JP2007501605A - Secreted polypeptide species associated with cardiovascular disease - Google Patents

Abstract

The present invention discloses human secreted polypeptides that circulate at increased levels in the plasma of patients with cardiovascular disease. The invention also provides methods of using compositions comprising these polypeptides, polynucleotides encoding them, and antibodies specific for these polypeptides for diagnosis, prognosis and drug discovery.

Description

Detailed Description of the Invention

  The present invention relates to polypeptide species that are differentially secreted in plasma of individuals with cardiovascular disease, isolated polynucleotides that encode such polypeptides, polymorphic variants thereof, and cardiovascular disease diagnostics. And the use of the nucleic acids and polypeptides or compositions thereof in detection assays for drug discovery.

  Cardiovascular disease is a serious health crisis in all industrialized countries. Coronary artery disease (CAD) is characterized by atherosclerosis or arteriosclerosis. Atherosclerosis is the most prevalent cardiovascular disease and the leading cause of heart attacks, strokes and limb gangrene, and is therefore the leading cause of death in the United States. Atherosclerosis is a complex disease involving many cell types and molecular factors (see, for example, Ross, 1993, Nature 362: 801-809). Under normal circumstances, the protective response to arterial wall endothelium and smooth muscle cell (SMC) damage consists of the formation of fiber fat and fiber lesions or plaques that precede and involve inflammation. The advanced lesions of atherosclerosis are related arterial blockages and are thought to result from an excessive inflammatory fibroproliferative response to many different forms of injury. Vascular endothelium damage or dysfunction is a common feature of many symptoms where an individual is susceptible to accelerated progression of atherosclerotic cardiovascular disease.

  Ischemia occurs because atherosclerotic plaques block the associated blood vessels and restrict blood flow. Ischemia is a condition characterized by a lack of oxygen supply to organ tissues that results from inadequate perfusion. Such inadequate perfusion can have many natural causes including atherosclerotic or restenotic lesions, anemia or stroke. The most common cause of cardiac ischemia is atherosclerotic disease of the epicardial coronary artery. In atherosclerosis, the smaller lumens of these vessels cause an absolute decrease in myocardial perfusion in the basal state or limit the appropriate increase in perfusion when flow demand increases. The Coronary blood flow can also be limited by arterial thrombosis, convulsions and, rarely, portal stenosis caused by coronary embolism and syphilitic aortitis. Congenital anomalies such as abnormal onset of the left anterior descending coronary artery from the pulmonary artery are thought to cause myocardial ischemia and infarction in early childhood, but this is very rare in adults .

  Myocardial ischemia can also occur when myocardial oxygen demands are abnormally elevated, as seen in severe ventricular hypertrophy due to hypertension or aortic stenosis. The latter may involve angina that is indistinguishable from that caused by coronary atherosclerosis. In rare cases, reduced oxygen carrying capacity of the blood, as seen in extremely severe anemia or in the presence of carboxyhemoglobin, is responsible for myocardial ischemia. Ischemia often coexists with two or more causes, such as increased oxygen demand due to left ventricular hypertrophy and decreased oxygen supply associated with coronary atherosclerosis.

  Extensive clinical studies have identified factors that increase the risk of cardiovascular disease. Some of these risk factors, such as age, gender, and family history, cannot be changed. Other risk factors include: smoking, high blood pressure, high fat and high cholesterol diet, diabetes, lack of exercise, obesity and stress.

  Fortunately, many incentives can be controlled through lifestyle changes. Smokers have more than twice the risk of cardiovascular disease than non-smokers. Regardless of the amount of smoking so far, quitting smoking rapidly reduces the risk of developing the disease. Serum cholesterol levels are directly related to the prevalence of cardiovascular disease, and hypertension or hypertension is an important risk factor. It is hypothesized that physical activity reduces the risk of developing cardiovascular disease through various mechanisms, which increases myocardial oxygen supply, decreases oxygen demand, and promotes myocardial contraction, Increases the stability of electrical impulses. Decreased oxygen demand and myocardial motion are indicated by a decrease in resting heart rate and blood pressure. Physical activity increases the diameter of the coronary arteries, increases the ability to stretch, increases collateral arteriogenesis, and decreases the rate of progression of coronary atherosclerosis. Activity reduces obesity and serum fatty acids.

  At rest, there are no noticeable symptoms of cardiovascular disease, but symptoms such as chest tightness can occur when activity or stress increases. Other first signs that appear include heartburn, nausea, vomiting, numbness, shortness of breath, severe cold sweat, unexplained fatigue and anxiety. More severe symptoms of cardiovascular disease are chest pain (angina), rhythm abnormalities (arrhythmia), stroke or heart attack (myocardial infarction). Strokes and heart attacks result from arterial occlusion in brain tissue and heart tissue, respectively. Because the symptoms are different, the tests and treatments chosen by the patients are quite different.

  Diagnostic tests useful for determining the extent and severity of cardiovascular disease include electrocardiogram (EKG), stress test, nuclear medicine examination, coronary angiography, resting EKG, EKG multifaceted diagnostic information index, Holter monitor, delay Potential, EKG mapping, echocardiogram, thallium scan, PET, MRI, CT, angiogram and IVUS. Additional risk factor countermeasures and useful diagnostic methods are also common and are best used by those with medical skill. There are many different therapeutic approaches depending on the severity of the disease. For many, cardiovascular disease is a disease managed by lifestyle changes and medications, but there are also more serious diagnoses that indicate the need for surgery.

  Surgical approaches to the treatment of ischemic atherosclerosis include bypass grafting, coronary angioplasty, laser angioplasty, atherectomy, endarterectomy and percutaneous transluminal angioplasty (PCTA) Is mentioned. The recurrence rate due to restenosis after these approaches (occlusions recur and often worsen) is very high (30-50%). Much of restenosis appears to be due to further inflammation, smooth muscle accumulation and thrombosis. Additional therapeutic approaches to cardiovascular disease also include treatments that promote angiogenesis in conditions such as ischemic heart disease and limb disease.

  A definitive diagnosis is difficult because the nature of the symptoms of most CAD and cardiovascular diseases is not clear. More quantitative diagnostic methods suffer from both inter-individual variation and individual-to-individual variation. Therefore, diagnostic measures must be standardized and applied to individuals with a well-proven and extensive medical history. Furthermore, current diagnostic methods often do not reveal the underlying cause for the observations or measurements obtained. Therefore, treatment strategies based on specific positive results may not solve the causative problem, and it may also be harmful to the individual.

  Diagnostic methods that utilize nucleotide detection include genetic approaches and expression profiling. For example, genes known to be associated with cardiovascular disease can be screened for mutations using common genotyping methods such as sequencing, hybridization-based techniques or PCR. In another example, the expression of known genes can be followed by standard techniques including RTPCR, various hybridization based techniques and sequencing. With these strategies, physicians often fail to detect differences in mRNA processing and splicing, translation rate, mRNA stability, and post-translational modifications such as proteolytic processing, phosphorylation, glycosylation and amidation.

  To address current shortcomings in the diagnostic context of the art relating to cardiovascular disease, the present invention provides specific plasma polypeptides that are differentially increased relative to control plasma in the plasma of individuals with coronary artery disease. Providing the actual polypeptide species reveals differences in mRNA processing and splicing, translation rate, mRNA stability, and post-translational modifications such as proteolytic processing, phosphorylation, glycosylation and amidation. Accordingly, the polypeptides of the present invention are referred to as “cardiovascular disease plasma polypeptides” or CPPs. These polypeptide sequences are designated as CPP 149-402 and comprise at least one amino acid sequence selected from the tryptic peptides of Table 3.

  The present invention discloses “cardiovascular disease plasma polypeptides” (CPP), fragments of CPP and post-translationally modified species that are present at high levels in plasma obtained from individuals with coronary artery disease (CAD). Thus, the CPPs of the present invention are at risk for CAD, coronary heart disease (CHD), peripheral vascular disease, cerebral ischemia (stroke), congestive heart failure, atherosclerosis, hypertension and other cardiovascular diseases. It is an important diagnostic tool for judging CPP is a secreted factor and as such can be easily detected and is useful for drug discovery, diagnosis, and prevention of cardiovascular disease.

SUMMARY OF THE INVENTION The present invention is preferably directed to compositions related to secreted polypeptide species that are increased in plasma from patients with cardiovascular disease. These polypeptide species are referred to herein as “Cardiovascular disorder Plasma Polypeptides” or CPPs. Such cardiovascular disease plasma polypeptides include a polypeptide selected from the group consisting of CPP 149-402. Preferred CPPs comprise a polypeptide selected from the group consisting of CPP 149-297. Even more preferred CPPs comprise a polypeptide selected from the group consisting of CPPs 149-194. Even more preferred CPPs comprise a polypeptide selected from the group consisting of CPPs 149-151. Compositions include CPP precursors, antibodies specific for CPP, including monoclonal antibodies, and other binding compositions derived therefrom. Further included are methods of making and using these compositions. Precursors of the present invention include unmodified precursors, proteolytic precursors of the tryptic peptides listed in Table 3, and precursors generated from other proteolytic sites of the amino acid sequence of CPP 149-402. .

  Preferred embodiments of the present invention include CPPs that have undergone post-translational modifications such as phosphorylation, glycosylation, acetylation, amidation, or have C-, N- or O-linked carbohydrate groups. Including. Further preferred are CPPs that have an intramolecular or intermolecular interaction, such as disulfide bonds and hydrogen bonds, resulting in higher order structures. Also preferred are CPPs generated by suggestive mRNA processing or splicing. Preferably, these CPPs are post-translationally modified species, structural variants, or splice variants that are present in the plasma of patients with cardiovascular disease.

  In another aspect, the invention includes a CPP comprising a sequence that is at least 75 percent identical to a sequence selected from the group consisting of CPP 149-402. Preferably, the invention comprises a polypeptide comprising at least 80 percent, preferably at least 85 percent, more preferably at least 90 percent identity with any one of sequences selected from CPP 149-402. Most preferably, the invention comprises a polypeptide comprising a sequence that is at least 95 percent identical to a sequence selected from the group consisting of CPP 149-402.

  In another aspect, the invention includes a natural variant of CPP that is present at a frequency of at least 2 percent in the extract population. More preferably, such natural variants are present at a frequency of at least 5 percent, even more preferably at least 10 percent in the extracted population. Most preferably, such natural variants are present at a frequency of at least 20 percent in the extract population. An extracted population can be any recognized population of tests in the field of population genetics. Preferably, the extracted population is white, black or Asian. More preferably, the extract is French, German, British, Spanish, Swiss, Japanese, Chinese, Irish, Korean, Singaporean, Icelandic, North American, Israeli, Arab Turkish, Greek, Italian, Polish, Pacific Islander, Finnish, Norwegian, Swedish, Estonian, Austrian or Indian. More preferably, the extract is an Islandic, Sami, Finnish, French White, Swiss, Chinese Singaporean, Korean, Japanese, Quebec, North American Pima Indian, Aman, Pennsylvania. Sect and Amish, Newfoundland or Polynesian.

  A preferred embodiment of the present invention is an isolated CPP, ie, a composition comprising a CPP having an isoelectric point significantly different from that of the CPP or having an apparent molecular weight significantly different from that of the CPP and free of protein isoforms. I will provide a. The isoelectric point and molecular weight of a CPP can be shown by separation chromatography based on affinity and size, two-dimensional gel analysis and mass spectrometry.

  In a preferred embodiment, the present invention provides a specific polypeptide species comprising a sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402). Preferably, CPPs 149-402 contain additional contiguous amino acids from the sequence of the corresponding polypeptide entry in the public database, as shown in Table 1. Preferred species include i) the amino acid sequence of any one of CPP 149-402; ii) found at higher levels in the plasma of individuals with cardiovascular disease; and iii) of the corresponding polypeptide entry shown in Table 1 A polypeptide comprising additional amino acids derived from the sequence.

  In a further aspect, the present invention includes a modified CPP. Such modifications include enzyme labels, fluorescent labels, isotope labels or affinity labels that allow group protection / blocking, conjugation with antibody molecules or other cellular ligands, and protein detection and isolation. Such detectable labels. Chemical modifications include but are not limited to cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage with NaBH4, acetylation, formylation, oxidation, reduction or metabolic synthesis in the presence of tunicamycin. It can implement by well-known techniques including these.

  Also provided by the present invention are chemically modified derivatives of the polypeptides of the present invention that may provide additional benefits such as increased solubility of the polypeptide, increased stability and increased circulation time, or decreased immunogenicity (e.g., , Polyethylene glycols, ethylene glycol / propylene glycol copolymers, water soluble polymers such as carboxymethyl cellulose, dextran, polyvinyl alcohol). A CPP is modified at any position in the molecule or is modified at a given position in the molecule and may contain one, two, three or more attached chemical moieties.

  In another embodiment, the present invention provides a method for identifying at least one modulator of CPP biological activity comprising: i) a test modulator of CPP biological activity, wherein the amino acid sequence listed in Table 3 ( Contacting with a polypeptide comprising a sequence selected from the group consisting of CPP 149-402), ii) detecting the level of CPP biological activity, and iii) determining the level of CPP biological activity, A method is provided that includes comparing to that of a control sample that does not include a test modulator. If the difference in the level of CPP protein biological activity is a decrease, the test modulator is an inhibitor of at least one CPP biological activity. If the difference in the level of CPP biological activity is an increase, the test substance is an activator of at least one CPP biological activity.

  In another aspect of the invention, a method of identifying a modulator of cardiovascular disease is provided, the method comprising: (a) providing a non-human test animal susceptible to or suffering from cardiovascular disease. Administering a candidate agent; (b) administering the candidate agent of (a) to a corresponding control non-human animal that is unlikely to suffer from cardiovascular disease or not suffering from cardiovascular disease; (c) step The group consisting of (i) the amino acid sequences (CPP 149 to 402) listed in Table 3 in the biological sample obtained from the non-human test animal of (a) and the biological sample obtained from the control animal of step (b) A polypeptide comprising an amino acid sequence selected from: (ii) 1 for an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) A variant having the above amino acid substitutions, deletions or insertions and having at least 75% sequence identity; and (iii) a fragment of a polypeptide as defined in i) or ii) above of at least 10 amino acids in length Detecting and / or quantifying the level of at least one polypeptide selected from: and (d) comparing the level of the polypeptide of step (c), a biological sample obtained from a non-human test animal The transition of the polypeptide level in to the level of the polypeptide in a biological sample obtained from a control animal indicates that the candidate agent is a modulator of cardiovascular disease. In a preferred embodiment of the present invention, the non-human test animal susceptible to cardiovascular disease or suffering from cardiovascular disease consists of (i) the amino acid sequence listed in Table 3 (CPP 149 to 402). A polypeptide comprising an amino acid sequence selected from the group; (ii) one or more amino acid substitutions, deletions or deletions relative to an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) A variant having an insertion and having at least 75% sequence identity; and (iii) at least 10 amino acids in length and at least one poly selected from fragments of a polypeptide as defined in i) or ii) above A case involving an increase in plasma levels of peptides is provided.

  In another aspect, the invention provides a polynucleotide encoding a polynucleotide encoding a CPP of the invention, a polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402). Includes nucleotides, oligonucleotides complementary to the CPP gene sequence for diagnostic and analytical assays (eg, PCR, hybridization-based techniques), and vectors for expressing CPPs.

  In another aspect, the present invention provides a vector comprising DNA encoding CPP. The invention also includes host cells and transgenic non-human animals comprising such vectors. Also provided is a method of making a CPP or CPP precursor. One preferred method includes (a) providing a host cell comprising the expression vector disclosed above, (b) culturing the host cell under conditions that allow expression of the DNA segment, and (c) Recovering the protein encoded by the DNA segment is included. Another preferred method comprises (a) providing a host cell capable of expressing CPP, (b) culturing the host cell under conditions that allow expression of said CPP, and (c) A step of recovering the CPP is included. In one embodiment, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, and the cell secretes the protein into the culture medium and recovers the protein from the medium. Particularly preferred methods of making CPPs include chemical synthesis using standard peptide synthesis techniques, as described in the section entitled “Chemical Production of CPP Compositions” and Example 2.

  In another aspect, the invention includes an isolated antibody specific for any of the polypeptides, peptide fragments or peptides described above. Preferably, the antibody of the present invention is a monoclonal antibody. Further preferred are antibodies that bind exclusively to CPP, ie, antibodies that do not recognize other polypeptides with high affinity. Anti-CPP antibodies have purification, diagnostic and prognostic applications. Preferred anti-CPP antibodies for purification and diagnosis are conjugated with a labeling group. Preferred CPP-related diseases for diagnosis include coronary artery disease (CAD), coronary heart disease (CHD), peripheral vascular disease, cerebral ischemia (stroke), congestive heart failure, atherosclerosis, hypertension and other hearts Examples include vascular diseases. Examples of the diagnostic method include, but are not limited to, a method using an antibody specific to CPP antigen or an antibody-derived composition. Both diagnostic methods for detecting CPP in specific tissue samples and body fluids (preferably plasma) as well as diagnostic methods for detecting CPP expression levels in tissues form part of the present invention. For example, compositions comprising a pharmaceutically acceptable carrier with one or more of the above-described antibodies for in vivo diagnostic methods and drug screening assays are also within the scope of the invention.

  The present invention further provides a method for the diagnosis of cardiovascular disease comprising detecting the level of at least one CPP in a sample of body fluid, preferably blood plasma. Most preferably, the diagnostic method is performed ex vivo. Further, a method of using a CPP composition comprising a CPP gene and / or messenger RNA and a primer complementary to an anti-CPP antibody to detect and measure the amount of CPP in tissues and body fluids, preferably plasma. included. These methods are also suitable for clinical screening, prognosis, therapeutic outcome monitoring, identification of patients most likely to respond to a particular therapeutic treatment, drug screening and drug discovery, and identification of new targets for drug treatment. Yes.

  A still further aspect of the invention relates to a method for monitoring the effectiveness of treatment with a drug in a subject having cardiovascular disease or at risk of developing cardiovascular disease, the method comprising: (A) obtaining a pre-administration biological sample from the subject prior to administration of the drug; (b) comprising the amino acid sequence (CPP 149-402) listed in Table 3 in the subject biological sample (i). A polypeptide comprising an amino acid sequence selected from the group; (ii) one or more amino acid substitutions, deletions or deletions relative to an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) Variants with insertions and at least 75% sequence identity; and (iii) at least 10 amino acids long, as defined in i) or ii) above Detecting and / or quantifying the level of at least one polypeptide selected from a particular polypeptide; and (c) obtaining one or more post-administration biological samples from the subject; (d) its in the post-administration sample Detecting the level of at least one polypeptide; (e) comparing the level of the at least one polypeptide in the pre-administration sample with the level of the at least one polypeptide in the post-administration sample; and ( f) adjusting the administration of the drug accordingly.

  The present invention provides kits that can include other reagents, labeling groups, substrates, and optionally instructions for use, as well as single or multiple preparations or antibodies that can be used in the above methods. The kit can be used for diagnosis of a disease or can be assayed for the identification of new diagnostic and / or therapeutic agents.

  In one embodiment, coronary artery disease (CAD) is defined by the development of at least one symptom. Such symptoms increase in severity as the disease progresses. CAD is often accompanied by a decrease in left ventricular volume and stroke volume. Early CAD symptoms include cholesterol and low density lipoprotein (particularly oxidized), and elevated plasma levels of platelet-rich plasma aggregates. The vascular endothelium responds to inflammation and, in response, increases plaque formation and increases levels of inflammatory and fibrinogen factors. In addition, CAD or atherosclerosis is characterized by vascular calcification and arteriosclerosis. The resulting partial blockage of blood vessels causes hypertension and ischemic heart disease. Ultimate complete vascular occlusion results in myocardial infarction, stroke or gangrene.

  In a preferred embodiment, detection of elevated plasma levels of at least one CPP of the present invention indicates an increased risk that an individual will develop CAD. Preferably, the detection indicates that the individual's likelihood of developing CAD is increased by at least 1.05, 1.1, 1.15, and more preferably at least 1.2. Alternatively, detection of elevated plasma levels of at least one CPP of the present invention indicates that the individual has CAD. The amount of increase observed when compared to the control sample correlates with the likelihood of CAD prediction or diagnosis. Because individual plasma CPP levels vary with family history and other risk factors, it is preferable to examine each individually. In a preferred embodiment, CPP is detected in a human plasma sample by the method of the present invention. Particularly preferred techniques are mass spectrometry and immunodetection. Preferably, CAD prediction or diagnosis is based on a test CPP level relative to a control that is at least 1.1-fold, 1.15-fold, 1.2-fold, 1.25-fold, more preferably 1.5-fold higher.

  The invention further includes methods of using CPP-modulating compositions to prevent or treat diseases associated with abnormal expression or processing of CPP149-402 CPP in an individual. Preferred CPP-related diseases include coronary artery disease (CAD), coronary heart disease (CHD), peripheral vascular disease, cerebral ischemia (stroke), congestive heart failure, atherosclerosis, hypertension and other cardiovascular diseases Is mentioned. A preferred embodiment of the present invention is a method of preventing or treating a CPP-related disease in an individual, the method comprising determining an individual suffering from or at risk for a CPP-related disease, And introducing a CPP modulating composition into the individual.

  Additional aspects of the invention are also described in the specification and claims.

Detailed Description of the Invention The present invention, described in detail below, is for screening, diagnosis and prognosis of cardiovascular disease in mammalian individuals; to identify individuals most likely to respond to a particular therapeutic treatment; Methods, compositions, and kits are provided that are useful for monitoring the outcome of treatment; for screening CPP modulators, and for drug discovery. The invention also includes administering a therapeutic composition to a mammalian individual to treat or prevent cardiovascular disease. The mammalian individual can be a non-human mammal, but is preferably a human, more preferably an adult. For purposes of clarity of disclosure, the present invention will be described with respect to the analysis of blood plasma samples, without limitation. However, one of ordinary skill in the art will appreciate that the assays and techniques described below may involve other body fluid samples (eg, cerebrospinal fluid, lymph fluid, bile, serum, etc.) from individuals who have or are at risk of developing cardiovascular disease. , Saliva or urine) or tissue samples. The methods and compositions of the invention are useful for screening, diagnosis and prognosis of live individuals, but also for postmortem diagnosis in individuals to identify family members at risk of developing the disease. Can be useful.

Definitions As used herein, “nucleic acid” and “nucleic acid molecule” refer to DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and analogs of DNA or RNA made using nucleotide analogs. Includes body. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. Throughout this specification, the expression “nucleotide sequence” is used to refer to polynucleotides or nucleic acids without distinction. More precisely, because the expression “nucleotide sequence” encompasses the nuclear material itself, sequence information that characterizes a specific DNA or RNA molecule biochemically (ie, a character selected from four base characters). Is not limited). “Nucleic acid”, “oligonucleotide” and “polynucleotide” are used interchangeably herein.

  An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid. Preferably, a sequence in which the “isolated” nucleic acid naturally flanks the nucleic acid within the genomic DNA of the organism from which the nucleic acid is obtained (ie, sequences present at the 5 ′ and 3 ′ ends of the nucleic acid). Not included. For example, in various embodiments, an isolated CPP nucleic acid molecule is less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb naturally flanking the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is obtained. , 0.5 kb or 0.1 kb nucleotide sequence. In addition, an “isolated” nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material, or may be substantially enriched in culture medium when made by recombinant techniques. Or, when chemically synthesized, may be substantially free of chemical precursors or other chemicals. CPP nucleic acid molecules use all or part of the nucleic acid as a hybridization probe, and standard hybridization and cloning techniques (eg, Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., As described in Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

As used herein, “hybridizes to” means that nucleotide sequences that are at least 60% homologous to each other under conditions for medium or high stringency hybridization, preferably hybridization and washing conditions, are hybridized to each other. It shall represent conditions that would result in a soy state. Preferably, the conditions are such that sequences that are at least about 70% homologous to each other, more preferably at least about 80%, more preferably at least about 85%, 90%, 95% or 98% homologous are generally hybridized to each other. It ’s like that. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. In a preferred non-limiting example, stringent hybridization conditions for the combined action of nucleic acids are as follows: Hybridization steps consisted of 6 × SSC buffer, 5 × Denhart solution, 0.5% SDS and 100 μg / Realized at 65 ° C. in the presence of ml salmon sperm DNA. Four washing steps following the hybridization step:
Preferably, two 5 minute washes with 2 × SSC and 0.1% SDS buffer at 65 ° C.
Preferably, one 30 minute wash with 2 × SSC and 0.1% SDS buffer at 65 ° C.
Preferably, one 10 minute wash with 0.1 × SSC and 0.1% SDS buffer at 65 ° C. is performed, and these hybridization conditions are suitable for nucleic acid molecules of about 20 nucleotides in length. . Of course, the hybridization conditions described above should be adjusted by techniques well known to those skilled in the art, depending on the desired nucleic acid length, for example, Hames BD and Higgins SJ (1985) Nucleic Acid Hybridization. A Practical Approach. Adjust according to the teachings disclosed in Hames and Higgins Ed., IRL Press, Oxford; and Current Protocols in Molecular Biology.

  “Percent homology” is used herein for both nucleic acid and amino acid sequences. Amino acid or nucleic acid “identity” is the same as amino acid or nucleic acid “homology”. To determine the percent homology of two amino acid sequences or two nucleic acids, align the sequences for the purpose of best comparison (e.g., to obtain the best alignment with a second amino acid or nucleic acid sequence Gaps can be inserted within the sequence of one amino acid or nucleic acid sequence, and non-homologous sequences can be ignored for purposes of comparison). The length of the reference sequence that is aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably the length of the reference sequence. Is at least 70%, 80%, 90% or 95%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If the same amino acid residue or nucleotide is present at a position in the first sequence as the corresponding position in the second sequence, then the molecules are homologous at that position. The percent homology between the two sequences is a function of the number of positions where the sequences match (ie,% homology = number of matching positions / total number of positions 100).

  Comparison of sequences and determination of percent homology between two sequences can be accomplished using mathematical algorithms. Preferred non-limiting examples of mathematical algorithms utilized for sequence comparison include the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68, and Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77, the disclosures of which are hereby incorporated by reference in their entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215: 403-10. A BLAST nucleotide search can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the sequences of the present invention. A BLAST protein search can be performed with the XBLAST program, score = 50, word length = 3 to obtain an amino acid sequence homologous to the polypeptide sequence of the present invention. Gapped BLAST may be used as described in Altschul et al., (1997) Nucleic Acids Research 25 (17): 3389-3402 to obtain a gapped alignment for comparison purposes. When using BLAST and Gapped BLAST programs, the default parameters of the programs (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov (the disclosures of which are hereby incorporated by reference in their entirety). Another preferred non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989), the disclosures of which are hereby incorporated by reference in their entirety. . Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using the ALIGN program for comparing amino acid sequences, the PAM120 weight residue table, gap length penalty 12 and gap penalty 4 can be used.

  “Polypeptide” refers to a polymer of amino acids, regardless of the length of the polymer. Thus, peptides, oligopeptides and proteins are included in the definition of polypeptide. The term also does not express or exclude post-translational modifications of the polypeptide, and includes, for example, covalent attachment of glycosyl groups, acetyl groups, phosphate groups, amide groups, lipid groups, carboxyl groups, acyl groups or carbohydrate groups. Polypeptides are clearly encompassed by the term polypeptide. Polypeptides containing one or more analogs of amino acids (eg, including unnatural amino acids, amino acids that are naturally occurring in unrelated biological systems, modified amino acids derived from mammalian systems, etc.), substitution bonds, and the art. Both natural and non-natural polypeptides with other known modifications are also included in the definition.

  “Protein” can be used interchangeably with “polypeptide” herein, and the term may further refer to a complex of two or more polypeptides that can be linked by bonds other than peptide bonds; For example, such a polypeptide constituting a protein may be linked by a disulfide bond. A “protein” is a family of polypeptides that have the same amino acid sequence, but in particular have different post-translational modifications that can be added when such proteins are expressed in eukaryotic hosts. Can also be included.

