METAL-BINDING THERAPEUTIC PEPTIDES
The present invention is related methods of delivering MBD peptide-linked agents into live cells. The methods described herein comprise contacting MBD peptide-linked agents to live cells under a condition of cellular stress. The methods of the invention may be used for therapeutic or diagnostic purposes.
This is a continuation of U.S. patent application Ser. No. 12/077,575, filed Mar. 19, 2008, which is a continuation-in-part of U.S. patent application Ser. No. 11/809,527, filed Jun. 1, 2007, abandoned, which is a continuation-in-part of U.S. patent application Ser. No. 11/725,672, filed Mar. 19, 2007, now U.S. Pat. No. 7,611,893, which is a continuation-in-part of U.S. patent application Ser. No. 11/595,367, filed Nov. 8, 2006, now U.S. Pat. No. 7,618,816, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/735,529, filed Nov. 9, 2005, and U.S. Provisional Application Ser. No. 60/789,100 filed Apr. 3, 2006, each application is hereby incorporated by reference in its entirety.
TECHNICAL FIELDThe invention relates to the field of medical diagnostics and therapeutics, and more particularly to methods for recognizing underlying mechanisms of disease and thereby identifying molecules that may be selectively active on human disease. The invention also relates to specific reagents of particular utility in the targeted delivery of drugs.
BACKGROUND ARTThe so-called diseases of western civilization (chronic conditions such as arthritis, lupus, asthma, and other immune-mediated diseases, osteoporosis, atherosclerosis, other cardiovascular diseases, cancers of the breast, prostate and colon, metabolic syndrome-related conditions such as cardiovascular dysfunctions, diabetes and polycystic ovary syndrome (PCOS), neurodegenerative conditions such as Parkinson's and Alzheimer's, and ophthalmic diseases such as macular degeneration) are now increasingly being viewed as secondary to chronic inflammatory conditions. A direct link between adiposity and inflammation has recently been demonstrated. Macrophages, potent donors of pro-inflammatory signals, are nominally responsible for this link: Obesity is marked by macrophage accumulation in adipose tissue (Weisberg S P et al [2003] J. Clin Invest 112: 1796-1808) and chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance (Xu H, et al [2003] J. Clin Invest. 112: 1821-1830). Inflammatory cytokine IL-18 is associated with PCOS, insulin resistance and adiposity (Escobar-Morreale H F, et al [2004] J. Clin Endo Metab 89: 806-811). Systemic inflammatory markers such as CRP are associated with unstable carotid plaque, specifically, the presence of macrophages in plaque, which is associated with instability can lead to the development of an ischemic event (Alvarez Garcia B et al [2003] J Vasc Surg 38: 1018-1024). There are documented cross-relationships between these risk factors. For example, there is higher than normal cardiovascular risk in patients with rheumatoid arthritis (RA) (Dessein P H et al [2002] Arthritis Res. 4: R5) and elevated C-peptide (insulin resistance) is associated with increased risk of colorectal cancer (Ma J et al [2004] J. Natl Cancer Inst 96:546-553) and breast cancer (Malin A. et al [2004] Cancer 100: 694-700). The genesis of macrophage involvement with diseased tissues is not yet fully understood, though various theories postulating the “triggering” effect of some secondary challenge (such as viral infection) have been advanced. What is observed is vigorous crosstalk between macrophages, T-cells, and resident cell types at the sites of disease. For example, the direct relationship of macrophages to tumor progression has been documented. In many solid tumor types, the abundance of macrophages is correlated with prognosis (Lin E Y and Pollard J W [2004] Novartis Found Symp 256: 158-168). Reduced macrophage population levels are associated with prostate tumor progression (Yang G et al [2004] Cancer Res 64:2076-2082) and the “tumor-like behavior of rheumatoid synovium” has also been noted (Firestein G S [2003] Nature 423: 356-361). At sites of inflammation, macrophages elaborate cytokines such as interleukin-1-beta and interleukin-6.
A ubiquitous observation in chronic inflammatory stress is the up-regulation of heat shock proteins (HSP) at the site of inflammation, followed by macrophage infiltration, oxidative stress and the elaboration of cytokines leading to stimulation of growth of local cell types. For example, this has been observed with unilateral obstructed kidneys, where the sequence results in tubulointerstitial fibrosis and is related to increases in HSP70 in human patients (Valles, P. et al [2003] Pediatr Nephrol. 18: 527-535). HSP70 is required for the survival of cancer cells (Nylandsted J et al [2000] Ann NY Acad Sci 926: 122-125). Eradication of glioblastoma, breast and colon xenografts by HSP70 depletion has been demonstrated (Nylansted J et al [2002] Cancer Res 62:7139-7142; Rashmi R et al [2004] Carcinogenesis 25: 179-187) and blocking HSF1 by expressing a dominant-negative mutant suppresses growth of a breast cancer cell line (Wang J H et al [2002] BBRC 290: 1454-1461). It is hypothesized that stress-induced extracellular HSP72 promotes immune responses and host defense systems. In vitro, rat macrophages are stimulated by HSP72, elevating NO, TNF-alpha, IL-1-beta and IL-6 (Campisi J et al [2003] Cell Stress Chaperones 8: 272-86). Significantly higher levels of (presumably secreted) HSP70 were found in the sera of patients with acute infection compared to healthy subjects and these levels correlated with levels of IL-6, TNF-alpha, IL-10 (Njemini R et al [2003] Scand. J. Immunol 58: 664-669). HSP70 is postulated to maintain the inflammatory state in asthma by stimulating pro-inflammatory cytokine production from macrophages (Harkins M S et al [2003] Ann Allergy Asthma Immunol 91: 567-574). In esophageal carcinoma, lymph node metastasis is associated with reduction in both macrophage populations and HSP70 expression (Noguchi T. et al [2003] Oncol. 10: 1161-1164). HSPs are a possible trigger for autoimmunity (Purcell A W et al [2003] Clin Exp Immunol. 132: 193-200). There is aberrant extracellular expression of HSP70 in rheumatoid joints (Martin C A et al [2003] J. Immunol 171: 5736-5742). Even heterologous HSPs can modulate macrophage behavior: H. pylori HSP60 mediates IL-6 production by macrophages in chronically inflamed gastric tissues (Gobert A P et al [2004] J. Biol. Chem 279: 245-250).
In addition to immunological stress, a variety of environmental conditions can trigger cellular stress programs. For example, heat shock (thermal stress), anoxia, high osmotic conditions, hyperglycemia, nutritional stress, endoplasmic reticulum (ER) stress and oxidative stress each can generate cellular responses, often involving the induction of stress proteins such as HSP70.
One common feature of nearly all of the emerging diseases in the Western world is the complexity of the underlying biochemical dysfunctions. New methodology for identifying the core biochemical lesions in disease conditions is needed. Such methodology would provide a first step to the development of predictive diagnostics and adequately targeted interventions.
About 40,000 women die annually from metastatic breast cancer in the U.S. Current interventions focus on the use of chemotherapeutic and biological agents to treat disseminated disease, but these treatments almost invariably fail in time. At earlier stages of the disease, treatment is demonstrably more successful: systemic adjuvant therapy has been studied in more than 400 randomized clinical trials, and has proven to reduce rates of recurrence and death more than 15 years after treatment (Hortobagyi G N. (1998) N Engl J Med. 339 (14): 974-984). The same studies have shown that combinations of drugs are more effective than just one drug alone for breast cancer treatment. However, such treatments significantly lower the patient's quality of life, and have limited efficacy. Moreover, they may not address slow-replicating tumor reservoirs that could serve as the source of subsequent disease recurrence and metastasis. A successful approach to the treatment of recurrent metastatic disease must address the genetic heterogeneity of the diseased cell population by simultaneously targeting multiple mechanisms of the disease such as dysregulated growth rates and enhanced survival from (a) up-regulated stress-coping and anti-apoptotic mechanisms, and (b) dispersion to sequestered and privileged sites such as spleen and bone marrow. Cellular diversification, which leads to metastasis, produces both rapid and slow growing cells. Slow-growing disseminated cancer cells may differ from normal cells in that they are located outside their ‘normal’ tissue context and may up-regulate both anti-apoptotic and stress-coping survival mechanisms. Global comparison of cancer cells to their normal counterparts reveals underlying distinctions in system logic. Cancer cells display up-regulated stress-coping and anti-apoptotic mechanisms (e.g. NF-kappa-B, Hsp-70, MDM2, survivin etc.) to successfully evade cell death (Chong Y P, et al. (2005) Growth Factors. September; 23 (3): 233-44; Rao R D, et al (2005) Neoplasia. October; 7 (10): 921-9; Nebbioso A, et al (2005) Nat Med. January; 11 (1): 77-84). Many tumor types contain high concentrations of heat-shock proteins (HSP) of the HSP27, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca at al 1993; Yano et al 1999; Cornford at al 2000; Strik et al 2000; Ricaniadis et al 2001; Ciocca and Vargas-Roig 2002). The role of HSPs in tumor development may be related to their function in the development of tolerance to stress (Li and Hahn 1981) and high levels of HSP expression seem to be a factor in tumor pathogenesis. Among other mechanisms individual HSPs can block pathways of apoptosis (Volloch and Sherman 1999). Studies show HSP70 is required for the survival of cancer cells (Nylandsted J, Brand K, Jaattela M. (2000) Ann N Y Acad Sci. 926: 122-125). Eradication of glioblastoma, breast and colon xenografts by HSP70 depletion has been demonstrated, but the same treatment had no effect on the survival or growth of fetal fibroblasts or non-tumorigenic epithelial cells of breast (Nylandsted J, et al (2002) Cancer Res. 62 (24): 7139-7142; Rashmi R, Kumar S, Karunagaran D. (2004) Carcinogenesis. 25 (2): 179-187; Barnes J A, et al. (2001) Cell Stress Chaperones. 6 (4): 316-325) and blocking HSF1 by expressing a dominant-negative mutant suppresses growth of a breast cancer cell line (Wang J H, et al. (2002) Biochem Biophys Res Commun. 290 (5): 1454-1461). Stress can also activate the nuclear factor kappa B (NF-kappa B) transcription factor family. NF-kappa-B is a central regulator of the inflammation response that regulates the expression of anti-apoptotic genes, such as cyclooxygenases (COX) and metalloproteinases (MMPs), thereby favoring tumor cell proliferation and dissemination. NF-kappa-B can be successfully inhibited by peptides interfering with its intracellular transport and/or stability (Butt A J, et al. (2005) Endocrinology. July; 146 (7): 3113-22). Human survivin, an inhibitor of apoptosis, is highly expressed in various tumors (Ambrosini G, Adida C, Altieri D C. (1997) Nat. Med. 3 (8): 917-921) aberrantly prolonging cell viability and contributing to cancer. It has been shown that ectopic expression of survivin can protect cells against apoptosis (Li F, et al. (1999) Nat. Cell Biol. 1 (8): 461-466). Tumor suppressor p53 is a transcription factor that induces growth arrest and/or apoptosis in response to cellular stress. Peptides modeled on the MDM2-binding pocket of p53 can inhibit the negative feedback of MDM2 on p53 commonly observed in cancer cells (Midgley C A, et al. (2000) Oncogene. May 4; 19 (19): 2312-23; Zhang R, et al. (2004) Anal Biochem. August 1; 331 (1): 138-46). The role of protein degradation rates and the proteasome in disease has recently come to light. Inhibitors of HSP90 (a key component of protein degradation complexes) such as bortezomib are in clinical testing and show promise as cancer therapeutics (Mitsiades C S, et al. 2006 Curr Drug Targets. 7(10):1341-1347). A C-terminal metal-binding domain (MBD) of insulin-like growth factor binding protein-3 (IGFBP-3) can rapidly (<10 min) mobilize large proteins from the extracellular milieu into the nuclei of target cells (Singh B K, et al. (2004) J Biol Chem. 279: 477-487). Here we extend these observations to show that MBD is a systemic ‘guidance system’ that attaches to the surface of red blood cells and can mediate rapid intracellular transport of its ‘payload’ into the cytoplasm and nucleus of target cells at privileged sites such as spleen and bone marrow in vivo. The amino acid sequence of these MBD peptides can be extended to include domains known to inhibit HSP, survivin, NF-kappa-B, proteasome and other intracellular mechanisms. The MBD mediates transport to privileged tissues and intracellular locations (such as the nucleus) in the target tissue. In this study we ask whether such MBD-tagged peptides might act as biological modifiers to selectively enhance the efficacy of existing treatment modalities against cancer cells. Patients presenting with metastatic disease generally face a poor prognosis. The median survival from the time of initial diagnosis of bone metastasis is 2 years with only 20% surviving 5 years (Antman et al. (1999) JAMA.; 282: 1701-1703; Lipton A. (2005) North American Pharmacotherapy: 109-112). A successful systemic treatment for recurrent metastatic disease is the primary unmet medical need in cancer.
Part of the lack of success in treating metastatic disease may have to do with a lack of understanding of the mestastatic disease process. Unlike the primary tumor event, which is primarily a dysfunction of unregulated growth, metastatic cells must generally adapt to unusual environments in body locations that are distant to the original tumor site. Thus, most traditional interventions designed to treat a primary tumor, which focus on controlling tumor cell growth, may be fundamentally unsuited to the treatment of metastatic disease, which is a disease of adaptation. Thus there is a need for identifying the biochemical correlates of cellular adaptivity.
Diabetes is a rapidly expanding epidemic in industrial societies. The disease is caused by the body's progressive inability to manage glucose metabolism appropriately. Insulin production by pancreatic islet cells is a highly regulated process that is essential for the body's management of carbohydrate metabolism. The primary economic and social damage of diabetes is from secondary complications that arise in the body after prolonged exposure to elevated blood sugar. These include cardiovascular complications, kidney disease and retinopathies. Most interventions so far developed for diabetic conditions focus on controlling blood sugar, the primary cause of subsequent complications. However, despite the availability of several agents for glycemic control, the population of individuals with poorly controlled blood sugar continues to explode. 40% of kidney failure is currently associated with diabetes, and that percentage is expected to rise.
One potential approach to treating the complications of diabetes is to focus on the cellular biochemistry of organs that are particularly sensitive to elevated blood sugar levels. Advanced glycosylation end products of proteins (AGEs) are non-enzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. Cellular actions of advanced glycation end-products (AGE) are mediated by a receptor for AGE (RAGE), a novel integral membrane protein (Neeper M et al [1992] J. Biol. Chem. 267: 14998-15004). Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The bioactivity of RAGE is governed by the settings in which these ligands accumulate, such as diabetes, inflammation and tumors. Vascular complications of diabetes such as nephropathy, cardiomyopathy and retinopathy, may be driven in part by the AGE-RAGE system (Wautier J-L, et al [1994] Proc. Nat. Acad. Sci. 91: 7742-7746; Barile G R et al [2005] Invest. Ophthalm. Vis. Sci. 46: 2916-2924; Yonekura H et al [2005] J. Pharmacol. Sci. 97: 305-311). Specific downstream cellular molecular events are now believed to mediate some of the damaging consequences of RAGE activation, and generate a rationale for chemical, biological and genetic interventions in these types of hypertrophic disease processes (Cohen M P et al [2005] Kidney Int. 68: 1554-1561; Cohen M P et al [2002] Kidney Int. 61: 2025-2032; Wendt T et al [2006] Atherosclerosis 185: 70-77; Wolf G et al [2005] Kidney Int. 68: 1583-1589). Soluble RAGE is associated with albuminuria in human diabetics (Humpert P M et al [2007] Cardiovasc. Diabetol. 6: 9) and in animal models of diabetic nephropathy such as the db/db mouse (Yamagishi S et al [2006] Curr. Drug Discov. Technol. 3: 83-88; Sharma K et al [2003] Am J. Physiol. Renal Physiol. 284: F1138-F1144). In the complex disease process of diabetic progression the causal interplay of hypertensive, glycemic, inflammatory and endocrinological factors is difficult to parse. Nevertheless, magnetic resonance imaging of the db/db mouse reveals progressive cardiomyopathic changes as diabetes progresses. Relatively early in the disease process (9 weeks), left ventricular hypertrophy (LVH) is observed. In human populations, LVH correlates with elevated levels of NT-pro-BNP and cardiac Troponin T (cTnT) in serum (Arteaga E et al [2005] Am Heart J. 150: 1228-1232; Lowbeer C et al [2004] Scand J. Clin. Lab Invest. 64: 667-676).
