| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | December 19, 1995 |
| PATENT TITLE |
Methods and compositions for the preparation and use of autocrine growth factors |
| PATENT ABSTRACT | Autocrine growth factors and isoforms of those factors have been identified, isolated, purified and manipulated. Nucleic acid segments coding for the factors, and antibodies directed to the factors are also aspects of the present invention. The effect of these growth factors on cells is to enhance their growth by increasing mitogenesis. In particular, the growth factors stimulate kidney epithelial cell growth. The growth factors differ from others previously reported in their molecular weights and other properties, for example, resistance to denaturation by dithiothreitol. Methods of preparation and use of the factors are also described. The growth factors are released from kidney epithelial cells by short exposures to a low-sodium environment. The factors have potential for treatment of kidney disease |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | September 22, 1993 |
| PATENT REFERENCES CITED |
Stnyer, L. 1975, in: Biochemistry, W. H. Freeman and Co. San Francisco, Calif. pp. 22-30. Cantor et al. 1980, in: Biophysical Chemistry, Part I. The Conformation of Biophysical Macromolecules, W. H. Freeman and Co. San Francisco, Calif., 54-56. Katz & Dong, BioTechniques, 8(5):546-555, 1990. National Kidney and Urologic Diseases Advisory Board Long-Range Plan, U.S. Department of Health and Human Services, 1990. Kato et al., J. Chromatog., 447:212-220, 1988. Walsh-Reitz et al., Proc. Natl. Acad. Sci., 83:4764-4768, 1986. Sporn & Roberts, Nature, 313:745-747, 1985. Mordan & Toback, Am. J. Physiol., 246:C351-C354, 1984. Walsh-Reitz et al., Am. J. Physiol., 247:C321-C326, 1984. Toback et al., Am. J. Physiol., 247:C14-C19, 1984. Moolenaar et al., Cell, 23:789-798, 1981. Holley et al., Proc. Natl. Acad. Sci., 77(10):5989-5992, 1980. Toback, Kidney International, 12:193-198, 1977. Toback et al., Am. J. Physiol., 232(2):E216-E222, 1977. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A purified and isolated autocrine growth factor which effects growth of kidney epithelial cells and has the following characteristics: (a) an apparent molecular weight of between 3,500 and 10,000 daltons as determined by SDS polyacrylamide gel electrophoresis and ultrafiltration; (b) resistant to inhibition by: (i) 65 mM dithiothreitol (DTT) for at least one hour; (ii) freezing for one week; or (iii) heating at 56.degree. C. for thirty minutes; (c) inhibited by: (i) heating at 100.degree. C. for fifteen minutes; (ii) exposure to 0.1% trifluoroacetic acid for one hour; or (iii) treatment with 100 .mu.g/ml of trypsin for three hours at 37.degree. C.; (d) stable in 1% acetic acid and over a pH range from 3.1 to 9.5; and (e) obtained from BSC-1 or MDCK cells or from cells which produce the autocrine growth factor having the characteristics of a factor obtained from BSC-1 or MDCK cells. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the identification, isolation and purification of autocrine growth factors, and to nucleic acid segments coding for the growth factors. The present invention also relates to methods of preparing the factors, including recombinant genetic technology, and to use of the growth factors to enhance cell growth, in particular, renal epithelial cell growth. Cellular growth enhancement is useful in treating kidney disease. 2. Description of the Related Art a. Kidney Disease is a Major Public Health Problem Acute renal failure refers to the abrupt disruption of previously normal kidney function. This serious clinical condition is due to a wide variety of mechanisms including circulatory failure (shock), vascular blockage, glomerulonephritis, and obstruction to urine flow. Acute renal failure frequently arises as a complication of abdominal or vascular surgery. Also, due to continued improvements in prenatal care, low birth weight, high-risk neonates may now survive lung and heart problems, only to die from complications of acute renal failure caused by infection or drug toxicity. Of particular clinical importance are cases of acute renal failure associated with trauma, sepsis, postoperative complications, or medication, particularly antibiotics. (National Center for Health Statistics, 1985, Table 1, National Institute of Health, 1990). Population data from the United States in 1985 further illustrate the nature of the problem. Acute renal failure was cited as a contributing cause in 26,922 deaths. The condition affects people of all ages, but those 65 years and older