Information
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Patent Application
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20030036633
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Publication Number
20030036633
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Date Filed
October 22, 200122 years ago
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Date Published
February 20, 200321 years ago
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Inventors
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Original Assignees
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CPC
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US Classifications
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International Classifications
- C07K014/705
- C12P021/02
- C12N005/06
- C07H021/04
Abstract
DNAs encoding bradykinin B1 receptors from mammalian cells have been cloned and characterized. The recombinant receptor is capable of forming receptors which bind des-Arg10 kallidin and other B1-specific ligands. The DNA has been expressed in recombinant host cells which produce active recombinant protein. In addition, the recombinant host cells are utilized to establish a method for identifying modulators of the receptor activity, and receptor modulators are identified.
Description
BACKGROUND OF THE INVENTION
[0001] Two mammalian bradykinin receptor subtypes, B1 and B2, have been defined based on their pharmacological properties (A. Dray, M. Perkins, TINS 16, 99-104 (1993), D. Proud, A. P. Kaplan, Annual Review Immunology 6, 49-83 (1988)). The nonapeptide bradykinin (BK) and the decapeptide Lys-BK (kallidin) are liberated from the large protein precursor kininogen by the proteolytic action of kallikreins. Because of their rapid degradation, these peptide hormones are presumed to mediate local effects. BK and kallidin both activate the B2 receptor. These B2 receptor agonists are then degraded by a carboxypeptidase to produce the B1 receptor agonists des-Arg9BK and des-Arg10kallidin or by the angiotensin converting enzyme (ACE) to yield inactive peptides. The phenomenon of the proteolytic transformation of a compound from B2 to B1 selectivity has been observed not only for the endogenous kinin agonists but also for several synthetic peptide antagonists (K. Wirth, et al., European Journal of Pharmacology 205, 217-218 (1991), D. Regoli, et al., European Journal of Pharmacology 127, 219-224 (1986)). BK and kallidin act as equipotent agonists at the B2 bradykinin receptor subtype. In contrast, BK is totally inactive at the B1 bradykinin receptor subtype. Although traditionally des-Arg9BK has been utilized to study the B1receptor, the most potent agonist for the B1 receptor appears to be des-Arg10kallidin (D. Regoli, et al., European Journal of Pharmacology 127, 219-224 (1986)).
[0002] Bradykinin and kallidin, acting through the B2 receptor present in smooth muscle and certain neurons, cause pronounced hypotension and are potent mediators of pain and inflammation (A. Dray, supra; D. Proud, supra). In contrast, the B1 receptor is not detected in most normal tissues and appears to act predominantly in pathophysiological conditions (A. Dray, supra). The B1 receptor was originally discovered through a contractile response to des-Arg9BK that was observed in rabbit aortic strips only after a prolonged ex vivo incubation (D. Regoli, J. Barabe, W. K. Park, Canadian Journal of Physiology and Pharmacology 55, 855-867 (1977); D. Regoli, F. Marceau, J. Barabe, Canadian Journal of Physiology and Pharmacology 56, 674-677 (1978); D. Regoli, J. Barabe, Pharmacological Reviews 32, 1-46 (1980)). The de novo synthesis of B1 receptors has been reported in vivo following treatment with bacterial lipopolysaccharide (LPS) (D. C. Regoli, R. Marceau, J. Lavigne, European Journal of Pharmacology 71, 105-115 (1981)), and in animal models of antigen arthritis (S. G. Farmer, B. A. McMillan, S. N. Meeker, R. M. Burch, Agents and Action 34, 191-193 (1991)). In vitro studies have implicated a number of cytokines, most notably interleukin-1 (IL-1) and IL-2, as mediators that induce the expression of B1 receptors (D. Regoli, supra; D. Deblois, J. Bouthillier, F. Marceau, British Journal of Pharmacology 93, 969-977 (1988); D. deBlois, J. Bouthillier, F. Marceau, British Journal of Pharmacology 103, 1057-1066 (1991); D. deBlois, J. Bouthillier, R. Marceau, Immunopharmacology 17, 187-198 (1989)). These results, in conjunction with the finding that activation of a B1 bradykinin receptor on mouse macrophages causes the release of cytokines (R. M. Burch, J. R. Connor, C. W. Tiffany, Agents and Action 27, 258-260 (1989); C. W. Tiffany, R. M. Burch, FEBS letters 247, 189-192 (1989)), suggest that the B1 receptor could be an important mediator of chronic inflammation. Significantly, the B1 bradykinin receptor antagonist des-Arg9[Leu8]BK was recently found to alleviate hyperalgesia in animal models of persistent inflammation (A. Dray, supra; M. N. Perkins, D. Kelly, British Journal of Pharmacology 110, 1441-1444 (1993); M. N. Perkins, E. Campbell, A. Dray, Pain 53, 191-197 (1993)). Thus, a body of evidence implicates the B1 bradykinin receptor in the pathophysiology of inflammation. Relatively little is known about the role for the B1 receptor in healthy tissues, although both B1 and B2 receptors may play a physiological role in renal function (N. -E. Rhaleb, et al., European Journal of Pharmacology 162,419-427 (1989); M. Lortie, D. Regoli, N. -E. Rhaleb, G. E. Plante, American Journal of Physiology 262, R72-R76 (1992)). The apparent inducibility of the B1 receptor under pathological conditions may provide a therapeutic window for the use of B1 receptor antagonists in treating chronic inflammation.