  An “isolated” or “purified” protein or biologically active portion thereof is derived from the cell or tissue from which the protein of the invention (ie, CPP or biologically active fragment thereof) is derived. Is substantially free of cellular material or other contaminating proteins of origin, or is chemically free of chemical precursors or other chemicals when chemically synthesized, or chemically synthesized Is substantially free of chemical precursors or other chemicals. “Substantially free of cellular material” includes a preparation of a protein of the invention in which the protein is separated from cellular components of the cell from which it is isolated or recombinantly produced. In one embodiment, “substantially free of cellular material” is less than about 30% (by dry weight) of proteins other than the protein of the present invention, more preferably proteins other than the protein of the present invention. Less than about 20%, more preferably less than about 10% of proteins other than the protein of the invention, most preferably less than about 5% of proteins other than the protein of the invention. Contains preparations. When recombinantly producing a protein of the invention or biologically active portion thereof, it is also preferred that it is substantially free of culture medium, ie, the culture medium is less than about 20% of the protein preparation amount, More preferably less than about 10%, most preferably less than about 5%.

  “Substantially free of chemical precursors or other chemicals” means a protein of the invention in which the protein is separated from chemical precursors or other chemicals involved in the synthesis of the protein Preparations. In one embodiment, “substantially free of chemical precursors or other chemicals” is less than about 30% (by dry weight) of chemical precursors or non-protein chemicals, more preferably The chemical precursor or non-protein chemical is less than about 20%, more preferably the chemical precursor or non-protein chemical is less than about 10%, most preferably the chemical precursor or non-protein. Includes preparations of the proteins of the invention that are less than about 5% chemical.

  A “recombinant polypeptide” as used herein is an artificially designed polypeptide and includes at least two polypeptide sequences that are not found in the original natural environment as a contiguous polypeptide sequence. Refers to a polypeptide or refers to a polypeptide expressed from a recombinant polynucleotide.

  “Cardiovascular disease plasma polypeptide” or “CPP” refers to a polypeptide comprising a sequence set forth in any one of the peptide sequences listed in Table 3. Each peptide listed in Table 3 corresponds to one of CPPs 149-402 as shown in Table 1. Accordingly, the polypeptide sequences of CPP 149-402 include the amino acid sequences of the corresponding peptides listed in Table 3. Preferably, CPPs 149-402 contain additional consecutive amino acids from the sequence of the corresponding polypeptide entry in the public database as shown in Table 1. Such polypeptides may have undergone post-translational modifications as described herein. CPPs may also have other structural or chemical modifications such as disulfide bonds or amino acid side chain interactions such as hydrogen and amide bonds resulting in complex secondary and tertiary structures. CPPs also include mutant polypeptides such as deletion mutants, addition mutants, exchange mutants or truncation mutants, fusion polypeptides comprising such polypeptides, and sequences of CPP 149-402. Also encompassed are polypeptide fragments of at least 3, preferably 8, 10, 12, 15 or 21 consecutive amino acids. Further included are CPP proteolytic precursors and intermediates of a sequence selected from the group consisting of CPP 149-402. The present invention encompasses a polypeptide encoded by the nucleic acid sequence of a CPP gene or CPP mRNA species, preferably human CPP gene and mRNA species, consisting of, consisting essentially of, or consisting of the sequence of CPP 149-402. An isolated CPP containing Preferred CPPs possess at least one biological activity of CPP 149-402.

  “Biological activity” as used herein refers to any one function performed by a CPP. These include, but are not limited to: (1) indicating that an individual has or will have cardiovascular disease; (2) through the blood flow of an individual having cardiovascular disease. Circulating at higher levels than normal; (3) ability to bind antigenic or anti-CPP specific antibodies; (4) ability to generate immunogenic or anti-CPP specific antibodies; (5) hydrogen bonds, amides; Form intramolecular amino acid side chain interactions such as bonds, or preferably disulfide bonds; (6) interact with CPP target molecules; better (7) undergo post-translational processing such as specific proteolysis Can be mentioned.

  As used herein, “CPP modulator” refers to a molecule (eg, polynucleotide, polypeptide, small molecule or antibody) that can modulate (ie, enhance or reduce) either the expression or biological activity of the CPP of the invention. It is. CPP modulators that enhance CPP expression or activity are referred to as CPP activators or agonists. Conversely, CPP modulators that suppress CPP expression or activity are said to be CPP inhibitors or antagonists. It is preferred that the CPP modulator enhances / reduces expression or activity by at least 5, 10 or 20%. CPP inhibitors include anti-CPP antibodies, fragments thereof, antisense polynucleotides, double stranded RNA, and molecules identified by screening assays as described herein. CPP agonists include polynucleotide expression vectors and molecules identified by screening assays as described herein.

  “CPP-related disease” or “CPP-related disease” refers to cardiovascular disease. Preferred diseases include coronary artery disease (CAD), coronary heart disease (CHD), peripheral vascular disease, cerebral ischemia (stroke), congestive heart failure, atherosclerosis, hypertension and other cardiovascular diseases. It is done. Preferably, the likelihood that an individual will develop such a disease or already has such a disease is indicated by having at least one CPP higher than normal plasma levels.

Another aspect of the invention pertains to anti-CPP antibodies. “Antibody” as used herein refers to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, ie, an antigen and specific such as CPP or a biologically active fragment or homologue thereof. Refers to a molecule having an antigen binding site that binds (immunoreacts with). Preferred antibodies bind exclusively to CPP and do not recognize other polypeptides with high affinity. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ′) ₂ fragments that can be generated by treating an antibody with an enzyme such as pepsin. The present invention provides polyclonal and monoclonal antibodies that bind to CPP or a biologically active fragment or homologue thereof. “Monoclonal antibody” or “monoclonal antibody composition” as used herein refers to a population of antibody molecules having only one type of antigen binding site capable of immunoreacting with a particular epitope of a CPP. Thus, a monoclonal antibody composition generally exhibits a single species of binding affinity for a particular CPP with which it immunoreacts. Preferred CPP antibodies are conjugated with a labeling group.

As used herein, a “labeling group” is any compound that binds to a polynucleotide or polypeptide (including antibodies) and allows detection or purification of the polynucleotide or polypeptide. The labeling group can be detected or purified directly, or it can be detected or purified indirectly by secondary compounds including antibodies specific for the labeling group. Useful labeling groups include radioisotopes (eg, ³² P, ³⁵ S, ³ H, ¹²⁵ I), fluorescent compounds (eg, 5-bromodeoxyuridine, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichloromethane. Triazinylamine fluorescein, dansyl chloride, phycoerythrin acetylaminofluorene, digoxigenin), luminescent compounds (eg luminol, GFP, luciferin, aequorin), enzyme or enzyme cofactor detection labels (eg peroxidase, luciferase, alkaline phosphatase, galactosidase or acetyl) Cholinesterase) or compounds recognized by secondary factors such as streptavidin, GST or biotin. Preferably, the labeling group is attached to the polynucleotide or polypeptide so as not to interfere with the biological activity of the polynucleotide or polypeptide.

  Radioisotopes can be detected, for example, by radioactive release, direct counting of film exposure, or by scintillation counting. Enzymatic labels can be detected by measuring the conversion of an appropriate substrate into a product (usually resulting in a fluorescent reaction). Fluorescent and luminescent compounds, and fluorescent and luminescent reactions can be detected, for example, by radioactive emission, fluorescence microscopy, fluorescence activated cell sorting or luminometer.

  As used herein, with respect to antibodies, the antibody recognizes and binds to the target of interest, but does not substantially recognize or bind to other molecules in the sample containing the target of interest, eg, a biological sample. In some cases, an antibody is said to “selectively bind” or “specifically bind” to a target.

  As used herein, “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid linked to the nucleic acid molecule. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector that can link additional DNA segments to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell, and as a result are replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” are used interchangeably so that the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors such as viral vectors that perform equivalent functions (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

  As used herein, “effective amount” refers to the amount of agent, preferably the CPP modulator of the invention, sufficient to provide the desired effect. For example, an effective amount of an anti-cardiovascular disease is the amount of drug required to reduce the symptoms of cardiovascular disease in an individual by at least 1%, 2%, 5%, 10%, 15% or preferably 25%. . The term can also represent the amount of drug required to ameliorate symptoms resulting from cardiovascular disease in an individual. Common symptoms of cardiovascular disease include chest tightness, heartburn, nausea, vomiting, numbness, shortness of breath, severe cold sweat, unexplained fatigue and anxiety. More severe symptoms of cardiovascular disease are chest pain (angina), rhythm abnormalities (arrhythmia), stroke or heart attack. The effective amount for a patient may vary depending on such factors as the method of diagnosis of the symptoms being assessed, the condition being treated, the general health of the patient, the method of administration and the severity of the side effects.

CPP of the present invention
The cardiovascular disease plasma polypeptide (CPP) of the present invention is represented by CPP 149-402 and comprises a sequence selected from the group consisting of the peptide sequences listed in Table 3. CPP 149-402 is secreted into the blood plasma of individuals with cardiovascular disease or at risk of developing cardiovascular disease and circulates at higher than normal levels. Preferred CPPs comprise a polypeptide selected from the group consisting of CPP 149-297. Even more preferred CPPs comprise a polypeptide selected from the group consisting of CPPs 149-194. Even more preferred CPPs comprise a polypeptide polypeptide selected from the group consisting of CPP 149-151.

  In addition, the CPP includes a polypeptide comprising an amino acid sequence selected from the group consisting of the peptide sequences listed in Table 3. Preferably, CPPs 149-402 contain additional contiguous amino acids from the sequence of the corresponding polypeptide entry shown in Table 1. Such additional amino acids are fused in frame with the selected CPP sequence to form a continuous amino acid sequence.

  Interestingly, the level of CPP of the present invention is dramatically increased in the plasma of individuals suffering from cardiovascular disease. The CPPs of the present invention themselves provide useful diagnostic tools, where CPPs above normal levels indicate an increased risk of developing cardiovascular disease or the presence of cardiovascular disease. In addition, CPPs are useful for drug design and therapeutic strategies for the prevention or treatment of cardiovascular disease.

  As used herein, “cardiovascular disease plasma polypeptide” and “CPP” are used to include any and all of the peptides, polypeptides and proteins of the invention. Polypeptides encoded by the polynucleotides of the present invention, as well as fusion polypeptides comprising such polypeptides, also form part of the present invention. The present invention relates to humans, including isolated or purified CPPs comprising, consisting essentially of, or comprising an amino acid sequence selected from the group consisting of the peptide sequences shown in Table 3. Includes derived CPP. Further included are unmodified precursors, proteolytic precursors and intermediates of sequences selected from the group consisting of the peptide sequences shown in Table 3.

  The invention encompasses isolated, purified and recombinant polypeptides comprising a continuous span of at least 3 amino acids, preferably at least 8-10 amino acids, having CPP biological activity. In a preferred embodiment, this stretch of contiguous amino acids includes sites of mutations or functional mutations involving amino acid deletions, additions, exchanges or truncations in the CPP sequence. The invention also relates to a polypeptide encoded by a CPP nucleotide sequence of the invention, or a complementary sequence thereof or a fragment thereof.

  One aspect of the present invention relates to isolated CPPs, and biologically active portions thereof, and polypeptide fragments suitable for use as immunogens to generate anti-CPP antibodies. In one embodiment, native CPP peptides can be isolated from plasma, cellular or tissue sources by an appropriate purification scheme utilizing standard protein purification techniques. In another embodiment, the CPP is made by recombinant DNA technology. Instead of recombinant expression, CPPs may be chemically synthesized utilizing peptide synthesis techniques such as those described in the section entitled “Chemical Production of CPP Compositions” and Example 2.

  In general, the biologically active portion comprises a domain or motif having at least one activity of CPP. Biologically active CPPs contain, for example, at least 1, 2, 3 or 5 amino acid changes in a sequence selected from the group consisting of the peptide sequences listed in Table 3, or Amino acids of such sequences may contain at least 1%, 2%, 3%, 5%, 8%, 10% or 15% changes.

CPP Characterization Polypeptides CPP149-402 of the present invention are shown in Tables 1 and 3. These polypeptides were isolated at high levels from the plasma of patients with coronary artery disease and characterized according to the MicroProt® method as described in Example 1.

For each CPP, Table 1 shows the following contents.
The accession number in the public database corresponding to the relevant polypeptide sequence, the type of protein, and the position of the amino acid defining the observed polypeptide with respect to the polypeptide in the public database.

  For a protein type, “parent” refers to a polypeptide sequence whose length is indicated by the position listed in the amino acid column, but otherwise otherwise known from the public database sequence. . “Fragment” refers to a specific newly defined fragment spanning the position indicated in the amino acid column. A “non-trypsin acting fragment” refers to a newly defined fragment that begins or ends with a non-trypsin acting portion. These themselves reflect in vivo processing of proteins that have not been known to date. “Isoform” indicates that the polypeptide species was detected as several different elution clusters by the method described in Example 1. These are evident in Table 2 and indicate the elution positions in the 1D and 2D fractions for trypsin acting peptides. Similarly, “fragment isoform” indicates that the polypeptide species, a newly defined fragment defined in the amino acid column, was detected as several different elution clusters. Finally, “variant” refers to polypeptides derived from public databases. The nature of this variant is described in parentheses.

Most of the accession numbers listed in Table 1 refer to the SwissPROT / TrEMBL database, both of which are disclosed, for example, at http://www.expasy.ch. However, CPP 195, 267, 285, 288, 289, 298-301, 320, 359, 367, 368, 396 and 401 are defined as sequences found in published patent applications, as detailed in Table 1. Has been. In addition, the accession numbers for CPPs 302-307, 389, 397 and 402 correspond to the putative protein sequences obtained by running some software on the GenBank entry, as detailed in Table 1. . Finally, the accession numbers for CPP196, 199, 381, 383 and 385 represent protein entries obtained from NCBI, http://www.ncbi.nlm.nih.gov/.

  The CPPs of the present invention all have a molecular weight of about 20 kD or less because plasma samples are first separated based on molecular weight. Higher molecular weight polypeptide species are separated and characterized in other ways. As described in Example 1, several chromatographic separations are performed on plasma samples. Details about these chromatographic methods are given in Example 1.

The initial separation is performed on a cation exchange chromatography column eluting with increasing salt concentration. Collect 18 fractions. The CEX column in Table 3 shows which fractions contained each trypsin acting peptide. Table 2 shows the NaCl concentration at which each fraction elutes according to the protocol described in Step 3 of Example 1 herein. Separation by cation exchange is indicative of the positive charge of an entire polypeptide species. After cation exchange, reverse phase HPLC separation is performed. The column of RP1 in Table 3 shows in which of the 30 fractions each trypsin acting peptide was eluted. Table 2 shows elution conditions (% B) according to the protocol described in Step 4 of Example 1. Separation by reverse phase indicates the hydrophobicity of an entire polypeptide species. The last column shows, for each trypsin acting sequence in the fifth column, the number of RP2 fractions in which trypsin acting sequences were found for the corresponding proteome in the corresponding CEX and RP1 fractions (second reverse phase HPLC separation). See Example 1). Olav scores are shown in parentheses next to each RP2 fraction in the last column, and these scores represent, among other things, the test MS-MS signal intensity against noise as detected by the MS-MS data identification software. Therefore, the protein concentration in the sample is shown.

  As shown in Table 1, CPPs 191 and 192 are isoforms of polypeptide fragments, which themselves exhibit a unique elution profile. The isoform represented by CPP191 elutes from the RP1 column at fraction 24 or before (see Table 3), while CPP192 elutes from the RP1 column to fraction 29. Similarly, CPP 193 and 194 are isoforms of the same parent polypeptide. CPP193 differs from CPP194 in elution from the CEX column. CPP193 elutes at or before fraction 9, while CPP194 elutes at or before fraction 5. Similarly, CPPs 271 and 272 are isoforms of the same parent polypeptide and as such exhibit a unique elution profile. The isoform represented by CPP271 elutes from the CEX column into fraction 15 (see Table 3), while CPP272 elutes from the CEX column into fraction 10. Similarly, CPPs 275 and 276 are newly defined isoforms of the same polypeptide fragment. CPP275 elutes from the CEX column into fraction 6, while CPP276 elutes into fraction 1. This indicates differences in the chemical properties of each isoform resulting from post-translational processing such as differential proteolysis or modification (eg, phosphorylation, glycosylation, amidation) of this plasma polypeptide.

  CPP274, 290, 291 and 293 are isoforms with specific elution profiles. For each of these, one or more additional isoforms exist and were detected by the method of the present invention, but are not listed in Table 1 or 3. CPP274 elutes in RP1 fraction 26 and above, while other isoforms detected for the same parent polypeptide elute in RP1 fraction 23 and below. CPP290 elutes in CEX fraction 11 and above, while other isoforms detected for the same parent polypeptide elute in CEX fractions 12 and 13. CPP291 elutes at CEX fraction 16 and above, while other isoforms detected for the same parent polypeptide elute at CEX fraction 15 and below. CPP293 elutes at CEX fraction 13 and below, while other isoforms detected for the same parent polypeptide elute at CEX fraction 14 and below. Finally, CPP187 is a newly defined fragment isoform that elutes at CEX fraction 14 or later. Although there are other isoforms of the same fragment eluting in CEX fraction 10, these other isoforms were not found to be differentially expressed by the method of the present invention, so Tables 1 and 3 show Not listed.

  Where applicable, the ratio of protein level in CAD to control plasma sample is calculated in two ways. The first method calculates the CAD / control ratio by the number of fractions from each sample containing CPP. For example, for CPP151, this calculation is 7/1 (see Table 3), indicating that CPP151 is 7 times higher in CAD plasma; for CPP170, this calculation is 7/2 (see Table 3), and CPP170 in CAD plasma For CPP231, this calculation is 16/7 (see Table 3), indicating that CPP231 is 2.3 times higher in CAD plasma; for CPP345, this calculation is 13/4 (See Table 3), indicating that CPP345 is 3.25 times higher in CAD plasma. Alternatively, more precisely, the weight ratio is obtained using the Olav score obtained for each peptide in the mass spectrometry data analysis software. In CPP151, the calculation is 286.69 / 16.81, which is 17.05. Thus, CPP151 is present at levels 17.05 times higher than the control in CAD plasma. In CPP170, the calculation is 241.02 / 44.98, which is 5.4. Thus, CPP170 is present at a level 5.4 times higher than the control in CAD plasma. In CPP231, the calculation is 832.6 / 3122.4, which is 2.7. Thus, CPP231 is present in 2.7 times higher concentrations in CAD plasma than controls. In CPP345, the calculation is 326.4 / 110.7, which is 2.95. Thus, CPP345 is present in CAD plasma at a concentration 2.95 times higher than the control. CPP149, 153-155, 173-174, 183, 185-188, 191-192, 194, 196, 198-203, 205, 208, 215, 217, 221-226, 228-230, 232, 233, 238, 240, 241, 243, 244, 246, 248, 252-258, 260, 262, 266, 268, 269-273, 275, 280-283, 287, 297, 299-304, 306, 308-313, 315 318, 320, 322, 324-331, 333, 334, 336, 337, 340, 342-344, 346-348, 350-355, 357-373, 375, 376, and 378-402 are only CAD plasma samples Not detected. The MicroProt® process can detect very small abundance proteins with plasma concentrations in the range of several hundred pM. Thus, this polypeptide is present at low levels that are rarely present in normal plasma. CPP itself provides a useful diagnostic tool, where elevated CPP levels indicate a high risk of developing cardiovascular disease or the presence of cardiovascular disease.

CPP Nucleic Acid One aspect of the invention pertains to purified or isolated nucleic acid molecules encoding CPPs or biologically active portions thereof as further described herein, as well as nucleic acid fragments thereof. Such nucleic acids can be used, for example, in therapeutic (DNA vaccine) and diagnostic methods and drug screening assays as described further herein.

  The object of the present invention is a purified, isolated or recombinant nucleic acid encoding CPP, sequences complementary thereto, and fragments thereof. The present invention also provides a polynucleotide having at least 95% nucleotide identity with a polynucleotide encoding CPP, advantageously a polynucleotide encoding CPP, or a sequence complementary thereto or a biologically active fragment thereof. It relates to a purified or isolated nucleic acid comprising a polynucleotide having a nucleotide identity of%, preferably having a nucleotide identity of 99.5%, most preferably having a nucleotide identity of 99.8%. Another object of the invention is a polynucleotide that encodes a CPP, or a sequence complementary thereto, or a variant thereof, or a biologically active fragment thereof under stringent hybridization conditions as defined herein. It relates to a purified, isolated or recombinant nucleic acid containing a polynucleotide that hybridizes with.

  In another preferred embodiment, the present invention relates to a purified or isolated nucleic acid molecule encoding a CPP portion or variant, wherein the portion or variant exhibits CPP biological activity. Preferably, this portion or variant is that of a naturally occurring CPP, or a precursor thereof.

  Another object of the present invention is a purification, isolation or recombination encoding a CPP comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of the peptide sequences listed in Table 3 Body nucleic acid, wherein the isolated nucleic acid molecule encodes one or more motifs such as target binding sites or disulfide bonds.

  Depending on the nucleotide sequence determined from the cloning of the CPP-encoding gene, for use in identifying and / or cloning other CPPs (eg sharing a novel functional domain), as well as CPP homologues from other species Designed probes and primers can be produced.

  A nucleic acid fragment encoding a “biologically active portion of a CPP” can be isolated by isolating a portion of a nucleotide sequence encoding a CPP that encodes a polypeptide having CPP biological activity, and expressing the coding site of the CPP. (Eg, by recombinant expression in vitro or in vivo) and by evaluating the activity of the coding portion of the CPP.

  The present invention further includes nucleic acid sequences that encode the same CPP of the present invention, unlike the CPP nucleotide sequences of the present invention, due to the degeneracy of the genetic code.

  One skilled in the art will appreciate that in addition to the CPP nucleotide sequences described above, DNA sequence polymorphisms that result in changes in the amino acid sequence of CPP may exist within a population (eg, the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. Such natural allelic variations can generally result in 1-5% change in the nucleotide sequence of a CPP-encoding gene or nucleic acid sequence.

  Nucleic acid molecules corresponding to naturally occurring allelic variants and homologues of the CPP nucleic acids of the invention can be obtained by subjecting the cDNA disclosed herein or a portion thereof to stringent hybridization conditions according to standard hybridization techniques. They can be used as hybridization probes and isolated based on their homology with the CPP nucleic acids disclosed herein.

  Of course, the present invention includes polypeptides having an amino acid sequence encoded by any of the polynucleotides of the present invention.

Use of CPP Nucleic Acids Polynucleotide sequences encoding CPP (or its complement) have a variety of uses including use as hybridization probes, use in chromosome and gene mapping, and production of antisense RNA and DNA. . Furthermore, as described herein, CPP-encoding nucleic acids are useful as targets for pharmacological intervention, eg, for the development of DNA vaccines, and for the production of CPPs by recombinant techniques. The polynucleotides described herein, including their sequence variants, can be used in diagnostic assays. Accordingly, diagnostic methods based on detecting the presence of such polynucleotides in body fluids or tissue samples are a feature of the invention. Examples of nucleic acid based diagnostic assays according to the present invention include, but are not limited to, for example, hybridization assays such as in situ hybridization and PCR based assays. Polynucleotides, including extended polynucleotides, sequence variants and fragments thereof, as described herein can be used to make hybridization probes or PCR primers for use in such assays. Such probes and primers can detect polynucleotide sequences comprising genomic sequences that are similar to or complementary to the CPP polynucleotides described herein.

  The present invention includes primer pairs for performing PCR for the purpose of amplifying a segment of the polynucleotide of the present invention. Each primer of a primer pair forms i) a duplex in which one primer in the pair is perfectly matched with one strand of the polynucleotide of the invention, and the other primer in the pair is complementary to the same polynucleotide. Duplexes that perfectly match the strands, and ii) the primer of the primer pair is such a perfect match at a position on that polynucleotide that is separated by a distance of between 10 and 2500 nucleotides An oligonucleotide having a length between 15 and 30 nucleotides so as to form a double strand. Preferably, the annealing temperature of each primer of the primer pair with its respective complementary sequence is substantially the same.

  Hybridization probes obtained from the polynucleotides of the invention are used to perform in situ hybridization on a tissue sample such as, for example, a fixed or frozen tissue section or cell suspension prepared on a microscope slide. be able to. In short, a labeled DNA or RNA probe is allowed to bind to its DNA or RNA target sample in a tissue section on a microscope preparation under controlled conditions. In general, dsDNA probes composed of DNA of interest cloned into a plasmid or bacteriophage DNA vector are used for this purpose, but ssDNA or ssRNA probes may also be used. Probes are generally oligonucleotides between about 15-40 nucleotides in length. Alternatively, these probes can be polynucleotide probes made by PCR random priming primer extension or in vitro transcription of RNA from a plasmid (riboprobe). These latter probes are generally several hundred base pairs long. These probes can be labeled with any of a number of labeling groups, and the particular detection method will depend on the type of label used for the probe (eg, autoradiography, if desired). X-ray detection, fluorescence or visual microscopic analysis). The reaction can also be further amplified in situ using immunocytochemical techniques for labeling the detection molecule used, such as an antibody against the fluorescein moiety present in the fluorescently labeled probe. Specific labeling and in situ detection methods are described in, for example, Howard, GC, Ed., Methods in Nonradioactive Detection, Appleton & Lange, Norwalk, Conn., (1993), which is incorporated herein by reference. ).

  Hybridization probes and PCR primers may also be selected from genomic sequences corresponding to full-length proteins identified according to the present invention, including promoters, enhancer elements and introns of genes encoding naturally occurring polypeptides. Nucleotide sequences encoding CPPs can also be used to construct hybridization probes for mapping genes encoding CPPs and for genetic analysis of individuals. Various techniques can detect individuals at the DNA level having variations or mutations in the gene encoding the CPP of the present invention. Nucleic acids used for diagnosis can be obtained from patient cells, including, for example, tissue biopsies and autopsy samples. Genomic DNA may be used directly for detection or may be amplified enzymatically using PCR (Saiki, et al. Nature 324: 163-166 (1986)) prior to analysis. RNA or cDNA can also be used for the same purpose. As an example, PCR primers complementary to the nucleic acids of the invention can be used to identify and analyze mutations in the genes of the invention. Deletions and insertions can be detected by changes in the size of the amplified product when compared to the normal genotype. Point mutations can be confirmed by hybridizing amplified DNA to radiolabeled RNA of the present invention or radiolabeled antisense DNA sequences of the present invention. Nucleotide protection assays such as RNase and S1 protection or chemical cleavage methods (eg, Cotton, et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985)) Or by the difference in melting temperature. “Molecular beacons” (Kostrikis LG et al., Science 279: 1228-1229 (1998)), ie, a hairpin single-stranded synthetic oligonucleotide comprising a probe sequence complementary to the nucleic acid of the present invention, It can be used to detect point mutations or other sequence changes, as well as to monitor CPP expression levels.