are almost five times more likely to be hospitalized for acute renal failure than those ages 45 to 64. Nearly two-thirds of all hospitalizations for acute renal failure occur in persons 65 years and older. Of those in that age group, black Americans were nearly twice as likely as white Americans to be hospitalized for acute renal failure. Acute renal failure is the most costly kidney or urologic condition requiring hospitalization. In 1985 there were 139,134 hospitalizations for the disease at a cost of $1.3 billion, or $9,329 per hospital discharge. In recent years there has been an increase in cases of acute renal failure, which can be attributed in part to medical progress. Most cases today result from the ability to perform complicated surgery in older patients, which can lead to post-operative complications, and the use of complex drugs such as antibiotics that successfully overcome previously fatal diseases. Unfortunately, these same drugs can be toxic to the kidneys, particularly in elderly persons. Because of the increasing age of the hospital population and advances in complicated medical and surgical techniques, cases of acute renal failure are expected to increase still more in number and significance unless significant advances in treatment modalities are made. b. Treatment Modalities for Kidney Disease are Inadequate Some advances have been made in understanding the pathophysiology of acute renal failure, including the toxicity of drugs to the kidney and the effects of oxygen deficit and reintroduction. Current treatment of the disorder depends on recognition of the underlying causes. Rapid fluid resuscitation of trauma and burn victims undoubtedly has prevented some cases. Carefully monitored administration of nephrotoxic drugs also has the potential to reduce the incidence of acute renal failure. Dialysis may be required to prevent death due to accumulating waste products or from fluid overload or chemical imbalance. Despite some advances, the mortality rate associated with kidney disease still has not changed in many years. These treatment modalities have focussed on preventing further deterioration rather than promoting organ repair. The latter approach would benefit from harnessing cell growth and repair processes. c. Growth Factors to Harness Cell Growth and Repair Processes Growth factors produced by cultured cells have been identified which, when present in tissue culture medium, will enhance the rate of division of normal, untransformed cells. Identification, isolation and purification of autocrine growth factors, that is, factors produced by a specific cell which are capable of regulating that cell's growth and division, would have important clinical applications. Certain cells might be targeted for growth enhancement by use of specific factors without disrupting the growth rates of other types of cells. Mitchell et al. (1977), Roberts et al. (1981), and Stoker et al. (1971) isolated growth stimulating proteins from animal cells and transformed cells. Transformed cells are abnormal cells which are no longer responsive to temporal and behavioral growth limits on normal cells. There is an association between transformed and malignant cancer cells. In other reports, factors were merely suspected by observing cell behavior. Mordan and Toback (1984) attempted to determine why accelerated kidney growth was observed empirically by several investigators, when potassium (K) was relatively low. The hypothesis explored was that cell growth was subject to autocrine control. These authors developed an in vitro model to identify factors that mediate renal growth. Molecules of an apparent molecular weight greater than 12-14 kD and less than 30 kD were separated during dialysis of medium conditioned by cells while exposed to a concentration of potassium that was lower (3.2 mM) than control (5.4 mM). Aliquots of this dialysate were tested for their effect on cell proliferation. The effects of a previously reported growth inhibitor were also tested. However, the factors that stimulated growth in K.sup.