[0003] The cloning of the B2 bradykinin receptor revealed that this receptor is a member of the superfamily of G-protein coupled receptors (A. E. McEachern, et al., Proc. Natl. Acad. Sci. 88, 7724-7728 (1991); S. J. Powell, et al., Genomics 15, 435-438 (1993); J. F. Hess, J. A. Borkowski, G. S. Young, C. D. Strader, R. W. Ransom, Biochem. and Biophys. Res. Comm. 184, 260-268 (1992); D. Eggerickx, E. Raspe, D. Bertrand, G. Vassart, M. Parmentier, Biochem. Biophys. Res. Comm. 187, 1306-1313 (1992)). The rat B2 bradykinin receptor was cloned using a Xenopus oocyte expression system (A. E. McEachern, et al., supra) that exploited the ability of the B2 receptor to act through G-proteins to activate phospholipase C and mobilize Ca2+ (R. M. Burch, J. Axelrod, Proc. Natl. Acad. Sci. 84, 6374-6378 (1987); S. R. Slivka, P. A. Insel, J. Biol. Chem 263, 14640-14647 (1988)). Recently, the B1 bradykinin receptor in rabbit aorta and rat mesangial cultured cells has also been shown to activate phospholipase C leading to Ca2+ mobilization (K. A. Schneck, supra; M. Issandou, J. -M. Darbon, Journal of Biological Chemistry 259, 9263-9268 (1991); M. M. Tropea, D. Gummelt, M. S. Herzig, L. M. F. Leeb-Lundberg, Journal of Pharmacology and Experimental Therapeutics 264, 930-937 (1993)). Furthermore, both B1 and B2 bradykinin receptor activities were detected when mRNA from the human fibroblast cell line WI-38 was injected into Xenopus laevis oocytes (E. Phillips, M. J. Conder, S. Bevan, P. McIntyre, M. Webb, Journal of Neurochemistry 58, 243-249 (1992). The similarity of ligands for the two bradykinin receptor subtypes suggests a similarity between the B1 and B2 receptor genes. However, the results of genomic Southern analyses indicated that these two receptors are not highly homologous (A. E. McEachern, et al., supra; J. F. Hess, et al., Molecular Pharmacology 45, 1-8 (1994). Therefore, in order to clone the human B1 receptor, we pursued an expression cloning strategy in Xenopus oocytes utilizing the photoprotein aequorin as an indicator of Ca2+ mobilization (K. Sandberg, A. J. Markwick, D. P. Trinh, K. J. Catt, FEBS Letters 241, 177-180 (1988); E. Giladi, E. R. Spindel, BioTechniques 10, 744-747 (1991). We isolated a cDNA clone that encodes a G-protein coupled receptor with an amino acid sequence that is 36% identical to that of the B2 bradykinin receptor. The pharmacological properties of this cloned receptor expressed in mammalian cells demonstrate that it is a B1 bradykinin receptor.
SUMMARY OF THE INVENTION
[0004] The bradykinin B1 receptor has been cloned, expressed and characterized. Using a recombinant expression system functional DNA clones encoding the receptor have been isolated. The pharmacological and structural properties of the protein are disclosed, as are the amino acid and nucleotide sequences. The recombinant protein is useful to identify modulators of the receptor. Modulators identified in this process are useful as therapeutic agents. Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005]
FIG. 1—The DNA sequence of the human bradykinin receptor B1 is shown.
[0006]
FIG. 2—The amino acid sequence of the human bradykinin receptor B1 is shown.
[0007]
FIG. 3—Relative light units are presented in response to 100 nM bradykinin (open bars) and 20 nM des-Arg10kallidin (closed bars) with increasing fraction number (decreasing in size). Oocytes were injected with an equal volume of sucrose-gradient size fractionated IL-1B induced IMR-90 poly(A)+ mRNA and challenged with bradykin agonists. The relative light units obtained with 100 nM bradykinin were divided by 50 to allow plotting both data sets on the same graph. A trace indicating the RNA size markers that were fractionated in a parallel gradient is shown in the upper portion of the figure.
[0008]
FIG. 4 panels A and B—Luminometer tracings of individual oocytes in response to 20 nM des-Arg10kallidin are shown. Panel A shows the signal obtained using total poly(A)+ mRNA isolated from IL-1β induced IMR-90 cells. Panel B shows the signal obtained using clone 33E9 cRNA.
[0009]
FIG. 5—Displacements of 1 nM [3H]des-Arg10kallidin from COS-7 cells expressing the human B1 bradykinin receptor by increasing concentrations of compounds are shown. Symbols for compounds: des-Arg10kallidin (q), des-Arg10[Leu9]kallidin (Δ), kallidin (*), HOE 140 (u), des-Arg9 bradykinin (O), and bradykinin (X).
DETAILED DESCRIPTION
[0010] The present invention relates to DNA encoding a bradykinin B1 receptor (B 1) which is isolated from B1 producing cells. B1, as used herein, refers to protein which can specifically function as the B1 bradykinin receptor subtype.
[0011] The amino acid and DNA sequences of B1 were not previously known. Cells capable of producing B1 include, but are not limited to, aortic smooth muscle cells. Cell lines which may produce B1 include, but are not limited to, IMR-90 and WI-38. The preferred cells for the present invention are IMR-90 cells.
[0012] Other cells and cell lines may also be suitable for use to isolate B1 cDNA. Selection of suitable cells may be done by screening for B1 activity in whole cells or cell extracts. Methods for detecting B1 activity are known in the art and either measure the ability to bind ligands known to interact at the B1 receptor subtype, such as des-Arg10kallidin, des-Arg9 bradykinin, des-Arg10Leu9kallidin, and des-Arg9Leu8 bradykinin, or measure phosphatidyl inositol hydrolysis, release of intracellular Ca2+ stores, or arachidonic acid release mediated by des-Arg10kallidin or des-Arg9 bradykinin. Cells which possess B1 activity in such assays may be suitable for the isolation of B1 DNA.
[0013] Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the B1 sequence but will be capable of hybridizing to B1 DNA even in the presence of DNA oligonucleotides with mismatches under appropriate conditions. Under alternate conditions, the mismatched DNA oligonucleotides may still hybridize to the B1 DNA to permit identification and isolation of B1 encoding DNA.
[0014] DNA encoding B1 from a particular organism may be used to isolate and purify homologues of B1 from other organisms. To accomplish this, the first B1 DNA may be mixed with a sample containing DNA encoding homologues of B1 under appropriate hybridization conditions. The hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified therefrom.
[0015] It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
[0016] It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
[0017] As used herein, a “functional derivative” of B1 is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of B1. The term “functional derivatives” is intended to include the “fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to “chemical derivatives” of B1. The term “fragment” is meant to refer to any polypeptide subset of B1. The term “variant” is meant to refer to a molecule substantially similar in structure and function to either the entire B1 molecule or to a fragment thereof. A molecule is “substantially similar” to B1 if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
[0018] The term “analog” refers to a molecule substantially similar in function to either the entire B1 molecule or to a fragment thereof.