PRR5 and related proteins are a new class of molecules found in association to mTOR complex, a central regulator of cellular biochemistry. The PRR5 gene encodes a conserved proline-rich protein predominant in kidney (Johnstone C N et al [2005] Genomics 85: 338-351). The PRR5 class of proteins is believed to physically associate with mTORC2 and regulate aspects of growth factor signaling and apoptosis (Woo S Y et al [2007] J. Biol. Chem. 282: 25604-25612; Thedieck K et al [2007] PLoS ONE 2: e1217). In this invention, the importance of a particular domain within PRR5 (referred to as the PRR5D sequence) comprising the sequence HESRGVTEDYLRLETLVQKVVSPYLGTYGL (SEQ ID NO: 234) is demonstrated. This sequence is conserved in human PRR5 isoforms and PRR5L as well as in rat and mouse.
In diabetic humans and db/db mice the receptor for advanced glycated end products (RAGE) is activated by systemic ligands such as amphoterin and glycated hemoglobin (Goldin A et al [2006] Circulation 114: 597-605). RAGE has been implicated in the development of kidney dysfunction consequent to elevated blood sugar (Tan A L et al [2007] Semin. Nephrol. 27:130-143). The intracellular biochemical events downstream of RAGE activation leading to the loss of kidney function and albuminuria in db/db mice are not well understood. RAGE blockade through the use of soluble RAGE decoys has been proposed as a method for controlling complications of diabetes in humans (Yamagishi S et al [2007] Curr. Drug Targets 8:1138-1143; Koyama H et al [2007] Mol Med 13:625-635). Kidney mesangial cell matrix expansion characterized by excessive deposition of collage-IV and fibronectin is an often-cited correlate of disease progression (Tsilibary E C et al [2003] J. Pathol. 200: 537-546). However, effective interventions based on this hypothesis have yet to be developed. Recently, the inhibition of protein kinase C (PKC) isoforms has been proposed as a possible therapeutic intervention for kidney disease (Tuttle K R et al [2003] Am. J. Kidney Dis. 42: 456-465). A peptide capable of inhibiting PKC beta II in cultured cells has been described (Ron D et al [1995] J. Biol. Chem. 270: 24180-24187). Correlation matrices or dendograms (Peterson L E [2003] Comput. Methods Programs Biomed. 70: 107-119) constructed from RAGE-adaptive datasets gathered in cultured kidney cell and kidney tissue extracts can help identify reliable biochemical correlates of disease, and can guide the development of effective therapeutic interventions. Although correlations do not reveal causative links, the clustering of biochemical correlates can help define ‘virtual dysregulons’ around which hypothesis-driven interventions can be designed and tested. This invention describes methods for surveying a panel of intracellular biochemical readouts in cultured 293 kidney cells challenged with glycated hemoglobin and various chemical and peptide inhibitors. From these data a method is described for selecting a subset of readouts that are significantly impacted by RAGE ligand in these cells. Taken together, these readouts are referred to as an “adaptive signature”. In this context, RAGE ligand is referred to as a “provocative agent” for the derivation of adaptive signatures. Adaptive signature refers to the delta, or difference in readouts, between cells that are treated with a specific provocative agent and cells that are treated with control, such as saline. Similar methodology can be applied to tissues from animals or humans that have been exposed to varying levels of a provocative agent. As an example, kidney extracts from albuminuric db/db mice can be assayed for these selected biochemical markers and compared with a group of control animals who have not developed albuminuria. Correlation matrices constructed from these data can subsequently suggest possible modifications to our current understanding of diabetic kidney disease, based on the adaptive signatures revealed. Three key features of this methodology are (a) the choice of provocative agent (b) the use of delta values as opposed to the more traditional approach of using actual biochemical assay values in profiling, and (c) the use of correlation matrices or dendograms to generate virtual dysregulon clusters based on related adaptive response, rather than logical pathway analysis.
Despite the worldwide epidemic of chronic kidney disease complicating diabetes mellitus, current therapies directed against nephroprogression are limited to angiotensin conversion or receptor blockade. Nonetheless, additional therapeutic possibilities are slowly emerging. The diversity of therapies currently in development reflects the pathogenic complexity of diabetic nephropathy. The three most important candidate drugs currently in development include a glycosaminoglycan, a protein kinase C (PKC) inhibitor and an inhibitor of advanced glycation (Williams M E [2006] Drugs. 66: 2287-2298). Treatment of hypertrophic conditions of the heart and kidney using protein kinase C-beta inhibitors (Koya D et al [2000] FASEB J. 14: 439-447) represents an alternative to RAGE blockade and TGF-beta-1 blockade approaches to new interventions in hypertrophic disease states.
Renal failure characterized by proteinuria and mesangial cell expansion is observed in a number of non-diabetic states. Many forms of renal disease that progress to renal failure are characterized histologically by mesangial cell proliferation and accumulation of mesangial matrix. These diseases include IgA nephropathy and lupus nephritis. Bone marrow transplantation (BMT) is an effective therapeutic strategy for leukemic malignancies and depressed bone marrow following cancer. However, its side effects on kidneys have been reported. (Otani M et al [2005] Nephrology 10: 530-536). Some hematological malignancies associated with nephrotic syndrome include Hodgkin's and non-Hodgkin's lymphomas and chronic lymphocytic leukemia (Levi I [2002] Lymphoma. 43: 1133-1136). Cancer drugs such as mitomycin, cisplatin, bleomycin, and gemcitabine (Saif M W and McGee P J [2005] JOP. 6: 369-374) and the anti-angiogenic agent bevacizumab (Avastin) (Gordon M S and Cunningham D [2005] Oncology. 69 Suppl 3: 25-33) and irradiation are also suggested to be nephrotoxic. Moreover, the observed cardiotoxicity of drugs such a 5-fluorouracil and capecitabine may be secondary to renal toxicity of these drugs (Jensen S A and Sorensen J B [2006] Cancer Chemother Pharmacol. 58: 487-493). There are a large number of glomerular diseases that may be responsible for a nephrotic syndrome, the most frequent in childhood being minimal change disease. Denys-Drash syndrome and Frasier syndrome are related diseases caused by mutations in the WT1 gene. Familial forms of idiopathic nephrotic syndrome with focal and segmental glomerular sclerosis/hyalinosis have been identified with an autosomal dominant or recessive mode of inheritance and linkage analysis have allowed to localize several genes on chromosomes 1, 11 and 17. The gene responsible for the Finnish type congenital nephrotic syndrome has been identified. This gene, named NPHS1, codes for nephrin, which is located at the slit diaphragm of the glomerular podocytes and is thought to play an essential role in the normal glomerular filtration barrier (Salomon R et al [2000] Curr. Opin. Pediatr. 12: 129-134).
Thymosin-beta-4 and its N-terminal tetrapeptide (Ac-SDKP (SEQ ID NO: 190)) have been implicated as powerful inhibitors of the proliferative TGF-beta signal observed in renal mesangial cell expansion, a precursor to renal dysfunction in diabetic nephropathy (Cavasin M A [2006] Am. J. Cardiovasc. Drugs 6: 305-311). Ac-SDKP is cleaved from prothymosin by prolyl oligopeptidase and is subsequently hydrolysed by angiotensin-converting enzyme (Cavasin M A et al [2004] Hypertension 43: 1140-1145). Therapeutic application of Ac-SDKP has shown promise in reversing hypertrophy in a number of renal and cardiovascular models (Yang F et al [2004] Hypertension 43: 229-236; Omata M et al [2006] J. Am. Soc. Nephrol. 17: 674-685; Shibuya K et al [2005] Diabetes 54: 838-845; Peng et al [2001] Hypertension 37: 794-800; Raleb N-E et al [2001] Circulation 103: 3136-3141).
Familial mutations in parkin gene are associated with early-onset PD. Parkinson's disease (PD) is characterized by the selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). A combination of genetic and environmental factors contributes to such a specific loss, which is characterized by the accumulation of misfolded protein within dopaminergic neurons. Among the five PD-linked genes identified so far, parkin, a 52 kD protein-ubiquitin E3 ligase, appears to be the most prevalent genetic factor in PD. Mutations in parkin cause autosomal recessive juvenile parkinsonism (AR-JP). The current therapy for Parkinson's disease is aimed to replace the lost transmitter, dopamine. But the ultimate objective in neurodegenerative therapy is the functional restoration and/or cessation of progression of neuronal loss (Jiang H, et al [2004] Hum Mol Genet. 13 (16): 1745-54; Muqit M M, et al [2004] Hum Mol Genet. 13 (1): 117-135; Goldberg M S, et al [2003] J Biol Chem. 278 (44): 43628-43635). Over-expressed parkin protein alleviates PD pathology in experimental systems. Recent molecular dissection of the genetic requirements for hypoxia, excitotoxicity and death in models of Alzheimer disease, polyglutamine-expansion disorders, Parkinson disease and more, is providing mechanistic insights into neurotoxicity and suggesting new therapeutic interventions. An emerging theme is that neuronal crises of distinct origins might converge to disrupt common cellular functions, such as protein folding and turnover (Driscoll M, and Gerstbrein B. [2003] Nat Rev Genet. 4(3): 181-194). In PC12 cells, neuronally differentiated by nerve growth factor, parkin overproduction protected against cell death mediated by ceramide Protection was abrogated by the proteasome inhibitor epoxomicin and disease-causing variants, indicating that it was mediated by the E3 ubiquitin ligase activity of parkin. (Darios F. et al [2003] Hum Mol Genet. 12 (5): 517-526). Overexpressed parkin suppresses toxicity induced by mutant (A53T) and wt alpha-synuclein in SHSY-5Y cells (Oluwatosin-Chigbu Y. et al [2003] Biochem Biophys Res Commun. 309 (3): 679-684) and also reverses synucleinopathies in invertebrates (Haywood A F and Staveley B E. [2004] BMC Neurosci. 5(1): 14) and rodents (Yamada M, Mizuno Y, Mochizuki H. (2005) Parkin gene therapy for alpha-synucleinopathy: a rat model of Parkinson's disease. Hum Gene Ther. 16(2): 262-270; Lo Bianco C. et al [2004] Proc Natl Acad Sci USA. 101(50): 17510-17515). On the other hand, a recent report claims that parkin-deficient mice are not themselves a robust model for the disease (Perez F A and Palmiter R D [2005] Proc Natl Acad Sci USA. 102 (6): 2174-2179). Nevertheless, parkin therapy has been suggested for PD (Butcher J. [2005] Lancet Neurol. 4(2): 82).
Variability within patient populations creates numerous problems for medical treatment. Without reliable means for determining which individuals will respond to a given treatment, physicians are forced to resort to trial and error. Because not all patients will respond to a given therapy, the trial and error approach means that some portion of the patients must suffer the side effects (as well as the economic costs) of a treatment that is not effective in that patient.
For some therapeutics targeted to specific molecules within the body, screening to determine eligibility for the treatment is routinely performed. For example, the estrogen antagonist tamoxifen targets the estrogen receptor, so it is normal practice to only administer tamoxifen to those patients whose tumors express the estrogen receptor Likewise, the anti-tumor agent trastuzumab (HERCEPTIN®) acts by binding to a cell surface molecule known as HER2/neu; patients with HER2/neu negative tumors are not normally eligible for treatment with trastuzumab. Methods for predicting whether a patient will respond to treatment with IGF-I/IGFBP-3 complex have also been disclosed (U.S. Pat. No. 5,824,467), as well as methods for creating predictive models of responsiveness to a particular treatment (U.S. Pat. No. 6,087,090).
IGFBP-3 is a master regulator of cellular function and viability. As the primary carrier of IGFs in the circulation, it plays a central role in sequestering, delivering and releasing IGFs to target tissues in response to physiological parameters such as nutrition, trauma, and pregnancy. IGFs, in turn, modulate cell growth, survival and differentiation, additionally; IGFBP-3 can sensitize selected target cells to apoptosis in an IGF-independent manner. The mechanisms by which it accomplishes the latter class of effects is not well understood but appears to involve selective cell internalization mechanisms and vesicular transport to specific cellular compartments (such as the nucleus, where it may interact with transcriptional elements) that is at least partially dependent on transferrin receptor, integrins and caveolin.
The inventor has previously disclosed certain IGFBP-derived peptides known as “MBD” peptides (U.S. patent application publication nos. 2003/0059430, 2003/0161829, and 2003/0224990). These peptides have a number of properties, which are distinct from the IGF-binding properties of IGFBPs, that make them useful as therapeutic agents. MBD peptides are internalized some cells, and the peptides can be used as cell internalization signals to direct the uptake of molecules joined to the MBD peptides (such as proteins fused to the MBD peptide).
Combination treatments are increasingly being viewed as appropriate strategic options for designed interventions in complex disease conditions such as cancer, metabolic diseases, vascular diseases and neurodegenerative conditions. For example, the use of combination pills containing two different agents to treat the same condition (e.g. metformin plus a thiazolidinedione to treat diabetes, a statin plus a fibrate to treat hypercholesterolemia) is on the rise. It is therefore appropriate to envisage combination treatments that include moieties such as MBD in combination with other agents such as other peptides, antibodies, nucleic acids, chemotherapeutic agents and dietary supplements. Combinations may take the form of covalent extensions to the MBD peptide sequence, other types of conjugates, or co-administration of agents simultaneously or by staggering the treatments i.e. administration at alternating times.
Humanin (HN) is a novel neuroprotective factor that consists of 24 amino acid residues. HN suppresses neuronal cell death caused by Alzheimer's disease (AD)-specific insults, including both amyloid-beta (betaAbeta) peptides and familial AD-causative genes. Cerebrovascular smooth muscle cells are also protected from Abeta toxicity by HN, suggesting that HN affects both neuronal and non-neuronal cells when they are exposed to AD-related cytotoxicity. HN peptide exerts a neuroprotective effect through the cell surface via putative receptors (Nishimoto I et al [2004] Trends Mol Med 10: 102-105). Humanin is also a neuroprotective agent against stroke (Xu X et al [2006] Stroke 37: 2613-2619). As has previously been demonstrated, it is possible to generate both single-residue variants of humanin with altered biological activity and peptide fusions of humanin to other moieties (Tajima H et al [2005] J. Neurosci Res. 79 (5): 714-723; Chiba T et al. [2005] J. Neurosci. 25: 10252-10261).
This indicates the feasibility of combining humanin peptide sequences with, for example, MBD-based therapeutic peptides or, alternatively, the therapeutic segments of previously described MBD-linked therapeutic peptides. The solution structures of both native humanin and its S14G variant have been described (Benaki D et al [2005] Biochem Biophys Res Comm 329: 152-160; Benaki D et al [2006] Biochem Biophys Res Comm 349: 634-642) thereby potentially facilitating the design of mutant or derivative sequences. The amino acid sequence of humanin is MAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 188) and the amino acid sequence of the variant is MAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO:189). Humanin binds a C-terminal domain of IGFBP-3 (Ikonen M et al [2003] Proc Nat Acad Sci. 100: 13042-13047). The binding of Zinc(II) to humanin was recently described (Armas A et al [2006] J. Inorg Biochem 100: 1672-1678). Therefore humanin may be considered a metal-binding therapeutic peptide.
Potentially therapeutic peptide sequences have been disclosed in the scientific literature. Many of these require cell internalization for action, which limits their in vivo utility without an appropriate delivery system. Peptide sequences that bind and possibly inhibit MDM2 (Picksley S M et al [1994] Oncogene. 9: 2523-2529), protein kinase C-beta (Ron D et al [1995] J Biol Chem. 270: 24180-24187), p38 MAP kinase (Barsyte-Lovejoy D et al [2002] J Biol Chem. 277: 9896-9903), DOK1 (Ling Y et al [2005] J Biol Chem. 280: 3151-3158), NF-kappa-B nuclear localization complex (Lin Y Z et al [1995] J Biol Chem. 270: 14255-14258), IKK complex (May M J et al [2000] Science. 289:1550-1554) and calcineurin (Aramburu J et al [1999] Science. 285: 2129-33) have been described.
IRS-1 and IRS-2 are master traffic regulators in intracellular signal transduction pathways associated with growth and metabolism, playing key roles in the docking of accessory proteins to phosphorylated insulin and IGF receptors. Although similar in function, activated IRS-1 and IRS-2 proteins generate subtly different cellular outcomes, at least in part through the phosphorylation of different Akt (especially Akt 1 and Akt 2) and MAP kinase isoforms.