Oligonucleotides and Antisense Compounds Oligonucleotides of the invention comprising PCR primers and antisense compounds can be synthesized by conventional methods using commercially available automated DNA synthesizers, such as Applied Biosystems (Foster City, CA) type 380B, 392 or 394 DNA / Synthesized with an RNA synthesizer or equivalent equipment. Preferably, for example, phosphoramidite chemistry disclosed in the following references is used: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, US Pat. No. 4,980,460. Koster et al, US Pat. No. 4,725,677; Caruthers et al, US Pat. No. 4,415,732; US Pat. No. 4,458,066; and US Pat. No. 4,973,679. A nuclease resistant backbone is preferred for therapeutic use. Many types of modified oligonucleotides that confer nuclease resistance, such as many references such as phosphorothioates: Stec et al, US Pat. No. 5,151,510; Hirschbein, US Pat. No. 5,166,387; Bergot U.S. Pat. No. 5,183,885; phosphoramidates: Froehler et al, international application PCT / US90 / 03138; and further review of applicable chemistry: phosphorothioates described in Uhlmann and Peyman (supra) , Phosphorodithioate, phosphoramidate and the like can be used. The length of the antisense oligonucleotide must be large enough to ensure that specific binding occurs only at the desired target polynucleotide and not at other unintended sites. The upper limit of length is determined by several factors including the inconvenience and cost of synthesis and purification of oligomers greater than about 30-40 nucleotides in length, the longer oligonucleotides are more resistant to mismatches than shorter oligonucleotides, and the like. Preferably, the length of the antisense oligonucleotide of the present invention ranges from about 15 to 40 nucleotides. More preferably, the length of the oligonucleotide portion ranges from about 18-25 nucleotides.

Double stranded RNA, ie sense-antisense RNA (also called small interfering RNA (siRNA) molecules), can also be used to inhibit the expression of nucleic acids encoding CPP. RNA interference is a method in which an exogenous short RNA duplex, one strand of which corresponds to the coding region of the target mRNA, is administered (Elbashir et al., Nature 2001, 411: 494-498). Upon entering the cell, siRNA molecules not only cause degradation of exogenous RNA duplexes, but also cause degradation of single-stranded RNA with the same sequence, including endogenous messenger RNA. Thus, since this technology is believed to work through a catalytic mechanism, conventional siRNA may be more powerful and effective than antisense RNA methodology. Preferred siRNA molecules are generally 19-25 nucleotides in length, preferably about 21 nucleotides in length, and include the CPP nucleic acid sequence. Effective strategies for delivering siRNA to target cells include, for example, transduction using physical or chemical transfection. Alternatively, siRNA can be expressed in cells using, for example, various PolIII promoter expression cassettes that allow transcription of functional siRNA or precursors thereof. For example, Scherr et al., Curr. Med. Chem. 2003, 10 (3): 245-256; Turki et al., Hum. Gene Ther. 2002, 13 (18): 2197-2201; Cornell et al., Nat. Struct. Biol. 2003, 10 (2): 91-92.

Primers and Probes The primers and probes of the present invention are, for example, the phosphodiester method of Narang SA et al (Methods Enzymol 1979; 68: 90-98) and the phosphodiester method of Brown EL et al (Methods Enzymol 1979; 68: 109-151). The diester method, the diethyl phosphoramidite method of Beaucage et al (Tetrahedron Lett 1981, 22: 1859-1862) and the solid support method described in EP 0 707 592 (the disclosures of which are incorporated herein by reference) May be prepared by any suitable method, including cloning and restriction of appropriate sequences by methods such as

  The detection probe is generally a nucleic acid sequence or, for example, a peptide nucleic acid disclosed in International Patent Application WO 92/20702, US Pat. Nos. 5,185,444; 5,034,506 and 5 , 142,047, such as morpholino analogs. If desired, the probe may be “non-extensible” in the sense that no further dNTPs can be added to the probe. Analogs in essence are usually non-extensible, and nucleic acid probes can be rendered non-extendable by modifying the 3 ′ end of the probe so that the hydroxyl group cannot participate in the extension. . For example, the 3 'end of the probe can be functionalized with a capture or detection label, thereby consuming the hydroxyl group or otherwise blocking the hydroxyl group.

  Any of the polynucleotides of the present invention can be optionally incorporated by incorporating any labeling group known in the art that is detectable by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Can be labeled. Further examples are described in Urdea et al. (Nucleic Acids Research. 11: 4937-4957, 1988) or Sanchez-Pescador et al. (J. Clin. Microbiol. 26 (10): 1934-1938, 1988). Non-radioactive labeling of nucleic acid fragments such as Furthermore, the probes of the present invention may have structural characteristics that allow them to amplify signals, such structural characteristics being, for example, Urdea et al (Nucleic Acids Symp. Ser. 24: 197-200, 1991) or as described in European Patent No. EP 0225807 (Chiron).

  The label can also be used to capture the primer to facilitate immobilization of either the primer or a primer extension product such as amplified DNA to a solid support. A capture label can be a specific binding member that is attached to a primer or probe and forms a binding pair with a specific binding member of a solid phase reagent (eg, biotin and streptavidin). Thus, capture labels can be used to capture or detect target DNA, depending on the type of label attached to the polynucleotide or probe. Furthermore, it will be appreciated that the polynucleotides, primers or probes provided herein can themselves serve as capture labels. For example, if the binding member of the solid phase reagent is a nucleic acid sequence, it is selected such that it binds to the complementary portion of the primer or probe, thereby immobilizing the primer or probe to the solid phase. One skilled in the art will appreciate that when a polynucleotide probe itself serves as a binding member, the probe contains a sequence or “tail” that is not complementary to the target. When the polynucleotide primer itself serves as a capture label, at least a portion of the primer is free to hybridize with the nucleic acid on the solid phase. The DNA labeling technique is a technique well known to those skilled in the art.

  The probes of the present invention are useful for many applications. They can be used in particular for Southern hybridization with genomic DNA. These probes can also be used to detect PCR amplification products. They can also be used to detect mismatches in genes or mRNA encoding CPP using other techniques.

  Any of the nucleic acids, polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid phase supports are known to those skilled in the art and include well walls of reaction trays, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or Other animals) such as red blood cells and duracyte. The solid support is not critical and can be selected by one skilled in the art. Thus latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, microtiter well walls, glass or silicon chips, sheep (or other suitable animal) red blood cells and durasite are all suitable examples. . Suitable methods for immobilizing nucleic acids on a solid phase include ionic interactions, hydrophobic interactions, covalent interactions, and the like. A solid support as used herein refers to any substance that is insoluble or can become insoluble by a subsequent reaction. The solid support can be selected for its inherent ability to attract and immobilize the capture reagent. Alternatively, the solid phase may carry additional receptors that have the ability to attract and immobilize capture reagents. Additional receptors can include charged substances that are oppositely charged to the capture reagent itself or to charged substances conjugated to the capture reagent. As yet another example, the receptor molecule can be a specific binding member that is bound to a solid support that has the ability to immobilize a capture reagent through a specific binding reaction. The receptor molecule allows indirect binding of the capture reagent and the solid support material prior to or during the performance of the assay. Thus, the solid phase can be plastic, derivatized plastic, magnetic or non-magnetic metal, test tube glass or silicon surface, microtiter wells, sheets, beads, microparticles, chips, sheep (or other suitable animal) red blood cells, It can be durasite and other forms known to those skilled in the art. At least 2, 5, 8, 10, 12, 15, 20, or 25 of the nucleic acids, polynucleotides, primers and probes of the invention individually or on one solid support Different polynucleotides of the invention can be combined and bound to a solid support or can be immobilized to a solid support. Furthermore, polynucleotides other than those of the present invention may be bound to the same solid support as one or more polynucleotides of the present invention.

  Any of the polynucleotides provided herein may be bound to overlapping regions on the solid support or may be bound at any position. Alternatively, the polynucleotides of the invention may be bound in an array that is aligned such that each polynucleotide is bound to a different region of the solid support that does not overlap with the binding site of the other polynucleotide. Preferably, such an aligned array of polynucleotides is designed to be “addressable” where different positions are recorded and accessible as part of the assay procedure. Addressable polynucleotide arrays generally comprise a plurality of different oligonucleotide probes attached to the surface of the support at various known locations. Recognition of the exact position of each polynucleotide position makes these “addressable” arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be used with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as Genechips, US Pat. No. 5,143,854, the disclosure of which is hereby incorporated by reference in its entirety; PCT Publication An overview is given in WO 90/15070 and 92/10092.

Methods for Obtaining Nucleic Acid and Polypeptide Variants The skilled artisan will introduce mutations into the nucleotide sequence encoding CPP, as well as naturally occurring allelic variants of the CPP sequence that may be present in the population, and so on. By doing so, it will be appreciated that changes in the amino acid sequence of the encoded CPP may result in alterations that alter or not alter the functional activity of the CPP.

  1) One or more amino acid residues are replaced with conserved or non-conserved amino acid residues, and such substituted amino acid residues may or may not be encoded by the genetic code. Or 2) one or more amino acid residues contain a substituent, or 3) a mutant CPP is fused with another compound such as a compound that extends the half-life of the polypeptide (eg, polyethylene glycol). Or 4) additional amino acids such as leader sequences, signal sequences or anchor sequences, sequences used to purify CPPs or sequences derived from precursor proteins are fused to CPPs, Several types of variants are contemplated. Such variants are considered to be within the scope of those skilled in the art.

  For example, nucleotide substitutions leading to amino acid substitutions can be made within sequences that do not substantially alter the biological activity of the protein. Amino acid residues can be modified from the wild-type sequence encoding CPP or a biologically active fragment or homologue thereof without altering biological activity. In general, amino acid residues common to the CPPs of the present invention are expected to be less susceptible to modification.

  In another aspect, the invention relates to nucleic acid molecules encoding CPPs that contain changes in amino acid residues that enhance or alter biological activity. In another aspect, the present invention relates to nucleic acid molecules encoding CPPs that contain changes in amino acid residues essential for CPP biological activity. Such CPPs differ in the amino acid sequence of CPP 149-402 and exhibit reduced activity or are essentially deficient in one or more CPP biological activities.

  Mutations, substitutions, additions or deletions can be introduced into any of CPPs 149-402 by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. For example, conservative amino acid substitutions can be made at one or more putative non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. A family of amino acid residues having similar side chains has been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), amino acids with uncharged polar side chains (eg, glycine) , Asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains ( Examples include threonine, valine, isoleucine) and amino acids having aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, a putative nonessential amino acid residue of CPP or a biologically active fragment or homologue thereof can be replaced with another amino acid residue of the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the CPP coding sequence, such as by saturation mutagenesis, and the resulting mutant screened for CPP biological activity, Mutants that retain activity can be identified. After mutagenesis of a nucleotide encoding one of CPP149-402, the encoded protein is recombinantly expressed and the activity of the protein is determined, for example, by a suitable assay as provided herein. Can do.

  The present invention also provides CPP chimeric or fusion proteins. As used herein, a CPP “chimeric protein” or “fusion protein” includes a CPP of the invention or fragment thereof operably linked to a non-CPP polypeptide sequence or fused in-frame. It is a waste. In preferred embodiments, the CPP fusion protein comprises at least one biologically active portion of CPP. In another embodiment, the CPP fusion protein comprises at least two biologically active portions of CPP. For example, in one embodiment, the fusion protein is a GST-CPP fusion protein in which the CPP domain sequence is fused to the C-terminus of the GST sequence. Such fusion proteins can facilitate the purification of recombinant CPP. In another embodiment, the fusion protein is a CPP containing a heterologous signal sequence at its N-terminus, eg, allowing for desired cellular localization in a particular host cell. In yet another embodiment, the fusion is a CPP bioactive fragment and an immunoglobulin molecule. Such fusion proteins are useful, for example, to increase the valency of the CPP binding site. For example, a bivalent CPP binding site can be formed by fusing a biologically active CPP fragment and an IgG Fc protein.

  The CPP fusion proteins of the invention can be used as immunogens to produce anti-CPP antibodies in a subject, to purify CPP or CPP ligands, and in screens to identify CPP modulators.

  Furthermore, isolated fragments of CPP can also be obtained by screening peptides produced recombinantly from corresponding fragments of nucleic acids encoding such peptides. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, the CPP of the present invention may be arbitrarily divided into fragments of a desired length without overlapping of the fragments, or preferably may be divided into overlapping fragments of a desired length. These fragments can be made (by recombinant or chemical synthesis), and these peptidyl fragments having CPP biological activity can be identified, for example, by microinjection assays or in vitro protein binding assays. In certain exemplary embodiments, a peptidyl portion of a CPP, such as a CPP target binding region, can be tested for CPP activity by expressing it as a thioredoxin fusion protein, each comprising a separate CPP fragment (eg, US Pat. Nos. 5,270,181 and 5,292,646; and PCT Publication No. WO 94/02502, the disclosures of which are hereby incorporated by reference.

  In addition, a library of CPP coding sequence fragments can be used to create a CPP fragment-rich population for screening and subsequent selection of CPP variants. In one embodiment, a double-stranded PCR fragment of a CPP coding sequence is treated with a nuclease under conditions that cause nicking only about once per molecule to denature the double-stranded DNA and regenerate the DNA. A double stranded DNA that may contain sense / antisense pairs derived from different nick products is formed, the single stranded portion is removed from the reshaped duplex by S1 nuclease treatment, and the resulting fragment live By linking the library to an expression vector, a library of coding sequence fragments can be generated. By this method, an expression library can be obtained that encodes N-terminal, C-terminal and internal fragments of various sizes of CPP.

  Whether a change in the amino acid sequence of a peptide results in a functional CPP homolog can be readily determined by assessing at least one CPP biological activity of the mutant peptide. Peptides in which more than one substitution has occurred can be easily tested as well.

Chemical Production of CPP Compositions Peptides of the invention are synthesized by standard techniques (eg, Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, IL, 1984). Preferably, a commercially available peptide synthesizer, such as Applied Biosystems, Inc. (Foster City, Calif.) Type 430A, is used to convert the polypeptide of the invention into a convergent synthesis approach, such as Kent et al, US Pat. No. 6,184. 344, and Dawson and Kent, Annu. Rev. Biochem., 69: 923-960 (2000), may be composed of a plurality of peptides synthesized and purified separately. The peptides of the present invention may be constructed on a cross-linked polystyrene support by solid phase synthesis, starting with a carboxyl terminal residue and adding amino acids stepwise until a complete peptide is formed. The following references provide guidance on the chemistry used during the synthesis: Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992); Merrifield, J. Amer. Chem. Soc. , Vol. 85, pg. 2149 (1963); Kent et al., Pg 185, in Peptides 1984, Ragnarsson, Ed. (Almquist and Weksell, Stockholm, 1984); Kent et al., Pg. 217 in Peptide Chemistry 84 , Izumiya, Ed. (Protein Research Foundation, BH Osaka, 1985); Merrifield, Science, Vol. 232, pgs. 341-347 (1986); Kent, Ann. Rev. Biochem., Vol. 57, pgs. 957- References cited in 989 (1988) and these last two references.

  Preferably, the chemical synthesis of the polypeptides of the present invention is performed by natural chemistry as described by Dawson et al, Science, 266: 776-779 (1994) and Kent el al, US Pat. No. 6,184,344. This is done by constructing peptide fragments by linking. In short, in the above approach, the first peptide fragment is provided with an N-terminal cysteine having a non-oxidized sulfhydryl side chain, and the second peptide fragment is provided with a C-terminal thioester. The non-oxidized sulfhydryl side chain of the N-terminal cysteine is condensed with the C-terminal thioester to yield an intermediate peptide fragment in which the first peptide fragment and the second peptide fragment are linked by a β-aminothioester bond. The β-aminothioester bond of the intermediate peptide fragment is then subjected to intramolecular transfer, resulting in a peptide fragment product in which the first peptide fragment and the second peptide fragment are linked by an amide bond. Preferably, the N-terminal cysteine of the internal fragment is protected from unwanted cyclization and / or chain reaction with a cyclic thiazolidine protecting group, as described below. Preferably such a cyclic thiazolidine protecting group is a thioprolinyl group.

Peptide fragments having a C-terminal thioester can be made as described in the following references, which are hereby incorporated by reference: Kent et al, US Pat. No. 6,184,344; Tam et al, Proc. Natl. Acad. Sci., 92: 12485-12489 (1995); Blake, Int. J. Peptide Protein Res., 17: 273 (1981); Canne et al, Tetrahedron Letters, 36: 1217 -1220 (1995); Hackeng et al, Proc. Natl. Acad. Sci., 94: 7845-7850 (1997); or Hackeng et al, Proc. Natl. Acad. Sci., 96: 10068-10073 (1999). . Preferably, the method described by Hackeng et al (1999) is used. In short, peptide fragments are obtained in situ for Boc chemistry as disclosed by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992), which is incorporated herein by reference. By using the sum / HBTU activation approach, it is generally synthesized on a solid support on a 0.25 mmol scale (described below). (HBTU is 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate and Boc is t-butoxycarbonyl). Each synthesis cycle consists of N ^α -Boc removal by treatment with pure TFA for 1-2 minutes, 1 minute DMF flow wash, 10-20 with 1.0 mmol pre-activated Boc-amino acid in the presence of DIEA. Consists of a minute binding time and a second DMF flow wash. (TFA is trifluoroacetic acid, DMF is N, N-dimethylformamide, and DIEA is N, N-diisopropylethylamine). N ^α -Boc-amino acid (1.1 mmol) is preactivated for 3 minutes with 1.0 mmol HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol). After each binding step, the yield is measured using a conventional quantitative ninhydrin assay as disclosed, for example, in Sarin et al, Anal. Biochem., 117: 147-157 (1981). Determined by After conjugation of the Gln residue, DCM flow washing is used before and after deprotection by using TFA to prevent possible high temperature (TFA / DMF) catalyzed pyrrolidone formation. After chain construction is complete, the peptide fragment is deprotected and cleaved from the resin by treatment with anhydrous HF at 0 ° C. for 1 hour using 4% p-cresol as a scavenger. Since the dnp removal procedure is not compatible with the C-terminal thioester group, the imidazole side chain 2,4-dinitrophenyl (dnp) protecting group remains on the His residue. However, dnp is gradually removed by thiols during the ligation reaction. After cleavage, the peptide fragments are precipitated with ice cold diethyl ether, dissolved in aqueous acetonitrile and lyophilized.

The thioester peptide fragments described above are preferably synthesized on a trityl-linked mercaptopropionic acid-leucine (TAMPAL) resin made as disclosed by Hackeng et al (1999) or equivalent protocol. In short, the presence of DIEA in N α ^-Boc-Leu and (4 mmol) 6 mmol, activated with HBTU of 3.6 mmol, therewith to bind and p- methylbenzhydrylamine (MBHA) resin or equivalent 16 minutes 2mmol . Next, 3 mmol of S-trityl mercaptopropionic acid is activated with 2.7 mmol of HBTU in the presence of 6 mmol of DIEA and allowed to bind to Leu-MBHA resin for 16 minutes. The obtained TAMPAL resin was subjected to treatment with 3.5% triisopropylsilane and 2.5% H ₂ O in TFA twice for 1 minute to remove the trityl protecting group, and then the starting resin having a polypeptide chain structure. Can be used as As disclosed in Schnolzer et al (supra), a standard in situ neutralizing peptide binding protocol can be used for 1 hour to form a thioester bond with any desired amino acid. Treatment of the final peptide fragment with anhydrous HF yields a C-terminal activated mercaptopropionic acid-leucine (MPAL) thioester peptide fragment.

  Preferably, a thiazolidine protected thioester peptide fragment intermediate is used for natural chemical ligation under conditions described by Hackeng et al (1999) or equivalent conditions. In short, 0.1M phosphate buffer (pH 8.5) containing 6M guanidine, 4% (vol / vol) benzyl mercaptan and 4% (vol / vol) thiophenol was added to the dried peptide to be ligated, The final peptide concentration is 1 to 3 mM at about pH 7. This pH is lowered by the addition of thiol and TFA from the lyophilized peptide. Preferably, the ligation reaction is performed at 37 ° C. with a heating block, which is periodically stirred to equilibrate the thiol additive. The progress of this reaction can be monitored by MALDI-MS or HPLC and electrospray ionization MS.

  After the natural chemical ligation reaction is complete or terminated, the N-terminal thiazolidine ring of the product is brought to pH 3.5-4.5, 37 ° C. with a cysteine deprotecting agent (0.5 M) such as O-methylhydroxylamine. Ring opening by treatment for 2 hours, and then prior to purification of the product by conventional preparative HPLC, a 10-fold excess of Tris- (2-carboxyethyl) -phosphine was added to the reaction mixture to oxidize the reaction components. Is completely reduced. Preferably, the fractions containing the ligation product are confirmed by electrospray MS and they are pooled and lyophilized.

  After synthesis is complete and the final product is purified, the final polypeptide product can be purified using conventional techniques such as Creighton, Meth. Enzymol., 107: 305-329 (1984); White, Meth. Enzymol., 11: 481- 484 (1967); Wetlaufer, Meth. Enzymol., 107: 301-304 (1984). Preferably, the final product is refolded by air oxidation, such as by: The reduced lyophilized product is dissolved in 1M guanidine hydrochloride (or equivalent chaotropic agent) pH 8.6 containing 100 mM Tris, 10 mM methionine (approximately 0.1 mg). / ML). After stirring slowly overnight, the refolded product is isolated by reverse phase HPLC using a conventional protocol.

Recombinant expression vectors and host cells The polynucleotide sequences described herein can be used in recombinant DNA molecules that direct the expression of the corresponding polypeptide in a suitable host cell. Because of the degeneracy of the genetic code, other DNA sequences may encode equivalent amino acid sequences, so they can also be used for CPP cloning and expression. Preferred codons for a particular host cell may be selected and used for naturally occurring nucleotide sequences to increase expression rate and / or efficiency. Nucleic acid (eg, cDNA or genomic DNA) encoding the desired CPP can be inserted into a replicable vector for cloning (amplifying DNA) or for expression. Recombinant expression of the polypeptide in any of a number of expression systems according to methods known in the art (Ausubel, et al., Editors, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1990) be able to. Suitable host cells include yeast, bacteria, primordial bacteria, fungi, and animal cells including insect cells and mammalian cells, such as, but not limited to, primary cells including stem cells including bone marrow stem cells. It is done. More particularly, these include, but are not limited to, recombinant bacteriophage, bacteria transformed with plasmid or cosmid DNA expression vectors, and yeast transformed with yeast expression vectors. Microorganisms. Also included are insect cells infected with recombinant insect viruses (such as baculovirus) and mammalian expression systems. The nucleic acid sequence to be expressed can be inserted into the vector by various techniques. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Vector components generally include, but are not limited to, one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. For the construction of suitable vectors containing one or more of these components, standard ligation techniques known to those skilled in the art are used.

  The CPP of the present invention is produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding CPP under appropriate conditions for inducing or causing protein expression. Appropriate conditions for expression of CPP will vary with the choice of expression vector and host cell, as will be ascertained by one skilled in the art. For example, using a constitutive promoter in an expression vector requires regular optimization of host cell growth and proliferation, while using an inducible promoter requires appropriate growth conditions for induction. is there. Furthermore, in some embodiments, the timing of recovery is important. For example, since the baculovirus system used for insect cell expression is a lytic virus, harvest time selection can be important for product yield.

  A host cell line is chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the protein include, but are not limited to, glycosyl groups, acetyl groups, phosphate groups, amide groups, lipid groups, carboxyl groups, acyl groups or carbohydrate groups. Post-translational processing that disrupts the “prepro” form of the protein may also be important for correct insertion, folding and / or function. As an example, host cells such as CHO, HeLa, BHK, MDCK, 293, W138, etc. have specific cellular and characteristic mechanisms for such post-translational activity, and the accuracy of the introduced heterologous protein. Are selected to ensure proper modification and processing. Of particular note are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, Cl29 cells, 293 cells, There are Neurospora, BHK, CHO, COS and HeLa cells, fibroblasts, schwannoma cell lines, immortalized mammalian bone marrow and lymphoid cell lines, Jurkat cells, human cells and other primary cells.

  A nucleic acid encoding a CPP must be “operably linked” by making it functionally related to another nucleic acid sequence. For example, if the presequence or secretory leader DNA is expressed as a preprotein that is involved in the secretion of the polypeptide, it is operably linked to the polypeptide DNA; a promoter or enhancer may transcribe the sequence. It is operably linked to the coding sequence; or if the ribosome binding site is positioned to facilitate translation, it is operably linked to the coding sequence. It is connected. In general, DNA sequences that are “operably linked” are contiguous, and in the case of secretory leaders or other polypeptide sequences, are adjacent in the reading phase. However, enhancers do not have to be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional practice. The promoter sequence encodes either a constitutive promoter or an inducible promoter. The promoter may be a naturally occurring promoter or a hybrid promoter. Hybrid promoters having two or more promoter elements are also known in the art and are useful in the present invention. An expression vector may contain additional elements, for example, an expression vector may have two replication systems, so that it is used for cloning and amplification with two organisms, eg, mammalian or insect cells for expression. It can be maintained in a prokaryotic host. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences for various bacteria, yeasts and viruses are well known. The origin of replication of plasmid pBR322 is suitable for most gram-negative bacteria; 2: the plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are found in mammalian cells. Useful for cloning vectors. In addition, in the case of an integrative expression vector, the expression vector contains at least one sequence that is homologous to the host cell genome, preferably two homologous sequences that flank the expression construct. By selecting appropriate homologous sequences for inclusion in the integration vector, the integration vector can be directed to a specific locus in the host cell. Integration vector constructs are well known in the art. In further embodiments, the heterologous expression control element can be operably linked to an endogenous gene of the host cell by homologous recombination (the disclosure of which is hereby incorporated by reference in its entirety). 6410266 and 6361972). This technique allows for the regulation of expression to the desired level by the selected control elements while ensuring proper processing and modification of the CPP endogenously expressed by the host cell. Useful heterologous expression control elements include, but are not limited to, CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, promoter of retroviral LTR such as that of Rous sarcoma virus (RSV), and metallothionein A promoter is mentioned.

  The expression vector preferably contains a selectable marker gene that allows selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Expression and cloning vectors generally contain a selection gene, also termed a selectable marker. Typical selection genes are (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement the lack of auxotrophy, or (c) natural media It encodes a protein that supplies important nutrients that cannot be used from the plant, for example, a gene encoding D-alanine racemase of Bacillus.

  Host cells transformed with a nucleotide sequence encoding CPP can be cultured under conditions suitable for expression of the encoded protein and recovery from the cell culture. Proteins produced by recombinant cells are secreted, membrane bound, or contained intracellularly, depending on the sequence and / or vector used. As will be apparent to those skilled in the art, expression vectors containing a polynucleotide encoding CPP can be designed to have a signal sequence that directs secretion of CPP through prokaryotic or eukaryotic cell membranes. The desired CPP can be generated not only directly by recombination, but also with a heterologous polypeptide that can be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. It can also be produced as a fusion polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the DNA encoding CPP that is inserted into the vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leader. For yeast secretion, signal sequences include, for example, the yeast invertase leader, alpha factor leader (Saccharomyces and Kluyveromyces a-factor leader, the latter of which is US Pat. No. 5,010,182. Or acid phosphatase leader, C.I. Signals described in the C. albicans glucoamylase leader (EP 362,179 published April 4, 1990) or WO901113646 published November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct secretion of the protein, for example, signal sequences of secreted polypeptides of the same or related species, as well as viral secretion leaders. Depending on the expression system chosen, once the coding sequence is inserted into the appropriate vector, it then requires the presence of a “control element” or “regulatory sequence” with specific characteristics. Suitable constructs are generally known in the art (Ausubel, et al., 1990) and often are Invitrogen (San Diego, Calif.), Stratagene (La Jolla, Calif.), Gibco BRL (Rockville , Md.) Or Clontech (Palo Alto, Calif.)