[0019] Any of a variety of procedures known in the art may be used to molecularly clone B1 DNA. These methods include, but are not limited to, direct functional expression of the B1 genes following the construction of a B1-containing cDNA or genomic DNA library in an appropriate expression vector system. Another method is to screen B 1-containing cDNA or genomic DNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled oligonucleotide probe designed from the amino acid sequence of the B1 subunits. An additional method consists of screening a B1-containing cDNA or genomic DNA library constructed in a bacteriophage or plasmid shuttle vector with a partial DNA encoding the B1 receptor. This partial DNA is obtained by the specific PCR amplification of B1 DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified B1 receptor. Another method is to isolate RNA from B1-producing cells and translate the RNA into protein via an in vitro or an in vivo translation system. The translation of the RNA into a peptide or a protein will result in the production of at least a portion of the B1 protein which can be identified by, for example, by the activity of B1 receptor protein or by immunological reactivity with an anti-B1 antibody. In this method, pools of RNA isolated from B1-producing cells can be analyzed for the presence of an RNA which encodes at least a portion of the B1 protein. Further fractionation of the RNA pool can be done to purify the B1 RNA from non-B1 RNA. The peptide or protein produced by this method may be analyzed to provide amino acid sequences which in turn are used to provide primers for production of B1 cDNA, or the RNA used for translation can be analyzed to provide nucleotide sequences encoding B1 and produce probes for the production of B1 cDNA. These methods are known in the art and can be found in, for example, Sambrook, J., Fritsch, E. F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989.
[0020] It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating B1-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than IMR-90 cells, and genomic DNA libraries.
[0021] It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have B1 activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate B1 cDNA may be done by first measuring cell associated B1 activity using the ligand binding assay described fully herein.
[0022] Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, Sambrook, J., Fritsch, E. F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
[0023] It is also readily apparent to those skilled in the art that DNA encoding B1 may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Sambrook, J., Fritsch, E. F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
[0024] In order to clone the B1 gene by the above methods, the amino acid sequence of B1 may be necessary. To accomplish this, B1 protein may be purified and partial amino acid sequence determined by manual sequencing or automated sequenators. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from the protein is determined for the production of primers for PCR amplification of a partial B1 DNA fragment.
[0025] Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the B1 sequence but will be capable of hybridizing to B1 DNA even in the presence of DNA oligonucleotides with mismatches under appropriate conditions. Under alternate conditions, the mismatched DNA oligonucleotides may still sufficiently hybridize to the B1 DNA to permit identification and isolation of B1 encoding DNA. Purified biologically active B1 may have several different physical forms. B1 may exist as a full-length nascent or unprocessed polypeptide, or as partially processed polypeptides or combinations of processed polypeptides. The full-length nascent B1 polypeptide may be postranslationally modified by specific proteolytic cleavage events which result in the formation of fragments of the full length nascent polypeptide. A fragment, or physical association of fragments may have the full biological activity associated with B1 (specific binding of des-Arg10kallidin, and des-Arg9 bradykinin) however, the degree of B1 activity may vary between individual B1 fragments and physically associated B1 polypeptide fragments.
[0026] B1 in substantially pure form derived from natural sources or from recombinant host cells according to the purification processes described herein, is found to be a polypeptide encoded by a single mRNA. The B1 polypeptide was found to have an apparent molecular weight of about 40.4 kDa.
[0027] The cloned B1 DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant B1. Techniques for such manipulations are fully described in Sambrook, J., et al., supra, and are well known in the art.
[0028] Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and animal cells.
[0029] Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria-invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
[0030] A variety of mammalian expression vectors may be used to express recombinant B1 in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant B1 expression, include but are not limited to, pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and XZD35 (ATCC 37565).
[0031] A variety of bacterial expression vectors may be used to express recombinant B1 in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant B1 expression include, but are not limited to pET11a (Novagen), lambda gt11 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
[0032] A variety of fungal cell expression vectors may be used to express recombinant B1 in fungal cells. Commercially available fungal cell expression vectors which may be suitable for recombinant B1 expression include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen).
[0033] A variety of insect cell expression vectors may be used to express recombinant B1 in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of B1 include but are not limited to pBlue Bac III (Invitrogen).
[0034] An expression vector containing DNA encoding B1 may be used for expression of B1 in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK−) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
[0035] The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce B1 protein. Identification of B1 expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-B1 antibodies, and the presence of host cell-associated B1 activity, such as B1-specific ligand binding or signal transduction defined as a response mediated by the interaction of B1-specific ligands at the receptor.
[0036] Expression of B1 DNA may also be performed using in vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolated from B1 producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
[0037] To determine the B1 DNA sequence(s) that yields optimal levels of B1 activity and/or B1 protein, B1 DNA molecules including, but not limited to, the following can be constructed: the full-length open reading frame of the B1 DNA (encoding a protein of about 40.4 kDa =about base 209 to about base 1267) and several constructs containing portions of the DNA encoding B1 protein. All constructs can be designed to contain none, all or portions of the 5′ or the 3′ untranslated region of B1 DNA. B1 activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the B1 DNA cassette yielding optimal expression in transient assays, this B1 DNA construct is transferred to a variety of expression vectors, for expression in host cells including, but not limited to, mammalian cells, insect cells, E. coli, and yeast cells such as S. cerevisiae.
[0038] Host cell transfectants and microinjected oocytes may be assayed for both the levels of B1 receptor activity and levels of B1 protein by the following methods. One method for assessing B1 receptor activity involves the direct measurement of binding of specific ligands. In the case of recombinant host cells, this involves the transfection or co-transfection of plasmids containing the B1 DNA. In the case of oocytes, this involves the injection of synthetic or native RNAs for B1. Following an appropriate period of time to allow for expression, B1 activity is measured. One method for detecting B1 activity involves the direct measurement of B1 activity in whole cells or cellular lysates prepared from host cells transfected with B1 DNA or oocytes injected with B1 RNA. B1 activity is measured by the specific ligand binding characteristics of the B1 receptor or the response to calcium mobilization or phosphatidyl inositol hydrolysis mediated by B1-specific modulators in host cells expressing B1 DNA or RNA. Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
[0039] Following expression of B1 in a recombinant host cell, B1 protein may be recovered to provide B1 in purified form. Several B1 purification procedures are available and suitable for use. As described herein, recombinant B1 may be purified from cell lysates and extracts by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
[0040] In addition, recombinant B1 can be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full length nascent B1, or polypeptide fragments of B1.
[0041] Monospecific antibodies to B1 are purified from mammalian antisera containing antibodies reactive against B1 or are prepared as monoclonal antibodies reactive with B1 using the technique of Kohler and Milstein, Nature 256, 495-497 (1975). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for B1. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the B1, as described above. B specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of B1 either with or without an immune adjuvant.