+ deficient animals were neither identified nor isolated. Some growth enhancers and inhibitors were associated with transformed cells which cannot be assumed to be the same as those that control normal cells, because growth of transformed cells, many of which are malignant, is aberrant by definition. Sporn and Roberts (1985) speculated that abnormally growing cells such as transformed or malignant cells may owe their enhanced growth abilities to a growth factor. These authors hypothesized that proliferation of transformed fibroblasts is under autocrine control and that autocrine secretion occurred in cancer cells. The role of oncogenes in growth was also addressed by these authors. In discussing their hypothesis, growth factors referred to included some previously reported, transforming growth factor-type alpha (TGF-.alpha.), TGF-.beta., platelet-derived growth factor (PDGF), and bombesin. The rapid stimulation by serum of electrical and ionic membrane properties of cells, and the relationship of these effects to the initiation of DNA synthesis and cell division were investigated in neuroblastoma cells (derived from malignant tissue). This stimulation was observed to be accompanied by sodium influx. However, "A crucial but unresolved question in the study of growth control concerns the molecular mechanisms by which growth factors exert their mitogenic effect." (Moolenaar et al., 1981, p. 789.) On the negative side of cell growth control, Holley et al. (1980) isolated two high molecular weight growth inhibitors that reversibly arrested the growth of BSC-1 cells (non transformed epithelial cells of African green monkey kidney) in the G.sub.1 phase of the cell cycle. Medium conditioned by crowded BSC-1 cell cultures was said to contain several growth inhibitors. A "cryptic" growth factor activated by shaking or heating was suspected but not identified. No characteristics specific to a growth factor were presented. The authors summarized their findings as follows: "The results presented here demonstrated that extremely active growth inhibitors can be recovered from the culture medium of BSC-1 cells." p. 5992. The mechanism of inhibition was not identified, and could potentially be at any of a large number of steps in cell division. Furthermore, assuming these inhibitors would act to oppose as yet unidentified growth stimulatory factors, would be merely speculation. Several determinants of growth regulation have been identified which affect BSC-1 cells (Holley et al., 1977, 1978 a, b). These include growth factors and hormones generally present in normal serum and their receptors on the cell surface. Specific growth-stimulatory molecules contained in serum include vasopressin, glucagon, and epidermal growth factor (EGF) (Walsh-Reitz and Toback, 1983). The growth of BSC-1 cells in culture also appears to be regulated by the cellular production of several inhibitors. These include lactate, ammonium ion, and a secreted protein which has an M.sub.r of 24,000 (Holley et al., 1978). This inhibitor protein, which has been identified as transforming growth factor (TGF)-.beta.2, is active at very low concentrations (1 ng/ml) on epithelial cells grown in culture, impedes exit of cells from the G.sub.0 /G.sub.1 (resting) phase of the cell cycle, and can be overcome by the stimulatory action of serum or EGF (Holley et al., 1978; Tucker et al., 1984; Hanks et al., 1988). This inhibitor protein appears to exert its growth inhibitory effect by interfering with cell sodium Na flux (Walsh-Reitz et al., 1984). Although the substratum upon which cells grow in culture is often considered a critical determinant of replication and differentiation (Martin et al, 1983; Rabito et al., 1980; Gospodarowicz et al., 1984), the contribution of collagen, fibronectin and other extracellular factors to the growth of BSC-1 cells has not yet been defined in detail. d. The Roles of Growth Factors in Growth and Repair of the Urological System Have Not Been Established Whereas no growth factor specific to kidney tissue has been previously described, several factors initially described in other types of cells have also been identified in kidney tissue or have been shown to have actions on renal cells in culture. Growth factors isolated from kidney tissue include EGF, TGF-.alpha.and .beta., platelet-derived growth factor (PDGF), insulin-like growth factors (IGF)-I and II, acidic and basic fibroblast growth factors (FGF), and interleukin-1 (Mendley and Toback, 1989). EGF is found in abundance in urine and renal cyst fluid (Gattone et al., 1990), and its precursor protein, preproEGF, is made by cells of the distal nephron in the mouse (Atkin et al., 1990). TGF-.beta.1 has been isolated from bovine kidney (Roberts et al., 1983), and TGF-.beta.2 has been reported to be an autocrine growth inhibitor for kidney epithelial cells in culture (Hanks et al., 1988). PDGF is produced by mesangial cells, stimulates mesangial cell contraction, and is an autocrine and paracrine mitogen for different types of renal cells (Silver et al., 1989). IGF-I and II are likely synthesized in the kidney, although their physiologic functions in the organ are unknown (Mendley and