The significance of IRS-2 to IRS-1 ratios in proliferative and inflammatory disease processes has never been explicitly cited. The possibility of using specific modulators of the IRS-2:IRS-1 to intervene in such disease processes has not been explicitly proposed. Such modulators might include, for example, treatments or compounds that preferentially reduce IRS-2 (versus IRS-1) signaling, or preferentially increase IRS-1 (versus IRS-2) signaling. Some unrelated observations of potential significance here are the use of a KRLB domain-specific inhibitor for IRS-2, the use of selected HIV protease inhibitors such as nelfinavir, saquinavir and ritonavir (previously shown to selectively inhibit IRS-2 over IRS-1). In this invention, the modulating effects of certain peptides such as humanin, PRR5 domain (PRR5D), and NPKC on IRS-2 versus IRS-1, both in vitro and in vivo, are described. The specific induction of IRS-2 in human kidney cells by a ligand of RAGE, first demonstrated here, and the modulation of that induction by humanin and NPKC peptides, further suggests the involvement of similar mechanisms of pathology in other RAGE-related proliferative or inflammatory conditions such as metastatic breast cancer, Alzheimer's disease, atherosclerosis, other cardiovascular conditions, arthritis, other autoimmune conditions and sepsis. Also shown here for the first time is the direct correlation between kidney IRS-2 levels, kidney collagen-IV levels and kidney function in diabetic db/db mice. Other peptides may also modulate IRS-2:IRS-1 ratios, including but not limited to MBD-KRLB (SEQ ID NO:216).
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
SUMMARY OF THE INVENTIONThe present invention provides compositions comprising a polypeptide having an amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO:2) or PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249) linked to a second polypeptide which exhibits binding affinity to a substantially purified intracellular molecular target. Administration of said composition to a mammal causes a clinically useful outcome.
In preferred embodiments of the invention the intracellular molecular target of the second polypeptide is selected from but is not limited to PRR5D sequence, NF-kappa-B regulator domain, p53 regulator domain, IGF-signaling regulator domain, survivin dimerization domain, proteasome subunit regulator domain, RAS active site domain, MYC regulator domain, HSP regulator domain and HIF1-alpha oxygen-dependent regulator domain.
In some embodiments of the invention, the first polypeptide is fused to the second polypeptide and in other embodiments of the invention the first polypeptide is conjugated to the second polypeptide.
In a preferred embodiment of the invention, the second polypeptide is an antibody or a fragment thereof.
The present invention provides methods of treating inflammatory disease conditions by administering an effective amount of the composition of the invention to a mammal. Inflammatory disease conditions include but are not limited to cancer, diabetes, cardiovascular disease, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, autoimmune disease, rheumatological disease and infectious disease.
In embodiments of the invention, the composition can be administered via any route including but not limited to intravenous, oral, subcutaneous, intraarterial, intramuscular, intracardial, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal, and inhalation.
The present invention also provides nucleic acids encoding a fusion polypeptide which includes the amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2) and/or PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249) and an additional polypeptide which exhibits binding affinity to a substantially purified intracellular molecular target.
In an embodiment of the invention, nucleic acids encoding fusion proteins are used in methods of treating an inflammatory disease condition. Inflammatory disease conditions include but are not limited to cancer, diabetes, cardiovascular disease, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, autoimmune disease, rheumatological disease and infectious disease.
The present invention provides the administration of dietary compounds curcumin and lycopene to treat subjects with an inflammatory disease condition including but not limited to cancer, diabetes, cardiovascular disease, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, autoimmune disease, rheumatological disease and infectious disease.
In a preferred embodiment, compositions of the invention comprised of the amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2) or PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249) linked to a second polypeptide which exhibits binding affinity to a substantially purified intracellular molecular target is administered in conjunction with the dietary compounds curcumin and lycopene to treat subjects with an inflammatory disease condition.
The invention provides a composition comprising a first metal-binding domain peptide selected from the group consisting of QCRPSKGRKRGFCW (SEQ ID NO: 2), SDKPDMAPRGFSCLLLLTSEIDLP (SEQ ID NO: 216), SDKPDMAPRGFSCLLLLTGEIDLP (SEQ ID NO: 217), SDKPDMAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 193), SDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192), PRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO:247), PRGFSCLLLLTSEIDLPVKRR (SEQ ID NO:248), PRGFSCLLLLTSEIDLPVKR (SEQ ID NO:246), PRGFSCLLLLTSEIDLPVK (SEQ ID NO:249), PRGFSCLLLLTGEIDLPVK (SEQ ID NO:250), PRGFSRLLLLTSEIDLPVKRRA (SEQ ID NO:251), PRGFSRLLLLTSEIDLPVKRR (SEQ ID NO:252), PRGFSRLLLLTSEIDLPVKR (SEQ ID NO:253), PRGFSRLLLLTSEIDLPVK (SEQ ID NO:230), and PRGFSRLLLLTGEIDLPVK (SEQ ID NO:254), wherein the first metal-binding domain peptide is linked to a second polypeptide that has less than 15% identity with the amino acid sequence of any naturally-occurring IGF-binding protein, exhibits binding affinity of micromolar or better to a substantially purified intracellular molecular target, and administration of said composition to a mammal causes a clinically useful outcome.
In some embodiments of the invention, the first metal-binding domain peptide is fused to said second polypeptide. In other embodiments of the invention the metal-binding domain peptide is conjugated to the second polypeptide. In some embodiments of the invention the second polypeptide is an antibody or a fragment thereof or a protein.
In some embodiments the invention provides nucleic acids of the fusion polypeptide and vectors comprising nucleic acids encoding the polypeptides of the invention.
In aspects of the invention, the intracellular molecular targets of the second polypeptide include but are not limited to PRR5D sequence, NF-kappa-B regulator domain, IKK complex, P53 regulator domain, MDM2, IGF-signaling regulator domain, survivin dimerization domain, proteasome subunit regulator domain, RAS active site domain, MYC regulator domain, HSP regulator domain, Smad2, Smad3, MAP kinase, Protein Kinase C, calcineurin, Src family kinases, DOK1, and HIF1-alpha oxygen-dependent regulator domain.
In some aspects of the invention the second polypeptide is comprised of an amino acid sequence selected from the group of sequences listed in Table 19 or Table 20.
In another aspect the invention provides methods of treating an inflammatory disease condition comprising administering an effective amount a polypeptide of the invention to a mammal. Inflammatory disease conditions include but are not limited to cancer, diabetes, cardiovascular disease, kidney disease, retinopathy, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, lupus, autoimmune disease, rheumatological disease and infectious disease.
In certain aspects the invention provides method of treating an inflammatory disease condition comprising administering an effective amount of humanin or humanin-S14G to a mammal. Inflammatory disease conditions include but are not limited to cancer, cardiomyopathy, nephropathy, retinopathy, obesity, lupus, autoimmune disease, rheumatological disease and infectious disease.
The compositions of the invention may be administered by means which include but are not limited to intravenous, oral, subcutaneous, intraarterial, intramuscular, intracardial, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal, and inhalation. In some embodiments, the composition is administered to a mammal at less than about 20 mg/kg/day.
The invention includes methods to treat inflammatory diseases conditions by administering nucleic acids and/or vectors encoding polypeptides of the invention to a mammal.
Another aspect of the invention includes methods of treating an inflammatory disease conditions in a mammal wherein a combination of two or more dietary compounds curcumin, lycopene and berberine are administered in said mammal at doses that produce peak blood levels of at least 1 nM for each selected compound.
In some embodiments of the invention the polypeptides of the invention are used in conjunction with curcumin, lycopene or berberine or any combination thereof, for the treatment of inflammatory disease conditions.
Inflammatory disease conditions include but are not limited to cancer, diabetes, cardiovascular disease, kidney disease, retinopathy, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, autoimmune disease, rheumatological disease and infectious disease.
One aspect of the invention includes methods of treating an inflammatory disease condition in a mammal comprising administering a therapeutic agent to a mammal, wherein the agent modulates the ratio of IRS-2 to IRS-1 in said mammal. Agents of this aspect of the invention include peptides; for example but not limited to humanin (SEQ ID NO: 188), humanin-S14G (SEQ ID NO: 189), peptides comprising the PRR5D sequence (RGVTEDYLRLETLVQKVVS; SEQ ID NO:256), NPKC (SEQ ID NO: 195) or MBD-KRLB (SEQ ID NO: 216). In another aspect of the invention, the IRS-2:IRS-1 modulating agent is a protease inhibitor; for example but not limited to nelfinavir, saquinavir and ritonavir. In a further aspect of the invention, the IRS-2:IRS-1 modulating agent is a nucleic acid; for example but not limited to nucleic acid encoding an IRS-2:IRS-1 modulating agent, siRNA, dsRNA, antisense RNA, RNAzymes, DNAzymes, and the like. Inflammatory disease conditions include but are not limited to cancer, diabetes, cardiovascular disease, kidney disease, retinopathy, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, lupus, autoimmune disease, rheumatological disease and infectious disease.
The invention also provides a method for modifying a disease process or a cellular process, said method comprising the steps of: (a) administering a provocative agent to live cells and generating an adaptive signature; (b) selecting a candidate therapeutic agent by co-administering various test compounds with the provocative agent, to test their ability to modify the adaptive signature caused by the provocative agent; and (b) delivering said candidate therapeutic agent into said live cells, whereby said disease process or said cellular process in said live cells is modified. In some embodiments, the disease process is selected from the group consisting of neurodegenerative, cancer, autoimmune, inflammatory, cardiovascular, diabetes, osteoporosis and ophthalmic diseases. In some embodiments, the cellular process is selected from the group consisting of transcriptional, translational, protein folding, protein degradation and protein phosphorylation events.
DISCLOSURE OF THE INVENTIONThe present invention provides a method for delivering an MBD peptide-linked agent into live cells, said method comprising contacting said MBD peptide-linked agent to live cells that are under a condition of cellular stress, whereby said contact results in cellular uptake of said MBD-peptide-linked agent.
The invention also provides a method for obtaining diagnostic information from live cells comprising the steps of: (a) administering an MBD peptide-linked agent to live cells that are under a condition of cellular stress; and (b) measuring a diagnostic readout. The diagnostic readout can be an enzymatic, a colorimetric, or a fluorimetric readout.
The invention also provides a method for modifying in a disease process or a cellular process, said method comprising the steps of: (a) administering an MBD peptide-linked agent to live cells that are under a condition of cellular stress, wherein the agent is capable of modifying the disease process or the cellular process within said live cells; and (b) delivering said MBD peptide-linked agent into said live cells, whereby said disease process or said cellular process in said live cells is modified. In some embodiments, the disease process is selected from the group consisting of neurodegenerative, cancer, autoimmune, inflammatory, cardiovascular, diabetes, osteoporosis and ophthalmic diseases. In some embodiments, the cellular process is selected from the group consisting of transcriptional, translational, protein folding, protein degradation and protein phosphorylation events.
In some embodiments, the condition of cellular stress is selected from the group consisting of thermal, immunological, cytokine, oxidative, metabolic, anoxic, endoplasmic reticulum, protein unfolding, nutritional, chemical, mechanical, osmotic and glycemic stress. In some embodiments, the condition of cellular stress is associated with upregulation of at least about 1.5-fold of at least one of the genes shown in
In some embodiments, the methods described herein further comprise a step or steps for identifying the cells for delivering the MBD peptide-linked agent into the cells. Such steps may include comparing levels of gene expression of one or more of the genes shown in
The agent linked to the MBD peptide may be a diagnostic agent or a therapeutic agent. In some embodiments, the agent is a protein or a peptide. In some embodiments, the agent is a nucleic acid. In some embodiments, the agent is a small molecule.
In some embodiments, the live cells are in a subject, such as a mammal. For example, the live cells are in a human. In some embodiments, the live cells are in a tissue or in cell culture.
Any MBD peptide described in U.S. Patent Application Nos. 2003/0059430, 2003/0161829, and 2003/0224990 (which are incorporated by reference in their entirety) may be used. In some embodiments, the MBD peptide comprises the amino acid sequence QCRPSKGRKRGFCW (SEQ ID NO: 2), QCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 3), or KKGFYKKKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 4).
The invention provides methods for identifying individuals who are candidates for treatment with MBD peptide-based therapies. MBD peptide-based therapies have been previously described in U.S. patent application publication nos. 2003/0059430, 2003/0161829, and 2003/0224990. However, the inventor has noted that there is variability in cellular internalization of MBD peptides. The invention provides methods for identifying which patients would be candidates for treatment with MBD peptide-based therapies, by predicting whether the relevant tissue(s) in the individual will take up MBD peptides.
In this invention I show that the physiological cellular state for which up-regulation of HSPs is emblematic is also the preferred state recognized by the MBD for cellular uptake and nuclear localization. MBD-mediated transport of appropriate macromolecules into cell nuclei at the sites of disease could allow for fine-tuned control of the disease process and for the design of very specific interventions. The possibility of delivery to sites of injury is also attractive. Liver injury leads to transcription of HSPs (Schiaffonati L and Tiberio L [1997] Liver. 17: 183-191) as does ischemia in isolated hearts (Nitta-Komatsubara Y et al [2000] 66:1261-1270). HSF1 is cardioprotective for ischemia/reperfusion injury (Zou Y et al [2003] Circulation 108: 3024-3030). This invention also provides for treatment of disorders characterized by secreted HSP70 and macrophage co-localized at the site of disease.
Privileged sites in the body also up-regulate HSPs constitutively, though most other cell types only induce HSPs as a specific response to stress. HSFs are required for spermatogenesis (Wang G et al [2004] Genesis 38: 66-80). Neuronal cells also display altered regulation of HSPs (Kaarniranta K et al [2002] Mol Brain Res 101:136-140). Longevity in C. elegans is regulated by HSF and chaperones (Morley J F and Morimoto R I. [2004] Mol Biol Cell 15:657-664). MBD-mediated transport of regulatory macromolecules to such sites offers opportunities for interventions in neuroprotection and reproductive biology.
It is interesting that Kupffer cells (macrophage-like) are the major site of synthesis of IGFBP-3 in the liver (Scharf J et al [1996] Hepatology 23: 818-827; Zimmermann E M et al [2000] Am J. Physiol. Gastro. Liver Phys. 278: G447-457). Exogenously administered radiolabelled IGFBP-3 selectively accumulates in rat liver Kupffer cells (Arany E et al [1996] Growth Regul 6:32-41). Our earlier work suggested that caveolin and transferrin receptor were implicated in MBD-mediated cellular uptake. Caveolin is expressed in macrophages (Kiss A L et al [2002] Micron. 33: 75-93). Macrophage caveolin-1 is up-regulated in response to apoptotic stressors (Gargalovic P and Dory L [2003] J Lipid Res 44: 1622-1632). Macrophages express transferrin receptor (Mulero V and Brock J H [1999] Blood 94:2383-2389).
We are interested in elucidating the physiological and biochemical correlates of cellular receptivity to IGFBP-3, uptake and intracellular localization. We have recently localized and characterized the minimal sequence determinants of cellular recognition, uptake and intracellular localization to a C-terminal metal-binding domain in the IGFBP-3 molecule. This domain, when to covalently linked to unrelated protein molecules such as GFP, can mediate specific cellular uptake and intracellular localization of such markers in selected cell systems. As a surrogate for the homing mechanism of IGFBP-3 itself, MBD-linked marker proteins can serve to elucidate patterns of cellular receptivity that might otherwise be difficult or impossible to discern against a background of endogenous IGFBP-3.
Heat shock proteins are molecular chaperones, involved in many cellular functions such as protein folding, transport, maturation and degradation. Since they control the quality of newly synthesized proteins, HSP take part in cellular homeostasis. The Hsp70 family in particular exerts these functions in an adenosine triphosphate (ATP)-dependent manner. ATP is the main energy source used by cells to assume fundamental functions (respiration, proliferation, differentiation, apoptosis). Therefore, ATP levels have to be adapted to the requirements of the cells and ATP generation must constantly compensate ATP consumption. Nevertheless, under particular stress conditions, ATP levels decrease, threatening cell homeostasis and integrity. Cells have developed adaptive and protective mechanisms, among which Hsp70 synthesis and over-expression is one.
Transferrin serves as the iron source for hemoglobin-synthesizing immature red blood cells. A cell surface receptor, transferrin receptor 1, is required for iron delivery from transferrin to cells. Transferrin receptor 1 has been established as a gatekeeper for regulating iron uptake by most cells. Iron uptake is viewed as an indicator of cellular oxidative metabolism and ATP-dependent metabolic rates.
In this study, we have dissected the molecular signatures of cells that selectively take up MBD-tagged markers.
By gene array and cellular protein analysis we have demonstrated that MBD-mediated protein uptake is linked to target cell physiological states resembling cellular responses to stress or injury. Thermal stress dramatically up-regulates uptake of MBD-tagged proteins. In vivo, inflammatory stress in an adjuvant arthritis rat model did not change the biodistribution of systemically administered MBD-tagged proteins. We are currently evaluating other in vivo and in vitro models of cellular stress.
Therapeutic peptides incorporating the MBD motif can be created by making fusions of peptide sequences known to have appropriate intracellular biological activities with either the N- or C-terminus of the core MBD sequence. Based on prior studies, peptide sequences can be selected to target up-regulated stress proteins (such as hsp70) in cancer, as well as MDM2 interactions with P53, inflammation (NF-kappa-B, NEMO, CSK), and previously characterized cancer-specific targets such as survivin and bcl-2.