[0042] Preimmune serum is collected prior to the first immunization. Each animal receives between about 0.1 μg and about 1000 μg of B1 associated with an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The initial immunization consists of B1 in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about −20° C.
[0043] Monoclonal antibodies (mAb) reactive with B1 are prepared by immunizing inbred mice, preferably Balb/c, with B1. The mice are immunized by the IP or SC route with about 0.1 μg to about 10 μg, preferably about 1 μg, of B1 in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 μg of B1 in a buffer solution such as phosphate buffered saline by the intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 molecular weight, at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using B1 as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.
[0044] Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press, 1973.
[0045] Monoclonal antibodies are produced in vivo by injection of pristane primed Balb/c mice, approximately 0.5 ml per mouse, with about 2×106 to about 6×106 hybridoma cells about 4 days after priming. Ascites fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
[0046] In vitro production of anti-B1 mAb is carried out by growing the hydridoma in DMEM containing about 2% fetal calf serum to obtain sufficient quantities of the specific mAb. The mAb are purified by techniques known in the art.
[0047] Antibody titers of ascites or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, precipitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of B1 in body fluids or tissue and cell extracts.
[0048] It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for B1 polypeptide fragments, or full-length nascent B1 polypeptide. Specifically, it is readily apparent to those skilled in the art that monospecific antibodies may be generated which are specific for the fully functional receptor or fragments thereof.
[0049] B1 antibody affinity columns are made by adding the antibodies to Affigel-10 (Biorad), a gel support which is activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing B1 are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). The purified B1 protein is then dialyzed against phosphate buffered saline.
[0050] The human embryonic fibroblast cell line IMR-90 had been shown previously to express the B1 bradykinin receptor subtype (R. H. Goldstein, M. Wall, Journal of Biological Chemistry 259, 9263-9268 (1984). A more detailed pharmacological characterization revealed the presence of approximately 5000 high affinity binding sites for the B1 agonist [3H]des-Arg10kallidin and approximately 70,000 high affinity binding sites for the B2 agonist [3H]bradykinin per cell. The ability of the B1 receptor expressed in IMR-90 cells to mobilize Ca2+ in response to des-Arg10kallidin was demonstrated using FURA-2 as an indicator. Furthermore, treatment of IMR-90 cells with the cytokine IL-1β was found to stimulate the number of B1 bradykinin receptors approximately 7-fold. Based on these data IL-1β induced IMR-90 cells were used as the source of mRNA for expression cloning.
[0051] A cDNA clone encoding a human B1 bradykinin receptor was isolated from an IMR-90 fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist des-Arg10kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with IMR-90 derived RNA. A single clone was isolated with an approximately 1307 bp insert which contains an open reading frame encoding an approximately 353 amino acid protein with the characteristics of a G-protein coupled receptor. The amino acid sequence of the B1 bradykinin receptor is about 36% identical to the amino acid sequence of the B2 bradykinin receptor. The B1 bradykinin receptor expressed in COS-7 cells exhibits high affinity binding for [3H]des-Arg10kallidin and low affinity for bradykinin. The B1 receptor antagonist des-Arg10[Leu9]kallidin effectively displaces [3H]des-Arg10kallidin from the cloned receptor, whereas, the B2 receptor antagonist HOE 140 (Hock, F.J. et al. British Journal of Pharmacology 102, 769-773 (1991)) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The B1 bradykinin receptor has been implicated in chronic inflammation and hyperalgesia, whereas the B2 receptor appears to mediate acute inflammatory and algesic responses. The availability of the cloned human B1 bradykinin receptor allows the identification of modulators useful in B1-mediated diseases and conditions such as hyperalgesia, as well as acute and chronic inflammatory processes.
[0052] The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding B1 as well as the function of B1 protein in vivo. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding B1, or the function of B1 protein. Compounds that modulate the expression of DNA or RNA encoding B1 or the function of B1 protein may be detected by a variety of assays. The assay may be a simple “yes/no” assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
[0053] Kits containing B1 DNA, antibodies to B1, or B1 protein may be prepared. Such kits are used to detect DNA which hybridizes to B1 DNA or to detect the presence of B1 protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses and epidemiological studies.
[0054] The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen and measure levels of B1 DNA, B1 RNA or B1 protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of B1. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant B1 protein or anti-B1 antibodies suitable for detecting B1. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
[0055] Nucleotide sequences that are complementary to the B1 encoding DNA sequence can be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2′-O-alkylRNA, or other B1 antisense oligonucleotide mimetics. B1 antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. B1 antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce B1 activity.
[0056] B1 gene therapy may be used to introduce B1 into the cells of target organs. The B14 gene can be ligated into viral vectors which mediate transfer of the B1 DNA by infection of recipient host cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like.
[0057] Alternatively, B1 DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo B1 gene therapy. B1 gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate B1 activity.
[0058] Pharmaceutically useful compositions comprising B1 DNA, B1 RNA, or B1 protein, or modulators of B1 receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modulator.
[0059] Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose B1 related disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
[0060] The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
[0061] The term “chemical derivative” describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
[0062] Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the B1 receptor or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.
[0063] The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of B1 receptors can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as a B1 modulating agent.
[0064] The daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult human/per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. The dosages of the B1 receptor modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
[0065] Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
[0066] For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.
[0067] The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
[0068] In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
[0069] For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
[0070] For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
[0071] Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
[0072] The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
[0073] Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
[0074] The following examples illustrate the present invention without, however, limiting the same thereto.