Expression in bacterial systems Transformation of bacterial cells may be achieved using an inducible promoter such as the hybrid lacZ promoter of “BLUESCRIPT” phagemid (Stratagene) or “pSPORT1” (Gibco BRL). In addition, but not limited to “BLUESCRIPT” (a-galactosidase; Stratagene) or used in bacterial cells to create cleavable fusion proteins that can be easily detected and / or purified A number of expression vectors may also be selected, including pGEX (glutathione S-transferase; Promega, Madison, Wis.). A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating downstream (3 ') transcription of the CPP coding sequence into mRNA. Bacterial promoters usually have a transcription initiation region located proximal to the 5 ′ end of the coding sequence. This transcription initiation region generally includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Bacteriophage promoters can also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tat promoter is a hybrid of trp and lac promoter sequences. Furthermore, bacterial promoters can also include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. An efficient ribosome binding site is also desirable. The expression vector may also include a signal peptide sequence that provides for secretion of CPP in bacteria. As is well known in the art, the signal sequence generally encodes a signal peptide consisting of hydrophobic amino acids that direct the secretion of the protein from the cell. The protein is secreted into the growth medium (Gram positive bacteria) or secreted into the periplasmic space and exists between the inner and outer membranes of the cells (Gram negative bacteria). Bacterial expression vectors can also include a selectable marker gene that allows for selection of transformed bacterial strains. Suitable selection genes include drug resistance genes such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes such as those of the histidine, tryptophan and leucine biosynthetic pathways. For example, if a large amount of NPP is required for antibody induction, vectors that direct high expression of easily purified fusion proteins may be desirable. Such vectors include, but are not limited to, BLUESCRIPT (Stratagene) (in BLUESCRIPT, the CPP coding sequence comprises the amino terminal Met of β-galactosidase followed by 7 residues so that a hybrid protein is produced. Multifunctional E. coli cloning and expression such as PIN vectors (Van Heeke & Schuster J Biol Chem 264: 5503-5509 1989); PET vectors (Novagen, Madison Wis.), Etc. Vector. Bacterial expression vectors include the various components described above, and these vectors are well known in the art. Examples include, inter alia, Bacillus subtilis, E. coli, Streptococcus cremoris and Streptococcus lividans vectors. Bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art such as calcium chloride mediated transfection or electroporation.

Yeast Expression Yeast expression systems are well known in the art and include Saccharomyces cerevisiae, Candida albicans and C. albicans. C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. cerevisiae. K. lactis, Pichia guillermondii and P. a. Examples include expression vectors for P. pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Examples of suitable promoters for use in yeast hosts include 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073 (1980)) or enolase, glyceraldehyde-3-phosphorus Acid dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, glucose phosphate isomerase, alpha factor, ADH2IGAPDH promoter, gluco Examples include promoters of other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149 (1968); Holland, Biochemistry 17: 4900 (1978)), such as kinase alcohol oxidase and PGH. See, for example, Ausubel, et al., 1990; Grant et al., Methods in Enzymology 153: 516-544, (1987). Other inducible yeast promoters have the additional advantage that transcription is controlled by growth conditions, including alcohol dehydrogenase 2, isocytochrome c, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein , Glyceraldehyde-3-phosphate dehydrogenase, and the promoter region of enzymes involved in the utilization of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. As a yeast selection marker, ADE2. HIS4. LEU2. TRPl. And ALG7; a neomycin phosphotransferase gene that confers resistance to G418; and the CUP1 gene that allows yeast to grow in the presence of copper ions. A yeast expression vector for intracellular production and secretion of CPP from the DNA encoding the target CPP can be constructed. For example, a selected signal peptide and a suitable constitutive or inducible promoter may be inserted into a suitable restriction site of a selected plasmid for direct intracellular expression of CPP. In order to secrete CPP, the DNA encoding CPP is selected with a promoter encoding DNA, yeast α-factor secretion signal / leader sequence and linker sequence (if necessary) to express CPP. Can be cloned into. Thus, yeast cells can be transformed with the expression plasmids described above and cultured in a suitable fermentation medium. The protein produced by such a transformed yeast can then be concentrated by precipitation with 10% trichloroacetic acid, and analyzed by SDS-PAGE separation and gel staining with Coomassie Blue stain. The recombinant CPP can then be isolated and purified from the fermentation medium by techniques known to those skilled in the art.

Expression in mammalian systems CPPs may be expressed in mammalian cells. Mammalian expression systems are known in the art and include expression systems with retroviral vectors. Mammalian host cells may be transformed with any expression system based on a number of different viruses, such as adenovirus, in which the coding region consists of a late promoter and a three-part leader sequence. It can be linked to a transcription / translation complex. By inserting into the nonessential E1 or E3 region of the viral genome, a live virus capable of expressing the polypeptide of interest in an infected host cell is obtained. Preferred expression vector systems are the retroviral vector systems as outlined in PCT / US97 / 01019 and PCT / US97 / 101048. Suitable mammalian expression vectors include a mammalian promoter that is any DNA sequence capable of binding to mammalian RNA polymerase and initiating downstream (3 ′) transcription of the CPP coding sequence into mRNA. A promoter usually has a TATA box that uses a transcription initiation region located proximal to the 5 'end of the coding sequence and 25-30 base pairs located upstream of the transcription start site. The TATA box is thought to direct RNA polymerase II to initiate RNA synthesis at the correct site. Mammalian promoters also include an upstream promoter element (enhancer element), generally located within 100-200 base pairs upstream of the TATA box. The upstream promoter element determines the rate at which transcription is initiated and can act in either direction. Since viral genes are often highly expressed and correspond to a wide range of hosts, promoters derived from mammalian viral genes are particularly useful as mammalian promoters. Examples include polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, Promoters derived from the genomes of viruses such as retrovirus, hepatitis B virus and simian virus 40 (SV40), heterologous mammalian promoters, such as actin promoters or immunoglobulin promoters, and heat shock promoters, as indicated above Such promoters are compatible with the host cell system. Transcription of DNA encoding NPP by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are usually cis-acting elements of about 10-300 bp of DNA that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein and insulin). However, in general, eukaryotic cell enhancers are used. Examples include the SV40 enhancer, cytomegalovirus early promoter enhancer, late-origin polyoma enhancer and adenovirus enhancer. The enhancer is preferably located at site 5 ′ of the promoter. In general, transcription termination and polyadenylation sequences recognized by mammalian cells are located 3 'to the translation stop codon and are therefore regulatory regions present with promoter elements and flanking the coding sequence. The 3 ′ end of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminators and polyadenylation signals include those obtained from SV40. Long-term, high-yield production of recombinant proteins can be performed with a stable expression system. For this purpose, expression vectors containing viral origins of replication or endogenous expression elements and a selectable marker gene may be used. Appropriate vectors containing selectable markers for use in mammalian cells are readily available commercially and are known to those skilled in the art. Examples of such selectable markers include, but are not limited to, herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase for use in tk- or hprt-cells, respectively. Methods for introducing exogenous nucleic acid into mammalian hosts and other hosts are well known in the art and will vary with the host cell used. These techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of polynucleotides by liposomes and direct microinjection of DNA into the nucleus. .

CPP can be purified from culture supernatants of mammalian cells transiently transfected or stably transformed with an expression vector having a sequence encoding CPP. Preferably, CPP is purified from the culture supernatant of COS7 cells transiently transfected with a pcD expression vector. Transfection with pcD of COS7 cells, proceed as follows: One day prior to transfection, COS7 monkey cells approximately 10 ^6, Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2mM glutamine (DME) Seed each 100 mm plate containing. To perform the transfection, the medium is aspirated from each plate and 50 mM Tris. Replace with 4 ml DME containing HCl pH 7.4, 400 mg / ml DEAE-dextran and 50 μg plasmid DNA. After incubating the plates at 37 ° C. for 4 hours, the medium containing the DNA is removed and the plates are washed twice with 5 ml of serum free DME. DME is returned to the plate, after which it is incubated at 37 ° C. for an additional 3 hours. The plates are washed once with DME, after which DME containing 4% fetal calf serum, 2 mM glutamine, penicillin (100 U / L) and streptomycin (100 μg / L) is added at standard concentrations. The cells are then incubated for 72 hours at 37 ° C., after which the growth medium is collected to purify the CPP. Plasmid DNA for transfection is the E. coli MC1061 (described by Casadaban and Cohen, J. Mol. Biol., Vol. 138, pgs. 179-207 (1980)) or a cDNA insert encoding CPP in a similar organism. It is obtained by propagating the contained pcD (SRα) or similar expression vector. The plasmid DNA is removed from the culture by standard techniques such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) or Ausubel et al (1990, supra). Isolate.

Expression in insect cells CPPs may be made in insect cells. Expression vectors for transforming insect cells, particularly expression vectors based on baculovirus, are well known in the art. In one such system, DNA encoding CPP is fused upstream of an epitope tag contained in a baculovirus expression vector. Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes in Spodoptera frugiperda Sf9 cells or Trichoplusia larvae. The sequence encoding CPP is cloned into a nonessential region of the virus such as the polyhedrin gene and placed under the control of the polyhedrin promoter. If the CPP-encoding sequence is successfully inserted, the polyhedrin gene will become inactive, resulting in a recombinant virus that does not have an outer protein protein. The recombinant virus is then used to Fulgipeda cells or Trichopulcia larvae are infected, where CPP is expressed (Smith et al., J. Wol. 46: 584 (1994); Engelhard EK et al., Proc. Nat. Acad. Sci. 91: 3224- 3227 (1994)). Suitable epitope tags for fusion with DNA encoding CPP include poly-his tags and immunoglobulin tags (such as the Fc region of IgG). A variety of plasmids can be used, including commercially available plasmids such as pVL1393 (Novagen). In short, DNA encoding CPP or a desired portion of DNA encoding CPP is amplified by PCR using primers complementary to the 5 ′ and 3 ′ regions. A flanking restriction site may be incorporated into the 5 ′ primer. The PCR product is then digested with the selected restriction enzyme and subcloned into an expression vector. The above plasmid and BaculoGoldTM viral DNA (Pharmingen) using Lipofectin (commercially available from GIBCO-BRL) or other methods known to those skilled in the art are cotransfected into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) To produce recombinant baculovirus. Virus is produced by culturing Sf9 cells at 28 ° C. for 4-5 days and used for further amplification. The procedure is as described further in O'Reilley et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, Oxford University Press (1994). Extracts may be prepared from Sf9 cells infected with recombinant viruses as described in Rupert et al., Nature 362: 175-179 (1993). The expressed epitope-tagged CPP can also be purified by affinity chromatography. For example, IgG-tagged (or Fc-tagged) CPP can be purified by chromatography including A protein or G protein column chromatography. Can be done using technology.

Assessing gene expression Using appropriately labeled probes and based on the sequences provided herein, eg, standard techniques known in the art, eg, Northern to determine transcription of mRNA Gene expression may be assessed directly in the sample by blotting, dot blotting (DNA or RNA) or in situ hybridization. Alternatively, polypeptides such as DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or specific duplexes including DNA-protein duplexes, using antibodies for nucleic acid detection assays Also good. When such an antibody is labeled and a double chain is formed on the surface, the location where the double chain is bound to the surface so that the presence of the antibody bound to the double chain can be detected. Perform the assay. Alternatively, gene expression may be measured directly by immunohistochemical staining of cells or tissue sections and cell culture or body fluid assays to directly assess CPP polypeptide or polynucleotide expression. Antibodies useful for such immunological assays may be monoclonal antibodies, polyclonal antibodies, or prepared against native sequence CPP. Protein levels may be detected by mass spectrometry. A further protein detection method uses a protein chip.

Purification of Expressed Protein After expression, the expressed CPP can be purified or isolated by any of a variety of methods known to those skilled in the art. The appropriate technique depends on what other components are present in the sample. Contaminating components that are removed by isolation or purification are generally substances that would interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones and other solutes. The choice of purification stage depends, for example, on the nature of the production process used and the particular CPP being produced. Since CPPs are secreted, they can be recovered from the culture medium. Alternatively, CPP can also be recovered from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable cleaning solution (eg, Triton-X 100) or by enzymatic cleavage. Alternatively, the cells used for CPP expression can be disrupted by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical disruption, or using cytolytic agents. Typical purification methods include, but are not limited to, ion exchange column chromatography; chromatography using a cation exchange resin such as silica gel or DEAE; for example, gel filtration using Sephadex G-75 A protein sepharose column to remove contaminants such as IgG; chromatography using a metal chelate column to bind epitope-tagged forms of CPP; ethanol precipitation; reverse phase HPLC; isoelectric point chromatography; SDS-PAGE; and ammonium sulfate precipitation. Usually, an isolated CPP is prepared by at least one purification step. For example, a standard anti-CPP antibody column can be used to purify the CPP. Combinations of ultrafiltration and dialysis techniques with protein concentration are also useful (see, for example, Scopes, R., PROTEIN PURIFICATION, Springer-Verlag, New York, NY, 1982). The required purity depends on the use of CPP. There may be no need for purification. Once the CPPs and nucleic acids of the invention are expressed and optionally purified, they are useful in many applications detailed herein.

Transgenic animals Non-human transgenic animals can also be produced using the host cells of the present invention. For example, in one embodiment, the host cell of the invention is a fertilized oocyte or embryonic stem cell into which a sequence encoding CPP has been introduced. Such host cells can further be used to create non-human transgenic animals in which exogenous CPP sequences have been introduced into the genome or homologous recombinant animals in which endogenous CPP sequences have been modified. Such animals are useful for studying the function and / or activity of CPP or fragments thereof, and for identifying and / or evaluating modulators of CPP biological activity. As used herein, “transgenic animal” refers to a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more cells of the animal contain a transgene. It is. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians and the like. Since the transgene is integrated into the genome of the cell from which the transgenic animal originates and is present in the genome of the mature animal, expression of the encoded gene product in one or more cell types or tissues of the transgenic animal Exogenous DNA to command. As used herein, a “homologous recombinant animal” refers to an endogenous gene between an endogenous gene and an exogenous DNA molecule introduced into an animal cell, eg, an animal embryonic cell, prior to animal development. A non-human animal, preferably a mammal, more preferably a mouse, which has been modified by homologous recombination.

  The transgenic animals of the invention can be obtained by introducing a nucleic acid encoding CPP into the male pronucleus of a fertilized oocyte, for example, by microinjection or retroviral infection, and developing the oocyte in a pseudopregnant female foster animal. Can be created. CPP cDNA sequences or fragments thereof can also be introduced into the genome of non-human animals as a transgene. Alternatively, a non-human homologue derived from mouse or rat of a gene encoding human CPP can also be used as a transgene. In addition, an intron sequence and a polyadenylation signal can be included in the transgene in order to increase the expression efficiency of the transgene. Tissue-specific regulatory sequences can also be operably linked to the CPP transgene to direct expression of CPP to specific cells. Methods for producing transgenic animals, particularly animals such as mice, through embryo manipulation and microinjection are conventional in the art, for example, US Pat. No. 4,736,866 by Leder et al. No. and 4,870,009, U.S. Pat. No. 4,873,191 by Wagner et al. And in Hogan, B., Manipulating the Mouse Embryo, (the disclosure of which is incorporated herein by reference in its entirety) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Similar methods are used for the production of other transgenic animals. Transgenic primary animals can be identified based on the presence of a CPP transgene in the animal's genome and / or expression of CPP mRNA in an animal tissue or cell. Such transgenic primary animals can be used to breed additional animals carrying the transgene. In addition, transgenic animals having transgenes encoding CPPs can be further crossed with other transgenic animals having other transgenes.

  CPPs that modify the CPP coding sequence, eg, deletions, additions, or substitutions that are functionally disrupted to produce animals in which the desired nucleic acid has been introduced into the genome via homologous recombination A vector containing at least part of the coding sequence is generated. The CPP coding sequence may be a human gene, but more preferably in a non-human homologue of the human CPP coding sequence (eg, cDNA isolated by stringent hybridization with a nucleotide sequence encoding CPP). is there. For example, a mouse CPP coding sequence can be used to construct a homologous recombination vector suitable for modification of an endogenous gene in the mouse genome. In a preferred embodiment, upon homologous recombination, the endogenous CPP coding sequence is functionally disrupted (ie, the vector is designed to no longer encode a functional protein; also referred to as a “knockout” vector). Alternatively, during homologous recombination, the vector can be designed to encode a functional protein even if the endogenous CPP coding sequence is mutated or changed (eg, modifying the upstream regulatory region). Can alter the expression of the endogenous CPP coding sequence). In the homologous recombination vector, an additional nucleic acid sequence of the CPP gene is flanked at the 5 ′ and 3 ′ ends of the modified portion of the CPP coding sequence, so that the foreign sequence contained in the vector and the endogenous gene of the embryonic stem cell Cause homologous recombination between. The additional flanking nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. In general, vectors will contain several kilobases of flanking DNA (both at the 5 ′ and 3 ′ ends) (for the description of homologous recombination vectors, for example, the disclosure of which is incorporated herein by reference in its entirety. Thomas, KR and Capecchi, MR (1987) Cell 51: 503). A vector is introduced into an embryonic stem cell line (for example, by electroporation), and a cell in which the introduced CPP coding sequence is homologously recombined with an endogenous gene is selected (for example, the content of the disclosure is directly incorporated by reference). Li, E. et al. (1992) Cell 69: 915, which is part of the specification). The selected cells are then injected into the blastocyst of an animal (eg, a mouse) to form an aggregate chimera (eg, Bradley, A., the disclosure of which is incorporated herein by reference in its entirety). in Teratocarcinomas and Embryonic Stem Cells. A Practical Approach, EJ Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). Such a chimeric embryo can be transferred to a suitable pseudopregnant female foster animal and the embryo can be born. Using progeny having DNA homologously recombined into germ cells, germline transmission of the transgene can be used to breed animals that contain DNA that is homologously recombined in all cells of the animal. Methods for constructing homologous recombination vectors and homologous recombination animals are described in Bradley, A. (1991) Current Opinion in Biotechnology 2: 823-829 and Le, the disclosures of which are incorporated herein by reference in their entirety. PCT International Publication No. WO 90/11354 by Mouellec et al .; WO 91/01140 by Smithies et al .; WO 92/0968 by Zijlstra et al .; and WO 93/04169 by Berns et al.

  In another embodiment, transgenic non-human animals can be generated that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, for example, Lakso et al. (1992) PNAS 89: 6232-6236, the disclosure of which is incorporated herein by reference in its entirety. Another example of the recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al. (1991) Science 251: 1351-, whose disclosure is incorporated herein by reference in its entirety. 1355). When using the cre / loxP recombinase system to regulate transgene expression, animals containing transgenes encoding both the Cre recombinase and the selected protein are required. Such animals are, for example, through the construction of “double” transgenic animals by crossing two types of transgenic animals: an animal containing a transgene encoding a selected protein and an animal containing a transgene encoding a recombinase. Can be provided.

Assessing CPP activity It will be appreciated that the present invention further provides a method for testing the activity of functional fragments and variants of CPP and CPP sequences or obtaining functional fragments and variants of CPP and CPP sequences. provide. Such methods include providing a mutated or modified CPP-encoding nucleic acid and assessing whether the encoded polypeptide exhibits CPP biological activity. Thus, (a) providing CPP or a biologically active fragment or homologue thereof; and (b) converting said CPP or biologically active fragment or homologue thereof to CPP activity for CPP biological activity. Also encompassed are methods of assessing the function of CPP, including testing under suitable conditions. Cell-free assays, cell-based assays and in vivo assays can be used to test CPP activity. For example, the assay includes expressing CPP nucleic acid in a host cell and observing CPP activity in the cell and other diseased cells. In another example, CPP, or a biologically active fragment or homologue thereof, is contacted with a cell and CPP biological activity is observed.

  CPP bioactivity indicates (1) that an individual has or will have a cardiovascular disease; (2) circulates at higher than normal levels through the bloodstream of an individual with cardiovascular disease. (3) ability to bind antigenic or anti-CPP specific antibodies; (4) ability to make immunogenic or anti-CPP specific antibodies; (5) hydrogen bonds, amide bonds or, preferably, disulfide bonds. (6) interaction with a CPP target molecule; and (7) post-translational processing, eg, subjecting to specific proteolysis, Also good.

  CPP biological activity can be assayed by any suitable method known in the art. For example, antigenicity and immunogenicity may be detected as described in the sections entitled “Anti-CPP antibodies” and “Use of CPP antibodies”. Circulation in plasma may be detected as described in “Diagnostic and Prognostic Use”. Determining the ability of a CPP to bind to or interact with a CPP target molecule can be done by methods that determine binding directly or indirectly, as is common in the art. Such methods are further described in the section entitled “Drug Screening Assays”.

  Cardiovascular disease can be diagnosed by methods determined by those skilled in the art to be appropriate for an individual. Further examples of symptoms and diagnosis can be found in the background section, and the best one is appropriately determined by one skilled in the art based on the patient's profile.

  Intramolecular interactions can be detected by structure estimation based on sequences. Such estimation is generally based on X-ray crystallography or NMR structural data for polypeptides having similar sequences. Detection of intramolecular interactions can also be performed using SDS-PAGE. In the disulfide bond example, the apparent molecular weight is reduced because the bonds formed between different sites of a protein result in a more compact protein. Disulfide bonds can be broken by reducing agents such as dithiothreitol (DTT). In this way, a protein sample treated with a reducing agent and an untreated control can be compared by SDS-PAGE to detect changes in apparent molecular weight. Such methods are common in the art.

  Specific proteolysis may be detected by comparing the molecular weight of the sample peptide with that of a peptide of known molecular weight. Molecular weights are easily compared by any method common in the art, such as SDS-PAGE, gel chromatography or mass spectrometry. Preferably, the molecular weight of the test peptide is obtained by mass spectrometry and compared to a database containing the molecular weight of peptides having post-translational modifications. Examples of databases include Genpept, SWISSPROT, EMBL, and protein sequence databases.

Anti-CPP Antibodies The present invention provides CPP specific antibodies and binding compositions. Such antibodies and binding compositions include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv fragments thereof, bispecific antibodies, heteroconjugates and humanized antibodies. Such antibodies and binding compositions can be made by a variety of methods including hybridoma culture, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. A wealth of guidance for selecting specific production methodologies can be found in the literature, for example, Chad and Chamow, Curr. Opin. Biotechnol., 12: 188-194 (2001).

  The choice of manufacturing methodology depends on several factors, including the desired antibody structure, the importance of the carbohydrate portion of the antibody, ease of culture and purification, and cost. Many different antibody structures can be made using standard expression techniques, including full length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies comprising components of different species. Small size antibody fragments with no effector function and little pharmacokinetic activity, such as Fab and Fv fragments, can be made in bacterial expression systems. Single-chain Fv fragments are highly selective for in vivo tumors, exhibit excellent tumor penetration and low immunogenicity, and are rapidly cleared from the blood. For example, Freyre et al, J. Biotechnol., 76: 157-163 (2000). Thus, such molecules are desirable for radioimmunodetection methods.

Polyclonal antibody The anti-CPP antibody of the present invention may be a polyclonal antibody. Such polyclonal antibodies can be produced in mammals by, for example, one or more injections of an immunizing agent, preferably an adjuvant. In general, immunizing agents and / or adjuvants are injected into a mammal by a series of subcutaneous or intraperitoneal injections. The immunizing agent may comprise CPP or a fusion protein thereof, which may be useful for conjugating antigens with proteins that are known to be immunogenic in immunized mammals. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), hepatitis B These include surface antigens, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of the adjuvant include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicoryno-mycolate). One skilled in the art can also determine the immunization protocol based on standard protocols or by routine testing.

  Alternatively, antibodies can be made using crude protein preparations enriched for CPP or portions thereof. Such proteins, fragments or preparations are introduced into non-human mammals in the presence of a suitable adjuvant. If the serum contains polyclonal antibodies against unwanted epitopes, the polyclonal antibodies are purified by immunoaffinity chromatography.

  Effective polyclonal antibody production is affected by many factors related to both antigen and host species. Host animals also vary depending on the site and dose of inoculation, resulting in low titers of sera whether the antigen dose is insufficient or excessive. It may be optimal to administer small doses (ng level) of antigen to multiple intradermal sites. Techniques for producing and processing polyclonal sera are known in the art, see, for example, Mayer and Walker (1987), the disclosure of which is incorporated herein by reference in its entirety. An effective immunization protocol for rabbits is disclosed in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33: 988-991 (1971), the disclosure of which is incorporated herein by reference in its entirety. Can be found. Semi-quantitatively, for example, when its antibody titer measured by an agar double immunodiffusion method contrasted with a known concentration of antigen begins to decline, booster injections are given periodically and antisera collected can do. See, for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). The plateau concentration of the antibody is usually in the range of 0.1 to 0.2 mg / ml of serum. For example, as described by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, DC (1980) The affinity of the antiserum for the antigen is determined by generating a competitive binding curve.

Monoclonal antibody Alternatively, the anti-CPP antibody may be a monoclonal antibody. Monoclonal antibodies can be produced by hybridomas, where mice, hamsters or other suitable host animals are immunized with an immunizing agent to produce or produce antibodies that specifically bind to the immunizing agent. Can induce lymphocytes, eg, Kohler and Milstein, Nature 256: 495 (1975). The immunizing agent generally comprises a CPP or fusion protein thereof, optionally a carrier. Alternatively, lymphocytes may be immunized in vitro. In general, spleen cells or lymph node cells are used when non-human mammalian origin is desired, and peripheral blood lymphocytes ("PBL") are used when cells of human origin are desired. A suitable fusion agent such as polyethylene glycol is used to fuse lymphocytes with an immortal cell line to produce hybridoma cells, e.g., Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, pp. 59- 103 (1986); Liddell and Cryer, A Practical Guide to Monoclonal Antibodies (John Wiley & Sons, New York, 1991); Malik and Lillenoj, Editors, Antibody Techniques (Academic Press, New York, 1994). In general, the immortal cell line is a transformed mammalian cell, for example a myeloma cell of rat, mouse, bovine or human origin. The hybridoma cells are preferably cultured in a suitable culture medium containing one or more substances that inhibit the growth or survival of non-fused immortalized cells. For example, if the parent cell does not have the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT), the culture medium of the hybridoma generally contains hypoxanthine, aminopterin and thymidine (HAT), a substance that prevents the growth of HGPRT deficient cells. Including. Preferred immortal cell lines are those that fuse efficiently, support stable high-level production of antibodies, and are susceptible to a medium such as HAT medium. A more preferred immortal cell line is the mouse or human myeloma line available from, for example, the American Type Culture Collection (ATCC), Rockville, MD. For the production of human monoclonal antibodies, human myeloma and mouse-human heteromyeloma cell lines have also been described, eg, Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, pp. 51-63 (1987).

  Culture medium (supernatant) in which hybridoma cells are cultured is assayed for the presence of monoclonal antibodies directed against CPP. Preferably, the binding specificity of the monoclonal antibody present in the hybridoma supernatant is measured by immunoprecipitation or by in vitro binding experiments such as radioimmunoassay (RIA) or enzyme immunoassay (ELISA). Suitable techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be measured, for example, by the Scatchard analysis method of Munson and Pollard, Anal. Biochem. 107: 220 (1980). After identifying the desired antibody-producing hybridoma cells, the cells may be cloned by limiting dilution techniques and grown by standard methods (Goding, 1986, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as mammalian ascites. Monoclonal antibodies secreted by selected clones are cultured by immunoglobulin purification techniques commonly used by those skilled in the art, such as, for example, protein A-sepharose, hydroxyl-apatite chromatography, gel electrophoresis, dialysis or affinity chromatography. It can be isolated or purified from the medium or ascites.

  Monoclonal antibodies may be produced by recombinant DNA methods such as those described in US Pat. No. 4,816,567. Isolating DNA encoding the monoclonal antibody of the present invention from CPP-specific hybridoma cells and using, for example, an oligonucleotide probe capable of specifically binding to a gene encoding the heavy and light chains of a mouse antibody Can be sequenced. Once isolated, the DNA is inserted into an expression vector which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells or myeloma cells (otherwise immunoglobulin protein In the recombinant host cell may be obtained. DNA can also be obtained, for example, by using the coding sequences of mouse heavy and light chain constant domains instead of homologous human sequences (Morrison et al., Proc. Nat. Acad. Sci. 81: 6851-6855 (1984 Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)), or all of the coding sequences of immunoglobulin and non-immunoglobulin polypeptides or You may modify | change by covalently bonding one part. Non-immunoglobulin polypeptides can be used to generate chimeric bivalent antibodies in place of the constant domains of the antibodies of the invention or in place of the variable domains of one antigen binding site of the antibodies of the invention. The antibody may be a monovalent antibody. Methods for making monovalent antibodies are well known in the art. For example, in vitro methods are suitable for producing monovalent antibodies. Digestion of the antibody to produce its fragments, particularly Fab fragments, can be performed using conventional techniques known in the art.