Metastasis is the primary cause of cancer-related mortality in the world. Our goal is to address this unmet need by enhancing existing chemotherapeutic cocktails with the addition of synergistic biological modifiers. We show that intracardiac injection of CCRF-CEM (T-cell leukemia), MDA-MB-435 or MDA-MB-231 (breast cancer) cells into Rag-2 mice establishes disseminated disease within a few days. The 22-amino acid MBD transporter, derived from IGFBP-3, targets malignant cancer cells via cell surface transferrin receptors and beta integrins. In vitro data show that MBD-linked peptides can inhibit stress-coping and anti-apoptotic mechanisms, commonly up-regulated in cancer (e.g. NF-kappa-B, Hsp-70, MDM2, survivin). The discriminant validity of these peptides as potential therapeutic agents was investigated by comparing their cytotoxicity to cancer cell lines versus normal human cell counterparts. In cell culture, synergies between these peptides as well as in combination with dietary supplements (lycopene and curcumin) and paclitaxel or 5-FU have been shown. 25-day intravenous administration of a 3-peptide cocktail (3 mg/kg) in combination with dietary lycopene and curcumin in Rag-2 mice with established CCRF-CEM leukemia significantly reduces splenomegaly from human cell burden, and improves survival. Similarly, 25-day administration of a 3-peptide cocktail and dietary supplement optimized for breast cancer reduces MDA-MB-231 human cell burden in bone marrow. Our data suggest that MBD-tagged peptides can be used to treat hematological and disseminated malignancies.
The human cancer and corresponding normal cell lines to be used in testing can be obtained from the American Type Culture Collection (ATCC). They are well characterized and have been extensively used in vitro and in vivo. Breast cancer cell lines (MCF7, MDA-MB-231, MX-1), leukemia cell lines (RPMI-8226, CCRF-CEM, MOLT-4), and prostate cancer cell lines (PC3, DU145, LNCAPs) were cultured in RPMI-1640 media supplemented with 10% FBS. Paired breast cancer and non-cancer cell lines (CRL7364/CRL7365, CRL7481/CRL7482, HTB-125/Hs578T) were cultured in DMEM media supplemented with 10% FBS. Normal cell lines such as MCF-10A, HMEC human T-cells were cultured in medias specified by the manufacturer.
Animal models of metastatic disease are described in this invention. Successful engraftment of both human hematopoietic and non-hematopoietic xenografts requires the use of severe combined immunodeficient (SCID) mice as neither bone marrow involvement nor disseminated growth are regularly observed using thymectomized, irradiated or nude mice. The mice used to establish a human-mouse xenograft model were purchased from Taconic. Mice were bred by crossing C57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient mice. The gc KO is a deletion of the X-chromosome linked gc gene resulting in a loss of NK cells, a loss of the common g receptor unit shared by an array of cytokines that include IL-2, IL-4, IL-7, IL-9, and IL-15, and as a result only a residual number of T and B cells are produced. To eliminate this residual number of T and B cells, the gc mouse KO mouse was crossed with a C57BL/10SgSnAi recombinase activating-2 (Rag-2) deficient mouse (a loss of the Rag-2 gene results in an inability to initiate V(D)J lymphocyte receptor rearrangements, and mice will lack mature lymphocytes). CCRF-CEM, MDA-MB-231 or MDA-MB-435 xenograft-bearing Rag-2 mice (10 mice per group, 3 groups, approx. 5×105 to 1×107 cancer cells injected per animal per group) are established through intra-cardiac injection. MBD-tagged peptide cocktails (“enhancers”) and paclitaxel combinations are intraperitonially (IP) injected into the animals. The groups are divided as follows: saline (group 1), peptide (group 2), and peptide/paclitaxel combination (group 3). Treatment is started on Day 4 with a one-time IP dosage of paclitaxel (group 3). On Day 6, the paclitaxel dose (0.5 mg/kg) is followed by peptide treatment for 7 days (groups 2 and 3). On a daily basis, each mouse receives IP injection of MBD peptide cocktails (in one embodiment, 3 peptide sequences are combined in one cocktail, each peptide administered at a dose of 0.1-5.0 mg/kg). Blood sampling and PCR analysis are carried out at weekly intervals. Approximately 100 ul blood is collected from the saphenous vein. PCR analysis is used on peripheral blood (PB) on Days 3-7 post-injection to determine whether animals have successfully established leukemia/cancer. Cancer cell count levels are monitored during and after treatment as well as at termination. PCR analysis on PB, bone marrow, spleen, liver and lung is used to quantify the cancer cells. At Day 3, prior to treatment, high levels of cancer cells may be seen in PB in the case of leukemia models and low levels of human cancer cells in peripheral organs. Blood and peripheral organs are collected at termination and stored for further analysis (Day 18-45, depending on the experiment). If dietary compounds such as curcumin or lycopene are to be used in the experiment they may be included in the animal diet or force-fed daily or at other specified intervals. It has been shown that blood levels exceeding 20 nM can be achieved for these compounds when fed orally. Dietary supplements curcumin and lycopene were purchased from Sigma. Chemotherapeutics paclitaxel and 5-fluorouracil (5-FU) can be purchased from Sigma. Biphosphonates (Alendronate, Clodronate) have been obtained from EMD Biosciences. At termination of each animal experiment blood and organs are collected and stored at −80° C. To isolate genomic DNA (gDNA) from blood samples the blood & cell culture DNA kit (purchased from Qiagen Inc., Carlsbad, Calif.) can be used to isolate gDNA from tissue samples. gDNA concentrations are established based on spectrophotometer OD260 readings. To determine human genomic DNA human-specific primers 5′-TAGCAATAATCCCCATCCTCCATATAT-3′ (SEQ ID NO: 5) and 5′-ACTTGTCCAATGATGGTAAAAGG-3′ (SEQ ID NO: 6), which amplify a 157-bp portion of the human mitochondrial cytochrome b region can be used with 100-500 ng input genomic DNA per PCR reaction, depending on type of tissue. Good results can be achieved using the KOD hot start PCR kit (Novagen, Inc., Madison, Wis.). PCR is performed in a thermal cycler (Perkin Elmer) for 25 or 32 cycles of 30 s at 96° C., 40 s at 59° C., and 1 min at 72° C. The program can be optimized for genomic DNA isolated from mouse tissue.
FIG. 27A—Left graph. Successful establishment of a leukemia model: Intracardial HL-60 cell injection into Rag-2 mice. Small but significant human cell-counts observed by day 23 post-inoculation. A 3% increase of human cells in PB was observed by FACS analysis and confirmed by anti-human HLA MAb staining. No increase of human cells was detected in BM or SP. At day 27 post HL-60 inoculation there were minimal levels of human cells in BM and SP, but an average increase of leukemia cells of about 60% compared to BM, SP or non-injected Rag-2 mice. Intracardial injection into Rag-2 mice with human leukemia cell lines (CCRF-CEM, MOLT-4, RPMI-8226) led to the establishment of an in vivo leukemia model appropriate for testing MBD-peptide cocktails.
FIG. 27A—Right graph. CCRF-CEM injection induces severe splenomegaly and death in Rag-2 mice at 21 days post injection. Three human leukemia lines induced splenomegaly in Rag-2 mice in proportion to cellular growth rates. CCRF-CEM is the fastest growing line and induces severe splenomegaly within three weeks.
The invention provides methods for identifying candidates for treatment with MBD peptide-based therapies.
Candidates for treatment with MBD peptide-based therapies are individuals (a) for whom MBD peptide-based therapy has been proposed (such as individuals who have been diagnosed with a disorder treatable with an MBD peptide-based therapy) and whose relevant tissue is predicted to have relatively high uptake of MBD peptide(s).
MBD peptide based therapy has been previously disclosed for a number of different indications, including cancer (such as breast, prostate, colon, ovarian, pancreatic, gastric and lung cancer), autoimmune disease, cardiovascular indications, arthritis, asthma, allergy, reproductive indications, retinal proliferative disease, bone disease, inflammatory disease, inflammatory bowel disease, and fibrotic disease. MBD peptides and therapies based thereon are further described in U.S. patent application publication nos. 2003/0059430, 2003/0161829, and 2003/0224990.
The inventor has discovered a number of different genes which are differentially regulated between cells that have low uptake of MBD peptides and those that have high uptake of MBD peptides. These genes, referred to herein as “MBD uptake indicator genes”, include GDF15, SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5, CEBPB, IL6R, ABCP2, CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAIL, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, and IL7R. Of these genes, GDF15, SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2 are up-regulated in cells which have high uptake of MBD peptides. It should be noted that at least one third of these up-regulated genes have been previously associated with cellular responses to stress (e.g. GDF15, ATF3, HSPF3, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1). Down-regulated genes include CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAIL, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, and IL7R. The inventor further notes that specific formulae for identifying candidates for MBD peptide therapy may be developed using the data and techniques described herein.
Accordingly, the invention provides methods of identifying candidates for MBD peptide-based therapy by obtaining a measured level for at least one MBD uptake indicator gene in a tissue sample from an individual and comparing that measured level with a reference level. For up-regulated genes, a comparison that indicates that the measured level is higher than the reference level identifies a candidate for MBD peptide-based therapy. Likewise, a comparison that indicates that the measured level is lower than a reference level for a down-regulated MBD uptake indicator gene is lower than the reference level identifies a candidate for MBD peptide-based therapy.
Levels of the particular genes which are differentially regulated may be measured using any technology known in the art. Generally, mRNA is extracted from a sample of the relevant tissue (e.g., where the individual has been diagnosed with cancer, a biopsy sample of the tumor will generally be the sample tested). Direct quantitation methods (methods which measure the level of transcripts from a particular gene without conversion of the RNA into DNA or any amplification) may be used, but it is believed that measurement will be more commonly performed using technology which utilizes an amplification step (thereby reducing the minimum size sample necessary for testing).
Amplification methods generally involve a preliminary step of conversion of the mRNA into cDNA by extension of a primer (commonly one including an oligo-dT portion) hybridized to the mRNA in the sample with a RNA-dependent DNA polymerase. Additionally, a second cDNA strand (complementary to the first synthesized strand) may be synthesized when desired or necessary. Second strand cDNA is normally synthesized by extension of a primer hybridized to the first cDNA strand using a DNA-dependent DNA polymerase. The primer for second strand synthesis may be a primer that is added to the reaction (such as random hexamers) or may be ‘endogenous’ to the reaction (i.e., provided by the original RNA template, such as by cleavage with an enzyme or agent that cleaves RNA in a RNA/DNA hybrid, such as RNase H).
Amplification may be carried out separately from quantitation (e.g., amplification by single primer isothermal amplification, followed by quantitation of the amplification product by probe hybridization), or may be part of the quantitation process, such as in real time PCR.
Measured levels may be obtained by the practitioner of the instant invention, or may be obtained by a third party (e.g., a clinical testing laboratory) who supplies the measured value(s) to the practitioner.
Reference levels are generally obtained from “normal” tissues. Normal tissues are those which are not afflicted with the particular disease or disorder which is the subject of the MBD peptide-based therapy. For example, when the disease to be treated with MBD peptide-based therapy is ductal breast carcinoma, the reference value is normally obtained from normal breast duct tissue. Likewise, for cardiovascular disorders, the “normal” tissue might be normal arterial wall tissue (e.g., when the disorder is atherosclerosis). Alternately, values from cells (which may be tissue culture cells or cell lines) which have low MBD peptide uptake may also be used to derive a reference value.
The process of comparing a measured value and a reference value can be carried out in any convenient manner appropriate to the type of measured value and reference value for the MBD uptake indicator gene at issue. It should be noted that the measured values obtained for the MBD uptake indicator gene(s) can be quantitative or qualitative measurement techniques, thus the mode of comparing a measured value and a reference value can vary depending on the measurement technology employed. For example, when a qualitative colorimetric assay is used to measure MBD uptake indicator gene levels, the levels may be compared by visually comparing the intensity of the colored reaction product, or by comparing data from densitometric or spectrometric measurements of the colored reaction product (e.g., comparing numerical data or graphical data, such as bar charts, derived from the measuring device). Quantitative values (e.g., transcripts/cell or transcripts/unit of RNA, or even arbitrary units) may also be used. As with qualitative measurements, the comparison can be made by inspecting the numerical data, by inspecting representations of the data (e.g., inspecting graphical representations such as bar or line graphs).
As will be understood by those of skill in the art, the mode of detection of the signal will depend on the exact detection system utilized in the assay. For example, if a radiolabeled detection reagent is utilized, the signal will be measured using a technology capable of quantitating the signal from the biological sample or of comparing the signal from the biological sample with the signal from a reference sample, such as scintillation counting, autoradiography (typically combined with scanning densitometry), and the like. If a chemiluminescent detection system is used, then the signal will typically be detected using a luminometer. Methods for detecting signal from detection systems are well known in the art and need not be further described here.
When more than one MBD uptake indicator gene is measured (i.e., measured values for two or more MBD uptake indicator genes are obtained), the sample may be divided into a number of aliquots, with separate aliquots used to measure different MBD uptake indicator gene (although division of the biological sample into multiple aliquots to allow multiple determinations of the levels of the MBD uptake indicator gene(s) in a particular sample are also contemplated). Alternately the sample (or an aliquot therefrom) may be tested to determine the levels of multiple MBD uptake indicator genes in a single reaction using an assay capable of measuring the individual levels of different MBD uptake indicator genes in a single assay, such as an array-type assay or assay utilizing multiplexed detection technology (e.g., an assay utilizing detection reagents labeled with different fluorescent dye markers).
As will be understood by those in the art, the exact identity of a reference value will depend on the tissue that is the target of treatment and the particular measuring technology used. In some embodiments, the comparison determines whether the measured value for the MBD uptake indicator gene is above or below the reference value. In some embodiments, the comparison is performed by finding the “fold difference” between the reference value and the measured value (i.e., dividing the measured value by the reference value). Table 1 lists certain exemplary fold differences for use in the instant invention.
Candidates suitable for treatment with MBD peptide-based therapies are identified when at least a simple majority of the comparisons between the measured values and the reference values indicate that the cells in the sample (and thus the diseased cells in the individual) have relatively high uptake of MBD peptides. For up-regulated MBD uptake indicator genes (GDF15, SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2), a measured value that is greater than the reference value (which may be a simple “above or below” comparison or a comparison to find a minimum fold difference) indicates that the cells in the sample have relatively high uptake of MBD peptides. For down-regulated MBD uptake indicator genes (CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAIL, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, and IL7R), a measured value that is less than the reference value (which may be a simple “above or below” comparison or a comparison to find a minimum fold difference) indicates that the cells in the sample have relatively high uptake of MBD peptides.
Additionally, because certain of the MBD uptake indicator genes are found in serum (e.g. HSP70, GFP15), the invention also provides methods of identifying candidates for MBD peptide-based therapy by obtaining a measured level for at least one MBD uptake indicator gene in a biological fluid sample from an individual and comparing that measured level with a reference level. For up-regulated genes, a comparison that indicates that the measured level is higher than the reference level identifies a candidate for MBD peptide-based therapy. Likewise, a comparison that indicates that the measured level is lower than a reference level for a down-regulated MBD uptake indicator gene is lower than the reference level identifies a candidate for MBD peptide-based therapy.
A measured level is obtained for the relevant tissue for at least one MBD uptake indicator protein (i.e., the protein encoded by an MBD uptake marker gene), although multiple MBP uptake indicator proteins may be measured in the practice of the invention. Generally, it is preferred that measured levels are obtained for more than one MBD uptake indicator protein. Accordingly, the invention may be practiced using at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten MBD uptake indicator proteins. In certain embodiments, at least one of the measured values is obtained for a MBD uptake indicator protein that is up-regulated in cells which have high MBD peptide uptake levels and at least one of the measured values is obtained for a MBD uptake indicator protein that is down-regulated in cells which have high MBD peptide uptake levels. As will be apparent to those of skill in the art, the MBD uptake indicator proteins for which measured values are obtained are most commonly MBD uptake indicator proteins which may be secreted (e.g., HSP70, GDF15).
The MBD uptake indicator protein(s) may be measured using any available measurement technology that is capable of specifically determining the level of the MBD uptake indicator protein in a biological sample. In certain embodiments, the measurement may be either quantitative or qualitative, so long as the measurement is capable of indicating whether the level of the MBD uptake indicator protein in the biological sample is above or below the reference value.
Although some assay formats will allow testing of biological samples without prior processing of the sample, it is expected that most biological samples will be processed prior to testing. Processing generally takes the form of elimination of cells (nucleated and non-nucleated), such as erythrocytes, leukocytes, and platelets in blood samples, and may also include the elimination of certain proteins, such as certain clotting cascade proteins from blood.