EXAMPLE 1
[0075] RNA Isolation
[0076] Oocyte Injections:
[0077] Injection of mRNA or CRNA into Xenopus oocytes was done by a modification of established protocols (A. Coleman, Transcription and Translation: A Practical Approach. B. D. Hanes, S. J. Higgins, Eds., 1984); Y. Masu, et al., Nature 329, 836-838 (1987)). Oocytes were surgically removed from either albino or pigmented 0. 17% tricaine anesthetized Xenopus laevis (Xenopus One). The excised ovarian lobes were teased apart with jeweler's forceps and then placed into OR-2 (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES pH 7.4) containing 2 mg/ml collagenase B (Boehringer Mannhiem) for 2 hours at room temperature with a change of the buffer at 1 hour. Isolated oocytes were repeatedly washed in OR-2 until the supernatant remained clear. Stage 5 and 6 oocytes were selected and cultured overnight in supplemented OR-2 (OR-2 containing 1.8 mM CaCl2, 0.5 mg/ml gentamycin and 0.5 mM theophylline). Initially, oocytes were injected with 46 nl of RNA at a concentration of 1 or 2 mg/ml in H2O, once the pool size became less than 30 clones the cRNA concentration was decreased to 40 ng/ml. RNA was injected using a Nanoject automatic oocyte injector (Drummond Scientific) and injection needles were pulled from 3.5″ Drummond capillaries using a Flaming/Brown Micropipette puller (Sutter Instruments). Two to three days after the RNA injection, oocytes were injected with 92 ng of aequorin (Friday Harbor Photoproteins) resuspended in 46 nl of 1 mM EDTA, as previously described (K. Sandberg, A. J. Markwick, D. P. Trinh, K. J. Catt, FEBS Letters 241, 177-180 (1988); E. Giladi, E. R. Spindel, BioTechniques 10, 744-747 (1991)). The following day, individual oocytes placed in wells of a microtiter dish containing 225 μl OR-2 were challenged with peptide agonists and the aequorin photo response was measured using a ML3000 Microtiter plate luminometer (Dynatech). Oocytes were challenged with several peptides (Peninsula Laboratories) as described in the text and figure legends.
[0078] Injection of poly(A)+ mRNA prepared from IL-1β induced IMR-90 cells into Xenopus laevis oocytes resulted in aequorin mediated luminescence in response to either the B1 agonist des-Arg10kallidin or the B2 agonist BK (FIG. 3). The poly(A)+ mRNA was size-fractionated over a sucrose gradient, fractions were injected into oocytes, and the oocytes were assayed for their ability to respond to either BK or des-Arg10kallidin. The B1 and B2 receptor transcripts were clearly separated by the size fractionation. The mRNA mediating the response to des-Arg10kallidin exhibited an apparent size of 1.6-1.8 kb, whereas, the mRNA mediating the response to BK had an apparent size of 4.4-4.6 kb. The apparent size of the mRNA encoding bradykinin response is consistent with the previously determined size of the B2 bradykinin receptor transcript (A. E. McEachern, et al., Proc. Natl. Acad. Sci. 88, 7724-7728 (1991); J. F. Hess, et al., Molecular Pharmacology 45, 1-8 (1994)).
EXAMPLE 2
[0079] RNA Fractionation
[0080] IMR-90 cells (ATCC CCL 186) were grown in MEM supplemented with 10% fetal calf serum, glutamine, nonessential amino acids, sodium pyruvate, penicillin and streptomycin (Gibco). Two and a half hours prior to mRNA extraction, IMR-90 cells were exposed to 200 pg/ml IL-1β (R & D Systems). mRNA was purified from these cells using the PolyAtract mRNA isolation system (Promega) and resuspended in H2O at a concentration of 2 mg/ml.
[0081] IMR-90 mRNA was size fractionated on a continuous 6 to 20% sucrose gradient in 15 mM PIPES (pH 6.5), 5 mM EDTA and 0.25% N-lauroylsarcosine. 480 μg of mRNA from the IL-1β induced IMR-90 cells was heated to 65° C. for 3 minutes, quickly cooled on ice, and loaded onto the gradient. The RNA was size fractionated by centrifugation at 18° C. for 19 hours at 77,000× g. Fractions (450 μl) from each gradient were collected from the bottom of the tube and their absorbance at OD 260 monitored. Fractions were ethanol precipitated twice and resuspended to a final concentration of 1 μg/μl RNA sizes determination was based on the migration pattern of 80 μg of 9.49-0.24 Kb RNA markers (BRL) loaded on a parallel gradient.
[0082] Library Construction:
[0083] A peak RNA fraction from the sucrose gradient which gave a response to des-Arg10kallidin was utilized to generate a cDNA library. The library was constructed in the mammalian expression vector pcDNA3 using a combination of random hexamers and oligo dT to prime the first strand cDNA synthesis. First strand cDNA synthesis of approximately 3 μg of fraction 14 size-selected mRNA was primed with 50 ng random hexamers and 400 ng of a Not I oligo dT oligonucleotide and synthesized with the BRL Superscript II reverse transcriptase (Gibco-BRL). Following second strand synthesis Bst XI/Eco RI adaptors (Invitrogen) were ligated onto the ends and the cDNA was passed over a BRL cDNA sizing column (Gibco-BRL). The cDNA was cloned into the Bst XI site of pcDNA3 (Invitrogen, San Diego, Calif.) containing a modified polylinker. Plasmid DNA was transformed into XL-1 Blue cells (Stratagene). The library contained greater than 90% inserts with an average insert size of 1.9 kb.
[0084] The library was plated in pools of approximately 5000 clones that were used to synthesize cRNA using the T7 RNA polymerase. Colonies were plated on Colony/Plaque screen filters (NEN/Dupont) that were placed on Luria-Bertani (LB) agar plates supplemented with 100 ug/ml ampicillin (Sigma). Replica filters were made, the bacterial colonies were scraped in LB broth, and a frozen glycerol stock was prepared. Plasmid DNA was made using the Wizard DNA purification system (Promega). Plasmid DNA was linearized with Not I and cRNA was synthesized using T7 RNA polymerase with the mCAP RNA capping kit (Stratagene).
[0085] Of the twenty-five pools of cRNA that were injected into Xenopus oocytes, 11 exhibited aequorin-mediated luminescence in response to des-Arg10kallidin. The pool that gave the most robust response was replated and fractionated into 25 pools of approximately 800 clones. Eight pools exhibited a response to des-Arg10kallidin. The strongest positive pool was further examined using electrophysiology to monitor the Ca2+ sensitive Cl− channel. Des-Arg10kallidin produced a response that was blocked by the B1 receptor antagonist des-Arg10[Leu]9kallidin. This pool was then subdivided into 32 pools of approximately 25 individual clones. Two positive pools, containing 14 and 34 clones, were identified. cRNA was prepared from individual clones and analyzed in Xenopus oocytes. Three individual clones were found to elicit a des-Arg10kallidin response (FIGS. 4A&B). Restriction analysis, Southern blotting, and preliminary DNA sequence analysis indicated that these individual clones were similar. One clone, 33E9, was chosen for further DNA sequence analysis, expression, and pharmacological characterization. The DNA sequence of both strands was determined by either manual sequencing using Sequenase (USB) or automated sequencing using an ABI 373A sequencing unit (Perkin Elmer).