Toback, 1989). Both acidic and basic FGF are found in the kidney and may play a role in angiogenesis during renal embryonic development (Risau and Ekblom, 1986). The metabolism and excretion of growth factors in acute and chronic renal failure have not yet been studied in detail. Although most of the factors probably act locally, their presence has also been detected in the circulation and in urine. Some may be filtered at the glomerulus or require intact tubular cells for degradation and might accumulate progressively in the extracellular fluid as glomerular and tubular function diminish. In this scenario, cells of the injured kidney could be exposed to abnormal concentrations of growth factors and subjected to nonphysiologic growth-regulatory signals (Klahr et al., 1988; Kujubu and Fine, 1989). It was noted that renal epithelial cells responded to perturbations in the extracellular concentration of K or Na by initiating DNA synthesis (Walsh-Reitz et al., 1983, 1984, 1986). The approach taken in the present invention is to develop methods and compositions for the repair of kidney damage by stimulating kidney cells to grow and divide. The use of growth factors capable of stimulating renal cell growth as disclosed in the present specification offers new therapeutic avenues for treatment of kidney disease, and new insights into mechanisms of kidney damage. SUMMARY OF THE INVENTION This invention relates to the identification, purification and manipulation of autocrine growth factors which are capable of stimulating cells to grow and the use of inhibitors of the autocrine growth factors to control cell division. The autocrine factors of the present invention are further defined as having an apparent molecular weight of approximately 3,000-10,000 daltons as determined by SDS-polyacrylamide gel electrophoresis and ultrafiltration, and relative resistance to inhibition by dithiothreitol (DTT). The present invention relates more particularly to the identification, purification and manipulation of autocrine growth factors having an apparent molecular weight of approximately 6,000-7,000 daltons as determined by purification by reversed-phase HPLC and SDS-polyacrylamide gel electrophoresis. This invention provides a means for attacking the serious problems associated with acute renal failure and urological disease which are costing millions of dollars, resulting in time extensive lost from human lives, and causing large numbers of deaths in the United States. The autocrine growth factors of the present invention have been derived from non-transformed cells placed in tissue culture medium having a low sodium concentration by methods which will be described in subsequent sections. Consequently, the autocrine growth factors have been designated by the term "LSGF", that is "low sodium growth factors." This designation refers to the initial method that identified these autocrine growth factors. It does not imply any mechanisms of action for the growth factors, nor does it limit the scope of the compositions of the present invention to growth factors prepared by use of low sodium concentration. Because sodium influx is an important early signal during the onset of mitogenesis in many types of cells, it was unexpected that a decrease in extracellular Na.sup.+ concentration would enhance cell proliferation. In an illustrative embodiment, the two autocrine growth factors comprise an amino acid composition as shown in Table 1. Another characteristic of this embodiment is a biological activity of up to 2.6.times.10.sup.6 activity units per mg of protein (Table 2). One activity unit of biological activity has been defined as an increased cell number of .about.25-30% over a period of time. The effect of the autocrine growth factors on epithelial cells in culture is to enhance their growth by stimulating the cultures. Biological activity is defined as the response of cells in culture to the addition of autocrine growth factors. This invention is also directed to antibodies which are directed against the autocrine growth factors described in the present application, in particular to monoclonal antibodies. These antibodies, when complexed with the growth factors, provide a means of controlling cell growth by acting as inhibitors. With regard to the apparent molecular weights of the autocrine growth factors, they fall in the range 3,000-10,000 daltons which distinguishes them from previously reported autocrine growth factors. In particular, the apparent molecular weight of these autocrine growth factors as determined by SDS-polyacrylamide gel electrophoresis is approximately 