  In addition, antibodies and antibody fragments unique to the hybridomas of the invention can also be produced by recombinant means by extracting messenger RNA, constructing a cDNA library, and selecting clones that encode segments of the antibody molecule. . The following are typical references disclosing recombinant techniques for generating antibodies: Wall et al., Nucleic Acids Research, Vol. 5, pgs. 3113-3128 (1978); Zakut et al , Nucleic Acids Research, Vol. 8, pgs. 3591-3601 (1980); Cabilly et al., Proc. Natl. Acad. Sci., Vol. 81, pgs. 3273-3277 (1984); , Nucleic Acids Research, Vol. 12, pgs. 3791-3806 (1984); Amster et al., Nucleic Acids Research, Vol. 8, pgs. 2055-2065 (1980); Moore et al., US Pat. No. 4, Skerra et al, Science, Vol. 240, pgs. 1038-1041 (1988); Huse et al, Science, Vol. 246, pgs. 1275-1281 (1989); and US Pat. No. 6,054 No. 5,530,101; No. 4,816,567; No. 5,750,105; and No. 5,648,237 (these patents are incorporated herein by reference) Part of the book). In particular, such techniques can be used to generate interspecies monoclonal antibodies, in which one binding region is combined with the non-binding region of another antibody to reduce immunogenicity. For example, Liu et al., Proc. Natl. Acad. Sci., Vol. 84, pgs. 3439-3443 (1987) and US Patent Nos. 6,054,297 and 5,530,101. Preferably, recombinantly produced Fab and Fv fragments are expressed in bacterial host systems. Preferably, full length antibodies are produced by mammalian cell culture techniques. More preferably, full length antibodies are expressed in Chinese hamster ovary (CHO) cells or NSO cells.

Both polyclonal and monoclonal antibodies can be screened by ELISA. In other solid phase immunoassays and the like, the test is based on the tendency to adsorb nonspecifically to polymeric plastics. This irreversibility of the reaction allows formation of antigen-antibody complexes and easy separation from unbound material of such complexes without loss of immunological activity. To measure the titer of anti-peptide serum, a peptide conjugated with a carrier different from that used for immunization is adsorbed to the wells of a 96-well microtiter plate. The adsorbed antigen is then reacted with a dilution of anti-peptide serum in the well. Unbound antibody is washed away and the remaining antigen-antibody complex is reacted with an antibody specific for IgG in the immunized animal. This second antibody is conjugated with an enzyme such as alkaline phosphatase. When an enzyme substrate is added, a visible color reaction occurs, indicating which well is bound to the anti-peptide antibody. By using a spectrophotometer measurement, a better quantification of the amount of bound peptide-specific antibody is possible. For high titer antisera, a linear titration curve is obtained between 10 ^{−3 and} 10 ⁻⁵ dilutions.

CPP Peptide Carriers The present invention includes immunogens obtained from CPPs and immunogens comprising conjugates of carriers and peptides of the present invention. An immunogen is a term used herein to refer to a substance that can elicit an immune response. A carrier, as used herein, is conjugated to a host organism that has been immunized with the resulting conjugate when chemically conjugated to a peptide of the invention. It is a term that refers to any substance that allows the production of an antibody specific to a peptide. Carriers include red blood cells, bacteriophages, proteins or synthetic particles such as agarose beads. Preferably, the carrier is a protein such as serum albumin, γ-globulin, keyhole limpet hemocyanin (KLH), thyroglobulin, ovalbumin, or fibrinogen.

  General techniques for linking synthetic peptides to carriers are described in several references, e.g., Walter and Doolittle, `` Antibodies Against Synthetic Peptides, '' in Setlow et al., Eds., Genetic Engineering Vol. 5, pgs. 61-91 (Plenum Press, NY, 1983); Green et al. Cell, Vol. 28, pgs. 477-487 (1982); Lerner et al., Proc. Natl. Acad. Sci , Vol. 78: pgs. 3403-3407 (1981); Shimizu et al., US Pat. No. 4,474,754; and Ganfield et al., US Pat. No. 4,311,639. Accordingly, these references are hereby incorporated by reference. Also, the technology used to link the hapten to the carrier is essentially the same as described above, for example, chapter 20 in Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, New York, 1985). The four most commonly used schemes for attaching peptides to carriers are (1) for example Kagan and Glick, in Jaffe and Behrman, eds. Methods of Hormone Radioimmunoassay, pgs. 328-329 (Academic Press, NY, 1979) and Walter et al. Proc. Natl. Acad. Sci., Vol. 77: pgf. 5197-5200 (1980); (2) for example Hoare et al., J. Biol. Chem., Vol. 242: pgs. 2447-2453 (1967), water soluble carbodiimides for amino coupling of carboxyls; (3) For example, Bassiri et al., pgs 46-47, in Jaffe and Behrman, eds. (Supra) and Walter et al. (Supra), bis-diazobenzidine (BDB) for tyrosine side chain coupling of tyrosine; and (4 ) For example, Kitagawa et al., J. Biochem. (Tokyo), Vol. 79: pgs. 233-239 (1976) and Lerner et al. (Supra), maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) for amino group coupling of cysteine (or other sulfhydryls). General rules for selecting an appropriate method for coupling a specific peptide to a protein carrier are as follows: the group involved in the binding is only once in the sequence, preferably at the appropriate end of the segment. Should occur. For example, BDB should not be used if tyrosine residues are present in the major portion of the sequence selected for its potential antigenic characteristics. Similarly, the glutaraldehyde method is often excluded when lysine is centrally located, and the carbodiimide approach is often excluded when aspartic acid and glutamic acid are generated. On the other hand, suitable residues may be present at either end of the sequence segment selected as the binding site, regardless of whether they are present in the “native” protein sequence. Unlike the amino and carboxy termini, the internal segment differs greatly in the “unbound end” of the same sequence, as found in a native protein with a continuous polypeptide backbone. The problem can be remedied to some extent by acetylating the α-amino group and then coupling to the peptide through its carboxy terminus. Coupling efficiency with the carrier protein is expedient by using a radiolabeled peptide prepared by using a radioactive amino acid in one step of the synthesis or by labeling the complete peptide by iodination of tyrosine residues. Is measured. If desired, a sensitive radioimmunoassay can be planned due to the presence of tyrosine in the peptide. Therefore, if tyrosine is not part of the peptide sequence defined by the natural polypeptide, it can be introduced as a terminal residue.

  Preferred carriers are proteins, and preferred protein carriers include bovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH) and the like. Peptides can be linked to KLH through cysteine by MBS disclosed by Liu et al., Biochemistry, Vol. 18, pgs. 690-697 (1979). The peptide is dissolved in phosphate buffered saline (pH 7.5), 0.1 M sodium borate buffer (pH 9.0) or 1.0 M sodium acetate buffer (pH 4.0). The dissolution pH of the peptide is selected to optimize peptide solubility. The free cysteine content of the soluble peptide is determined by the method of Ellman, Ellman, Arch. Biochem. Biophys., Vol. 82, pg. 7077 (1959). For each peptide, 4 mg of KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg of MBS (dissolved in dimethylformamide) and stirred at room temperature for 30 minutes. Since KLH becomes insoluble with> 30% formamide, MBS is added dropwise so that the local concentration of formamide does not become too high. The reactive organism, KLH-MBS, was then passed through Sephadex G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free MBS and the peak fraction of the column eluate (at OD280). KLH recovery from (monitored) is estimated to be approximately 80%. KLH-MBS is further reacted with 5 mg peptide dissolved in 1 ml of the selected buffer. The pH is adjusted to 7-7.5 and the reaction is stirred at room temperature for 3 hours. Coupling efficiency is monitored by dialyzing a sample of the conjugate with phosphate buffered saline using a radioactive peptide, and the efficiency can be between 8% and 60%. If peptide-carrier conjugates can be obtained, for example, Campbell, Monoclonal Antibody Technology (Elsevier, New York, 1984); Hurrell, ed. Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, FL, 1982); Polyclonal or monoclonal antibodies are made by standard techniques such as those disclosed by Schreier et al. Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); US Pat. No. 4,562,003. In particular, US Pat. No. 4,562,003 is hereby incorporated by reference.

Humanized antibody The anti-CPP antibody of the present invention may further comprise a humanized antibody or a human antibody. “Humanized antibody” refers to a chimeric antibody, immunoglobulin chain or fragment thereof (Fv, Fab, Fab ′, F (ab ′) of an antibody or other antigen) containing a portion of a sequence derived from a non-human antibody. It is a term that refers to a humanized non-human (eg, murine) antibody that is a binding moiety sequence or the like. A humanized antibody is a residue of a CDR of a non-human species, such as a mouse, rat or rabbit, in which the complementarity determining region (CDR) residues of a human immunoglobulin have the desired binding specificity, affinity and ability. Including human immunoglobulins that have been replaced by In general, a humanized antibody comprises substantially all of at least one, generally two variable domains, all or substantially all of its CDR regions corresponding to those of a non-human immunoglobulin, All or substantially all of the human immunoglobulin consensus sequence. Humanized antibodies optimally also comprise an immunoglobulin constant region (Fc), generally at least part of that of a human immunoglobulin (Jones et al., Nature 321: 522-525 (1986) and Presta, Curr. Op Struct. Biol. 2: 593-596 (1992)). Methods for humanizing non-human antibodies are well known in the art. In general, humanized antibodies retain the binding activity inherent in antibodies, while one or more amino acids have been introduced from a non-human source so that it more closely resembles human antibodies. Methods for humanizing antibodies are described in Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); and Verhoeyen et al., Science 239: 1534-1536 ( 1988). Such “humanized” antibodies are chimeric antibodies in which less than a fully human variable domain has been replaced by the corresponding sequence in a non-human species.

Heteroconjugate antibodies Heteroconjugate antibodies comprising two covalently linked antibodies are also within the scope of the present invention. Heteroconjugate antibodies may be prepared in vitro using known methods by synthetic protein chemistry, including those requiring cross-linking agents. For example, immunotoxin conjugates can be prepared utilizing a disulfide exchange reaction or by formation of a thioether bond.

Bispecific antibodies Bispecific antibodies have binding specificities for at least two different antigens. Such antibodies are monoclonal antibodies, preferably human or humanized antibodies. One of the binding specificities of the bispecific antibody of the invention is directed to the CPP and the other is preferably directed to a cell surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art, and in general, recombinant production of bispecific antibodies is performed in hybridoma cells in which the two heavy chains have different specificities. Based on the co-expression of two immunoglobulin heavy / light chain pairs, eg, Milstein and Cuello, Nature 305: 537-539 (1983). Given that any combination of immunoglobulin heavy and light chains can produce 10 different antibody molecules by hybridomas, accurate molecular purification usually involves some kind of affinity purification, For example, affinity chromatography is required.

Use of CPP antibodies Since CPP antibodies are preferably specific for the CPPs of the present invention, they themselves do not bind to peptides derived from other proteins with high affinity. In the present specification, the “heavy chain variable region” means (1) 110 to 125 amino acids in length from the N-terminal amino acid of the heavy chain, and (2) the amino acid sequence of the heavy chain of the antibody of the present invention Corresponding term for polypeptide. Similarly, the “light chain variable region” is (1) 95-115 amino acids in length from the N-terminal amino acid of the light chain, and (2) its amino acid sequence corresponds to that of the light chain of the antibody of the present invention. , A term that refers to a polypeptide. As used herein, “monoclonal antibody” is a term that refers to a homogeneous population of immunoglobulins that can specifically bind to CPP.

  CPP antibodies can be used as functional modulators. Preferably, the antibody modulator of the present invention is derived from a monoclonal antibody specific for CPP. Monoclonal antibodies that can block or neutralize CPP are selected for their ability to inhibit CPP biological activity.

  The use of antibody fragments is also well known, eg, Fab fragment: Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and Fv fragment: Hochman et al. Biochemistry, Vo. 12: pgs. 1130-1135 (1973), Sharon et al., Biochemistry, Vol. 15: pgs. 1591-1594 (1976) and Ehrlich et al., US Pat. No. 4,355,023; and antibody half-molecules: Auditore-Hargreaves, US patent No. 4,470,925.

Preferably, the monoclonal antibody, Fv fragment, Fab fragment or other binding composition obtained from the monoclonal antibody of the present invention has a high affinity for CPP. The affinity of monoclonal antibodies and related molecules for CPP can be measured by conventional techniques including plasmon resonance, ELISA or equilibrium dialysis. For example, affinity measurements by plasmon resonance technique may be performed using a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden) according to the manufacturer recommended protocol. Preferably, affinity is measured by ELISA, as described, for example, in US Pat. No. 6,235,883. Preferably, the dissociation constant between the CPP and the monoclonal antibody of the present invention is less than 10 ⁻⁵ molar. More preferably, the dissociation constant is less than 10 ⁻⁸ molar; more preferably, the dissociation constant is less than 10 ⁻⁹ molar; most preferably, the dissociation constant is 10 ⁻⁹ to 10 ⁻¹¹ mole. A range of concentrations.

  Furthermore, the antibodies of the present invention are useful for the detection of CPP. Such a detection method is advantageously applied to the diagnosis of cardiovascular diseases, particularly coronary artery diseases. The antibodies of the present invention may be used in most assays involving antigen-antibody reactions. The assay may be a homogeneous assay or a heterogeneous assay. In a homogeneous assay approach, the sample can be a biological sample or body fluid such as serum, urine, whole blood, lymph, plasma, saliva, cells, tissues and substances secreted from cells or tissues cultured in vitro. If necessary, the sample can be pretreated to remove unwanted material. An immunological reaction usually requires a sample suspected of containing a specific antibody, a labeled analyte, and an antigen. When the antibody and labeled analyte are bound, the signal resulting from the label is directly or indirectly modified. Both the immune response and the detection of the range are performed in a homogeneous solution. Usable immunochemical labels include free radicals, fluorescent dyes, enzymes, bacteriophages, coenzymes and the like.

  In a heterogeneous assay approach, the reagent is usually a means for generating a sample, a specific antibody and a detectable signal. In general, the sample is placed on a support such as a plate or slide and contacted with the antibody in the liquid phase. The support is then released from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal using means for generating a signal or a signal generating system. The signal is related to the presence of the antigen in the sample. Means for generating a detectable signal include the use of radioactive labels, fluorescent compounds, enzymes, and the like. Examples of heterogeneous immunoassays include radioimmunoassays, immunofluorescence methods, enzyme-linked immunoassays, and the like.

  For a more detailed discussion of the above immunoassay techniques, see “Enzyme-Immunoassay” by Edward T. Maggio, CRC Press, Inc., Boca Raton, Fla., 1980. Also, for example, U.S. Pat. Nos. 3,690,834; 3,791,932; 3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074; 3,984,533; 3,966,345; and 4, See also 098,876 (this list is not intended to be exhaustive). Methods for conjugating labels with antibodies and antibody fragments are well known in the art. Such methods can be found in US Pat. Nos. 4,220,450; 4,235,869; 3,935,974; and 3,966,345. Another example of a technique in which the antibodies of the present invention can be used is immunoperoxidase labeling (Sternberger, Immunocytochemistry (1979) pp. 104-169). Alternatively, these antibodies may be conjugated with radioactive substances or drugs to create radiopharmaceuticals or drugs, respectively (Carrasquillo, et al., Cancer Treatment Reports (1984) 68: 317-328).

  One embodiment of an assay using the antibodies of the invention involves the use of a surface to which the monoclonal antibodies of the invention are bound. The basic structure of the surface may take different forms, may have different compositions, may be a composition or a laminate or a mixture of combinations thereof. The surface takes various shapes and forms and has various characteristics depending on the use and measurement method. Illustrative surfaces may be flat, concave or convex pads, beads, disks or strips. The thickness is not critical and is generally about 0.1-2 mm thick, any convenient diameter, or other dimensions. Surfaces are generally on rods, tubes, capillaries, fibers, strips, disks, plates, cuvettes, and other compounds that are generally part of the means for covalently binding antibodies and producing a detectable signal Can be porous and multifunctional, or can be multifunctional. A wide variety of organic and inorganic polymers can be used as solid surface materials, both natural and synthetic, and combinations thereof. Illustrative polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), rayon, nylon, poly (vinyl butyrate), silicon, polyformaldehyde, cellulose, cellulose acetate, Examples include nitrocellulose and latex. Other surfaces include paper, glass, ceramic, metal, metalloid, semiconductor, cement, silicate, and the like. Of surfactants such as dextran, polyalkylene glycols (alkylenes of 2 to 3 carbon atoms) or phospholipids that form gels, gelatin, lipopolysaccharides, silicates, agarose and polyacrylamides or several aqueous phases Materials that form such polymers are also included. Binding to the surface of the antibody may be accomplished by well-known techniques commonly available in the literature (eg, “Immobilized Enzyme,” Ichiro Chibata, Press, New York (1978) and Cuatrecasas, J. Bio. Chem., 245 : 3059 (1970)). When performing an assay according to this aspect of the invention, the sample is mixed with an aqueous medium and the medium is contacted with the surface to which the antibody is bound. To provide a detectable signal that binds to the surface, the label may be added to the aqueous medium at the same time or later. Means for generating a detectable signal may involve the incorporation of a labeled analyte or may use a second monoclonal antibody to which the label is conjugated. Perform separation and washing steps as necessary. The detected signal is related to the presence of CPP in the sample. It is within the scope of the present invention to include assays on the same support. Certain embodiments of the assay according to the present invention include, but are not limited to, the use of a support such as, for example, a slide or a well of a Petri dish. In that technique, the sample to be analyzed is fixed to a support with a suitable fixing material, and the sample on the slide is incubated with the monoclonal antibody. For example, after washing with a suitable buffer such as phosphate buffered saline, the support is contacted with a labeled binding partner specific for the antibody. If necessary, after the incubation, the slide is washed a second time with an aqueous buffer, and the binding between the labeled monoclonal antibody and the antigen is measured. If the label is fluorescent, a fluorescent antibody-encapsulating solution is placed on the slide, a cover slip is put on the slide, and then examined by a fluorescent microscope to determine the degree of binding. In addition, if the label is an enzyme conjugated with a monoclonal antibody, the extent of binding can be determined by examining the slide for the presence of enzyme activity, including the formation of precipitate, color, etc. Indicated by. A specific example of an assay utilizing this antibody is the dual determinant ELISA assay. For example, a support such as a glass or vinyl plate is coated with antibodies specific for CPP by conventional techniques. The support is contacted with a sample suspected of containing CPP, usually in an aqueous medium. After an incubation period of 30 seconds to 12 hours, the support is removed from the medium and washed with, for example, water or an aqueous buffer medium to remove unbound CPP, which is usually converted to CPP in aqueous medium. It is contacted with a specific antibody. The antibody is directly or indirectly labeled with an enzyme such as, for example, horseradish peroxidase or alkaline phosphatase. After incubation, the support is released from the medium and washed as described above. The enzyme activity of the support or aqueous medium is measured. This enzyme activity is related to the amount of CPP in the sample.

  The invention also includes kits, eg, diagnostic assay kits for performing the methods disclosed above. In one embodiment, the kit is detectable as a packaged combination (a) a monoclonal antibody, more particularly a monoclonal antibody as defined above and (b) a binding partner specific for the monoclonal antibody. It includes a conjugate of a label that can generate a signal. Reagents may also include adjuvants such as buffers and proteolytic inhibitors such as polysaccharides. The kit may optionally further include other members of the signal generation system of which the label is a member, agents for reducing background interference in the test, control reagents, devices for performing the test, etc. Good. In another embodiment, the diagnostic kit comprises a conjugate of a label capable of generating a detectable signal with a monoclonal antibody of the invention. Adjuvants such as those described above may also be included.

  In addition, anti-CPP antibodies (eg, monoclonal antibodies) can be used to isolate CPPs by standard techniques such as affinity chromatography or immunoprecipitation. For example, anti-CPP antibodies may facilitate the purification of natural CPP from cells and recombinantly produced CPP expressed in host cells. In addition, anti-CPP antibodies for the purpose of isolating CPPs that aid in the detection of low concentrations of CPPs (eg, in plasma, cell lysates or cell supernatants) or for assessing the amount and pattern of CPP expression Can be used. For example, anti-CPP antibodies can be used diagnostically to monitor protein levels in tissues as part of a clinical trial procedure to determine the effectiveness of a particular treatment plan. Detection can be facilitated by linking (ie, physically linking) the antibody and a labeling group.

Protein array Retentate chromatography (preferably protein, as described by US Pat. No. 6,225,027 and US patent application 20010014461, the disclosure of which is hereby incorporated by reference in its entirety. Arrays or chips) may be used to detect, purify and screen the polypeptides of the invention. In short, retentate chromatography is a method in which a polypeptide (and / or other sample components) is retained on an adsorbent (eg, an array or chip) and then detected. In such a method, (1) a sample-derived polypeptide is selectively adsorbed to a substrate under a plurality of different adsorbent / eluent combinations (“selectivity conditions”), and (2) the adsorbed polypeptide Is detected by desorption spectroscopy (eg, by mass spectrometry). In conventional chromatographic methods, the polypeptide is eluted from the adsorbent prior to detection. The combination of adsorption chromatography and detection by desorption spectroscopic analysis provides surprising sensitivity, the ability to rapidly analyze retained components with a variety of different selectivity conditions, and different sites on the array (ie, “ Parallel processing of the components adsorbed on the “affinity site” or “spot”) is provided.

  These methods include combinatorial, biochemical separation and purification of CPP; differential gene expression studies; detection of protein level differences (eg, diagnostic purposes); and detection of molecular recognition events (eg, screening and creation). Useful for medicinal purposes). Therefore, the present invention provides a molecular discovery diagnostic device characterized by having the ability to process multiple polypeptides in parallel. The substance to which the polypeptide of the present invention and CPP bind is preferably bound to a labeling group so that they are directly detected and the same “circuit” (ie, addressable “chip”) during unit operation. Simultaneous transmission of two or more signals from

Detection of CPP by Mass Spectrometry According to the present invention, the identity and abundance of a protein in a sample can be measured using any instrument, method, process, etc. A preferred method of obtaining identity is by mass spectrometry in which protein molecules in the sample are ionized and then the mass and charge of the resulting protein ions are detected and measured.

  In order to use mass spectrometry for protein analysis, it is preferred that the protein is converted to a gas-ion phase. Various methods of protein ionization are useful, such as, for example, fast ion irradiation (FAB), plasma desorption, laser desorption, thermal desorption, preferably electrospray ionization (ESI) and matrix assisted lasers Desorption / ionization (MALDI). Many different mass analyzers are available for peptide and protein analysis, including but not limited to time of flight (TOF), ion trap (ITMS), Fourier transform ion cyclotron (FTMS). , Quadrupole ion traps and sector (electrical and / or magnetic) spectrometers. See, for example, US Pat. No. 5,572,025 for ion trap MS. The mass analyzer can be used alone or in combination with other mass analyzers, and the mass spectrometers can be used in a vertical row. In the latter case, a first mass analyzer is used to separate protein ions (precursor ions) from each other and measure the molecular weight of the various protein constituents in the sample. A second mass analyzer is used to analyze each separated component, for example, by subdividing precursor ions into product ions (eg, by using an inert gas). Any desired combination of mass analyzers can be used, including, for example, triple quadrupole, tandem time-of-flight, ion trap and / or combinations thereof.

  Different types of detectors can be used to detect protein ions. For example, destructive detectors such as ion electron multipliers or cryogenic detectors can be utilized (eg, US Patent No. https://www.delphion.com/details?pn10=US05640010&s_all=1). In addition, non-destructive detectors such as quadrupole ion trap mass analyzers or ion traps used as ion current pick-up means in FTMS can be used.

  In MALDI-TOF, droplet drying (Karasand Hillenkamp, Anal. Chem., 60: 2299-2301, 1988), vacuum drying (Winberger et al., In Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco , May 31-June 4, 1993, pp. 775a-b), crystal grinding (Xiang et al., Rapid Comm. Mass Spectrom., 8: 199-204, 1994), crystal growth retardation (Xiang et al., Org Mass Spectrom, 28: 1424-1429, 1993); active film (Mock et al., Rapid Comm. Mass Spectrom., 6: 233-238, 1992; Bai et al., Anal. Chem., 66: 3423- 3430, 1994), air spray (Kochling et al., Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics; Atlanta, GA, May 21-26, 1995, p1225); electrospray (Hensel et al., Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics; Atlanta, GA, May 21-26, 1995, p947); Fast solvent evaporation (Vorm et al., Anal. Chem., 66: 3281-3287, 1994); Sandwich ( Li et al., J. Am. Chem. Soc., 11 8: 11662-11663, 1 996); and many sample preparation methods are available, including the two-layer method (Dal et al., Anal. Chem., 71: 1087-1091, 1999). See also, for example, Liang et al., Rapid Commun. Mass Spectrom., 10: 1219-1226, 1996; van Adrichem et al., Anal. Chem., 70: 923-930, 1998.

  In MALDI analysis, a sample is mixed with an energy absorbing compound or colloid (matrix) in the liquid phase, and finally the solution is dried on the surface of an inert probe, thereby rendering them as solid state co-crystals or thin films. Prepare. In some cases, energy absorbing molecules (EAM) may be an essential component of the sample presentation surface. Regardless of the EAM application approach, the probe contents are allowed to dry prior to introduction into the laser desorption / ionization time-of-flight mass spectrometer (LDIMS).

  Ion detection by TOF mass spectrometry is generally obtained through the use of an electron emission detector such as an electron multiplier (EMP) or a microchannel plate (MCP). Both of these devices function by converting primary incident charged particles into a series of secondary, tertiary, quaternary electrons, and the like. The possibility that secondary electrons are generated by the impact of a single incident charged particle is considered to be the ion-electron conversion efficiency (or simply conversion efficiency) of this charged particle. The total electron yield of a series of events compared to the total number of incident charged particles is generally said to be the detector gain. In general, MCP is the preferred electron emission detector for mass / charge resolution enhancement because the overall response time of MCP is much higher than that of EMP. However, EMP works well for the detection of ion populations with distributed kinetic energy that do not require rapid response time and wide frequency bandwidth.

  In a preferred embodiment, a liquid chromatography tandem mass spectrometer (LC-TMS) is used for digested protein analysis. This system provides an additional step of sample separation by using tandem mass spectrometry after liquid chromatography.

  In a preferred embodiment, proteins eluted from the column according to the system described in Example 1 are analyzed using both MS and MS-MS analysis. For example, a small portion of the complete protein eluting from RP2 may be diverted to on-line detection using LC-ESI MS. These proteins are dispensed into a number of plates, including plates that allow digestion or preparation of MALDI-MS with different matrices, as well as ESI-MS, and MALDI without trypsin. Thus, these methods allow for analysis by both peptide mass fingerprinting and MS-MS techniques as well as information on complete mass.

  The methods for separating and fractionating proteins described herein result in individual proteins or fractions containing a small number of different proteins. These proteins can be identified by mass spectral measurement of the molecular weight of the proteins and peptides obtained by the fractionation. Using the available information in the protein sequence database, a comparison can be made between the mass pattern of proteolytic peptides generated in silico and the mass of peptides observed by the test. Alternatively, a protein database can be constructed by performing 6-frame translation of a nucleotide sequence database (eg, Genbank). Based on the number of matches between theoretical and experimental proteolytic fragments (among other criteria), database candidate proteins can be ranked and a “hit list” can be created. Several websites providing online protein identification software based on peptide mapping and sequence database search techniques are accessible (eg http://www.expasy.ch). Peptide mapping and sequencing methods using MS are described in WO 95/252919, US Pat. No. 5,538,897, US Pat. No. 5,869,240, US Pat. No. 5,572,259 and U.S. Pat. No. 5,696,376. See also Yates, J. Mass Spec., 33: 1 (1998).