[0086] Primary Structure of the B1 Receptor
[0087] The DNA sequence of the approximately 1307 bp clone 33E9 contains an open reading frame of about 1059 bp (FIGS. 1 and 2). The proposed initiator methionine at nucleotide about 209 conforms to the Kozak sequence (M. Kozak, Journal of Cell Biology 108, 229-241 (1989)) at the +4 position but not at the −3 position. The approximately 208 nucleotide putative 5′ untranslated sequence contains a region of relatively high nucleotide identity to a wide variety of human DNA sequences, suggestive of a human repetitive element. The apparent repetitive element appears to be an authentic portion of the mRNA transcript since it was detected in several independently derived cDNA clones. The DNA sequence identity between the open reading frame in clone 33E9 and the DNA encoding the human B2 receptor (S. J. Powell, et al., Genomics 15, 435438 (1993); J. F. Hess, J. A. Borkowski, G. S. Young, C. D. Strader, R. W. Ransom, Biochem. and Biophys. Res. Comm. 184, 260-268 (1992); D. Eggerickx, E. Raspe, D. Bertrand, G. Vassart, M. Parmentier, Biochem. Biophys. Res. Comm. 187, 1306-1313 (1992)) is 54%. This is consistent with the inability of genomic Southern analyses to detect this gene (A. E. McEachem, et al., Proc. Natl. Acad. Sci. 88, 7724-7728 (1991); J. F. Hess, et al., Molecular Pharmacology 45,1-8 (1994)).
[0088] The proposed amino acid sequence encoded by the open reading frame is 36% identical to the B2 bradykinin receptor. A hydrophobicity plot of the amino acid sequence reveals the seven potential transmembrane spanning domains that are characteristic of G-protein coupled receptors (H. G. Dohlman, J. Thomer, M. G. Caron, R. J. Lefkowitz, Ann. Rev. Biochem. 60, 653-688 (1991); C. D. Strader, I. S. Sigal, R. A. F. Dixon, FASEB 3, 1825-1832 (1989)). Two conserved Cys residues that are proposed to form a disulfide bond between the first and second extracellular loops in nearly all G-protein coupled receptors are also present in the sequence. There are two potential N-linked glycosylation sites in the N-terminal domain of the sequence and one putative site for N-linked glycosylation in the second extracellular loop region. Potential protein kinase C phosphorylation sites are present in intracellular domain 2 and the carboxy-terminal tail. Similar potential phosphorylation sites in other G-protein coupled receptors have been implicated in short-term desensitization of the receptor following agonist stimulation.
EXAMPLE 3
[0089] Genomic DNA Cloning of the B1 Receptor
[0090] A human genomic DNA library is constructed in the cosmid sCOS vector using genomic DNA isolated from human foreskin fibroblast HSF42 cells (Chartrain, N. A., et al. Journal of Biological Chemistry 269, 6765-6772 (1994)). The recombinant DNA library constructed from genomic DNA of human cells is plated on Colony/Plaque screen hybridization transfer membrane (Dupont/NEN) at a density of approximately 30,000 colonies per plate. Replicas of master plates are lysed and processed for hybridization using standard protocols (Sambrook, J., Fritsch, E. F., Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). The DNA is UV crosslinked to the membrane with a Stratalinker (Stratagene). The filters are incubated overnight at 42° C. with radiolabeled probe in 50% formamide hybridization solution, [5× SSC, 5× Denhardt's, 100 μg/ml DNA (Sigma)]. The probe, a DNA fragment containing the coding sequence of the human B1 bradykinin receptor, is generated by random prime labeling (Boehringer Mannheim Biochemicals) in the presence of [α-32P]dCTP (3000 Ci/mmole). Filters are washed at a final stringency of 0.1× SSC, 0.1% SDS at 42° C. Positives are rescreened to isolate single colonies. DNA is prepared from positive colonies, digested with restriction enzymes, and Southern blot analysis is done to identify restriction fragments for subcloning. A fragment is subcloned into pSP72 (Promega).
EXAMPLE 4
[0091] Cloning of B1 cDNA into a Mammalian Expression Vector
[0092] Mammalian Cell Expression and Characterization
[0093] Transient expression of the cloned B1 bradykinin receptor in COS-7 cells was accomplished by electroporation using a Biorad Gene Pulser. Three days post-transfection, cells were processed for a whole cell binding assay as previously published (K. A. Schneck, J. F. Hess, G. Y. Stonesifer, R. W. Ransom, European Journal of Pharmacology—Molecular Pharmacology Section 266, 277-282 (1994)) or membranes were prepared as described previously (Huang, R. -R. C., Dehaven, R. N., Cheung, A. H., Diehl, R. E., Dixon, R. A. F., Strader, C. D., Molecular Pharmacology 37, 304-310 (1990)) and radioligand binding measured as follows. Displacement studies were done with 1 nM [des-Arg10],[3,4-3H(N)]-kallidin (NEN) in the presence of varying concentrations of competitor compounds. Binding assays were performed at room temperature for 45 minutes. Reactions were terminated by filtration using an Inotech cell harvester onto glass fiber filters (Inotech) that had been briefly soaked in 0.3% polyethyleneimine. The filters were washed five times with 1 ml per well of cold PBS and counted in a liquid scintillation counter. Alternatively, reactions were collected using a Tomtec cell harvester 96 onto 0.3% polyethyleneimine presoaked glass fiber filters (Pharmacia, printed filtermat for use with the 1205 Betaplate). The filter was washed five times with a total of 10 ml per well of cold 1× PBS, dried and sealed in a bag (Pharmacia) containing scintillation fluid. Counting was accomplished using a LKB1205 Betaplate liquid scintillation counter.
[0094] The B1 bradykinin receptor clone 33E9 was transfected into COS-7 cells and the pharmacological properties of the expressed receptor were determined. Scatchard analysis of saturation binding data with [3H]des-Arg10kallidin indicated a Kd of about 1.0 nM and a Bmax of approximately 120 fmol/mg protein. Mock transfected COS-7 cells do not contain any specific binding for [3H]des-Arg10kallidin.
