et al., BMC Medical Genetics, 7:11 (2006)).
Statistical data analysis revealed 360 genes that were at least 2-fold up- or down-regulated in all patients following IVIG treatment. The effect of IVIG treatment was most prominent at 21 days after the beginning of IVIG treatment. Genes mostly affected in expression by IVIG treatment included genes that encode proteins that regulate cell cycle, signal transduction, transcription, inflammation, cell-cell interactions and apoptosis. These processes are likely to be involved in the pathogenesis of MS. When we compared the effects on gene expression caused by IVIG treatment with the effects caused by IVMP treatment, we found 583 genes to be differentially regulated upon IVMP treatment. The majority of these genes was altered in expression at day 6 compared to day 0 after the beginning of therapy. These results indicate that IVMP might be a faster acting drug than IVIG.
We identified 17 genes that were significantly changed in expression in both groups of patients. Most of the proteins that are encoded by these 17 genes regulate cell cycle. These results strongly suggest that the regulation of cell proliferation, in particular the regulation of T cell proliferation, is a mechanism of action that both drugs have in common. These results agree with published data that indicate that IVIG suppresses the proliferation of activated T cells when given to patients with MS (Andersson U. et al., Immunol Rev, 139:21-42 (1994); Bayry J. et al., Intravenous immunoglobulin in autoimmune disorders: An insight into the immunregulatory mechanisms).
An important mechanism of action of IVIG in MS seems to be the modulation of chemokine expression. This conclusion is based on our findings that a number of genes that encode chemokines (CXCL3, CXCL5, CCL13 and XCL2) are differentially expressed upon IVIG treatment. These changes in gene expression were not found in patients treated with IVMP. Therefore, we believe that the modulation of chemokine expression in peripheral T cells might be a specific mechanism of action of IVIG in MS. Several studies have shown that chemokines and chemokine receptors are involved in the pathogenesis of MS (Trebst C. and Ransohoff R. M., Arch Neurol, 58:1975-80 (2001)). Chemokines have been shown to mediate trafficking of immune cells across the blood-brain barrier and to direct migration of immune cells towards sites of active lesions (Szczucinski A. and Losy J., Acta Neurol Scand, 115:137-146 (2007)). Moreover, chemokines were detected in active lesions and were found to be elevated in the cerebrospinal fluid of patients with MS during relapse (Sindern E. et al., J Neuroimmunol, 131:186-90 (2002)). Two of the chemokines (CXCL3 and CXCL5) that were significantly down-regulated in our study are known to specifically interact with the chemokine receptor CXCR2 (Omari K. et al., Brain, 128:1003-1015 (2005)). Previous studies have shown that CXCR2 is not only expressed on peripheral blood cells such as granulocytes, monocytes or lymphocytes (Murdoch C. et al., Brain, 128:1003-1015 (2005(?)); Murphy P. M. et al., Pharmacol Rev., 52:145-76 (2000)) but also on oligodendrocytes in the brain. Oligodendrocytes are most essential for the myelination of axons in the white matter of the Central Nervous System and for remyelination after demyelination of axons during inflammation in MS (Blakemore W. F., J Neurol Sci., (2007)). Recently it was shown that CXCR2 expressed on oligodendrocytes is essential for the development and maintenance of the oligodendrocyte lineage, myelination and white matter in the vertebrate CNS (Tsai H. H. et al., Cell, 110:373-83 (2002); Padovani-Claudio D. et al., Glia, 54:471-483 (2006)). The regulation of oligodendrocyte development and migration depends on the localized expression of the chemokine CXCL1 and its interaction with CXCR2 expressed on oligodendrocyte precursor cells and oligodendrocytes (Padovani-Claudio D. et al., Glia, 54:471-483 (2006)). Any event that disrupts the interaction between CXCL1 and CXCR2 expressed on oligodendrocytes or the signalling induced by this interaction could therefore cause a disruption of the remyelination processes in MS patients. Based on these findings we propose the following hypothesis for a new mechanism of action of IVIG in RRMS patients during relapse. Peripheral T cells and monocytes enter the CNS in response to chemokines produced by the inflammation in the brain. The disrupted blood-brain barrier (Man S. et al., Brain Pathol., 17:243-50 (2007)) facilitates this process. Both T cells and monocytes produce chemokines in the brain that interfere with the tightly regulated activity of oligodendrocyte precursor cells and oligodendrocytes. This interference could be caused by either a desensitization of the CXCR2 receptor expressed on oligodendrocytes or by interference with the interaction between locally expressed CXCL1 and CXCR2 on oligodendrocytes. IVIG down-regulates the expression of chemokines in peripheral T cells, monocytes or both. Consequently, the interference of chemokines produced by these cells with the function of oligodendrocytes would be prevented and the natural process of remyelination induced by oligodendrocytes would be re-established. It remains to be shown whether IVIG might not only modulate the expression of chemokines in peripheral T cells but also the expression of chemokines in cells of the CNS, e.g., in astrocytes.
The aim of our study was to identify genes that are likely to be associated with T cell responses in MS. The strategy that we used for positive cell selection does not exclude the possibility that some of the identified genes are associated with peripheral monocytes rather than T cells. This has to be taken into consideration when interpreting the above data. The genes that we found to be differentially expressed under IVIG treatment will be confirmed in a second clinical trial with a larger study group. Differentially expressed genes can be used as diagnostic markers for the therapeutic efficacy of IVIG treatment. Furthermore, some of the proteins encoded by the genes of interest will provide suitable targets for future drug development.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Drosophila) (EGR2) (NM_000399)
Drosophila) (EGR2) (NP_000390.2)
The present application is a continuation of U.S. Ser. No. 12/189,367, filed Aug. 11, 2008, which claims priority to U.S. Ser. No. 60/955,610, filed Aug. 13, 2007, herein incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60955610 | Aug 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12189367 | Aug 2008 | US |
| Child | 13026053 | US |