Commonly, MBD uptake indicator protein levels will be measured using an affinity-based measurement technology. Affinity-based measurement technology utilizes a molecule that specifically binds to the MBD uptake indicator protein being measured (an “affinity reagent,” such as an antibody or aptamer), although other technologies, such as spectroscopy-based technologies (e.g., matrix-assisted laser desorption ionization-time of flight, or MALDI-TOF, spectroscopy) or assays measuring bioactivity (e.g., assays measuring mitogenicity of growth factors) may be used.
Affinity-based technologies include antibody-based assays (immunoassays) and assays utilizing aptamers (nucleic acid molecules which specifically bind to other molecules), such as ELONA. Additionally, assays utilizing both antibodies and aptamers are also contemplated (e.g., a sandwich format assay utilizing an antibody for capture and an aptamer for detection).
If immunoassay technology is employed, any immunoassay technology which can quantitatively or qualitatively measure the level of a MBD uptake indicator protein in a biological sample may be used. Suitable immunoassay technology includes radioimmunoassay, immunofluorescent assay, enzyme immunoassay, chemiluminescent assay, ELISA, immuno-PCR, and western blot assay.
Likewise, aptamer-based assays which can quantitatively or qualitatively measure the level of a MBD uptake indicator protein in a biological sample may be used in the methods of the invention. Generally, aptamers may be substituted for antibodies in nearly all formats of immunoassay, although aptamers allow additional assay formats (such as amplification of bound aptamers using nucleic acid amplification technology such as PCR (U.S. Pat. No. 4,683,202) or isothermal amplification with composite primers (U.S. Pat. Nos. 6,251,639 and 6,692,918).
A wide variety of affinity-based assays are known in the art. Affinity-based assays will utilize at least one epitope derived from the MBD uptake indicator protein of interest, and many affinity-based assay formats utilize more than one epitope (e.g., two or more epitopes are involved in “sandwich” format assays; at least one epitope is used to capture the marker, and at least one different epitope is used to detect the marker).
Affinity-based assays may be in competition or direct reaction formats, utilize sandwich-type formats, and may further be heterogeneous (e.g., utilize solid supports) or homogenous (e.g., take place in a single phase) and/or utilize or immunoprecipitation. Most assays involve the use of labeled affinity reagent (e.g., antibody, polypeptide, or aptamer); the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA and ELONA assays.
In a heterogeneous format, the assay utilizes two phases (typically aqueous liquid and solid). Typically a MBD uptake indicator protein-specific affinity reagent is bound to a solid support to facilitate separation of the MBD uptake indicator protein from the bulk of the biological sample. After reaction for a time sufficient to allow for formation of affinity reagent/MBD uptake indicator protein complexes, the solid support containing the antibody is typically washed prior to detection of bound polypeptides. The affinity reagent in the assay for measurement of MBD uptake indicator proteins may be provided on a support (e.g., solid or semi-solid); alternatively, the polypeptides in the sample can be immobilized on a support. Examples of supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates), polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, and Protein A beads. Both standard and competitive formats for these assays are known in the art.
Array-type heterogeneous assays are suitable for measuring levels of MBD uptake indicator proteins when the methods of the invention are practiced utilizing multiple MBD uptake indicator proteins. Array-type assays used in the practice of the methods of the invention will commonly utilize a solid substrate with two or more capture reagents specific for different MBD uptake indicator proteins bound to the substrate a predetermined pattern (e.g., a grid). The biological sample is applied to the substrate and MBD uptake indicator proteins in the sample are bound by the capture reagents. After removal of the sample (and appropriate washing), the bound MBD uptake indicator proteins are detected using a mixture of appropriate detection reagents that specifically bind the various MBD uptake indicator proteins. Binding of the detection reagent is commonly accomplished using a visual system, such as a fluorescent dye-based system. Because the capture reagents are arranged on the substrate in a predetermined pattern, array-type assays provide the advantage of detection of multiple MBD uptake indicator proteins without the need for a multiplexed detection system.
In a homogeneous format the assay takes place in single phase (e.g., aqueous liquid phase). Typically, the biological sample is incubated with an affinity reagent specific for the MBD uptake indicator protein in solution. For example, it may be under conditions that will precipitate any affinity reagent/antibody complexes which are formed. Both standard and competitive formats for these assays are known in the art.
In a standard (direct reaction) format, the level of MBD uptake indicator protein/affinity reagent complex is directly monitored. This may be accomplished by, for example, determining the amount of a labeled detection reagent that forms is bound to MBD uptake indicator protein/affinity reagent complexes. In a competitive format, the amount of MBD uptake indicator protein in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled MBD uptake indicator protein (or other competing ligand) in the complex. Amounts of binding or complex formation can be determined either qualitatively or quantitatively.
Complexes formed comprising MBD uptake indicator protein and an affinity reagent are detected by any of a number of known techniques known in the art, depending on the format of the assay and the preference of the user. For example, unlabelled affinity reagents may be detected with DNA amplification technology (e.g., for aptamers and DNA-labeled antibodies) or labeled “secondary” antibodies which bind the affinity reagent. Alternately, the affinity reagent may be labeled, and the amount of complex may be determined directly (as for dye- (fluorescent or visible), bead-, or enzyme-labeled affinity reagent) or indirectly (as for affinity reagents “tagged” with biotin, expression tags, and the like).
As will be understood by those of skill in the art, the mode of detection of the signal will depend on the exact detection system utilized in the assay. For example, if a radiolabeled detection reagent is utilized, the signal will be measured using a technology capable of quantitating the signal from the biological sample or of comparing the signal from the biological sample with the signal from a reference sample, such as scintillation counting, autoradiography (typically combined with scanning densitometry), and the like. If a chemiluminescent detection system is used, then the signal will typically be detected using a luminometer. Methods for detecting signal from detection systems are well known in the art and need not be further described here.
When more than one MBD uptake indicator protein is measured, the biological sample may be divided into a number of aliquots, with separate aliquots used to measure different MBD uptake indicator proteins (although division of the biological sample into multiple aliquots to allow multiple determinations of the levels of the MBD uptake indicator protein in a particular sample are also contemplated). Alternately the biological sample (or an aliquot therefrom) may be tested to determine the levels of multiple MBD uptake indicator proteins in a single reaction using an assay capable of measuring the individual levels of different MBD uptake indicator proteins in a single assay, such as an array-type assay or assay utilizing multiplexed detection technology (e.g., an assay utilizing detection reagents labeled with different fluorescent dye markers).
It is common in the art to perform ‘replicate’ measurements when measuring MBD uptake indicator proteins. Replicate measurements are ordinarily obtained by splitting a sample into multiple aliquots, and separately measuring the MBD uptake indicator protein (s) in separate reactions of the same assay system. Replicate measurements are not necessary to the methods of the invention, but many embodiments of the invention will utilize replicate testing, particularly duplicate and triplicate testing.
Kits for Identification of Candidates for MBD Peptide TherapyThe invention provides kits for carrying out the methods of the invention. Kits of the invention comprise at least one probe specific for a MBD uptake indicator gene (and/or at least one affinity reagent specific for a MBD uptake indicator protein) and instructions for carrying out a method of the invention. More commonly, kits of the invention comprise at least two different MBD uptake indicator gene probes (or at least two affinity reagents specific for MBD uptake indicator proteins), where each probe/reagent is specific for a different MBD uptake indicator gene.
Kits comprising a single probe for a MBD uptake indicator gene (or affinity reagent specific for a MBD uptake indicator protein) will generally have the probe/reagent enclosed in a container (e.g., a vial, ampoule, or other suitable storage container), although kits including the probe/reagent bound to a substrate (e.g., an inner surface of an assay reaction vessel) are also contemplated. Likewise, kits including more than one probe/reagent may also have the probes/reagents in containers (separately or in a mixture) or may have the probes/affinity reagents bound to a substrate (e.g., such as an array or microarray).
A modified substrate or other system for capture of MBD uptake indicator gene transcripts or MBD uptake indicator proteins may also be included in the kits of the invention, particularly when the kit is designed for use in an array format assay.
In certain embodiments, kits according to the invention include the probes/reagents in the form of an array. The array includes at least two different probes/reagents specific for a MBD uptake indicator gene/protein (each probe/reagent specific for a different MBD uptake indicator gene/protein) bound to a substrate in a predetermined pattern (e.g., a grid). The localization of the different probes/reagents allows measurement of levels of a number of different MBD uptake indicator genes/.proteins in the same reaction.
The instructions relating to the use of the kit for carrying out the invention generally describe how the contents of the kit are used to carry out the methods of the invention. Instructions may include information as sample requirements (e.g., form, pre-assay processing, and size), steps necessary to measure the MBD uptake indicator gene(s), and interpretation of results.
Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. In certain embodiments, machine-readable instructions comprise software for a programmable digital computer for comparing the measured values obtained using the reagents included in the kit.
Therapeutic MethodsThe therapeutic methods of the invention utilize treatment of certain disorders (e.g., disorders characterized by secreted HSP70 and macrophage co-localized at the site of disease) with MBD peptide therapies. The invention provides methods of treating diseases characterized by measurable cellular stress responses (such as the induction of heat shock proteins) including, but not limited to, metabolic and oxidative stress, with MBD peptide therapies. MBD peptide therapies include treatment by administration of (a) MBD peptides, (b) MBD peptide fusions, and (c) MBD peptide conjugates.
The invention provides methods for delivering an MBD peptide-linked agent into live cells, said method comprising contacting said MBD peptide-linked agent to live cells that are under a condition of cellular stress, whereby said contact results in cellular uptake of said MBD-peptide-linked agent.
The condition of cellular stress can be any type of stress, such as thermal, immunological, cytokine, oxidative, metabolic, anoxic, endoplasmic reticulum, protein unfolding, nutritional, chemical, mechanical, osmotic and glycemic stress. In some embodiments, the condition of cellular stress is associated with upregulation of at least one, at least two, at least three, at least four, at least five, at least ten, at least fifteen, at least twenty, or all of the genes shown in
“Metal-binding domain peptide” or “MBD peptide” means an IGFBP-derived peptide or polypeptide from about 12 to about 60 amino acids long, preferably from about 13 to 40 amino acids long, comprising a segment of the CD-74-homology domain sequence in the carboxy-terminal 60-amino acids of IGFBP-3, comprising the sequence CRPSKGRKRGFC (SEQ ID NO: 7) and exhibiting metal-binding properties, but differing from intact IGFBP-3 by exhibiting distinct antigenic properties, lacking IGF-I-binding properties, and lacking the mid-region sequences (amino acids 88-148 of IGFBP-3 sequence). For example, the peptide GFYKKKQCRPSKGRKRGFCW (SEQ ID NO: 8) is an example of a metal-binding domain peptide. It binds metal ions but not IGF-I, and polyclonal antibodies raised to this peptide do not substantially cross-react with intact IGFBP-3, and vice versa. In certain embodiments, the MBD peptide includes a caveolin consensus binding sequence (#x#xxxx#, where ‘#’ is an aromatic amino acid) in addition to, or overlapping with, the MBD peptide sequence. The caveolin consensus sequence may be at the amino terminal or carboxy terminal end of the peptide. In certain preferred embodiments, the caveolin consensus binding sequence is at the carboxy terminal end of the peptide, and overlaps with the MBD core 14-mer sequence. Exemplary MBD peptides with caveolin consensus binding sequences include peptides comprising the sequence QCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 3) or KKGFYKKKQCRPSKGRKRGFCWAVDKYG (SEQ ID NO: 4). Metal-binding peptides comprising humanin sequences include SDKPDMAPRGFSCLLLLTSEIDLP (SEQ ID NO: 216), SDKPDMAPRGFSCLLLLTGEIDLP (SEQ ID NO: 217), SDKPDMAPRGFSCLLLLTSEIDLPVKRRA (SEQ ID NO: 193) and SDKPDMAPRGFSCLLLLTGEIDLPVKRRA (SEQ ID NO: 192). These peptides also include the N-terminal tetrapeptide of thymosin-beta-4.
MBD peptides may be modified, such as by making conservative substitutions for the natural amino acid residue at any position in the sequence, altering phosphorylation, acetylation, glycosylation or other chemical status found to occur at the corresponding sequence position of IGFBP-3 in the natural context, substituting D- for L-amino acids in the sequence, or modifying the chain backbone chemistry, such as protein-nucleic-acid (PNA).
“Conjugates” of an MBD peptide and a second molecule include both covalent and noncovalent conjugates between a MBD peptide and a second molecule (such as a transcriptional modulator or a therapeutic molecule). Noncovalent conjugates may be created by using a binding pair, such as biotin and avidin or streptavidin or an antibody (including Fab fragments, scFv, and other antibody fragments/modifications) and its cognate antigen.
Sequence “identity” and “homology”, as referred to herein, can be determined using BLAST (Altschul, et al., 1990, J. Mol. Biol. 215(3):403-410), particularly BLASTP 2 as implemented by the National Center for Biotechnology Information (NCBI), using default parameters (e.g., Matrix 0 BLOSUM62, gap open and extension penalties of 11 and 1, respectively, gap x_dropoff 50 and wordsize 3). Unless referred to as “consecutive” amino acids, a sequence optionally can contain a reasonable number of gaps or insertions that improve alignment.
An effective amount of the MBD therapy is administered to a subject having the disease. In some embodiments, the MBD therapy is administered at about 0.001 to about 40 milligrams per kilogram total body weight per day (mg/kg/day). In some embodiments the MBD therapy is administered at about 0.001 to about 40 mg/kg/day of MBD peptide (i.e., the MBD peptide portion of the therapy administered is about 0.001 to about 40 mg/kg/day).
The terms “subject” and “individual”, as used herein, refer to a vertebrate individual, including avian and mammalian individuals, and more particularly to sport animals (e.g., dogs, cats, and the like), agricultural animals (e.g., cows, horses, sheep, and the like), and primates (e.g., humans).
The term “treatment” is used herein as equivalent to the term “alleviating”, which, as used herein, refers to an improvement, lessening, stabilization, or diminution of a symptom of a disease. “Alleviating” also includes slowing or halting progression of a symptom.
For the purposes of this invention, a “clinically useful outcome” refers to a therapeutic or diagnostic outcome that leads to amelioration of the disease condition. “Inflammatory disease condition” means a disease condition that is typically accompanied by chronic elevation of transcriptionally active NF-kappa-B or other known intermediates of the cellular inflammatory response in diseased cells. The following intracellular molecular targets are suggested as examples:
“NF-kappa-B regulator domain” includes a binding domain that participates in transport of NF-kappa-B into the nucleus [Strnad J, et al. J Mol Recognit. 19(3):227-33, 2006; Takada Y, Singh S, Aggarwal B B. J Biol Chem. 279(15): 15096-104, 2004) and domains that participate in upstream signal transduction events to this transport. “P53 regulator domain” is the P53/MDM2 binding pocket for the regulatory protein MDM2 (Michl J, et al, Int J. Cancer. 119(7): 1577-85, 2006). “IGF-signalling regulator domain” refers to the SH domain of Dok-1 which participates critically in IGF receptor signal transduction (Clemmons D and Maile L. Mol Endocrinol. 19(1): 1-11, 2005). “RAS active site domain” refers to the catalytic domain of the cellular Ras enzyme. “MYC regulator domain” refers to the amino-terminal regulatory region of c-myc or to its DNA-binding domain, both of which have been well-characterized (Luscher B and Larson L G. Oncogene. 18(19):2955-66, 1999). “HSP regulator domain” includes trimerization inhibitors of HSF-1 (Tai L J et al. J Biol Chem. 277(1):735-45, 2002). “Survivin dimerization domain” refers to well-characterized sequences at the dimer interface of Survivin (Sun C, et al. Biochemistry. 44(1): 11-7, 2005). “Proteasome subunit regulator domain” refers to the target for hepatitis B virus-derived proteasome inhibitor which competes with PA28 for binding to the proteasome alpha4/MC6 subunit (Stohwasser R, et al. Biol Chem. 384(1): 39-49, 2003). “HIF1-alpha oxygen-dependent regulator domain” refers to the oxygen-dependent degradation domain within the HIF-1 protein (Lee J W, et al. Exp Mol Med. 36(1): 1-12, 2004). “Smad2” is mothers against decapentaplegic homolog 2 (Drosophila) (Konasakim K. et al. J. Am. Soc. Nephrol. 14:863-872, 2003; Omata, M. et al. J. Am. Soc. Nephrol. 17:674-685, 2006). “Smad3” is mothers against decapentaplegic homolog 3 (Drosophila) (Roberts, A B et al Cytokine Growth Factor Rev. 17:19-27, 2006). “Src family kinases” refers to a group of proto-oncogenic tyrosine kinases related to a tyrosine kinase originally identified in Rous sarcoma virus (Schenone, S et al. Mini Rev Med Chem 7:191-201, 2007).