[0095] The ability of several bradykinin receptor agonists and antagonists to displace 1 nM [3H]des-Arg10kallidin from the cloned receptor was assessed (FIG. 5, Table 1). The IC50 for displacement of 1 nM [3H]des-Arg10kallidin by bradykinin from the cloned receptor is >10 μM. The low affinity of this receptor for bradykinin and high affinity for des-Arg10kallidin argues strongly that this cloned bradykinin receptor is of the B1 subtype. Competition binding studies yielded a rank order of affinity for kinin agonists of des-Arg10kallidin>kallidin >des-Arg9 bradykinin>>bradykinin at the cloned human receptor. This is very similar to the rank order of potency reported for the rabbit B1 bradykinin receptor and identical to that observed for the B1 receptor in IMR-90 cells (Table 1). Both the cloned human receptor and the B1 receptor in IMR-90 cells exhibit a relatively low affinity for the “classical” B1 receptor agonist des-Arg9 bradykinin which has been reported to a have a high affinity for the B1 receptor in rabbit aorta (K. A. Schneck, J. F. Hess, G. Y. Stonesifer, R. W. Ransom, European Journal of Pharmacology—Molecular Pharmacology Section 266, 277-282 (1994); D. Regoli, J. Barabe, Pharmacological Reviews 32, 1-46 (1980)). The B1 bradykinin receptor present in human aorta also has a relatively low affinity for des-Arg9 bradykinin (>1000 nM, R.W.R, unpublished data). These results taken together with the lower affinity for des-Arg9 bradykinin relative to des-Arg10kallidin in the rabbit (K. A. Schneck, J. F. Hess, G. Y. Stonesifer, R. W. Ransom, European Journal of Pharmacology—Molecular Pharmacology Section 266, 277-282 (1994); D. Regoli, J. Barabe, Pharmacological Reviews 32, 1-46 (1980)) suggest that the most potent natural ligand for the B1 receptor is des-Arg10kallidin . Therefore, the B1 receptor cloned here appears to be the human homologue of the B1 receptor present in rabbit aorta, and the lower affinity of the human receptor for des-Arg9 bradykinin appears to be a consequence of species differences.
1TABLE 1
|
|
Comparison of the peptide binding properties of the cloned human
B1 bradykinin receptor in transiently transfected COS-7 cells (COS
33E9) with the B1 receptor in IMR-90 cells (IMR-90).
The IC50's were determined by the displacement of 1 nM [3H]des-
Arg10kallidin in the membrane binding assay described above.
CompetitorCOS 33E9IMR-90
|
des-Arg10kallidin2.6nM2.3nM
des-Arg10Leu9kallidin9nM7.8nM
Kallidin46nM44nM
Hoe 140>10μM>10μM
Bradykinin>10μM>10μM
des-Arg9bradykinin1500nM660nM
des-Arg9Leu8bradykinin605nM266nM
des-Arg10HOE 14094nM40nM
Met-Lys-Bradykinin71nM40nM
Substance P>10μM>10μM
Bradykinin(2-9)>10μM>10μM
Bradykinin(1-7)>10μM>10μM
Neurotensin>10μM>10μM
Leu-Enkephalin>10μM>10μM
Angiotensin II>10μM>10μM
Acetylated Neurotensin(8-13)>10μM>10μM
Arg Vasopressin>10μM>10μM
Oxytocin>10μM>10μM
Dynorphin(1-17)>7μM>7μM
Endothelin I>1μM>1μM
|
[0096] The ability of several bradykinin receptor antagonists to displace 1 nM [3H]des-Arg10kallidin was also analyzed. The cloned receptor has high affinity binding for the B1 specific antagonist des-Arg10[Leu9]kallidin and des-Arg9[Leu8]bradykinin (FIG. 5, Table 1). The potent B2 specific antagonist HOE 140 has a very low affinity for the cloned receptor. However, the removal of the C-terminal Arg from HOE 140 results in a significant increase in affinity, as expected for a B1 receptor (K. Wirth, et al., European Journal of Pharmacology 205, 217-218 (1991)). Therefore, the interaction of the cloned receptor with bradykinin receptor antagonists is consistent with the B1 receptor classification.
[0097] In summary, we have utilized an expression cloning strategy to isolate a clone encoding a human B1 bradykinin receptor. This receptor was isolated by its ability, when expressed in Xenopus oocytes, to functionally respond to the B1 receptor agonist des-Arg10kallidin. The cloned receptor is a G-protein coupled receptor that is most similar in amino acid sequence to the B2 bradykinin receptor. The pharmacological properties of the cloned receptor expressed in mammalian cells are characteristic of the B1 bradykinin receptor classification.
EXAMPLE 5
[0098] Cloning of the B1 cDNA into E. coli Expression Vectors
[0099] Recombinant B1 is produced in E. coli following the transfer of the B1 expression cassette into E. coli expression vectors, including but not limited to, the pET series (Novagen). The pET vectors place B1 expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this construct into an E. Coli host which contains a chromosomal copy of the T7 RNA polymerase gene driven by the inducible lac promoter, expression of B1 is induced when an appropriate lac substrate (IPTG) is added to the culture. The levels of expressed B1 are determined by the assays described herein.
[0100] The cDNA encoding the entire open reading frame for B1 is inserted into the NdeI site of pET 11a. Constructs in the positive orientation are identified by sequence analysis and used to transform the expression host strain BL21. Transformants are then used to inoculate cultures for the production of B1 protein. Cultures may be grown in M9 or ZB media, whose formulation is known to those skilled in the art. After growth to an OD600=1.5, expression of B1 is induced with 1 mM IPTG for 3 hours at 37° C.
EXAMPLE 6
[0101] Cloning of B1 cDNA into a Vectors for Expression in Insect Cells
[0102] Baculovirus vectors, which are derived from the genome of the AcNPV virus, are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL#1711). Recombinant baculoviruses expressing B1 cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the B1 cDNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P. A., Nuc. Acid. Res. 18, 5667 (1990)] into Sf9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of B-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, B1 expression is measured by the assays described herein.
[0103] The cDNA encoding the entire open reading frame for B1 is inserted into the BamHI site of pBlueBacII. Constructs in the positive orientation are identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV wild type DNA.
[0104] Authentic, active B1 is found in association with the infected cells. Active B1 is extracted from infected cells by hypotonic or detergent lysis.
[0105] Alternatively, the human B1 receptor is expressed in the Drosophila Schneider 2 cell line by cotransfection of the Schneider 2 cells with a vector containing the B1 receptor DNA downstream and under control of an inducible metallothionin promoter, and a vector encoding the G418 resistant neomycin gene. Following growth in the presence of G418, resistant cells are obtained and induced to express B1 receptor by the addition of CuSO4. Identification of modulators of the B1 receptor is accomplished by assays using either whole cells or membrane preparations.