6,000-7,000. The autocrine growth factors are further characterized as having an amino acid sequence of approximately 54 amino acids. However, these ranges are flexible. A method of preparing autocrine growth factors comprises the steps of 1) placing cells in medium containing a low-sodium concentration; 2) allowing the cells to remain in the low-sodium concentration environment until an autocrine growth factor is released; and 3) collecting the autocrine growth factor. In particular embodiments, the low-sodium concentration environment is further defined as a buffer having a sodium concentration of less than 150 millimolar and a pH range of about 6.6-8.2. In yet more specific embodiments, the sodium concentration of the buffer is further defined as about 120-130 millimolar. The method is generally accomplished by allowing the cells to remain in the low-sodium concentration environment for about 3-5 minutes, although longer times are within the scope of this invention. Determination of the times appropriate for collection of the growth factors from any particular cell type is made by simple calibrations of the time necessary to achieve a desired level of cell enhancement in terms of increased cell numbers. Cultured cells generally divide about every 24 hours, so calibration is straightforward and is not a time consuming process. Another aspect of this invention is the coding domain in the nucleic acid segment for autocrine growth factors as defined herein, or their biologically functional equivalents. In context, the biologically functional equivalents are defined as those nucleic acid segments that, because of codon equivalency, are capable of coding for essentially the same biologically functional autocrine growth factors. Biological function is defined as stimulation of epithelial cell division. The nucleic acid segments are further defined as DNA segments. TABLE 1 __________________________________________________________________________ AMINO ACID COMPOSITION OF 25-MINUTE AND 23-MINUTE LSGF ISOFORMS AND FIVE OTHER POLYPEPTIDE GROWTH FACTORS.sup.1 Monkey LSGF Mouse Human Human Human Human Amino Acid 25-min 23-min EGF TGF-.alpha. Insulin IGF-I IGF-II __________________________________________________________________________ Asp/Asn 5 3 4/3 4/1 0/3 4/1 3/0 Glu/Gln 7 16 2/1 2/2 4/3 4/2 6/1 Cys 1 1 6 6 6 6 6 Ser 8 7 6 3 3 5 7 Gly 10 9 6 3 4 7 5 His 1 1 1 5 2 0 0 Thr 2 2 2 2 3 3 4 Ala 3 3 0 4 1 6 5 Arg 3 2 4 2 1 6 8 Pro 2 2 2 2 1 5 3 Tyr 1 0 5 1 4 3 3 Val 2 2 2 5 4 3 4 Met 2 0 1 0 0 1 0 Ile 2 1 2 0 2 1 1 Leu 2 2 4 3 6 6 6 Phe 1 1 0 4 3 4 4 Lys 2 2 0 1 1 3 1 Trp ND ND 2 0 0 0 0 Mr .about.6500 .about.6500 6045 5600 5733 7649 7471 # residues .about.54 .about.54 53 50 51 70 67 __________________________________________________________________________ Abbreviations: LSGF = low sodium growth factor; EGF = epidermal growth factor; TGF.alpha. = transforming growth factortype alpha; IGF insulinlik growth factor; ND = not done .sup.1 Savage et al. (1973) ; Derynck et al. (1984; Rinderknecht and Humbel (1978). Methods of preparing the autocrine growth factors and of testing their growth enhancement abilities generally employ epithelial cells, in particular those derived from kidneys. Kidney cells from both the canine kidney and the African green monkey kidney cell line have been used successfully both to prepare the autocrine growth factor activity by placing cells in low-sodium concentration growth medium, as well as using the cells as an assay to test the biological activity as measured by cell growth enhancement. The observation that at least two completely different species respond in this fashion and produce the growth factors, indicates that this is a growth factor that is likely to be conserved. Other cell types likely to yield factors include pig kidney line LLC-PK.sub.1, and primary cultures of diploid rabbit proximal tubular cells. The invention also relates to amino acid receptor sequences which bind to the autocrine growth factors, and to antibodies directed to the amino acid sequences of these receptors, in particular monoclonal antibodies. This invention also relates to other inhibitors of the receptors. The inhibitors provide a negative means of controlling cell growth. In certain applications it is useful to clone the gene coding for the autocrine growth factors of the present invention. Three different cloning strategies are within the scope of the present invention; (i) if the sequence of a 25 amino acid fragment of LSGF is obtained, the mixed oligonucleotides primed amplification of cDNA (MOPAC) procedure to generate a probe is used to screen a BSC-1 cell