As used herein, “in conjunction with”, “concurrent”, or “concurrently”, as used interchangeably herein, refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during or after delivery of the other treatment modality to the subject.
The MBD peptide is normally produced by recombinant methods, which allow the production of all possible variants in peptide sequence. Techniques for the manipulation of recombinant DNA are well known in the art, as are techniques for recombinant production of proteins (see, for example, in Sambrook et al., M
Preferably, the MBD peptide is produced using a bacterial cell strain as the recombinant host cell. An expression construct (i.e., a DNA sequence comprising a sequence encoding the desired MBD peptide operably linked to the necessary DNA sequences for proper expression in the host cell, such as a promoter and/or enhancer elements at the 5′ end of the construct and terminator elements in the 3′ end of the construct) is introduced into the host cell. The DNA sequence encoding the MBD peptide may optionally linked to a sequence coding another protein (a “fusion partner”), to form a fusion protein. Preferably, the DNA sequence encoding the MBD peptide is linked to a sequence encoding a fusion partner as described in U.S. Pat. No. 5,914,254. The expression construct may be an extrachromosomal construct, such as a plasmid or cosmid, or it may be integrated into the chromosome of the host cell, for example as described in U.S. Pat. No. 5,861,273.
Accordingly, the invention provides methods of treatment with fusions and/or conjugates of MBD peptides with molecules (such as agents) which are desired to be internalized into cells. The fusion partner molecules may be polypeptides, nucleic acids, or small molecules which are not normally internalized (e.g., because of large size, hydrophilicity, etc.). The fusion partner can also be an antibody or a fragment of an antibody. As will be apparent to one of skill in the art, such fusions/conjugates will be useful in a number of different areas, including pharmaceuticals (to promote internalization of therapeutic molecules which do not normally become internalized), gene therapy (to promote internalization of gene therapy constructs), and research (allowing ‘marking’ of cells with an internalized marker protein). Preferred MBD peptides are peptides comprising the sequence KKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO:9) or a sequence having at least 80, 85, 90, 95, 98, or 99% homology to said sequence. Fusions of MBD peptides and polypeptides are preferably made by creation of a DNA construct encoding the fusion protein, but such fusions may also be made by chemical ligation of the MBD peptide and the polypeptide of interest. Conjugates of MBD peptides and nucleic acids or small molecules can be made using chemical crosslinking technology known in the art. Preferably, the conjugate is produced using a heterobifunctional crosslinker to avoid production of multimers of the MBD peptide.
Therapy in accordance with the invention may utilize MBD peptides and transcriptional modulators (e.g., transcription factors). For example, T-bet (Szabo et al., 2000, Cell 100(6):655-69), a transcription factor that appears to commit T lymphocytes to the Th1 lineage, can be fused to a MBD peptide to create a molecule a useful therapeutic. Likewise, therapy in accordance with the invention using conjugates of MBD peptides and therapeutic molecules is also provided. MBD peptides may be conjugated with any therapeutic molecule which is desired to be delivered to the interior of a cell, including antisense oligonucleotides and polynucleotide constructs (e.g., encoding therapeutic molecules such as growth factors and the like).
Peptides comprising an MBD peptide which includes a caveolin consensus binding sequence (MBD/caveolin peptides) may also be incorporated into conjugates. MBD/caveolin peptides may be conjugated with any therapeutic molecule that is desired to be delivered to the interior of a cell, including antisense oligonucleotides and polynucleotide constructs (e.g., encoding therapeutic molecules such as growth factors and the like).
Molecules comprising an MBD peptide are preferably administered via oral or parenteral administration, including but not limited to intravenous (IV), intra-arterial (IA), intraperitoneal (IP), intramuscular (IM), intracardial, subcutaneous (SC), intrathoracic, intraspinal, intradermal (ID), transdermal, oral, sublingual, inhaled, and intranasal routes. IV, IP, IM, and ID administration may be by bolus or infusion administration. For SC administration, administration may be by bolus, infusion, or by implantable device, such as an implantable minipump (e.g., osmotic or mechanical minipump) or slow release implant. The MBD peptide may also be delivered in a slow release formulation adapted for IV, IP, IM, ID or SC administration. Inhaled MBD peptide is preferably delivered in discrete doses (e.g., via a metered dose inhaler adapted for protein delivery). Administration of a molecule comprising a MBD peptide via the transdermal route may be continuous or pulsatile. Administration of MBD peptides may also occur orally.
For parenteral administration, compositions comprising a MBD peptide may be in dry powder, semi-solid or liquid formulations. For parenteral administration by routes other than inhalation, the composition comprising a MBD peptide is preferably administered in a liquid formulation. Compositions comprising a MBD peptide formulation may contain additional components such as salts, buffers, bulking agents, osmolytes, antioxidants, detergents, surfactants, and other pharmaceutical excipients as are known in the art.
A composition comprising a MBD peptide is administered to subjects at a dose of about 0.001 to about 40 mg/kg/day, more preferably about 0.01 to about 10 mg/kg/day, more preferably 0.05 to about 4 mg/kg/day, even more preferably about 0.1 to about 1 mg/kg/day.
As will be understood by those of skill in the art, the symptoms of disease alleviated by the instant methods, as well as the methods used to measure the symptom(s) will vary, depending on the particular disease and the individual patient.
Patients treated in accordance with the methods of the instant invention may experience alleviation of any of the symptoms of their disease.
EXAMPLES Example 1HEK293 kidney cell line and 54 tumor cell lines obtained from the National Cancer Institute and passaged in RPMI1640 cell culture medium supplemented with 10% fetal bovine serum and 10 uM FeCl2. Uptake of streptavidin-horseradish peroxidase (SA-HRP) conjugate and of various SA-HRP::MBD peptide complexes was determined as described (Singh et al. J Biol Chem. 279 (1):477-87 [2004]) using biotinylated MBD9 (KKGFYKKKQCRPSKGRKRGFCWNGRK) (SEQ ID NO: 10) and MBD21 (KKGFYKKKQCRPSKGRKRGFCWAVDKYG) (SEQ ID NO: 4) peptides and SA-HRP. Nuclear and cytoplasmic localization of these proteins was also determined in each case. The results of this survey are summarized in Table 2. They show that the rate of MBD-mediated uptake is highly variable across cell lines. In order to establish the underlying molecular mechanism for this variability, we cross-linked MBD21 peptide to the following cell surface markers at 4 degrees Celsius as previously described (Singh et al. J Biol Chem. 279 (1):477-87 [2004]): transferrin receptor 1, caveolin 1, PCNA, integrins alpha v, 2, 5 and 6, integrins beta 1, 3 and 5. Significant correlations (positive or negative) between crosslinking rates and the previously measured rates of MBD-mediated SA-HRP uptake were observed in the case of transferrin receptor 1, caveolin 1, integrins beta 3, beta 5 and alpha v. Based on the strength of these correlations, it was possible to derive crude predictive formulae for MBD-mediated uptake based on the rate of cross-linking to surface markers. Such predictive formulas could form the basis for a diagnostic procedure to select appropriate targets for MBD-based therapies.
Seven matched pairs of tumor cell lines (one MBD high-uptake and one MBD low-uptake line for each tissue) were selected for further study. Of these, six pairs (all except the leukemia lines) were selected for gene array analysis.
Total RNA was isolated using standard RNA purification protocols (Nucleospin RNA II). The RNA was quantified by photometrical measurement and the integrity checked by the Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, Calif.). Based on electropherogram profiles, the peak areas of 28S and 18S RNA were determined and the ratio of 28S/18S was calculated. In all samples this value was greater than 1.5, indicating qualitative integrity of the RNAs. 1 μg total RNA was used for linear amplification (PIQOR™ Instruction Manual). Amplified RNA (aRNAs) were subsequently checked with the Bioanalyzer 2100 system. Samples yielded in every case >20 μg aRNA and showed a Gaussian-like distribution of the aRNA transcript lengths as expected (average transcript length 1.5 kB). This indicates successful amplification of the total RNA samples and good quality of the obtained aRNAs. All aRNAs were used for fluorescent label in PIQOR™ (Parallel Identification and quantification of RNAs) cDNA microarrays (Memorec Biotec GmbH, Cologne, Germany). cDNA microarray production, hybridization and evaluation were carried out as previously described [Bosio, A., Knorr, C., Janssen, U., Gebel, S., Haussmann, H. J., Muller, T., 2002. Kinetics of gene expression profiling in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke. Carcinogenesis 23, 741-748.]. Samples were labeled with FluoroLink™ Cy3/Cy5-dCTP (Amersham Pharmacia Biotech, Freiburg, Germany). 1 μg of amplified RNA for validation experiments were labeled and hybridized. All hybridizations were performed in quadruplicate. Quality controls, external controls and hybridization procedures and parameters were performed according to the manufacturer's instructions and comply to the MIAME standards. The Cy3 (sample) and Cy5 (reference) fluorescent labeled probes were hybridized on customized PIQOR™ Microarrays and subjected to overnight hybridization using a hybridization station. The arrays are designed to query genes previously implicated in processes relevant to cancer. These include 110 transcription factors, 153 extracellular matrix-related, 207 enzymes, 120 cell-cycle-related, 171 ligands/surface markers, and 368 signal transduction genes. Equal amounts of aRNA from the 12 respective cell lines were pooled and served as a reference against which each of the individual cell lines were hybridized.
Correlation analysis was carried out to identify those genes that might be implicated in the cellular physiological state most permissive for MBD-mediated uptake. Briefly, genes were sorted based on the -fold change in expression (up or down) when pairwise comparison of the selected high and low MBD-mediated uptake lines was performed by tissue. Based on an average of these -fold changes across all pairs, approximately the top (up-regulated) and bottom (down-regulated) 3% of the gene list was selected for further analysis. The functional distribution of genes in these two groups is highly non-random, as shown in Table 4.
There is a notable difference in the functional distribution of up- and down-regulated genes. The former primarily include transcription factors and other select intracellular proteins whereas the latter are exclusively extracellular. Using correlation of expression patterns across all cell lines to further sort the subsets of up- and down-regulated genes, it is possible to identify 2-3 major groupings in each set. Up-regulated genes include GDF15, SRC, ATF3, HSPF3, FAPP2, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1, WDR9A, MAZ, NSG-X, KIAA1856, BRF2, COL9A3, TPD52, TAX40, PTPN3, CREM, HCA58, TCFL5, CEBPB, IL6R and ABCP2. It is remarkable that at least one third of these genes have been previously associated with cellular responses to stress (e.g. GDF15, ATF3, HSPF3, PSMB9, PSMB10, c-JUN, JUN-B, HSPA1A, HSPA6, NFKB2, IRF1). Down-regulated genes include CTGF, LAMA4, LAMB3, IL6, IL1B, UPA, MMP2, LOX, SPARC, FBN1, LUM, PAI1, TGFB2, URB, TSP1, CSPG2, DCN, ITGA5, TKT, CAV1, CAV2, COL1A1, COL4A1, COL4A2, COL5A1, COL5A2, COL6A2, COL6A3, COL7A1, COL8A1, and IL7R.
The patterns of up- or down-regulation of the following genes (shown in Table 5) serve as illustrations. Table 3 shows the fold expression difference in pairwise comparisons.
Low-uptake lines HCT-15, HOP-62, Hs578T, K562 and UO31 were heat-shocked at 42 degrees for 1 hour. HSP70 was induced by this treatment (
HEK293 cellular uptake of MBD9::SAHRP is stimulated by pre-treatment with stressors. Peroxidase activity was measured 20 minutes after addition of 100 ng/ml of MBD::SAHRP protein to the cell culture medium, as described in Example 1. All pretreatments were for 20 hours except for sample 5. The results of this experiment are shown in
Sample Key: (1) 293 control (2) 293+30 ng/ml TNF-a (3) 293+25 mM D-glucose (4) 293+700 mM NaCl (5) 293+42 deg C., 1 hour (6) 293+200 uM Cobalt chloride (7) 293+200 uM hydrogen peroxide (8) 293+low (1%) serum (9) 293+300 nM thapsigargin (10) 293+100 uM ethanol.
Example 5MBD-mediated protein mobilization into PC12 cells is stimulated by stressors used in models of PD. 6-OHDA or MPP+ treatment of PC12 cells dramatically stimulates uptake of MBD-mobilized horseradish peroxidase. PC12 cells cultured in RPMI 1640+FBS were pretreated with MPTP or 6-OHDA. Uptake of exogenously added MBD::SAHRP (100 ng/ml) was measured in nuclear and cytoplasmic extracts 20 minutes after addition of the protein to the cell culture medium. The results are shown in
Combinations of stressors can have novel effects on cellular uptake of MBD-tagged proteins in HEK293 cells and can be modulated by IGF-I. HEK293 cells were grown in 1% serum (nutritional stress) and peroxidase activity was measured 20 minutes after addition of 100 ng/ml of MBD::SAHRP protein to the cell culture medium, as described in Example 1. All pretreatments with growth factors IGF-I or EGF (100 ng/ml) were for 2 hours, followed by the indicated stress treatment (heat shock at 42 degrees Celsius for 60 minutes or 200 uM Cobalt Chloride for 60 minutes to simulate anoxia). Uptake was measured at the end of the stress treatment. The results are shown in Table 6 below (p values shown are relative to the control without growth factor treatment in each group; only significant p values are shown):
Combinations of stressors can have novel effects on cellular uptake of MBD-tagged proteins in MCF-7 cells and can be modulated by IGF-I. MCF-7 cells were grown in 1% serum (nutritional stress) and peroxidase activity was measured 20 minutes after addition of 100 ng/ml of MBD::SAHRP protein to the cell culture medium, as described in Example 1. All pretreatments with growth factors IGF-I or EGF (100 ng/ml) were for 2 hours, followed by the indicated stress treatment (heat shock at 42 degrees Celsius for 60 minutes or 200 uM Cobalt Chloride for 60 minutes to simulate anoxia). Uptake was measured at the end of the stress treatment. The results are shown in Table 7 below (p values shown are relative to the control without growth factor treatment in each group; only significant p values are shown):
Peptide Bio-KGF binds shRNA: Bio-KGF peptide was synthesized by Genemed Synthesis, Inc. (S. San Francisco, Calif.) as a 40-mer containing an MBD sequence and an RNA-hairpin binding domain from the N-terminus of bacteriophage lambda N protein: Bio-KGF: (“N”-terminal biotin) . . . KGF YKK KQC RPS KGR KRG FCW AQT RRR ERR AEK QAQ WKA A . . . (“C” terminus) (SEQ ID NO: 11)
An shRNA designed to silence the human beclin gene was designed to include a hairpin sequence corresponding to the NutR box of bacteriophage lambda mRNA (the binding target for the Bio-KGF peptide) and was amplified using the Silencer™ siRNA
Construction Kit (Ambion) using conditions specified by the manufacturer. The sequence of the DNA oligonucleotide used for the kit transcription reaction was: T7BECR: 5′ . . . AG TTT GGC ACA ATC AAT AAC TTTTTC AGT TAT TGA TTG TGC CAA ACT CCTGTCTC . . . 3′ (SEQ ID NO: 12)
As a vector control for in vivo confirmation of siRNA efficacy, the following oligonucleotides were designed for cloning into the pGSU6 vector (BamHI-EcoRI) BECF: 5′ . . . GAT CGG CAG TTT GGC ACA ATC AAT AAC TGAAAA AGT TAT TGA TTG TGC CAA ACT GTT TTT TGG AAG . . . 3′ (SEQ ID NO: 13). BECR: 5′ . . . AAT TCT TCC AAA AAA CAG TTT GGC ACA ATC AAT AAC TTTTTC AGT TAT TGA TTG TGC CAA ACT GCG . . . 3′ (SEQ ID NO: 14).
Various molar excess amounts of Bio-KGF (ranging from 63 pg to 2 ug per well; similar results were obtained across this range) were attached to a Ni-NTA plate (Qiagen Inc., Carlsbad, Calif.) for 1 hour and blocked overnight with 3% BSA at 4 degrees C. in the refrigerator, and washed with PBS/Tween and TE buffers. RNA dilutions were added in TE buffer, incubated for 30 min on shaker, then for 30 min on bench at room temperature. After one wash with TE buffer, Ribogreen reagent (Ribogreen RNA Quantitation Reagent and Kit from Molecular Probes/Invitrogen) was added to the wells, incubated 5 minutes, and fluorescence was read on a fluorescent plate reader. The results are listed in Table 8 (each number is a mean of eight readings):
The Bio-KGF peptide binds the shRNA containing the lambda nutR hairpin loop.