EXAMPLE 7
[0106] Cloning of B1 cDNA into a Yeast Expression Vector
[0107] Recombinant B1 is produced in the yeast S. cerevisiae following the insertion of the optimal B1 cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the B1 cistron [Rinas, U. et al., Biotechnology 8, 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265,4189-4192 (1989)]. For extracellular expression, the B1 cistron is ligated into yeast expression vectors which fuse a secretion signal. The levels of expressed B1 are determined by the assays described herein.
EXAMPLE 8
[0108] Purification of Recombinant B1
[0109] Recombinantly produced B1 may be purified by antibody affinity chromatography. B1 antibody affinity columns are made by adding the anti-B1 antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HCl (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents and the cell culture supernatants or cell extracts containing solubilized B1 or B1 subunits are slowly passed through the column. The column is then washed with phosphate-buffered saline together with detergents until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl (pH 2.6) together with detergents. The purified B1 protein is then dialyzed against phosphate buffered saline.
Claims
- 1. An isolated and purified DNA molecule which encodes a bradykinin B1 receptor or a functional derivative thereof.
- 2. The isolated and purified DNA molecule of claim 1, having a nucleotide sequence:
- 3. The isolated and purified DNA molecule of claim 1, wherein said DNA molecule is genomic DNA.
- 4. An expression vector for expression of a bradykinin B1 receptor in a recombinant host, wherein said vector contains a recombinant gene encoding a bradykinin B1 receptor or functional derivaative thereof.
- 5. The expression vector of claim 4, wherein the expression vector contains a cloned gene encoding the bradykinin B1 receptor, having a nucleotide sequence:
- 6. The expression vector of claim 4, wherein the expression vector contains genomic DNA encoding the bradykinin BI receptor.
- 7. A recombinant host cell containing a recombinantly cloned gene encoding a bradykinin B1 receptor or functional derivative thereof.
- 8. The recombinant host cell of claim 7, wherein said gene encoding the bradykinin B1 receptor has a nucleotide sequence:
- 9. The recombinant host cell of claim 7, wherein said cloned gene encoding the bradykinin B1 receptor is genomic DNA.
- 10. A protein, in substantially pure form which functions as a bradykinin B1 receptor.
- 11. The protein according to claim 10, having an amino acid sequence:
- 12. A monospecific antibody immunologically reactive with a bradykinin B1 receptor.
- 13. The antibody of claim 12, wherein the antibody blocks activity of the bradykinin B1 receptor.
- 14. A process for expression of a bradykinin B1 receptor protein in a recombinant host cell, comprising:
(a) transferring the expression vector of claim 4 into suitable host cells; and (b) culturing the host cells of step (a) under conditions which allow expression of the bradykinin B1 receptor protein from the expression vector.
- 15. A method of identifying compounds that modulate bradykinin B1 receptor activity, comprising:
(a) combining a suspected modulator of bradykinin B1 receptor activity with a bradykinin B1 receptor; and (b) measuring an effect of the modulator on the receptor.
- 16. The method of claim 15, wherein the effect of the modulator on the receptor is inhibiting or enhancing binding of B1 receptor ligands.
- 17. The method of claim 15, wherein the effect of the modulator on the receptor is stimulation or inhibition of signal transduction mediated by B1 receptors.
- 18. The method of claim 17, wherein the effect of the modulator on the receptor is signal transduction mediated by B1 receptors, and said signal transduction is selected from the group consisting of phosphatidyl inositol hydrolysis, release of intracellular Ca2+ stores, and arachidonic acid release.
- 19. A compound active in the method of claim 15, wherein said compound is a modulator of a bradykinin B1 receptor.
- 20. A compound active in the method of claim 15, wherein said compound is an agonist or antagonist of a bradykinin B1 receptor.
- 21. A compound active in the method of claim 15, wherein said compound is a modulator of expression of a bradykinin B1 receptor.
- 22. A pharmaceutical composition comprising a compound active in the method of claim 15, wherein said compound is a modulator of bradykinin B1 receptor activity.
- 23. A method of treating a patient in need of such treatment for a condition which is mediated by a bradykinin B1 receptor, comprising administration of a bradykinin B1 receptor modulating compound active in the method of claim 15.
- 24. A method of treating a patient in need of such treatment for a condition which is mediated by a bradykinin B1 receptor and is characterized by hyperalgesia, acute inflammation or chronic inflammation, comprising administration of a bradykinin B1 receptor modulating compound active in the method of claim 15.
- 25. A method of identifying compounds that modulate bradykinin B1 receptor activity, comprising:
(a) combining a suspected modulator of bradykinin B1 receptor activity with a cell expressing a recombinant bradykinin B1 receptor; and (b) measuring an effect of the modulator on the receptor.
- 26. The method of claim 25, wherein the effect of the modulator on the receptor in step (b) is inhibiting or or enhancing binding of B1 receptor ligands.
- 27. The method of claim 25, wherein the effect of the modulator on the receptor in step (b) is inhibition or enhancement of signal transduction mediated by B1 receptors.
- 28. The method of claim 27, wherein the signal transduction is selected from a group consisting of phosphatidyl inositol hydrolysis, release of intracellular Ca2+ stores, and arachidonic acid release.
- 29. A compound active in the method of claim 25, wherein said compound is a modulator of a bradykinin B1 receptor.
- 30. A compound active in the method of claim 25, wherein said compound is an agonist or antagonist of a bradykinin B1 receptor.
- 31. A compound active in the method of claim 25, wherein said compound is a modulator of expression of a bradykinin B1 receptor.
- 32. A pharmaceutical composition comprising a compound active in the method of claim 25, wherein said compound is a modulator of bradykinin B1 receptor activity.
- 33. A method of treating a patient in need of such treatment for a condition which is mediated by a bradykinin B1 receptor, comprising administration of a bradykinin B1 receptor modulating compound active in the method of claim 25.
- 34. A method of treating a patient in need of such treatment for a condition which is mediated by a bradykinin B1 receptor and is characterized by hyperalgesia, acute inflammation or chronic inflammation, comprising administration of a bradykinin B1 receptor modulating compound active in the method of claim 25.
Divisions (2)
|
Number |
Date |
Country |
Parent |
09307912 |
May 1999 |
US |
Child |
10007343 |
Oct 2001 |
US |
Parent |
08229418 |
Apr 1994 |
US |
Child |
08932761 |
Sep 1997 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
08932761 |
Sep 1997 |
US |
Child |
09307912 |
May 1999 |
US |