cDNA library in lambda gt10 (Lee et al., 1988); (ii) if a peptide sequence of less than 25 amino acids is defined, degenerate oligonucleotides are generally synthesized and used to screen a BSC-1 cDNA library in lambda gt10; or (iii) a polyclonal monospecific rabbit antiserum (prepared as described herein) raised against purified LSGF is used to screen a BSC-1 cDNA library in lambda gt11. Nucleic acid segments comprising nucleotide sequences which correspond to segments at least 14 nucleotide bases long which are capable of hybridizing to a nucleic acid segment capable of coding for at least the growth enhancement region of the autocrine growth factors of the present invention under stringent conditions, are other aspects contemplated for the present invention. These selected segments of the nucleic acid sequence, together with appropriate promoters and enhancers, may be incorporated into plasmids and other vectors to determine the actions and interactions of their genetic regions, their expression products, and mechanisms for control over their expression. Small segments such as these may be used as probes to detect the presence of the autocrine growth factor coding regions in various species. The nucleic acid segments may also be messenger RNA sequences for the autocrine growth factors. Nucleic acid segments may be carried in recombinant expression vectors capable of expressing the autocrine growth factors when present in a host cell. Examples of such nucleic acid segments are those derived from the coding region for the autocrine growth factors. These nucleic acid segments may be operably linked to heterologous components in various plasmids or their functional equivalents. These components may include promoters such as those capable of controlling the host cell gene expressions or their functional equivalents. The recombinant expression vectors may comprise as a promoter either a eurokaryotic or a prokaryotic promoter, and may include a polyadenylation signal at position 3' of the carboxyl terminal amino acid. The promoter may be within a transcriptional unit of the encoded protein. Examples of host cells are BHK cells, VERO cells, E. coli, or other eurokaryotic or prokaryotic cells known to those of skill in the art to permit expression of transferred vectors according the present invention. In an illustrative embodiment, the method of preparing an autocrine growth factor includes use of a host cell into which a nucleic acid segment has been transferred. The host cell is cultured under conditions suitable for expression, and the protein is thereby expressed. The method may also include a step wherein the autocrine growth factor is isolated and purified by methods well known to those of skill in the art. The degree of purification required will depend on the application for which the protein is intended. Alternatively, the nucleic acid segment may be expressed in a cell-free system such as a rabbit lysate, or the protein encoded by the nucleic acid segment synthesized by an automated protein synthesizer. Some of the methods of preparing autocrine growth factors make use of the nucleic acid segments described herein and their expression in selected host cells to produce the autocrine growth factors. A method for regenerating kidney tissue comprises the following steps: 1) preparing an autocrine growth factor as disclosed herein; 2) adding a pharmacologically acceptable carrier diluent to the growth factor preparation; and 3) contacting the kidney tissue with the growth factor-carrier composition. A therapeutic amount of the composition is administered. It is contemplated that the route of delivery of this combination to the tissue could be by intravenous infusion, localized injections or implants. Alternatively, damaged kidneys could be removed, treated in vitro, and returned to the host after the kidney is repaired. In other aspects of the present invention, inhibitors of the autocrine growth factors are disclosed. These inhibitors may be antibodies directed to the autocrine growth factors or any other inhibitors selected by the following method from a series of candidate inhibitor substances. To determine the ability of a candidate substance to inhibit the autocrine growth factor enhancing activity, autocrine growth factors are prepared and combined with the candidate inhibitor. Epithelial cells in culture are then contacted with the combination and it is determined whether there is reduced cell growth enhancement compared to that enhancement obtained by use of the composition without the inhibitor |
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