  Data collected with a mass spectrometer generally includes the intensity and mass to charge ratio of each detected event. Spectral data can be recorded in any suitable format including, for example, a graph format, numeric format, or electronic format in either digital or analog format. Preferably, the spectrum is recorded on a storage medium such as a magnetic medium such as a floppy disk, tape or hard disk; an optical medium such as a CD-ROM or laser disk; or a ROM-chip.

  A sample mass spectrum generally provides information about protein strength, mass to charge ratio, and molecular weight. In a preferred embodiment of the invention, the molecular weight of the protein in the sample is used as a matching criterion for querying the database. The molecular weight is usually calculated, for example, by subtracting the number of ionized protons by multiplying the measured mass / charge ratio by the number of charges of the multiply charged ion by reducing the mass of the ionized proton of the monovalent protonated molecular ion. .

  Various databases are useful in the present invention. Useful databases include databases that contain genomic sequences, expressed gene sequences and / or expressed protein sequences. Preferred databases contain molecular weights derived from the nucleotide sequences of proteins present in known organisms, organs, tissues or cell types. There are many algorithms for identifying open reading frames (ORFs) and converting nucleotide sequences into protein sequences and molecular weight information. Several public databases are available, including the SwissPROT / TrEMBL database (http://www.expasy.ch).

  In general, mass spectrometers are equipped with commercially available software that identifies peaks that exceed a certain threshold and calculates the mass, charge and intensity of the detected ions. The correlation between the molecular weight and the specific output peak can be done directly from the spectral data, i.e. here the charge of the ion is monovalent, so that the molecular weight is the value of the molecule minus the mass of the ionized proton. equal. However, protein ions can form complexes with various counter ions and adducts such as those of N, C and K. In such cases, it is expected that a particular protein ion will exhibit multiple peaks, such as a triple peak, thereby indicating different ionic states (or species) in the same protein. Thus, spectral data may need to be analyzed and processed in order to determine peak groups originating from the same protein. In general, this analysis can be performed as described, for example, by Mann et al., Anal. Chem., 61: 1702-1708 (1989).

  In matching the molecular weight calculated from the mass spectrometer with the molecular weight predicted from a database such as a genomic or expressed gene database, post-translational processing may have to be considered. There are a variety of processing events that alter protein structure, including proteolytic processing, N-terminal methionine removal, acetylation, methylation, glycosylation, phosphorylation, and the like.

  The database can be queried for a range of proteins that match the molecular weight of an unknown. The range window is determined by the accuracy of the instrument, the method for preparing the sample, and the like. Based on the number of hits in the spectrum (where the hit is a match), the unknown protein or peptide is identified or classified.

  Methods for identifying one or more CPPs by mass spectrometry are useful for the diagnosis and prognosis of cardiovascular disease. Preferably, such a method is used to detect one or more CPPs present in human plasma. Examples of the technology are described in U.S. Patent Application Nos. 02/0060290, 02/0137106, 02/0138208, 02/0142343, and 02/0155509, the disclosures of which are incorporated herein by reference in their entirety. .

Diagnostic and Prognostic Uses Nucleic acid molecules, proteins, protein homologues and antibodies described herein can be used in one or more of the following methods: diagnostic assays, prognostics, as further described herein Assays, monitoring clinical trials, drug screening and pharmacogenetics.

  The present invention provides diagnostic and prognostic assays for detecting CPP nucleic acids and proteins, as further described. Also provided are diagnostic and prognostic assays for detecting interactions between CPP and CPP target molecules, particularly natural agonists and antagonists.

  The present invention provides a method for identifying polypeptides that are differentially expressed between two or more samples. “Differential expression” refers to the difference in the amount or quality of a polypeptide between samples. Such differences can occur at any stage of protein expression from transcription to post-translational modification. For example, using protein array methods, two samples can be combined with affinity spots on different sets of adsorbents (eg, chips) and the recognition maps are compared and kept differentially on the two sets of adsorbents. Identify the polypeptide that has been identified. Differential retention includes not only quantitative retention of polypeptides but also qualitative differences. For example, recognition maps that can be detected as differences in binding properties (eg, glycosylated proteins that differentially bind to lectin adsorbents) or as mass differences (eg, post-translational cleavage products) due to differences in post-translational modification of proteins. Differences can occur. In certain embodiments, the adsorbent has an array of affinity spots selected for a combination of diagnostic markers for disease or syndrome.

  Confirmation of differences in polypeptide levels between samples (eg, CPP differentially expressed in plasma samples) by exposing the samples to various conditions for analysis by desorption spectroscopy (eg, mass spectrometry) can do. By detecting physicochemical properties (eg, molecular weight), unknown proteins can be identified, and this information can be used to search a database for proteins with similar profiles.

  A preferred method for detecting CPP utilizes mass spectrometry techniques. Such methods provide information about the size and characteristics of a particular CPP isoform present in a sample being examined for diagnosis or prognosis, eg, a biological sample. Mass spectrometry techniques are detailed in the section entitled “Detection of CPP by mass spectrometry”. Example 1 outlines a preferred detection scheme in which a biological sample is separated by chromatography prior to characterization by mass spectrometry. The present invention is a method for detecting CPP in a biological sample, wherein the biological sample (eg, plasma, serum, lymph, cerebrospinal fluid, cell lysate of a specific tissue) is fractionated by at least one chromatography step. Subjecting the fraction to mass spectrometry; and comparing the properties of the peptide species observed in mass spectrometry to the known properties of CPP.

  An isolated nucleic acid molecule of the invention can be used, for example, to detect genetic changes in CPP mRNA (eg, in a biological sample), or CPP-encoding gene, and to modulate CPP activity as described further below. Can be used for Further, CPPs can be used to screen for naturally occurring CPP target molecules and to screen for agents or compounds that modulate CPP activity. Furthermore, the anti-CPP antibodies of the present invention can be used to detect and isolate CPP, to modulate CPP bioavailability, and to modulate CPP activity.

  Thus, in one embodiment of the invention, a molecule of the invention (eg, CPP, CPP nucleic acid or CPP modulator, or antibody) is used, for example, for diagnosis and / or prognosis of a disease that exhibits any of the aforementioned CPP activities. The method of use is included. In another embodiment, the invention provides the use of a molecule of the invention for the diagnosis and / or prognosis of a subject, preferably a human subject, whose pathology is disrupted, for example, any of the activities described above. Methods are included.

  For example, the invention is a method of determining whether CPP is expressed in a biological sample, comprising: a) a polynucleotide that hybridizes with CPP nucleic acid under stringent conditions; i) said biological sample; or ii) contacting with a detectable polypeptide (eg, antibody) that selectively binds to CPP; and b) presence or absence of hybridization between said polynucleotide and an RNA species in said sample, or said detectable A method comprising detecting the presence or absence of binding between a polypeptide and a polypeptide in the sample. Detection of the hybridization or the binding indicates that the CPP is expressed in the sample. Preferably, the polynucleotide is a primer (wherein the hybridization is detected by detecting the presence of an amplification product containing the primer sequence) or the detectable polypeptide is an antibody.

  In certain embodiments, detection involves polymerase chain reaction (PCR) such as anchor PCR or RACE PCR (eg, US Pat. No. 4,683, the disclosure of which is hereby incorporated by reference in its entirety). 195 and 4,683,202), or ligation chain reaction (LCR) (eg, Landegren et al. (1988) Science, the disclosure of which is incorporated herein by reference in its entirety). 241: 1077-1080; and Nakazawa et al. (1994) PNAS 91: 360-364), and the use of the latter is particularly useful for detecting point mutations in CPP-encoding genes. (See Abravaya et al. (1995) Nucleic Acids Res. 23: 675-682, the disclosure of which is hereby incorporated by reference in its entirety).

  A method for determining whether the expression level of a mammalian, preferably human, CPP is increased or decreased, comprising: a) providing a biological sample of the mammal; and b) CPP in the biological sample. Alternatively, a method is devised that includes comparing the amount of CPP RNA species encoding CPP to a level detected in or predicted from a control sample. If the amount of the CPP or CPP RNA species in the biological sample is detected in the control sample or increased relative to the level expected from the control sample, the level of CPP in the mammal An expression level is shown to be elevated, and the amount of the CPP or CPP RNA species in the biological sample is detected in the control sample or decreased compared to the level expected from the control sample If so, it is shown that the expression level of CPP in the mammal is decreased.

  The present invention also relates to the field of predictive medicine using diagnostic assays, prognostic assays and monitoring clinical trials for prognostic purposes. Thus, one aspect of the present invention is to measure CPP and / or nucleic acid expression and CPP activity in a biological sample (eg, blood, plasma, cells, tissue) and accordingly the individual is associated with abnormal CPP expression or activity. The present invention relates to diagnostic assays for determining whether or not a person is at risk of developing a disease or disorder. The present invention also provides a prognostic (or prognostic) assay for determining whether an individual is at risk of developing a disease associated with CPP, nucleic acid expression or activity. For example, mutations in the CPP-encoding gene can be assayed in a biological sample. Such assays can be used for prognostic or prognostic purposes, and an individual can be treated prophylactically accordingly before the onset of a disease characterized by or associated with CPP expression or activity.

  A “biological sample” is intended to include tissues, cells and body fluids isolated from an individual, as well as tissues, cells and body fluids present within an individual. That is, by using the detection method of the present invention, CPP mRNA, protein or genomic DNA in a biological sample can be detected in vivo as well as in vitro. Preferred biological samples are body fluids such as lymph, cerebrospinal fluid, blood, especially plasma. For example, in vitro techniques for detecting CPP mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting CPP include mass spectrometry, enzyme immunoassay (ELISA), western blot, immunoprecipitation and immunofluorescence. In vitro techniques for detecting genomic DNA encoding CPP include Southern hybridization. Furthermore, in vivo techniques for detecting CPP include introduction of labeled anti-CPP antibodies into individuals.

  In preferred embodiments, the subject method generally comprises: (i) mutation of a gene encoding one of the subject CPPs or (ii) misexpression of a CPP-encoding gene in a tissue sample of an individual (eg, a human patient). And detecting the presence or absence of a genetic lesion characterized by at least one of the following: For example, (i) deletion of one or more nucleotides of a CPP-encoding gene, (ii) addition of one or more nucleotides to the gene, (iii) substitution of one or more nucleotides of the gene, (iv) substitution of the gene Significant chromosomal rearrangement or amplification, (v) significant change in the level of the messenger RNA transcript of the gene, (vi) abnormal modification of the gene, eg, abnormal methylation pattern of the genomic DNA, (vii) Verify that there is at least one of the presence of a non-wild type splicing pattern in the messenger RNA transcript, and (viii) decreased expression levels that indicate damage to regulatory elements or reduced stability of the CPP-encoding transcript. Thus, such a genetic lesion can be detected.

  In yet another exemplary embodiment, the genomic DNA of the patient sample is sensitive to methylation and the recognition site is present in the CPP-encoding gene (including in flanking and intron sequences) By digesting with a restriction endonuclease, an abnormal methylation pattern of a CPP nucleic acid can be detected. See, for example, Buiting et al. (1994) Human Mol Genet 3: 893-895. The digested DNA is separated by gel electrophoresis and it is hybridized with, for example, a probe derived from a genomic or cDNA sequence. The methylation status of the CPP-encoding gene can be determined by comparing the restriction pattern generated from the sample DNA with that of known methylation standards.

  In yet another embodiment, a diagnostic assay is provided that detects the ability of a CPP to bind to a cell surface or extracellular protein. For example, it may be desirable to detect a CPP mutant that is expressed at an appropriate level in a cell while being defective in binding of a CPP target protein (with reduced or enhanced binding affinity for the target). Such mutations may result from, for example, mutations such as point mutations and may be practical to detect by diagnostic DNA sequencing techniques or by the immunoassays described above. Thus, the present invention further typically clones one or more CPP-encoding genes from a sample tissue and the cloned genes under conditions that allow detection of the interaction between the recombinant gene product and the target protein. Diagnostic screening assays involving expression are contemplated. As will be apparent from the description of the various drug screening assays presented herein, a variety of techniques can be used to determine the ability of a CPP to bind to other components. These techniques are used to detect mutations in a CPP-encoding gene that result in a mutated protein that has a higher or lower binding affinity for a CPP target protein compared to wild-type CPP. Is possible. Conversely, this target assay is used to compare the CPP target protein and CPP to the wild type of the CPP target protein by switching which one is “bait” and which is derived from the patient sample. Thus, CPP target protein mutants with higher or lower binding affinity for CPP can also be detected.

  In an exemplary embodiment, the target protein can be provided as an immobilized protein (“target”), such as by using a GST fusion protein and a glutathione-treated microtiter plate, as described herein.

  In another embodiment, the method obtains a control biological sample from a control subject, and the control sample is CPP, mRNA or genomic DNA such that the level of CPP, mRNA or genomic DNA is measured in the biological sample. Contact with a detectable compound or agent, and comparing the level of CPP, mRNA or genomic DNA in the control sample to the level in the test sample. The invention also encompasses kits for detecting the presence of CPP, mRNA or genomic DNA in a biological sample. For example, the kit includes a labeled compound or agent capable of detecting CPP, mRNA or genomic DNA in a biological sample; a means for measuring the amount of CPP in the sample; and the amount of CPP in the sample as a standard. Means for comparing. The compound or agent can be packaged in a suitable container. The kit may further comprise instructions for using the kit for CPP or nucleic acid detection.

Drug Screening Assays The present invention relates to methods for identifying candidate modulators (eg, small molecules and peptides, antibodies, peptidomimetics or other drugs) that enhance or suppress CPP expression or CPP biological activity (herein “ Also referred to as a screening assay). In some embodiments, small molecules can be made using combinatorial chemistry or can be obtained from natural product libraries. The assay is a cell based assay or a non-cell based assay. The drug screening assay is a binding assay, more preferentially a functional assay, as further described.

  When the present invention is used in drug development, eg, to determine the ability of a CPP modulator or drug candidate to cause an anti-cardiovascular disease response, the body fluid analyzed for at least one level of CPP is preferably non-human. It is derived from mammals. Preferably, induction of such a response in humans is predicted by inducing an anti-cardiovascular disease response by endogenous and / or exogenous factors in a non-human mammal. Rodents (mouse, rat, etc.) and primates are particularly suitable for use in this aspect of the invention.

  Using the screening assays further described herein, CPP activity is at least 5%, more preferably at least 10%, more preferably at least 30%, more preferably at least 50%, more preferably at least 70%, More preferably, an agent found to modulate at least 90% may be selected for further testing as a prophylactic and / or therapeutic anti-cardiovascular disease agent.

  In another aspect, using the screening assays further described herein, CPP expression is at least 5%, more preferably at least 10%, more preferably at least 30%, more preferably at least 50%, more preferably Agents found to modulate at least 70%, more preferably at least 90%, can be selected for further testing as prophylactic and / or therapeutic anti-cardiovascular agents.

  Agents found to modulate CPP activity are used, for example, to adjust treatment plans for cardiovascular disease or to reduce symptoms of cardiovascular disease alone or in combination with other appropriate agents or treatments Can do.

  Protein array methods are useful for screening and drug discovery. For example, one member of a receptor / ligand pair is docked with an adsorbent and its ability to bind to its binding partner in the presence of a test substance is determined. Because of the rapidity of the adsorption test, combinatorial libraries of test substances can be easily screened for their ability to modulate their interactions. In a preferred screening method, CPP is docked with an adsorbent. Preferably, the binding partner is labeled to allow detection of the interaction. Alternatively, in certain embodiments, the test substance is docked with the adsorbent. A polypeptide of the invention is exposed to a test substance and screened for binding.

  In other embodiments, the assay is a cell-based assay, wherein the cell-based assay comprises contacting a cell that expresses CPP or a biologically active portion thereof with a test compound to modulate a test compound that modulates CPP activity. Determine ability. Determining the ability of a test compound to modulate CPP activity can be accomplished by monitoring the biological activity of CPP or a biologically active portion thereof. The cell is, for example, a cell of mammalian origin, a cell of insect origin, a cell of bacterial origin or a yeast cell.

  In one embodiment, the present invention provides an assay for screening candidate or test compounds that are target molecules of a CPP or biologically active portion thereof. In another embodiment, the invention provides an assay for screening a candidate or test compound that binds to or modulates the activity of a CPP or biologically active portion thereof. provide. Test compounds of the present invention can be obtained using any of a number of approaches by combinatorial library methods known in the art, including biological libraries; spatially addressed Possible parallel solid phase or liquid phase libraries; synthetic library methods requiring deconvolution; 1-bead 1-compound library method; and synthetic library methods utilizing affinity chromatography selection. The biological library approach is used in conjunction with peptide libraries, and the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (the disclosure of which is incorporated herein by reference in its entirety). Lam, KS (1997) Anticancer Drug Des. 12: 145).

  Examples of methods for the synthesis of molecular libraries are described in the art, for example, DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al. (1994). J. Med. Chem. 37: 2678; Cho et al. (1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059 and 2061; and Gallop et al. (1994) J. Med. Chem. 37: 1233 (the disclosures of which are incorporated herein by reference in their entirety).

  The library of compounds is either in solution (e.g., Houghten (1992) Biotechniques 13: 412-421) or on beads (Lam (1991) Nature 354: 82-84), on chip (Fodor (1993) Nature 364: 555-556), bacteria (Ladner US Pat. No. 5,223,409), spores (Ladner US Pat. No. '409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865- 1869) or phage (Scott and Smith (1990) Science 249: 386-390); (Devin (1990) Science 249: 404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87: 6378-6382); (Felici (1991) J. Mol. Biol. 222: 301-310); (Ladner supra).

  Determining the ability of a test compound to modulate CPP activity can also include, for example, CPP or biologically active portion thereof and its biologically active portion by detecting labeled CPP or biologically active portion thereof in the complex. It can also be done by attaching a CPP or biologically active portion thereof to a labeling group so that binding to a cognate target molecule can be determined. For example, the amount of complex formation may be measured by immunoprecipitation of the complex or gel electrophoresis.

  It is also within the scope of the present invention to determine the ability of a compound to interact with its cognate target molecule without labeling the reactant. For example, by using a microphysiometer, the interaction between a compound and its cognate target molecule can be detected without labeling the compound or the target molecule. McConnell, H. M. et al. (1992) Science 257: 1906-1912, the disclosure of which is incorporated herein by reference in its entirety. A microphysiometer (eg, a site sensor) is an analytical instrument that measures the rate at which cells acidify their environment by photoaddressable potential measurement (LAPS). This change in acidification rate can be used as an indicator of the interaction between the compound and the receptor.

  In a preferred embodiment, the assay involves contacting a cell expressing CPP or a biologically active portion thereof with a target molecule to create an assay mixture, contacting the assay mixture with a test compound, and Included is determining the ability of the test compound to modulate the activity of the CPP or biologically active portion thereof. Determining the ability of a test compound to modulate the activity of a CPP or biologically active portion thereof includes determining the ability of the test compound to modulate the biological activity of a CPP-expressing cell (eg, interacting with a CPP target molecule as described above). Action) is included.

  In another preferred embodiment, the assay comprises contacting a cell that reacts with CPP or a biologically active portion thereof with CPP or a biologically active portion thereof to create an assay mixture, the assay Contacting the mixture with the test compound and determining the ability of the test compound to modulate the activity of the CPP or biologically active portion thereof. Determining the ability of a test compound to modulate the activity of a CPP or biologically active portion thereof includes determining the ability of the test compound to modulate the biological activity of a CPP-responsive cell.

  In another embodiment, the assay is a cell-based assay, wherein the cell-based assay contacts a cell expressing a CPP target molecule (ie, a molecule with which CPP interacts) with a test compound, and the CPP target molecule The ability of the test compound to modulate activity is determined. Determining the ability of a test compound to modulate the activity of a CPP target molecule, for example, assessing the ability of a CPP to bind to or interact with a CPP target molecule by assessing the activity of the target molecule. Can be done.

  Determining the ability of a CPP to bind to or interact with a CPP target molecule can be made, for example, by one of the methods described above for determining binding directly or indirectly. In a preferred embodiment, the assay involves contacting CPP or a biologically active portion thereof with a known compound that binds to said CPP (eg, a CPP antibody or target molecule) to produce an assay mixture, said known Contacting the CPP with the test compound before and after the compound and determining the ability of the test compound to interact with the CPP. Determining the ability of a test compound to interact with a CPP includes determining the ability of the test compound to preferentially bind to CPP or a biologically active portion thereof by comparison with a known compound. Determination of the binding ability of CPP to a CPP target molecule can also be performed by a technique such as real-time biomolecule interaction analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin, the disclosure of which is incorporated herein by reference in its entirety. Struct. Biol. 5: 699-705. In the present specification, “BIA” is a technique for investigating a biospecific interaction in real time without labeling a reactant (for example, BIAcore). As an index of a real-time reaction between biomolecules, a change in an optical phenomenon caused by surface plasmon resonance (SPR) can be used.

  In another embodiment, the assay is a cell-free assay, in which the CPP or biologically active portion thereof is contacted with a test compound to modulate the activity of the CPP or biologically active portion thereof. Determine the ability of the test compound to perform. In a preferred embodiment, determining the ability of a CPP to modulate a CPP target molecule or interact with a CPP target molecule can be made by determining the activity of the target molecule. For example, the activity of a target molecule can be measured by contacting the target molecule with CPP or a fragment thereof, measuring the induction of a target second cell messenger (eg, cAMP, STAT3, Akt, intracellular Ca2 +, diacylglycerol, IP3, etc.) Detecting catalytic / enzymatic activity against the appropriate substrate and detecting induction of a reporter gene (including a target responsive regulatory element operably linked to a nucleic acid encoding a luciferase) Alternatively, it can be determined by detecting a cellular response modulated by the target, eg, signal transduction or protein: protein interaction.

The cell-free assays of the present invention are suitable for use with either soluble and / or membrane-bound isolated proteins (eg, CPP to which a CPP target binds or a biologically active portion or molecule thereof). . In the case of cell-free assays using membrane-bound isolated protein, it is desirable to use a solubilizer so that the membrane-bound isolated protein is maintained in solution. Examples of such solubilizers include nonionic detergents (eg, n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methyl glucamide, decanoyl-N-methyl glucamide, Triton ™ X-100, Triton ™ X-114, Thesit ™, isotridecipoly (ethylene glycol ether) _n , 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), 3-[( 3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO) or N-dodecyl = N, N-dimethyl-3-ammonio-1-propanesulfonate).

  In two or more embodiments of the above assay methods of the present invention, to facilitate separation of complexing species from one or both proteins that are not complexed, and to accommodate assay automation, CPP Or it is desirable to immobilize any of its target molecules. The binding of the test compound to the CPP or the interaction of the CPP with the target molecule in the presence and absence of the candidate compound is any of those described herein in any container suitable for containing reactants. It can also be performed by the following fixation protocol. Alternatively, the complex can be dissociated from the matrix and the level of CPP binding or activity measured using standard techniques.

  Other techniques for immobilizing proteins to the matrix can also be used in the screening assays of the present invention. For example, either CPP or CPP target molecules can be immobilized using the binding of biotin and streptavidin. A biotinylated CPP or target molecule is prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (eg, biotinylation kit, Pierce Chemicals, Rockford, Ill.) And streptavidin Can be fixed in wells of a coated 96 well plate (Pierce Chemical). Alternatively, the wells of the plate can be coated with an antibody that reacts well with the CPP or target molecule but does not interfere with the binding of the CPP to its target molecule, and unbound target or CPP can be captured in the well by antibody binding. . Methods for detecting such complexes include immunodetection of complexes using antibodies that react well with CPP or target molecules in addition to those described above for GST-immobilized complexes, and further CPP or target molecules There are also enzyme binding assays based on the detection of the activity of an enzyme that binds to.

  In another embodiment, a modulator of CPP expression is identified by a method of contacting a cell with a candidate compound and determining expression of CPP mRNA or protein in the cell. The expression level of CPP mRNA or protein in the presence of the candidate compound is compared to the expression level of CPP mRNA or protein in the absence of the candidate compound. Based on this comparison, it can be confirmed that the candidate compound is a modulator of CPP expression. For example, if CPP mRNA or protein expression in the presence of a candidate compound is higher (statistically significantly higher) than in the absence, the candidate compound is identified as a promoter of CPP mRNA or protein expression. A candidate compound is also confirmed to be an inhibitor of CPP mRNA or protein expression if the expression of CPP mRNA or protein in the presence of the candidate compound is lower (statistically significantly lower) than in the absence. The level of CPP mRNA or protein expression in the cell can be determined by the methods for detecting CPP mRNA or protein described herein.

  In yet another aspect of the invention, the CPP is a two-hybrid assay or a three-hybrid assay (eg, US Pat. No. 5,283,317, the disclosure of which is hereby incorporated by reference in its entirety). No .; Zervos et al. (1993) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent WO 94/10300), used as “beit protein” to relate to, bind to or interact with CPP activity. Other proteins that act ("CPP binding protein" or "CPP-bp") can be identified. Such CPP binding proteins are likely to be involved in signal propagation by CPP or CPP targets, for example, as downstream elements of CPP-mediated signaling pathways.

  The two-hybrid system is based on the modularity of most transcription factors composed of separable DNA binding and activation domains. In short, the assay utilizes two different DNA constructs. In one construct, the gene encoding for CPP or a fragment thereof is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In another construct, a DNA sequence encoding an unidentified protein (“prey” or “sample”) in a DNA sequence library is fused to a gene encoding for the activation domain of a known transcription factor. Yes. If the “bait” protein and the “prey” protein interact in vivo to form a CPP-dependent complex, the DNA binding domain and the activation domain of the transcription factor are brought into close proximity. Such proximity allows transcription of a reporter gene (eg, LacZ) operably linked to a transcriptional regulatory site that responds to a transcription factor. Reporter gene expression is detectable and cells containing functional transcription factors can be isolated and their colonies used to obtain cloned genes encoding proteins that interact with CPP.

  The present invention further relates to novel agents identified by the screening assays described above and techniques for making such agents by these assays. Accordingly, in one embodiment, the invention includes a compound or agent obtainable by a method comprising any one stage of the above-described screening assays (eg, cell-based assays or cell-free assays).

  Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (eg, a CPP modulating agent or CPP binding partner) may be used in an animal model to reduce the efficacy, toxicity or side effects of treatment with such an agent. Can be determined. Alternatively, agents identified as described herein can be used in animal models to elucidate the mechanism of action of such agents. Furthermore, the present invention relates to the use of novel agents identified by the screening assays described above for the treatment as described herein.

  The present invention also relates to the use of the novel agents identified by the screening assays described above for diagnosis, prognosis, prevention and treatment as described herein. Accordingly, the use of such agents in the design, formulation, synthesis, manufacture and / or production of a drug or pharmaceutical composition for use in diagnosis, prognosis or treatment as described herein is also contemplated by the present invention. Within range. For example, in one embodiment, the invention includes a method of synthesizing or producing a drug or pharmaceutical composition by reference to the structure and / or properties of a compound obtained by one of the above-described screening assays. For example, a drug or pharmaceutical composition is a compound obtained by contacting a cell expressing a CPP target molecule with a test compound and determining the ability of the test compound to bind to or modulate the activity of the CPP target molecule Can be synthesized based on the structure and / or properties of In another exemplary embodiment, the present invention contacts a CPP or biologically active portion thereof with a test compound and binds to or binds to CPP or biologically active portion thereof. Methods of synthesizing or producing a drug or pharmaceutical composition based on the structure and / or properties of a compound obtained by a method for determining the ability of a test compound to modulate the activity of a moiety.

Animal-based drug screening It is also advantageous to perform drug screening assays in vivo. In vivo screening assays are performed in non-human animals to find effective CPP modulators that may be involved in cardiovascular disease. Model systems based on animals of cardiovascular disease include, but are not limited to, non-recombinant animals and transgenic animals.