Example 9 Sequences of Therapeutic MBD PeptidesTherapeutic peptides incorporating the MBD motif can be created by making fusions of peptide sequences known to have appropriate intracellular biological activities with either the N- or C-terminus of the core MBD sequence. The following table (Table 9) lists peptides used in this study. Based on prior studies, peptide sequences were selected to target up-regulated stress proteins (such as hsp70) in cancer, as well as MDM2 interactions with P53, inflammation (NF-kappa-B, NEMO, CSK), and previously characterized cancer-specific targets such as survivin and bcl-2.
Peptides were added at 24 and 48 hours of culture and results of cytotoxicity measured at 96 hours using XTT assay according to the manufacturer's instructions. All measurements were made in triplicate or quadruplicate.
Peptides were added at 24 and 48 hours of culture and results of cytotoxicity measured at 96 hours using XTT assay according to the manufacturer's instructions. All measurements were made in triplicate or quadruplicate.
As shown in
As shown in
Effects of peptides were compared using primary HMEC cells versus MCF-7 breast cancer cells or primary isolated CD4+T-cells versus the CCRF-CEM leukemia line. All cells are human. Results of 48 hour cytotoxicity using 6.25 ug.ml added peptide are shown in Table 10 below:
In order to test the hypothesis that cancer cells are specifically susceptible to targeted disruption of constitutively up-regulated stress-coping and anti-apoptotic mechanisms, MBD-tagged peptides were designed to inhibit either the synthesis, transport or action of inflammatory and heat-shock response proteins, as well as molecules involved in anti-apoptotic actions within cancer cells. Table 11A lists the sequences of synthesized peptides. Peptides were synthesized by Genemed Synthesis, Inc. with N-terminal biotin, and purified by HPLC.
MBD-tagged peptides targeting stress-coping and anti-apoptotic mechanisms commonly upregulated in cancer exhibit selective cytoxicity to cancer cells without affecting their normal cell counterparts. Peptides shown to have a strong cytotoxic effect on cancer cells but not their human counterparts include PEP1, PEP2 and PEP3, which target the MDM2::P53 interface. Also, peptides such as NFKB and CSK are of interest, targeting stress-coping mechanisms such as inflammation. The breast cancer lines tested are HS578T, MX-1, MDA-MB231, MDA-MB435 and MCF7. Leukemia cell lines tested for cytotoxicity effects with these MBD-tagged peptides are CCRF-CEM, RPMI-8226 and MOLT-4. Overall, MCF-7 and CCRF-CEM yield the most consistent data and the strongest effect across the board (Table 12). In addition, elevated levels of cytotoxicity are observed when multiple peptides are combined while keeping the overall amount of peptide added constant. Cytotoxicity increases with the number of peptides added per cocktail and is further enhanced by combining peptide cocktail treatment with paclitaxel.
Additional peptides were synthesized by Pepscan Systems B.V. (Lelystad, Holland) for testing of mutant variations in the original peptide sequences, and new sequences. These peptides are listed in Table 11B.
As shown in
In order to maximize the effects of cytotoxic peptides, alanine scanning of one peptide (MDOKSH) was undertaken as an illustration. 48 mutants were synthesized, purified and tested in CCRF-CEM and MCF-7. The cytotoxicity of the 48 peptides was strongly correlated in the two cell line assay systems (r=0.606). Some of the mutants synthesized and tested in CCRF-CEM and MCF-7 cells are shown in Table 13. Mutant #27 exhibits greatly enhanced cytotoxicity in both cell line assays. This result illustrates the general applicability of simple substitution and addition of residues, for example, alanine substitution one residue at a time, addition of one (of the 20) amino acid to each end of the peptide sequence, and deletion of one residue at a time. The core MBD sequence may, if desired, be excluded from the region to be explored by mutagenesis, in order to expedite the experiment.
Eight week old diabetic (db/db) male mice were ordered from Jackson Laboratory (Bar Harbor, Me.). Sixty-eight animals were used in the study and had an initial glucose measurement in order to determine if they had developed diabetes (>200 mg/dL serum glucose). For five weeks, mice were injected once daily with peptides and once a week they were weighed, glucose was measured and blood was collected. An initial and terminal sample of urine was collected from all animals by placing them in metabolic cages for 24 hours. Upon termination left and right kidneys, brain, and pancreas were collected from all animals. Results of various measurements are shown in the table below. They demonstrate that humanin-S14G had distinct effects on reducing albuminuria, accompanied by corroborating changes in left kidney tissue collagen-IV, but without lowering serum glucose or insulin.
Metal-binding therapeutic peptides (12.5 ug/ml, 48 hours) differentially sensitize breast cancer versus normal cells to low dose (1 ng/ml) 5-Fluorouracil [5-FU].
Cytotoxicity assays were performed as previously described. Numbers in bold show significant (p<0.05) differences from control peptide (SMZ) treatment. PNPKC (SEQ ID NO:195, Table 20), MBDP38 (SEQ ID NO:194, Table 20).
The mice used were purchased from Taconic. Mice were bred by crossing C57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient mice. Approximately 1×106 MDA-MB231 breast cancer cells were injected into mice intracardially. Mice received once weekly intra-peritoneal injections of 5-fluorouracil (SFU; 1 mg/kg) and daily subcutaneous bolus injections of 4-peptide cocktail (4 mg/kg) or saline. One group additionally received a daily dietary supplement of curcumin/lycopene. Animals were sacrificed at Day 35 post-injection and scored based on visible liver metastasis, hindlimb paralysis and bone marrow (BM) MDA-MB231 metastatic cell burden based on PCR amplification index >1 of BM genomic DNA using primers specific for MDA-MB231 human sequences. “Confirmed metastasis” means the animal scored positive on at least 2 of these 3 criteria. At termination blood and organs were collected and stored at −80° C. The DNaesy Tissue Kit (Qiagen, Carlsbad, Calif.) was used and to isolate genomic DNA (gDNA) from tissue samples. gDNA concentrations were established based on spectrophotometer OD260 readings. PCR amplifications were performed with human-specific primers 5′-TAGCAATAATCCCCATCCTCCATATAT-3′ (SEQ ID NO: 5) and 5′-ACTTGTCCAATGATGGTAAAAGG-3′ (SEQ ID NO: 6), which amplify a 157-bp portion of the human mitochondrial cytochrome b region. 400-800 ng gDNA was used per PCR reaction, depending on type of tissue. Best results were achieved using the KOD hot start PCR kit (Novagen, Madison, Wis.). PCR was performed in a thermal cycler (Perkin Elmer) for 35 cycles (30 s at 96° C., 40 s at 59° C., and 60 s at 72° C.). Results are shown in the table below.
Humanin-S14G but not colivelin binds a Ni-NTA column. 1 ml Ni-NTA columns (Qiagen, Carlsbad, Calif.) were loaded with each protein. Flow-through was collected. Wash, Eluate 1 (imidazole) and Eluate 2 (EDTA) buffers of the manufacturer's specification were each applied 4×1 ml. Each set of fractions was pooled. A280 was read for each pool. Results listed in the table below show that Humanin-S14G but not colivelin binds the Ni-NTA column, and can be eluted. Colivelin is a derivative of humanin with the amino acid sequence SALLRSIPAPAGASRLLLLTGEIDLP (SEQ ID NO: 218) (Chiba, T. et al. J. Neurosci. 25:10252-10261, 2005).
Human embryonic kidney cells (HEK293) were treated with glycated hemoglobin or TNF-alpha for 24 hours and assayed for total IRS-1 or IRS-2. The results, shown in
Sixteen-week-old db/db mice exhibit significantly elevated blood glucose and albuminuria. Kidney mesangial cell matrix expansion and collagen-IV synthesis correlate with disease progression, but the underlying mechanism is unclear. Adaptive biochemical datasets were generated in cultured 293 kidney cells and in db/db mice.
Reagents: Humanin (WT) and S14G-Humanin were purchased from American Peptide Co, Sunnyvale, Calif. NPKC (AKKGFYKKKQCRPSKGRKRGFCWPSIQITSLNPEWNET; SEQ ID NO:195) and P38 (AKKGFYKKKQCRPSKGRKRGFCWAPSRKPALRVIIPQAGK; SEQ ID NO:194) peptides contain the MBD domain of IGFBP-3, which provides effective biodistribution, cell internalization and nuclear delivery for linked sequences were synthesized and purified by Genenmed Synthesis, Inc., S. San Francisco, Calif. Glycated-hemoglobin, amphoterin, TNF-alpha, EGF, resistin, insulin, SDKP, caffeine, rapamycin, and the antibodies anti-IRS1, anti-RAGE, anti-Fibronectin, anti-IRS1(Ser307) and anti-IRS2(Ser731) were purchased from Sigma Chemical Co., St Louis, Mo. The following reagents were obtained from EMD Chemicals, San Diego, Calif.: AKT(Ser473)-blocking peptide, AKT Inhibitors (II through IX), JNK Inhibitors II and III, SB203580, LY294002, PD98059. Phosphosafe tissue cell extract reagent was from Novagen, Madison, Wis. Cell culture reagents RPMI 1649, DMEM and FBS were from Hyclone, Logan, Utah. Protein Concentration Kit was purchased from Pierce Biotechnology, Rockford, Ill. Antibodies to the following antigens were purchased from the indicated suppliers: c-Jun(Ser63), c-Jun(ser73), c-myc(Ser62), c-myc(Thr58) (EMD Chemicals, San Diego Calif.); Erk1/2(Thr202/Tyr204), P38MAPK(T180/Y182), SAPK/JNK(Thr183/Ty185), P38-alpha/SAPK2a, c-myc(Thr58Ser52), PKC-betaII, phospho-PKC-alpha/betaII (Thr638/641), PKC-Delta, PKC-Delta/Theta, PKC-Theta, PKC-zeta/lambda, PKD/pKCmu (Ser916), PKD/PKCmu (Ser744/748), PKD/PKCmu, AKT(Thr308), AKT(Ser473), AKT1, AKT2, AKT3, MKK3/MKK6(Ser189/207), ATF2(Thr71), paxillin (Y118), GSK3B(Ser9) (Cell Signaling, Danvers, Mass.); Collagen-IV and IRS-2 (RnD Systems, Minneapolis, Minn.).
293 kidney cell culture: Cells were passaged in DMEM plus 10% FBS and plated in 6-well plates. When 90-95% confluent, they were treated with different reagents for 4 hours. Cells were collected off plates and washed twice with 1×PBS. Extracts were made in 200 ul phosphosafe and diluted in 1×PBS to set up ELISAs.
Human mesangial cell culture: Human kidney mesangial cells and media were purchased from Lonza (Walkersville, Md.). Cells grown in mesangial cell basal media that were quiescent for two days were treated with glycosylated hemoglobin and peptides, and cell extracts were prepared and assayed by ELISA in exactly the same manner as described for 293 cells.
Animal studies: db/db mice were purchased from Jackson Laboratories. Animals with blood glucose below 200 mg/dL in Week 8 were sacrificed and used as null controls. Remaining animals were randomized into 4-8 animals per treatment group and were injected by subcutaneous bolus daily from week 8 through 13 (first experiment) or week 9 through 15 (second experiment). At the beginning and end of each experiment, each mouse was housed in an individual metabolic cage for a 24-hour urine collection. The volume of urine collected was recorded. Urine samples were assayed for albumin by ELISA and the total amount of albumin excreted calculated by multiplying the volume of urine by the concentration of albumin in the urine. Diabetes progression was monitored weekly during treatment by measuring blood glucose levels. Animals were sacrificed at week 13 (first experiment) or week 15 (second experiment). At termination, plasma and organs (right and left kidneys, pancreas, brain, heart, liver) were collected for preparation of tissue extracts and ELISA assays. Organ slices were ground in cell lysis buffer and total protein concentration was measured using a BCA protein assay kit.
Measurement of plasma glucose and insulin. Insulin levels were determined in plasma samples with the UltraSensitive Mouse Insulin ELISA from ALPCO Diagnostics (Windham, N.H.). Blood was collected in heparin-coated capillary tubes and red blood cells were separated by centrifugation at 5000 rpm for 5 minutes. Plasma glucose was assessed by pipetting 5 ul samples on glucometer strips and reading in the One Touch Basic Glucometer (LifeScan Canada Ltd., Burnaby, BC). Mice were fasted overnight prior to the glucose test.
ELISA assays: Extracts were diluted 1/25 and 100 ul of each sample was added to a 96-well plate. After 1 hour the plate was washed (3 times with 1×PBS+Tween). 3% BSA was added to the plates and incubated for 1 hour. The wash step was repeated and then primary antibody was added for 1 hour. Another wash step was followed by treatment with secondary antibody for 1 hour. Wash was then repeated and 100 ul per well TMB added. After incubation for 15 minutes, the samples were read in a plate reader at 655 nm.
PI3-kinase-associated IRS-2 immunoprecipitation: Immunoprecipitation was done using the Catch and Release IP Kit (Millipore, Billerica Mass.) according to the manufacturer's specifications. Briefly, HEK 293 cells were treated with either saline, glycated hemoglobin or amphoterin for 4 hours. The cells were collected and washed 2 times and whole cell extracts were prepared in phosphosafe buffer. 300 ul of each extract was mixed with 10 ul anti-PI3-kinase antibody for 60 minutes at 4 degrees C. with gentle rocking. The samples were then applied to the column and centrifuged for 30 seconds. The column was washed 3 times and then 400 ul of elution buffer was added to the column and centrifuged at 5000 rpm for 30 seconds to collect all samples. The purified material was assayed for IRS-2 by ELISA.
Statistical analysis: Probability values (p values) were computed using Student T-test. Unless otherwise stated, p values are expressed relative to saline-treated controls.
RAGE-adaptive elevation of IRS-2 and collagen-IV in 293 kidney cells:
Modulation of RAGE-activated biochemical changes by bioactive peptides and chemical inhibitors: The influence of selected inhibitors (Akt inhibitor IV, rapamycin and LY290004) and of the bioactive peptides humanin, NPKC and Akt-Ser473-blocking peptide on a selected set of RAGE-activated biochemical events is shown in
Effects of Humanin and NPKC peptides in vivo: In order to test the effect of subcutaneously-injected peptides in diabetic mice, 8-week old db/db mice were treated daily for 5 weeks with the indicated subcutaneous bolus doses of humanin or NPKC peptide. Wild type humanin was compared with the S14G substitution mutant (previously reported by others to be more active) and the wild type peptide was surprisingly found to be more effective.
Uncoupling of collagen-IV synthesis from albuminuria in P38-peptide treated mice: In order to examine the possibility of an obligate relationship between collagen-IV synthesis and albuminuria, 9-week-old db/db mice were treated for 5 weeks with 40 ug/day subcutaneous bolus P38 peptide (an intracellular inhibitor of activated P38MAPK target ATF2 that includes an MBD domain sequence for cell internalization and nuclear delivery of the peptide in vivo) or humanin peptide. The results (Table 23) show a marked reduction of collagen-IV in P38 peptide-treated animals, but in these animals a significant exacerbation of albuminuria is observed. Kidney tissue IRS-2 is also elevated in P38-treated animals relative to saline treated controls (0.205±0.007 versus 0.184±0.009 arbitrary units; p=0.028). As in the first experiment, humanin reversed albuminuria.
Conclusions: Treatment of db/db mice with bioactive peptides humanin and NPKC ameliorates albuminuria. Kidney tissue extracts were used to generate an adaptive dataset of biochemical markers. Correlation matrices based on these datasets reveal tightly clustered readouts which may, in turn, provide potentially fundamental insights into the adaptive circuitry of kidney cells. Readout clusters may be considered ‘virtual regulons’ for the purpose of guiding the hypothesis-driven design and development of novel and targeted therapeutic approaches to disease. The underlying assumption of this approach is that cellular responses to environmental insults are adaptive (or maladaptive, in the case of disease) and may expose universal aspects of adaptive logic such as characteristic responses to stress, enhanced plasticity or increased internality of decision-making as revealed, for example, by the temporarily modified response to endocrine and metabolic signals summarized in
IRS-1 and IRS-2 proteins are central integrators of signaling traffic from cell membrane receptor tyrosine kinases responding to metabolic and growth signals, especially insulin and insulin-like growth factors and may be of particular relevance in diabetes. Although selective action of IRS isoforms has been proposed for specialized settings such as metastasis, the existence of a universal cellular logic switch based on the ratio of total active IRS-2 to IRS-1 has not been previously postulated. We show that in cultured 293 kidney cells challenged with glycated hemoglobin, as well as in kidney extracts from diabetic mice, a marked elevation in total IRS-2—but not IRS-1—levels is observed, accompanied by higher levels of phosphorylated IRS-1/Ser 307, which has been linked to insulin-resistance, but not of phosphorylated IRS-2/Ser 731. These types of changes would be expected to result in an increased involvement of IRS-2 in signaling events through the PI3 kinase pathway leading to activation of protein kinase B/Akt. We show a significantly elevated level of IRS-2 associated with PI3-kinase in cells treated with RAGE ligand.