  Non-recombinant animal models for cardiovascular disease can include, for example, genetic models. Genetic models for such cardiovascular diseases include apoB or apoR deficient pigs (Rapacz, et al., 1986, Science 234: 1573-1577) and Watanabe hereditary hyperlipidemia (WHHL) rabbits (Kita et al. 1987, Proc. Natl. Acad. Sci USA 84: 5928-5931). Non-recombinant non-genetic animal models of atherosclerosis include, for example, animals exposed to either chemical injury through LDL dietary supplementation or mechanical injury through balloon catheter angioplasty, For example, a pig, rabbit or rat model can be included.

  As shown in the art (Ferns, G. A. A. et al. (1991) Science, 253: 1129-1132), a rat carotid artery injury model of restenosis can be a useful indicator of a potent therapeutic effect. An example of this method is described in US Pat. No. 6,500,909, the disclosure of which is hereby incorporated by reference. In short, the protocol approved by the National Institute on Aging Animal Care and Use Committee is that of GRC colonies anesthetized with 20 mg / kg body weight pentobarbital, 2 mg / kg body weight ketamine and 4 mg / kg body weight xylazine intraperitoneally. Six month Wistar rats were used. The left external carotid artery is cannulated with a 2-French Fogarty embolectomy catheter, swollen with saline, passed 3 times up and down the common carotid artery, and started 2 hours after swollen endothelial loss injury Daily dose based on body weight in an appropriate solution such as 1: 2: 2: 165 DMSO: Cremophor EL: dehydrated ethanol: phosphate buffered saline by internal injection ) Treated the animals. The test substance or vehicle alone was administered as an intraperitoneal injection once a day for the next 4 days. After 11 days, the animals (8 and 10 vehicle treatments) were anesthetized as described above, the carotid artery was removed, fixed with 10% buffered formalin and embedded in paraffin. A transverse section of the carotid artery was mounted on a microscope slide and stained with hematoxylin and eosin stain. The image of the carotid artery was projected on a digital conversion board, and the intima and media cross sections were measured. A decrease in neointimal area (thickening) indicates that the test substance is an effective anti-restenosis drug.

Causal relationships have been shown to reduce serum cholesterol levels by interfering with bile acid recirculation from the gut lumen. Epidemiological data have been accumulated and these data have shown that such a reduction leads to an improvement in the pathology of atherosclerosis (Stedronsky, Biochimica et Biophysica Acta, 1210, 255-287 (1994)) . Inhibition of cholesteryl ester transfer protein (CETP) has been shown to effectively regulate the plasma HDL / LDL ratio and is expected to suppress the progression and / or formation of certain cardiovascular diseases. Inhibiting CETP should result in an increase in plasma HDL cholesterol and a decrease in plasma LDL cholesterol, which should provide a therapeutically beneficial plasma lipid profile (McCarthy, Medicinal Res. Revs., 13, 139-59 (1993)). ^In vivo assays for compounds that inhibit rat ileal uptake of ¹⁴ C-taurocholate into bile (CETP inhibition) are disclosed in US Pat. No. 6,489,366, the disclosure of which is hereby incorporated by reference. And Une, et al. Biochimica et Biophysica Acta, 833, 196-202 (1985).

Briefly, male Wistar rats (200-300 g) are anesthetized with inactin (100 mg / kg). The bile duct is catheterized using a 10 inch long PE10 tube. The small intestine is exposed and spread on a gauze pad. A cannula (1/8 "luer lock type, taper female adapter) is inserted 12 cm from the junction of the small intestine and caecum. A slit is cut at 4 cm from this same connection (using an 8 cm long ileum). Milliliters of warm Dulbecco's phosphate buffered saline pH 6.5 (PBS) is used to clean the intestinal segment, a 20 cm long silicone tube (0.02 ″ ID × 0.037 ″ O at the distal opening). D.) Connect the proximal cannula to a peristaltic pump and wash the intestine with warm PBS for 20 minutes at 0.25 ml / min .. Monitor the temperature of the intestinal segment continuously. sometimes, ^one 0.05mCi / mL containing control sample (5 mM non-radiolabeled taurocholate of 2.0ml in 3ml syringe C- taurocholate) was added to the intestinal segment, initiating the recovery of bile samples. Control sample is injected 21 minutes at 0.25 ml / min. Bile samples fractions, the first 27 minutes of treatment, The ileal loop is washed with 20 ml of warm PBS (using a 30 ml syringe) 21 minutes after sample injection and then washed with warm PBS at 0.25 ml / min for 21 minutes. Perfusion is initiated as described above, with the test compound administered similarly (21 minutes of administration followed by 21 minutes of irrigation) and bile sampled every 3 minutes for the first 27 minutes. In response, a third perfusion (generally containing a control sample) is performed as described above.

  Furthermore, measurement of liver cholesterol concentration is a useful assay for determining the effectiveness of a test substance against cardiovascular disease. In this assay, liver tissue is weighed and homogenized with chloroform: methanol (2: 1). After homogenization and centrifugation, the supernatant is separated and dried under nitrogen. The residue can be dissolved in isopropanol and enzymatically combined with cholesterol oxidase as described by Allain, CA et al., Clin. Chem., 20, 470 (1974), which is incorporated herein by reference. Cholesterol content is determined using a combination of peroxidases.

  Similarly, serum cholesterol can be measured as follows. Total serum cholesterol is measured enzymatically using a commercial kit from Wako Fine Chemicals (Richmond, Va.); Cholesterol C11, catalog number 276-64909. HDL cholesterol is assayed using this same kit after VLDL and LDL were precipitated using Sigma Chemical Co. HDL cholesterol reagent, catalog number 352-3 (dextran sulfate method). Total serum triglyceride (blank) (TGI) is also assayed enzymatically using Sigma Chemical Co. GPO-Trinder, catalog number 337-B. VLDL and LDL (VLDL + LDL) cholesterol concentrations are calculated as the difference between total cholesterol and HDL cholesterol. FIG. 3 shows that a reduction in VLDL + LDL cholesterol in a sample treated with a test substance compared to a control is an effective anti-cardiovascular drug.

  A canine model for evaluating lipid-lowering drugs can also be used, for example, as described in US Pat. No. 6,489,366.

  In short, male beagle dogs weighing 6-12 kg obtained from a specialist (eg Marshall farms) are fed once a day for 2 hours and given water ad libitum. A dosing group consisting of 6-12 dogs at random (eg, vehicle, ig; 1 mg / kg, ig; 2 mg / kg, ig; 4 mg / kg, i.e. g .; 2 mg / kg, po (powder in capsule)). Intragastric administration of therapeutic substance in aqueous solution (eg, 0.2% Tween 80 solution [polyoxyethylene monooleate, Sigma Chemical Co., St. Louis, Mo.]) is performed using a feeding tube To do. Prior to initiation of administration, blood samples are taken from the cephalic vein before feeding in the morning to assess serum cholesterol (total cholesterol and HDL cholesterol) and triglycerides. The animals are administered for several consecutive days in the morning before feeding. The animals are allowed to eat for 2 hours, after which the remaining food is removed. Feces are collected at the end of the study over 2 days and analyzed for bile acid or lipid content. A blood sample is also taken at the end of the treatment period for comparison with pre-test serum lipid levels. Statistical significance is determined using a standard student t test (p <0.05).

  Serum lipid content is also measured in the same way. Blood is collected from the cephalic vein of a fasted dog into a serum separator tube (Vacutainer SST, Becton Dickinson and Co., Franklin Lakes, NJ). Centrifuge the blood at 2000 rpm for 20 minutes and decant the serum. Total cholesterol uses a cholesterol oxidase reaction that produces hydrogen peroxide (colorimetrically quantifies the hydrogen peroxide) and is 96-well using a Wako enzyme diagnostic kit (cholesterol CII) (Wako Chemicals, Richmond, Va.). Measure in form. Create a standard curve of 0.5-10 ug cholesterol from the first two columns of the plate. Serum samples (20-40 μl, depending on estimated lipid concentration) or known serum control samples are added to independent wells (double test). Add water to each well to bring the volume to 100 μl. A 100 μl aliquot of color reagent is added to each well and after 15 minutes incubation at 37 ° C., the plate is read at 500 nm.

  HDL cholesterol is assayed using Sigma kit number 352-3 (Sigma Chemical Co., St. Louis, Mo.) (utilizing dextran sulfate and Mg ions to selectively precipitate LDL and VLDL). A 150 μl volume of each serum sample is added to each microfuge tube, followed by 15 μl of HDL cholesterol reagent (Sigma 352-3). Mix sample and centrifuge at 5000 rpm for 5 minutes. A 50 μl aliquot of the supernatant is then mixed with 200 μl of saline and assayed using the same procedure as for total cholesterol determination.

  Triglycerides are measured in 96-well plate format using Sigma kit number 337. In this procedure, glycerol is measured after it has been released by the reaction of triglycerides and lipoprotein lipase. A standard curve is generated using a standard solution of glycerol (Sigma 339-11) in the range of 1-24 μg. Serum samples (20-40 μl, depending on estimated lipid concentration) are added to the wells (double test). Water is added to bring the volume of each well to 100 μl, and 100 μl of color reagent is also added to each well. After mixing and incubation for 15 minutes, the plate is read at 540 nm and triglyceride values are calculated from the standard curve. One plate for replicates is also run with blank enzyme reagent to correct endogenous glycerol in serum samples.

  Test compounds against serum glucose and serum insulin in db / db mice (C578BL / KsJ-db / db Jcl) as described in US Pat. No. 6,462,046, the disclosure of which is hereby incorporated by reference. Evaluate their effects. The compound is dissolved in a vehicle (eg composed of 2% Tween 80 in distilled water) and administered orally. The dose is determined from the body weight. All aspects of work, including testing and animal disposal, are generally performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals (CIOMS Publication No. ISBN 92 90360194, 1985). A glucose-HA assay kit (Wako, Japan) is used for quantification of serum glucose, and an ELISA mouse insulin assay kit (SPI bio, France) is used for quantification of insulin. A suitable positive control is troglitazone (Helios Pharmaceutical, Louisville, Ky.).

  The animals are divided into 20 groups of 4 animals in each group. The animals weigh 52 +/− 5 grams and are 8-10 weeks old. During the test period, animals are given test food (Fwusow Industry Co., Taiwan) and water ad libitum. Prior to treatment, a blood sample is taken from each animal (pretreatment blood). Four groups of animals (vehicle group) receive only vehicle administration. Each vehicle group is orally administered with vehicle of 100, 30, 10 or 1 ml / kg body weight. Triglitazone solution (tween 80 / water, 10 ml / kg body weight) is orally administered to 4 positive control groups at doses of 100, 30, 10 and 1 ml / kg body weight, respectively. The test compound is similarly administered orally as a solution to four groups of animals, each group being given a different dose of compound. Vehicle, positive control and test compound solutions are administered to groups immediately 24 and 48 hours after pretreatment blood collection. Blood is drawn 1.5 hours after administration of the final dose (post-treatment blood). Serum glucose is measured enzymatically (Mutaratose-GOD) and insulin levels are measured by ELISA (mouse insulin assay kit). The mean SEM for each group is calculated and the percent inhibition of serum glucose and insulin is obtained by comparing pre-treatment blood and post-treatment blood. The rate of decrease in serum glucose and insulin levels in post-treatment blood relative to pre-treatment blood is determined, and a Student t test that does not correspond to the comparison between the control and test solution groups and the vehicle group is applied. Consider significant differences at P <0.05. Troglitazone as an effective anti-cardiovascular disease drug decreases glucose levels (25 +/- 2%) at 10 mg / kg body weight.

  US Pat. No. 6,121,319, the disclosure of which is hereby incorporated by reference, describes an assay for the progression of atherosclerosis in hypercholesterolemic rabbits. Rabbits are sacrificed to obtain the aorta. The aorta is stained with Sudan-4 and the degree of staining is analyzed. Graph the aortic surface area ratio of lesions in lipid-fed rabbits treated with test substance and untreated lipid-fed rabbits. Rabbit aorta treated with effective anti-atherosclerotic drugs are less stained, indicating a reduction in atherosclerosis. In addition, aortic sections are immunostained for VCAM-1 expression and macrophage accumulation using antibodies against VCAM-1 or Ram-11 antigens. A decrease in VCAM-1 expression and macrophage accumulation compared to control treated samples indicates an effective agent.

  LDL cholesterol reduction can also be measured in a primate model. For example, cynomolgus monkeys are rendered hypercholesterolemic by feeding a high fat cholesterol diet prior to administration of the test compound. The monkey is then orally dosed with test compound or control vehicle for 2 weeks. A decrease in the proportion of serum LDL cholesterol in monkeys over this period indicates an effective anti-atherosclerotic drug.

Pharmaceutical Compositions Where the polypeptides of the invention are expressed in soluble form, for example as a secreted product of transformed yeast or mammalian cells, they provide guidance in purification, eg, “Enzyme Purification and Related Techniques,” Methods in Enzymology, 22: 233-577 (1977), and Scopes, R., Protein Purification: Principles and Practice (Springer-Verlag, New York, 1982), ammonium sulfate precipitation, ion exchange chromatography, gel filtration, electrophoresis, Purification can be done according to standard procedures to those skilled in the art including affinity chromatography steps. Similarly, if the polypeptides of the invention are expressed in insoluble form, for example as aggregates or inclusion bodies, they are centrifuged, the inclusion bodies are solubilized with chaotropic agents and reducing agents, and the solubilized mixture By separating the encapsulated pair from the disrupted host cells by reducing the concentration of the chaotropic agent and reducing agent so that the polypeptide assumes a biologically active conformation. It can be purified by technology. The latter procedure is described in the following references, which are hereby incorporated by reference: Winkler et al, Biochemistry, 25: 4041-4045 (1986); Winkler et al, Biotechnology, 3: 992-998 (1985) Koths et al, US Pat. No. 4,569,790; and European Patent Applications 86306917.5 and 863066353.3.

  Compounds that can detect or modulate CPP or CPP biological activity, including small molecules, peptides, CPP nucleic acid molecules, and anti-CPP antibodies of the present invention, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions generally comprise a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. Shall be included. The use of such media and agents as pharmaceutically effective substances is well known in the art. Except where the usual vehicle or drug is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration such as intravenous administration, intradermal administration, subcutaneous administration, oral administration (eg, inhalation), transdermal administration (topical administration), transmucosal administration, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: water for injection, saline, hydrogenated oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents Aseptic diluents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid or phosphoric acid And tension modifiers such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In any case, the composition must be sterile and must be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Inhibition of active microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

  Where the active compound is a protein, eg, an anti-CPP antibody, a sterile injectable solution may contain the required amount of the active compound in a suitable solvent, optionally with one or a combination of ingredients listed above. And then sterilized by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other required ingredients from those enumerated above. In the case of a sterile powder for preparing a sterile injectable solution, preferred preparation methods are vacuum drying and lyophilization in which a powder of an active ingredient and an additional target ingredient is obtained from the solution previously filtered and sterilized.

  Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. Systemic administration is also possible by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to permeate the barrier are used in the formulation. Such penetrants are generally known in the art, for example, transmucosal administration includes surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, these active compounds are generally formulated into ointments, salves, gels, or creams known in the art. Most preferably, the active compound is delivered to the subject intravenously.

  In one embodiment, these active compounds are prepared with carriers that prevent the compound from being rapidly eliminated from the body, such as sustained release formulations, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also available from Alza Corporation and Nova Pharmaceuticals, Inc. Commercially available. Liposomal suspensions (including liposomes targeted to cells infected with monoclonal antibodies to viral antigens) can also be used as pharmacologically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811, the disclosure of which is hereby incorporated by reference in its entirety.

  In a further embodiment, the active compound can be coated on a microchip drug delivery device. Such devices are useful for the controlled transmission of proteinaceous compositions to the bloodstream, cerebrospinal fluid, lymph, or individual tissues without undergoing digestion or injection into an individual. US Pat. Nos. 6,123,861 and 5,797,898, the disclosures of which are incorporated herein by reference in their entirety, and US patent applications. No. 2002201176A1.

  It is especially advantageous to formulate oral or preferably parenteral compositions in dosage unit form for ease of dosage administration and homogenization. A unit dosage form as used herein means a physically discrete unit suitable as a unit dose for the subject to be treated, with each unit providing the desired therapeutic effect with the required pharmaceutical carrier. Contains a calculated amount of active compound. The unit dosage form specifications of the present invention are based on the unique properties of the active compounds and the specific therapeutic effects to be achieved and the limitations inherent in the art of combining such active compounds for the treatment of an individual. Determined and depend directly on them.

  Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or test animals such as LD50 (dose lethal to 50% of the population) and ED50 (50% of the population therapeutically effective). To determine the appropriate dose). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but delivery systems that target such compounds to affected tissue sites are designed to minimize damage to uninfected cells and thereby reduce side effects You have to be careful.

  Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for human use. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dose can vary within this range depending on the dosage form used and the route of administration used. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. To more accurately achieve the circulation used to determine useful doses in humans, dose formulations may be made in animal models. Plasma levels may be measured, for example, by high performance liquid chromatography.

  These pharmaceutical compositions can be included in a container, pack or dispenser together with instructions for administration.

Cardiovascular Disease Treatment The CPP modulators and CPP-related compositions of the present invention can be used in the treatment or prevention of CPP-related diseases. Accordingly, in one aspect, the invention relates to a pharmaceutical composition comprising an antibody, antibody fragment, or peptide modulator of CPP, preferably comprising a pharmacologically acceptable carrier or diluent. The carrier or diluent is preferably compatible with oral, intravenous, intramuscular, or subcutaneous administration. The pharmaceutical composition can comprise or consist essentially of the CPP-related compositions, CPP modulators, anti-CPP antibodies, or anti-CPP antibody fragments described herein.

  Many drugs are useful for the treatment and prevention of cardiovascular disease. Advantageously, such agents can be used in combination with CPP related compositions.

  For example, cell cycle inhibitors and proto-oncogenes (Simari and Nabel, Semin. Intervent. Cardiol. 1: 77-83 (1996)); NO (nitrogen monoxide) donors; bcl-x (Pollman et al., Nature Med) 2: 222-227 (1998)); herpesvirus thymidine kinase (tk) gene and systemic ganciclovir (Ohno et al., Science 265: 781-784 (1994); Guzman et al., Proc. Natl. Acad. Sci. USA 91: 10732-10736 (1994); Chang et al., Mol. Med. 1: 172-181 (1995); and Simari et al., Circulation 92: 1-501 (1995) )) Has been adopted for atherosclerosis, restenosis and neointimal smooth muscle proliferation. The disclosures of the above references are incorporated herein by reference in their entirety.

  Antithrombotic agents useful in combination with the compositions of the present invention include, for example, IIb / IIIa integrin inhibitors; tissue factor inhibitors; and antithrombin agents. In addition, the disclosure is directly incorporated herein by reference, Vukmir, Am. J. Emer. Med. 13: 459-470 (1995); Grant, PACE 20: 432-444 (1997) ; Assmann I., Curr. Med. Res. Opin. 13: 325-343 (1995); and Lipka et al., Am. Heart J. 130: 632-640 (1995). Antiarrhythmic drugs such as drugs (class I drugs), sympathetic antagonists (class II drugs), anti-atrial fibrillation drugs (class III drugs), calcium channel drugs (class IV drugs) or anion antagonists (class V drugs) Medicines can also be used. Examples of class I drugs include procainamide; quinidine or disopyramide; lidocaine; phenytoin; tocainide or mexiletine; encainide; flecainide; lorcainide; Sympathetic antagonists include propranolol, esmolol, metoprolol, ateneral or acebutolol. Examples of anti-atrial fibrillation agents include bretylium, amiodarone, soterol (II) or N-acetyl procainamide. Class IV drugs include verapamil, diltiazem, and bepridil, and anionic antagonists such as alinidine.

  Congestive heart failure drugs include Embrel ™ (Immunex Corp .; Seattle, Whsh.), TNF inhibitors such as TBC11251, or ACE (angiotensin converting enzyme) such as Natrecor (Nesiritide; Scios, Inc.). Inhibitors are mentioned. Angiogenic agents, eg, recombinant VEGF isoforms such as rhVEGF developed by Genentech; nucleic acid molecules encoding VEGF's 121 amino acid isoform (BioByPass ™; GenVec / Parke Davis); or VEGF-2 FIBLAST ™, a recombinant form of FGF-2 developed by Scios, Inc. (Mountain View, Calif.) And Wyeth Ayerst Laboratories (Radnor, Pa.) GENERX ™, see Collateral Therapeutics (San Diego, Calif.) And Schering AG (Miller and Abrams, Gen. Engin. News 18: 1 (1998), the disclosure of which is incorporated herein by reference in its entirety. Adenoviral gene therapy vectors encoding FGF-4 developed in part)) are also useful in combination with the CPP-related compositions of the present invention. Finally, amlodipine (Marche et al., Int. J. Cardiol. 62 (Suppl.): S17-S22 (1997); Schachter, Int. J. Cardiol. 62 (Suppl.): S85-S90 (1997)) Calcium antagonists such as: nicardipine; nifedipine; propanolol; isosorbide dinitrate; diltiazem; and isradipine (Nayler (Ed.) Calcium Antagonists pages 157-260 London: Academic Press (1988); Schachter, Int. J. Cardiol. 62 (Suppl.): S9-S15 (1997)) is also an advantageous treatment for cardiovascular diseases.

Treatment of Other Diseases The CPP provided by the present invention is useful in the treatment of cardiovascular diseases because it preferentially increases in the plasma of individuals with cardiovascular diseases. However, the usefulness of the CPP of the present invention is not limited to the treatment of cardiovascular diseases. As illustrated in Example 4, the CPPs of the present invention may be used to diagnose, prognose, and / or treat various other diseases or disorders caused or affected by, for example, increased or decreased levels of CPPs of the present invention. Can also be used. Example 4 provides a method suitable for the determination of diseases and disorders in which the CPP of the present invention can be used as a therapeutic, prognostic and / or diagnostic method. However, it will be appreciated that there are other suitable methods known in the art that can be used for this purpose. In a particularly preferred embodiment, the present invention provides CPP232 for the treatment of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motor dysfunction or for the prognosis or diagnosis of said disease. .

  Polypeptide CPP232 (SEQ ID NO: 706) is a peptide detected in human plasma according to the present invention and was found to have an amino acid sequence (accession number P12872) that is part of motilin. Motilin is a gastrointestinal regulatory polypeptide synthesized by motilin cells of the duodenal epithelium. This polypeptide is released into the systemic circulation at about 100 minute intervals during the fasting state and is the most important factor in controlling fasting propagating contractions. Motilin also stimulates endogenous release of the endocrine pancreas. It has been reported that plasma levels of motilin are elevated in celiac disease (Sjolund K, et al., Peptides 24 (2003) 483-486). Impaired proteolytic processing of promotilin into motilin produces numerous motilin fragments from promotilin. Therefore, celiac disease does not produce the most important biologically active motilin fragment. The present invention therefore provides CPP232 as a suitable therapeutic agent to supplement biologically active peptides that are deficient or reduced in celiac disease.

  In the celiac mucosa, the number of motilin producing cells is reduced (Johnston CF et al., Hepatogastroenterology. 35: 285-288 1988). Accordingly, the present invention therefore provides CPP232 as a suitable therapeutic agent to overcome the lack of motilin producing cells.

  It is known that gallstone formation is accompanied by apoptosis. For example, ursodeoxycholic acid (UDCA), one of the treatments for gallstones, suppresses apoptosis. The gene expression data in Table 4 indicates that CPP232 inhibits cecal apoptosis. Accordingly, the present invention therefore provides CPP232 as a suitable therapeutic agent for the treatment of gallstones.

  In accordance with the present invention, CPP232 down-regulates gene expression of dihydrolipoamide acetyltransferase and related genes (the last three genes in Table 4). Dihydrolipoamide acetyltransferase is the major autoantigen of biliary cirrhosis (Surh CD, et al., J Immunol 1990; 144: 3367-74). Accordingly, the present invention therefore provides CPP232 as a suitable therapeutic for reducing the expression of the major autoantigens of biliary cirrhosis. CPP232 further down-regulates the gene for β-2-microglobulin. Therefore, the present invention further provides CPP232 as a therapeutic agent suitable for suppressing antigen presentation (or autoantigen presentation). Furthermore, it has been found that dihydrolipoamide dehydrogenase is a self-antigen found in hepatitis C virus (HCV) infection (Wu Y.Y. et al., Clin Exp Immunol 2002; 128: 347-352). Thus, the present invention provides CPP232 as a therapeutic agent intended for the treatment of HCV infection or problems associated with HCV infection (eg, cirrhosis).

CPP232 (SEQ ID NO: 706) was synthesized by GeneProt, Inc (Geneva, Switzerland) by an approach as described in Example 4, in which the peptide was injected into mice and Gene expression profiling was performed. Table 4 shows that CPP232 affects gene expression and that the effect on gene expression is limited to the caecum, a potential target region of motilin. The main effect is seen in the differential expression of genes involved in apoptosis and biliary cirrhosis, and most of these genes are down-regulated, so dihydro, which is the main autoantigen of suppression of apoptosis and biliary cirrhosis. Reduction of the expression of lipoamide dehydrogenase occurs. Gallstone formation is accompanied by apoptosis, and plasma levels of motilin in gallstone patients are higher than in healthy patients. Thus, the present invention provides not only a therapeutic substance for the treatment of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motor dysfunction, but also a modulator of said disease, in particular biliary cirrhosis or cholesterol. CPP232 is provided as a target for identifying modulators of cholelithiasis. Examples of intestinal motility dysfunction include irritable bowel syndrome, diabetes, gastric insufficiency, esophageal spasm, Hirsch spring disease, chronic intestinal pseudo-obstruction, scleroderma, and achalasia. Symptoms related to intestinal motor dysfunction include, for example, difficulty swallowing, heartburn, gas, bloating, nausea, vomiting, constipation, and diarrhea. Also provided are methods for prognosis and / or diagnosis of the disease.

  According to one aspect of the present invention, there is provided a method for the treatment of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motor dysfunction, wherein an effective amount of CPP232 polypeptide is afflicted with the disease. There is provided a method comprising administering to a mammal, including a human. In a related aspect of the invention, there is provided use of a CPP232 polypeptide for the manufacture of a medicament for use in the treatment of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motor dysfunction. In a preferred embodiment, the disease is biliary cirrhosis or cholesterol cholelithiasis. In another preferred embodiment, the gastrointestinal motility disorder is selected from the group consisting of irritable bowel syndrome, diabetes, gastric insufficiency, esophageal spasm, Hirschspring disease, chronic intestinal pseudoocclusion, scleroderma, achalasia It is a dysfunction. In another preferred embodiment, the symptom associated with intestinal motility dysfunction is a symptom selected from the group consisting of difficulty swallowing, heartburn, gas, bloating, nausea, vomiting, constipation, diarrhea.

  According to another aspect, the present invention provides a method for identifying modulators of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility dysfunction, comprising i) against CPP232 biological activity Contacting a test compound with CPP232 under acceptable sample conditions; ii) determining a level of said at least one CPP232 biological activity; iii) comparing said level to that of a control sample without said test compound Providing a method comprising: In a preferred embodiment, the test compound that alters the level is selected for further testing as a CPP232 modulator for prophylactic and / or therapeutic treatment of gallstones or celiac disease. In a preferred embodiment, the level of CPP232 biological activity is measured by detecting the expression level of one or more genes listed in Table 4.