Akt is a central consolidator of cellular logic. Fully-activated Akt is phosphorylated at two key residues, Thr308 and Ser473. Differential phosphorylation of Akt at these residues has been previously described. RAGE-mediated changes in 293 kidney cells involve altered signaling in the IRS-Akt axis. In db/db mice exhibiting elevated albuminuria, Akt1 levels are coupled to albumin excretion which is, in turn, coupled to Akt/Ser473 (but not Akt/Thr308) phosphorylation. In cultured 293 cells challenged with glycated hemoglobin, similarly linked responses are observed, with differential phosphorylation at Ser473 (inhibited by Ser473-blocking peptide), and consequently altered responses to insulin and EGF signaling. LY20004, a specific inhibitor of PI3-kinase, enhances the preferential phosphorylation of Ser473, suggesting that the event is independent of the PI3-kinase cascade. Although the rapamycin-insensitive mTOR complex mTORC2, which contains Rictor, has been recently implicated as the elusive PDK2 responsible for the phosphorylation of Akt-Ser473, rapamycin appears to reduce Ser473 phosphorylation in kidney cells. Other enzymes, such as PKC, have also been implicated as potential kinases for Akt-Ser473. Preferential phosphorylation of Akt-Ser473 in a PI3-kinase-independent manner may be part of the adaptive response characterized by elevated IRS-2 levels.
In this work we have surveyed a panel of intracellular biochemical readouts in cultured 293 kidney cells challenged with glycated hemoglobin and various chemical and peptide inhibitors. As shown in Table 22, similar data can be obtained from cultured human kidney mesangial cells. We elected to use 293 cells for most experiments because of better assay reproducibility, ease of culture and handling, and lower cost of materials for routine assay use.
Treatment of db/db mice with 20 ug/day subcutaneous bolus humanin or 40 ug/day NPKC peptide for 5 weeks ameliorates albuminuria and lowers IRS-2 levels. In addition, humanin helps normalize a cluster of RAGE-mediated biochemical effects, without affecting circulatory levels of glucose or insulin. Similar effects of humanin on biochemical markers can be observed as a result of short-term treatment of cultured kidney cells, except that the modulation of Akt1 is in the reverse direction. Treatment with wild type humanin is more effective than with the S14G variant, which has been shown to be more active in models of neurodegenerative disease.
In order to further understand the linkage between albuminuria and the biochemical readouts that may be significantly altered by disease, correlation matrices were generated from a dataset derived from ELISAs of kidney extracts prepared from 30 db/db mice. In these matrices, biochemical readouts cluster into distinct ‘virtual regulons’. Humanin and NPKC appear to influence the readouts that correlate most closely with albuminuria.
Inhibition of PKC using the NPKC peptide ameliorates albuminuria and reduces IRS-2 levels in the kidneys of treated mice. However, unlike humanin, NPKC does not normalize the elevation in phospho-Akt-Ser473 and phospho-JNK-Thr183/Tyr185, two markers comprising the so-called “stress regulon”. In kidney extracts (r=0.419) and in 293 kidney cell culture (r=0.502), these two markers co-vary in response to environmental stimuli (data not shown). The uncoupling of responses to humanin and NPKC with respect to these markers suggests a distinction between indicia directly linked to albuminuria and other, more generalized, stress responses generated perhaps by exposure to hyperglycemic or hyperinsulinemic stress. Moreover, treatment of diabetic mice with peptide P38 (designed as an intracellular inhibitor of activated p38 MAPK), exacerbates albuminuria despite inhibiting collagen-IV production. This observation is consistent with the hypothesis that biochemical changes linked to a generalized stress response may not be as closely linked to albuminuria as are dysregulated IRS-2 levels.
Taken together, our data from kidney extracts and cultured kidney cells suggests that humanin acts by modifying biochemical parameters most closely associated with kidney disease as well as those associated with a more generalized stress response. On the other hand, NPKC may act on a more limited subset of biochemical indices. Collagen-IV synthesis, a canonical marker of matrix expansion, can be uncoupled from albuminuria in animals treated with P38 peptide: the peptide dramatically inhibits collagen synthesis but exacerbates protein excretion.
Although albuminuria is itself tightly linked to plasma glucose and body weight, humanin dramatically ameliorates protein excretion in the urine without exerting any significant impact on plasma glucose and insulin levels or body weight. Thus, markers driven by hyperglycemic or hyperinsulinemic stress may be separable from those that have a primary causal connection to kidney disease. Although a causal connection between IRS-2 elevation and albuminuria are not established by our data, we propose that the adaptive uncoupling of cellular IRS-2 levels from those of IRS-1 constitutes a potentially useful biochemical correlate of kidney disease in diabetic mice. The human peptide humanin, previously thought to have a function in neurodegenerative disease, has a profound effect on IRS-2 elevation both in vitro and in vivo, and may be a candidate for therapeutic intervention in kidney disease.
Example 23 Use of Adaptive Signatures to Select Therapeutic Candidate PeptidesThe methodology established in Example 22 is extended to the screening of therapeutic candidates. Human 293 kidney cells were exposed to glycated hemoglobin as a provocative agent for 4 hours, as described in Example 22, in the presence or absence of 20 ug/ml peptide. Various readouts, such as IRS-2 or IRS-2:IRS-1 ratios can be obtained by assaying cell extracts by ELISA. The table below shows one example of how peptide variants based on the humanin sequence can be assessed.
Note that the biochemical readouts are modified deltas i.e. adaptive changes caused by a provocative agent (in this case glycated hemoglobin, for 4 hours as in Example 22) and subsequently further modified by peptide exposure. Similar methodology, but using multiple biochemical readouts can also be used for greater confidence in the result. The following peptides were tested (Table 25).
Some of the peptide sequences shown in the above tables were added to human 293 kidney cells at 20 ug/ml in the presence of glycated hemoglobin as provocative agent. Cell extracts were assayed by ELISA and deltas (ratios of biochemical readouts) calculated. Table 26 shows some of the results obtained.
From the above results, it is possible to make strategic choices as to which peptide candidates should be used in further studies.
Example 24 Biodistribution of MBD-Tagged MoleculesIn order to establish the tissue distribution of genes favorable to MBD-mediated uptake of molecules, PCR was performed on cDNA from a range of human tissues. Based on gene array data GDF15 was chosen to evaluate biodistribution of peptides representing stress-coping and anti-apoptotic mechanisms via PCR. Human cDNA MTC panel I (#636742, Clontech) was tested against GDF15 (forward primer 5′-GGGCAAGAACTCAGGACGG-3′ (SEQ ID NO:275) and reverse primer 5′-TCTGGAGTCTTCGGAGTGCAA-3′) (SEQ ID NO: 276) and GAPDH control primers. The PCR was performed in a thermal cycler (Perkin Elmer). The optimized PCR conditions are: 28 cycles of 30 s at 96° C., 40 s at 59° C., and 1 min at 72° C. From a 50 ul PCR reaction 15 ul sample was loaded per well on a 1×TBE and 10% polyacrylamide gel (VWR) and run out at 90V for 1.5 hrs. Bands were visualized via Ethidium Bromide staining. The results are shown in the top two panels of
In the middle panel of the figure, results are shown for an experiment in which 2 mg/kg PEP2 peptide ETFSDVWKLLKKGFYKKKQCRPSKGRKRGFCW (SEQ ID NO. 17) was administered to mice by subcutaneous bolus injection and tissues were harvested two hours later. Tissue extracts were assayed by ELISA, using an MBD-specific antibody. The antibody does not recognize intact IGFBP-3 in rodents or humans. Kidney extracts contained significantly more MBD antigen that blood cells (leukocytes). Averages are shown for two animals.
In the bottom panel, results are shown for an identical biodistribution experiment done using rats (average of 6 animals). Two proteins were injected at 2 mg/kg by subcutaneous bolus injection: MBD-tagged GFP protein (MBDGFP) and a control protein thymidine kinase (TK). Pancreas contained a significantly elevated MBD signal, relative to TK control.
Example 25 Discriminant Chemosensitization of Two-Peptide Cocktail on Cancer CellsIn order to show the selective action of bioactive peptides on cancer cells versus their normal counterparts, paired cell lines were exposed to varying concentrations of peptide cocktail either in the presence or absence of what was previously determined to be a moderately toxic (LC20-LC50) concentration of 5-fluorouracil for each cell line. Peptide cocktail consisted of a 1:1 mixture of:
Cells and cell culture. All cell lines were obtained from Cambrex or the American Type Culture Collection (ATCC). They are well characterized and have been extensively used in vitro and in vivo. Breast cancer cell lines (MCF7, MDA-MB-435, MDA-MB-231, MX-1), leukemia cell lines (RPMI-8226, CCRF-CEM, MOLT-4), and prostate cancer cell lines (PC3, DU145, LNCAPs) were cultured in RPMI-1640 media supplemented with 5% FBS. Paired non-cancer and breast cancer cell lines (CRL-7481/CRL-7482, CRL-7364/CRL-7365) were cultured in DMEM media supplemented with 10% FBS. Normal cell lines such as MCF-10A, HMEC and HTB-125 were cultured in A, B, C media, serum-free, respectively. Cancer and metastatic cancer cell pairs (CCL-227/CCL-228, CRL-7425/CRL-7426, and CRL-1675/CRL-1676) were cultured in L-15 or MEM media with 10% FBS.
Cytotoxicity Assay. Cells are incubated 48 hrs with MBD peptide (fresh peptide is added to the plate every 24 hrs). MBD-domain-only peptide is used as a control in these experiments. PROMEGA's 96-well Cell Titer Cytotoxicity Assay Kit was optimized for use in breast cancer and leukemia cell lines and their non-cancerous counterparts, HMEC and CD4-T-cells (Cambrex), respectively. Using increasing doses of peptides (3.125, 6.25, 12.5 and 25.0 ug/ml) and a fixed number of cells (e.g. 104) per well, we measure cytotoxicity after a 48-hr incubation at 37° C. The 96-well format allows high-throughput data to determine enhanced and synergistic effects on cell-death, i.e. comparing mutant peptides singly or in various combinations.
The results are shown in
Cell lines were challenged with glycated hemoglobin as described for human kidney cells in Example 22. Deltas (difference readings) of selected biochemical readouts were collected and analyzed to generate adaptive signatures.
Cells and cell culture. All cell lines were obtained from Cambrex or the American Type Culture Collection (ATCC). They are well characterized and have been extensively used in vitro and in vivo. Breast cancer cell lines (MCF7, MDA-MB-435, MDA-MB-231, MX-1), leukemia cell lines (RPMI-8226, CCRF-CEM, MOLT-4), and prostate cancer cell lines (PC3, DU145, LNCAPs) were cultured in RPMI-1640 media supplemented with 5% FBS. Paired non-cancer and breast cancer cell lines (CRL-7481/CRL-7482, CRL-7364/CRL-7365) were cultured in DMEM media supplemented with 10% FBS. Normal cell lines such as MCF-10A, HMEC and HTB-125 were cultured in A, B, C media, serum-free, respectively. Cancer and metastatic cancer cell pairs (CCL-227/CCL-228, CRL-7425/CRL-7426, and CRL-1675/CRL-1676) were cultured in L-15 or MEM media with 10% FBS.
ELISA. Cells were lysed using cell lysis buffer (Clontech) or phospho-safe extraction reagent (Novagen) and lysate dilutions of 1:10 or 1:20 were loaded in triplicate in a 96-well plate format. Protein contained in the lysate was allowed to attach to coated plates for 1 hour at room temperature. The plates were then incubated for 1 hour at room temperature (or over night at 4° C.) in blocking buffer, consisting of 3% BSA in PBS with 0.05% Tween-20. The plates were washed and incubated with the diluted primary antibody for 1 hr on the shaker at room temperature. The plates were washed and then incubated with horseradish peroxidase—conjugated secondary antibody (Sigma Chemical Co, St. Louis, Mo.) for 45 minutes at room temperature. The antibody-antigen complex was visualized with Tetramethylbenzidine (TMB) liquid substrate system (Sigma) according to the manufacturer's protocol. Plates were read at 655 nm on the ELISA plate reader (Molecular Devices).
Mouse model. Successful engraftment of both human hematopoietic and non-hematopoietic xenografts requires the use of severe combined immuno deficient (scid) mice as neither bone marrow involvement nor disseminated growth are regularly observed using thymectomized, irradiated or nude mice. The mice used to establish a human-mouse xenograft model were purchased from Taconic. Mice were bred by crossing C57BL/6J gc KO mice to C57BL/10SgSnAi Rag-2 deficient mice. The gc KO is a deletion of the X-chromosome linked gc gene resulting in a loss of NK cells, a loss of the common g receptor unit shared by an array of cytokines that include IL-2, IL-4, IL-7, IL-9, and IL-15, and as a result only a residual number of T and B cells are produced. To eliminate this residual number of T and B cells, the gc mouse KO mouse was crossed with a C57BL/10SgSnAi recombinase activating-2 (Rag-2) deficient mouse (a loss of the Rag-2 gene results in an inability to initiate V(D)J lymphocyte receptor rearrangements, and mice will lack mature lymphocytes). MDA-MB-231 xenograft-bearing Rag-2 mice (10 mice per group, 3 groups, approx. 5×105 cancer cells injected per animal per group) are established through intra-cardial injection. Blood sampling and PCR analysis are carried out at weekly intervals. Approximately 100 ul blood is collected from the saphenous vein. PCR analysis is used on peripheral blood (PB) on Day 3 post-injection to determine whether animals have successfully established leukemia/cancer. Cancer cell count levels are monitored during and after treatment as well as at termination. PCR analysis on PB, bone marrow, spleen, liver and lung is used to quantify the cancer cells. At Day 3, prior to treatment, high levels of cancer cells should be seen in PB and low or no levels of human cancer cells in peripheral organs. Blood and peripheral organs were collected at termination and stored for further analysis (Day 18).
The results of an experiment comparing 3 matched pairs of primary tumor and metastatic cell lines derived from the same patient in each case are summarized in
As a control, the results of a similar experiment comparing 3 matched pairs of cancer/non-cancer cell lines are shown in
As a final control, MDA-MB-231 breast cancer cells were intracardially implanted in mice as described above. Visible liver metastases were recovered from 3 animals and cell extracts (assayed with human-specific antibodies) were compared with those from the original MDA-MB-231 cells in culture. The results of the comparison are shown in
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.
Claims
1-36. (canceled)
37. A polypeptide comprising the sequence SDKPDMAPRGFSCLLLLTGEIDLPV (SEQ ID NO:245).
38. A composition comprising the polypeptide of claim 37 and a pharmaceutical excipient.
39. A nucleic acid encoding the polypeptide of claim 37.
40. A vector comprising the nucleic acid of claim 39.
41. A method of treating an inflammatory disease condition comprising administering an effective amount of a polypeptide to a mammal, wherein said polypeptide comprises and amino acid sequence selected from the group consisting of SDKPDMAPRGFSCLLLLTGEIDLPV (SEQ ID NO:245), humanin (SEQ ID NO:188), and humanin-S14G (SEQ ID NO:189); and said inflammatory disease condition is selected from the group consisting of cancer, cardiomyopathy, nephropathy, retinopathy, obesity, lupus, autoimmune disease, rheumatological disease and infectious disease.
42. The method of claim 41, wherein the nephropathy is diabetic nephropathy.
43. The method of claims 41 wherein the polypeptide is administered via a route selected from the group consisting of intravenous, oral, subcutaneous, intraarterial, intramuscular, intracardial, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal, and inhalation.
44. A method of treating an inflammatory disease condition comprising administering the nucleic acid of claim 39 to a mammal; said inflammatory disease condition is selected from the group consisting of cancer, diabetes, cardiovascular disease, kidney disease, retinopathy, obesity, metabolic disease, neurodegenerative disease, gastrointestinal disease, lupus, autoimmune disease, rheumatological disease and infectious disease.
Type: Application
Filed: Nov 20, 2009
Publication Date: Jun 17, 2010
Inventor: Desmond Mascarenhas (Los Altos Hills, CA)
Application Number: 12/623,242
International Classification: A61K 38/16 (20060101); C07K 14/00 (20060101); C07H 21/00 (20060101); C12N 15/63 (20060101); A61P 29/00 (20060101); A61K 48/00 (20060101); A61P 35/00 (20060101); A61P 9/00 (20060101); A61P 3/10 (20060101); A61P 13/12 (20060101); A61P 3/04 (20060101); A61P 25/00 (20060101); A61P 1/00 (20060101); A61P 37/00 (20060101);