  According to another aspect, the present invention is a method for the prognosis or diagnosis of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility dysfunction, comprising detecting plasma levels of CPP232 Wherein the increased level indicates symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility dysfunction. An increase in plasma levels of CPP232 in the present invention is an increase relative to plasma levels of CPP232 that is observed in individuals not suffering from gallstones or celiac disease. The increase is preferably at least 1.2 times, more preferably at least 1.5 times, 2 times, 3 times, 5 times or 10 times. According to a preferred embodiment of the present invention, the method comprises i) detecting the expression level of at least one gene listed in Table 4 in a sample of suitable tissue obtained from a subject to obtain a first value And ii) comparing the first value to the expression level of said gene from a disease-free subject, wherein the increase or decrease in expression level in the subject sample compared to the sample from the disease-free subject is The subject has a predisposition to symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motor dysfunction, or suffers from symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motor dysfunction To show). Gene expression is detected at the mRNA or protein level. Suitable tissues include but are not limited to bile ducts, duodenum, gallbladder, cecum, colon, ileum, intestinal tissue, liver, stomach. The mRNA expression level can be detected by any suitable technique such as, for example, microarray analysis, Northern blot analysis, reverse transcription PCR and real time quantitative PCR. Similarly, protein levels can also be detected by any suitable technique, such as, for example, by Western blotting utilizing a labeled probe specific for the protein.

  In a preferred embodiment of the above aspect, the gene is selected from the genes that are up-regulated or down-regulated as listed in Table 4. Preferably, the gene is selected from the genes listed in Table 4, which are up-regulated 1.2-fold, 1.3-fold or 1.5-fold or more; or listed in Table 4. It is selected from genes that are down-regulated 0.8 times or less, 0.7 times or less, or 0.6 times or less. In yet another preferred embodiment of the above aspect, the expression of at least one, two, three, four or five genes is measured. In yet another preferred embodiment of the above aspect, the disease is cholesterol cholelithiasis.

  Having generally described the invention, a further understanding can be obtained by reference to specific embodiments. These specific examples are provided herein for illustrative purposes only, and are not intended to be limiting unless otherwise specified.

Example 1: Characterization of CPP levels in disease and control populations Subjects enrolled in the Duke data bank for cardiovascular disease were selected based on coronary artery disease (CAD). A total of 241 CAD patients and control individuals were matched for gender, age, and ethnicity, and individuals with abnormal plasma were excluded. A set of 53 CAD patients and a set of 53 control individuals were set up. Six liters of plasma from each set were pooled. Since a portion of plasma was retained from each individual, a positive result of the pool sample can be confirmed for each member of the population. Such confirmation is useful in eliminating the potential for confounding effects in individuals with abnormal levels of specific polypeptides not associated with cardiovascular disease. 2.5 liters of plasma pooled from each population was separated by multi-stage chromatography according to the Microprot® method as follows.
Step 1: HSA / IgG depletion 125 ml of frozen plasma was thawed and filtered through a 0.45 μm sterile filter in a sterile hood.

  Two filtrates in series: 300 ml HSA ligand Sepharose fast flow column (Amersham, Upsala, Sweden) each 5 cm ID and 5 cm length; and 100 ml G protein Sepharose fast flow column (Amersham, Upsala, Sweden) 5 cm ID and 5 cm length. The column was injected.

The column was equilibrated and washed with 50 mM PO ₄ buffer pH 7.1, 0.15 M NaCl. The flow rate was 5 ml.

  The fraction not retained (350 ml) was frozen until the second step. The test was performed 20 times.

Step 2: Gel filtration / reverse phase capture step The sample from Step 1 was thawed and filtered through a 0.45 μm sterile filter in a sterile hood.

The filtrate was injected into two in-line gel filtration columns: two 9.5 liter Spadex 75 (Amersham, UK) columns with an inner diameter of 14 cm and a length of 62 cm. The column was equilibrated with 50 mM PO ₄ buffer pH 7.4, 0.1 M NaCl, 8 M urea. Hydrophobic impurities were retained in reverse phase pre-lum: 150 ml PLRPS (Polymer Labs, UK). The precolumn was switched for sample injection. Gel filtration was performed at a flow rate of 40 ml / min.

Low molecular weight protein (<20 kDa) was fed into a series reverse phase capture column: 50 ml PLRPS 100 angstrom (Polymer Labs, UK). The three-way valve controlling the injection into the PLRPS column was switched with a cut-off value of 33 mAU (280 nm) and the gel filtration eluate was sent to the reverse phase capture column. This cut-off value is obtained by first determining the evaluation range of the OD value using SDS-PAGE, and then evaluating three types of cut-off values (high value, intermediate value, and low value OD range). Set. The final cut-off value was chosen so that the low molecular weight protein population was at least 85% to maximize the resulting low molecular weight protein. Low molecular weight proteins and peptides were eluted from the reverse phase capture PLRPS column with a gradient of 1 column volume of 0.1% TFA in water, 80% CH ₃ CN.

  The eluate fraction (50 ml) was frozen until the next step. The test was performed 20 times. At the end of this step, all reverse phase eluates were thawed, pooled (1 liter) and divided into 7 polypropylene containers (143 ml). The vessel was maintained at -20 ° C until used for the next step.

Step 3: Cation Exchange The sample from step 2 (147 ml) was thawed and mixed with the same amount of cation exchange buffer A (Gly / HCl buffer 50 mM, pH 2.7, urea 8M).

  The sample was injected onto a 100 ml sucrose 15S column (Amersham, Upsala, Sweden) with an inner diameter of 35 mm and a length of 100 mm. The column was equilibrated and washed with buffer A. The flow rate was 10 ml / min.

Step gradient from 100% buffer A to 100% buffer B (buffer A containing 1M NaCl):
3 column volumes of 7.5% B (75 mM NaCl)
3 column volumes of 10% B (100 mM NaCl)
3 column volumes of 17.5% B (175 mM NaCl)
2 column volumes of 22.5% B (225 mM NaCl)
2 column volumes of 27.5% B (275 mM NaCl)
2 column volumes of 100% B (1M NaCl)
And eluted.

  45-60 fractions were collected based on the peak. 7 times. After the seventh round, the fractions were pooled inside and outside the run to obtain 18 fractions. Fractions were maintained at −20 ° C. until used for the next step.

Step 4: Reduction / alkylation and reverse phase HPLC fraction 1
After adjusting the pH to 8.5 with concentrated Tris-HCl, each of these 18 cation exchange fractions was reduced with dithioerythritol (DTE, 30 mM, 3 hours at 37 ° C.) and iodoacetamide (120 mM, 25 ° C. in the dark). For 1 hour). The latter reaction was stopped by adding DTE (30 mM) followed by acidification (TFA, 0.1%). These fractions were then injected onto an Uptiper C8, 5 μm, 300 Å column (Interchim, France) with an inner diameter of 21 mm and a length of 150 mm. The injection was performed at a flow rate of 10 ml / min.

The C8 column was equilibrated and washed with 0.1% TFA in water (solution A). Proteins and peptides were eluted with a biphasic gradient from 100% A to 100% B (0.1% TFA in water, 80% CH ₃ CN) in 60 minutes. The flow rate was 20 ml / min. Thirty fractions of 40 ml were collected.

  Based on the optical density (OD) measured at 280 nm of each fraction, reflecting the protein concentration of that fraction, aliquots of equivalent protein content were made for each fraction.

  All aliquots were frozen except one aliquot for each fraction was added to each fraction with 500 μl 10% glycerol in water followed by Speed Vac (Savant, Fischer, Geneva) to prevent excessive drying. Maintained until use. The dried fraction was kept at −20 ° C. until used in the next step.

Step 5: Reversed phase HPLC fraction 2
The dried sample obtained from step 4 was suspended in 1 ml of solution A (0.03% TFA in water) and injected into a Vydac LCMS C4 column with an inner diameter of 4.6 mm and a length of 150 mm, 5 μm, 300 Å (Vydac, USA). did. The flow rate was 0.8 ml / min.

The C4 column was equilibrated and washed with Solution A, and the protein and peptide were eluted with a biphasic gradient adapted to the elution point of the sample in reverse phase HPLC fraction 1. Complete mass data was obtained using electrospray ion trap mass spectrometry. Sixteen different gradients were used with CH ₃ CN in the CH ₃ CN concentration range ± 5% of the RP1 fraction corresponding to the solvent concentration. For proteins that eluted in RP1 at a solvent concentration corresponding to or higher than 30% CH ₃ CN, the elution start condition of the RP2 gradient was set to RP1 elution concentration −30% in CH ₃ CN percentage. Twenty-four elution fractions were collected in deep well plates corresponding to various optimized collection formats designed for optimal SpeedVac concentration and further robotic processing.

Step 6: Mass Detection After reverse phase HPLC fraction 2, approximately 13,000 fractions were collected in a 96 well deep well plate (DWP). A small amount (2.5%) of this was sent to online analysis using LC-ESI-MS (Bruker Esquire). Undigested protein aliquots were mixed with a MALDI matrix and spotted on a MALDI plate with a mass calibration standard and a sensitivity standard. An automatic spot device (Bruker MALDI sample preparation robot) was used. Synapic acid (SA), also known as synapinic acid, ie, trans-3,5-dimethoxy-4-hydroxycinnamic acid and α-cyano-4-hydroxycinnamic acid (HCCA) 2 Different MALDI matrices were used. Mass detection was performed on the MALDI plate using a Bruker Reflex III MALDI MS apparatus. These 96-well plates were stored at + 4 ° C.

  A 96-well plate (DWP) was collected and subjected to a series of two concentration steps. The volume was reduced from 0.8 ml per well to about 50 μl by drying with SpeedVac, then redissolved to about 200 μl, concentrated again to about 50 μl per well and stored at + 4 ° C. The protein was then rebuffered, trypsin was added to each well, sealed, and the plate was incubated at 37 ° C. for 12 hours before digestion by quenching (adding formic acid until pH dropped to 2.0). . The concentration of trypsin added to the wells was adjusted based on the OD at 280 nm recorded for each specific fraction. This ensured optimal use of trypsin and complete digestion of the highest concentration fraction. Using an automatic spot device (Bruker MALDI sample preparation robot), a pre-mix of aliquots from each well with HCCA was deposited on the MALDI plate along with the sensitivity and mass calibration standards. MALDI plates were analyzed using a Bruker Reflex III MALDI MS instrument. The content of each well of the 96-well plate was analyzed with an LC-ESI-MS-MS Bruker Esquire ESI Ion-Trap MS apparatus.

Step 7: Detection and identification of small peptides in human plasma The separated fractions were further subjected to mass spectrometry (both matrix assisted laser desorption / ionization (MALDI) and MS-MS) for separation and detection.

  Complete mass data, peptide mass fingerprint and peptide sequence data were integrated for protein identification and characterization. The protein was identified using Mascot software (Matrix Science Ltd., London, UK) and the results from peptide identification were confirmed by manual analysis of the spectra.

  Of the proteins identified by this method, calgranulin A (S100 calcium binding protein A8 with SwissProt accession number P05109) is larger in pooled samples from controls than in pooled samples from CAD patients. Peptides from this protein were observed, for example, twice in many control fractions compared to the disease fraction, and the accumulation obtained during mass spectral identification of this protein. The score was 2.5 times higher for the control sample). Calgranulin A has been characterized as an inflammatory protein (Odink, et al., Nature 330 (6143), 80-82 (1987) and numerous subsequent references). It is expressed by spinal cord cells that exude during the inflammatory response, where they bind to glucosaminoglycan structures on epithelial cells (Robinson, et al., JBC 277: 3658-65 (2002)). Interestingly, PCT publication WO 00/61742 discloses the use of calgranulin A for the treatment of heart failure caused, for example, by atherosclerosis. Furthermore, PCR publication WO 00/18970 discloses the use of calgranulin A as an inhibitor of vascular membrane growth for the prevention of myocardial infarction and hypertension. Thus, providing a protein that has a beneficial effect in treating the disease under study when the protein separation and identification approach described herein is detected at a higher level in the control sample than in the disease sample. It is clear that this is effective.

  In contrast, the protein isolation and identification methods described in this example were compared to Matrix Gla Protein (SwissProt accession number P08493) as overexpressed in pooled samples from CAD patients by comparison with samples pooled from controls. (For example, peptides from the protein were observed almost twice in many disease fractions compared to the control fraction, and the accumulation score obtained during mass spectral identification of this protein) Was twice as high for disease samples). MGP is a vitamin K-dependent protein associated with the organic matrix of bone and cartilage. Mori, et al. Has demonstrated that MGP can inhibit vascular calcification (FEBS Letters 433: 19-22 (1998)). MGP levels increase within atherosclerotic plaques as a possible feedback response to vascular calcification. PCT publications WO01 / 02863 and WO01 / 25427 describe MGP as a biomarker for atherosclerosis and cardiovascular disease. Thus, it is clear that the protein separation and identification approach described herein is effective in providing proteins with a recognized use in the diagnosis of the disease under study.

  Finally, the tryptic peptides listed in Table 3 were observed by tandem mass spectrometry at high levels in coronary artery disease samples. The presence of a trypsin acting peptide suggests that a polypeptide comprising the peptide's amino acid sequence was present at high levels in the starting plasma sample from an individual with CAD. Such polypeptides include CPP 149-402. In addition to that observed by MALDI mass spectrometry, the sequence of the peptide defines the CPP peptide CPP 149-402 of the present invention.

  The method of protein separation and identification according to the present invention is extremely sensitive. The MicroProt® method can detect very small amounts of protein, with plasma concentrations in the range of several hundred pM. The accuracy has been confirmed during the implementation of the methods described so far. In particular, proteins with well-characterized roles in atherosclerosis and CAD were detected differently in CAD and control samples (supra).

Example 2: Chemical synthesis of CPP In this example, the CPP of the present invention is synthesized. Peptide fragment intermediates are first synthesized and then assembled into the desired polypeptide.

  CPPs can be prepared, for example, with 5 fragments first selected to have an N-terminal Cys residue of the fragment to be ligated. Fragment 1 is first bound to fragment 2 to give a first product, and after preparative HPLC purification, the first product is then bound to fragment 3 to give a second product. After preparative HPLC purification, the second product is coupled to fragment 4 to obtain the third product. Finally, after preparative HPLC purification, the third product is coupled to fragment 5 to obtain the desired polypeptide, which is purified and refolded.

Thioester formation Fragments 2, 3, 4 and 5 are synthesized on a thioester producing resin as described above. For this purpose the following resin is prepared: S-acetylthioglycolic acid pentafluorophenyl ester is prepared under the conditions essentially as described by Hackeng et al (1999) Leu-PAM resin. To join. In the first case, the resulting resin was used as the starting resin for peptide chain extension on the 0.2 mmol scale after removal of the acetyl protecting group by treatment with 10% mercaptoethanol, 10% piperidine in DMF for 30 minutes. Use as The N ^alpha N-terminal Cys of the fragments 2 to 5, Boc-thioproline (Boc-SPr, i.e. Boc-L-thioproline), and at the end of each strand in place of Cys with conventional N ^alpha or S ^beta Protection Protect by binding (eg, Brik et al, J. Org. Chem, 65: 3829-3825 (2000)).

Peptide synthesis Solid phase synthesis was performed in situ neutralization / 2 for stepwise Boc chemical chain extension as described by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992). Custom modified 433A peptide synthesis from Applied Biosystems using the (1H-benzotriazol-1-yl) -1,1,1,3,3-tetriethyluronium hexafluoro-phosphate (HBTU) activation protocol Perform on the device. Each synthesis cycle consists of treating with neat TFA for 1-2 minutes, 1 minute DMF flow wash, 10 minutes binding time with pre-activated 2.0 mmol Boc-amino acid in the presence of excess DIEA, and Consisting of N ^α -Boc-removal by washing with two DMF streams. Nα-Boc-amino acid (2 mmol) is preactivated with 1.8 mmol HBTU (0.5 M in DMF) for 3 min in the presence of excess DIEA (6 mmol). After conjugation of Gln residues, washing with a stream of dichloromethane is used before and after deprotection with TFA to prevent the formation of pyrrolidone carboxylic acid catalyzed by possible high temperature (TFA / DMF). Side chain protected amino acids are Boc-Arg (p-toluenesulfonyl) -OH, Boc-Asn (xanthyl) -OH, Boc-Asp (O-cyclohexyl) -OH, Boc-Cys (4-methylbenzyl) -OH, Boc-Glu (O-cyclohexyl) -OH, Boc-His (dinitrophenylbenzyl) -OH, Boc-Lys (2-Cl-Z) -OH, Boc-Ser (benzyl) -OH, Boc-Thr (benzyl) -OH, Boc-Trp (cyclohexylcarbonyl) -OH, and Boc-Tyr (2-Br-Z) -OH (Orpagen Pharma, Heidelberg, Germany). Other amino acids are used without side chain protection. The C-terminal fragment 1 was synthesized on Boc-Leu-O—CH ₂ -Pam resin (0.71 mmol / g relative to the loaded resin), while the machine-assisted synthesis of fragments 2-5 was Boc-Xaa _-S-CH 2 begins on -CO-Leu-Pam resin. This resin is obtained by coupling S-acetylthioglycolic acid pentafluorophenyl ester to Leu-PAM resin under standard conditions. The resulting resin is used as the starting resin for peptide chain elongation on a 0.2 mmol scale after removal of the acetyl protecting group by treatment with 10% mercaptoethanol, 10% piperidine in DMF for 30 minutes. .

After chain assembly is complete, the peptide fragment is deprotected and cleaved from the resin by treatment with anhydrous hydrogen fluoride for 1 hour at 0 ° C. with 5% p-cresol as a scavenger. In all cases except fragment 1, the imidazole side chain 2,4-dinitrophenyl (DNP) protecting group remains on the His residue because the DNP removal procedure is incompatible with the C-terminal thioester group. However, DNP is gradually removed by thiols during the ligation reaction to yield unprotected His. After cleavage, the peptide fragment is precipitated with ice-cold diethyl ether, dissolved in aqueous acetonitrile and lyophilized. Peptide fragments were obtained from Waters by using a linear gradient of buffer B (acetonitrile / 0.1% trifluoroacetic acid) in buffer A (H ₂ O / 0.1% trifluoroacetic acid) and UV detection at 214 nm. Purify by RP-HPLC on a C18 column. Samples are analyzed by electrospray mass spectrometry (ESMS) using an Esquire instrument (Bruker, Bremen, Germany) or similar instrument.

Natural Chemical Ligation Reaction As described in more detail below, the ligation reaction of unprotected fragments is performed as follows: To obtain a final peptide concentration of 1-8 mM around pH 7, the dried peptide is 6 M guanidine hydrochloride. (GuHCl), dissolved in equimolar amounts in 0.2M phosphoric acid pH 7.5, add 1% benzyl mercaptan, 1% thiophenol. Typically, the reaction is performed overnight and monitored by HPLC and electrospray mass spectrometry. The ligation product is subsequently processed to remove protecting groups still present. Opening the N-terminal thiazolidine ring further requires the addition of solid methoxamine to a final concentration of 0.5 M at pH 3.5 and a further incubation at 37 ° C. for 2 hours. A 10-fold excess of tris (2-carboxyethyl) phosphine is added prior to preparative HPLC purification. Fractions containing the polypeptide chain are identified by ESMS, pooled and lyophilized.

The ligation reaction of fragments 4 and 5 is performed in 6M GuHCl at pH 7.0. The concentration of each reactant is 8 mM, and 1% benzyl mercaptan and 1% thiophenol are added to create a reducing environment and promote the ligation reaction. Most quantitative ligation reactions are observed after overnight stirring at 37 ° C. At this point in the reaction, CH ₃ —O—NH ₂ HCl is added to this solution to a final concentration of 0.5M and the pH is adjusted to 3.5 to open the N-terminal thiazolidine ring. After 2 hours incubation at 37 ° C., ESMS is used to confirm the completion of the reaction. Subsequently, the reaction mixture is treated with a 10-fold excess of tris (2-carboxyethylphosphine) relative to the peptide fragment, and after 15 minutes, the ligation product is purified by preparative HPLC (eg, C4 20-60% CH ₃ CN 0.5% / min), lyophilized and stored at -20 ° C.

  The same procedure is repeated for the remaining ligation reactions with minor modifications.

Polypeptide folding Full-length peptides are refolded by air oxidation by dissolving reduced lyophilized protein (approximately 0.1 mg / mL) in 1 M GuHCl, 100 mM Tris, 10 mM methionine, pH 8.6. After stirring gently overnight, the protein solution is purified by RP-HPLC as described above.

Example 3: Preparation of CPP antibody composition A substantially pure CPP or portion thereof is obtained. The concentration of protein in the final preparation is adjusted to a level of several micrograms / ml, for example by concentration on an Amicon filter device. Next, monoclonal or polyclonal antibodies against the protein are prepared as described in the sections entitled “Monoclonal Antibodies” and “Polyclonal Antibodies”.

  In short, to produce anti-CPP monoclonal antibodies, mice are repeatedly inoculated with several micrograms of CPP or fragments thereof over a period of several weeks. The mouse is then sacrificed and spleen antibody-producing cells are isolated. The spleen cells are fused to mouse bone marrow cells by means of polyethylene glycol, and excess non-fused cells are destroyed by growth of the system on selective medium containing aminopterin (HAT medium). The well fused cells are diluted and an aliquot of the dilution is placed in the well of a microtiter plate where the culture continues to grow. Antibody-producing clones can be prepared as described in ELISA, as originally described by Engvall, E., Meth. Enzymol. 70: 419 (1980), the disclosure of which is hereby incorporated by reference in its entirety. Identification by detection of antibodies in the well supernatants by simple immunoassay methods. Selected positive clones can be expanded and the monoclonal antibody product can be recovered for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York, Section 21-2, the disclosure of which is hereby incorporated by reference in its entirety. Has been.

  For the production of polyclonal antibodies by immunization, polyclonal antisera containing antibodies against heterologous epitopes in CPP or a portion thereof cannot or cannot be modified to enhance immunogenicity, and mice are immunized with CPP or a portion thereof. To prepare. Any suitable non-human animal, preferably a non-human mammal, including a rat, rabbit, goat or horse can be selected.

  Antibody preparations prepared according to either monoclonal or polyclonal protocols are useful in quantitative immunoassays to determine the concentration of CPP in a biological sample, or they can be used to identify the presence of an antigen in a biological sample. It can also be used semi-quantitatively or qualitatively. The antibodies can also be used in therapeutic compositions to kill cells expressing the protein or to reduce the level of protein in the body.

Example 4: Identification of CPP polypeptides in vivo CPP232 is administered subcutaneously to male C57BL / 6 mice at a dose of 600 μg / day for 7 days. At the end of this treatment period, samples from all organs are snap frozen at necropsy and analyzed using GeneChip® expression profiling.

  Total RNA is extracted from these frozen tissues using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Total RNA is quantified by absorbance at λ = 260 nm (A260 nm) and purity is estimated by the A260 nm / A280 nm ratio. Integrity is confirmed by denaturing gel electrophoresis. RNA is stored at −80 ° C. until analysis.

  A sufficient amount of total RNA is used to synthesize double-stranded cDNA using the Superscript Choice System (Life Technologies). The cDNA is then transcribed in vitro (MEGAscript ™ T7 kit, Ambion) to form biotin-labeled cRNA. Next, 12-15 mg of labeled cRNA is hybridized with the Affymetrix mouse MOE430A expression probe array for 16 hours at 45 ° C. The array is then washed according to the EukGE-WS2 protocol (Affymetrix) and stained with 10 mg / ml streptavidin-phycoerythrin conjugate (Molecular Probs). Signals were 2 mg / ml acetylated BSA (Life Technologies), 100 mM MES, 1 M [Na +], 0.05% Tween 20, 0.005% Antifoam (Sigma), 0.1 mg / ml goat IgG and 0.5 mg / ml. Antibody is amplified using ml biotinylated antibody and re-stained with streptavidin solution. After washing, the array is scanned with a Gene Array® scanner (Affymetrix).

  The expression level is evaluated by averaging the difference in signal intensity measured by the oligonucleotide pair of a given probe (AvgDiff value). The image acquisition and quantity translation software used for this study is Affymetrix Microarray Suite version 5 (MAS5).

  In order to identify the genes affected by the treatment, these data sets are first screened, and the values are systematically in the first-wave analysis gene in the low expression range where the experimental noise is high (for any experimental point) At least the AvgDiff value 80) in the number of experiments corresponding to the minimum number of replicates. In the second selection, the threshold t-test p-value (0.05) is based on a two-component error model (Global Error Model) and, if possible, a multiple hypothesis test (Benjamini and Hochberg false discovery rate) step Down correction is used to identify genes with different values between treatment and no treatment.

  The selected gene list is then compared to the established gene list for pathways and cellular components using Fisher's exact test. Venn diagrams are used to identify common genetic changes between various organs. Using highly related gene expression profiles, several distance metrics (standard, Pearson) are used to find genes with correlated changes at individual experimental points.

  The decision to consider a particular genetic association is the relationship between exploratory screening as described above and the quantitative changes identified by statistical algorithms, as well as with other regulated genes pointing to a common biological theme. It is based on relationships.

Table 4 shows that CPP232 RNA levels affect the apoptotic pathway, proteosome, ubiquitin pathway and genes associated with ribosomal RNA and proteins. Most genes are up-regulated at least 1.2 fold or down-regulated at least 0.8 fold.

Claims (27)

A method of screening and / or diagnosing cardiovascular disease in a subject comprising:
a) detecting and / or quantifying the level of polypeptide in said biological sample of interest, wherein said polypeptide is i) an amino acid sequence selected from the group consisting of cardiovascular disease plasma polypeptide (CPP) 149-402 A polypeptide comprising:
ii) a variant having one or more amino acid substitutions, deletions or insertions relative to an amino acid sequence selected from the group consisting of CPP 149-402 and having at least 75% sequence identity; and iii) at least 10 amino acids long Selected from fragments of a polypeptide as defined in i) or ii) above;
b) A method comprising the step of comparing said level with the level of a control sample, wherein said increased level relative to the control level is indicative of cardiovascular disease. A method for predicting cardiovascular disease in a subject comprising:
a) detecting and / or quantifying the level of polypeptide in said biological sample of interest, wherein said polypeptide is i) an amino acid sequence selected from the group consisting of cardiovascular disease plasma polypeptide (CPP) 149-402 A polypeptide comprising:
ii) variants having at least 75% sequence identity with one or more amino acid substitutions, deletions or insertions relative to an amino acid sequence selected from the group consisting of CPP 149-402; and iii) at least 10 amino acids long Selected from fragments of a polypeptide as defined in i) or ii) above;
b) comparing the level to the level of a control sample, wherein the increase relative to the control level indicates a risk of developing cardiovascular disease.   The method according to claim 1 or 2, wherein the cardiovascular disease is coronary artery disease (CAD).   The method according to claim 1, wherein the biological sample is plasma.   The method according to claim 1, wherein the method is performed ex vivo.   6. The method according to any one of claims 1 to 5, wherein the polypeptide is detected and / or quantified by mass spectrometry.   6. The method according to any one of claims 1 to 5, wherein the polypeptide is detected and / or quantified by enzyme immunoassay.   An isolated polypeptide comprising an amino acid sequence selected from the group consisting of cardiovascular disease plasma polypeptide (CPP) 149-402 and fused to a heterologous polypeptide sequence.   An anti-cardiovascular disease plasma polypeptide (CPP) antibody that selectively binds to a polypeptide comprising an amino acid sequence selected from the group consisting of CPP149-402. A method of binding an antibody to a cardiovascular disease plasma polypeptide (CPP) comprising:
i) contacting the antibody of claim 9 with a biological sample under conditions that allow antibody binding;
ii) removing the contaminants.   The method of claim 10, wherein the antibody is conjugated to a labeling group.   The method of claim 10, wherein the biological sample is human plasma. A method of identifying a cardiovascular disease plasma polypeptide (CPP) modulator comprising:
i) contacting a test compound with a polypeptide selected from the group consisting of CPP 149-402 under conditions that permit at least one CPP biological activity;
ii) determining the level of said at least one CPP biological activity;
iii) comparing the level to the level of a control sample without the test compound; and iv) changing the level for further testing as a CPP modulator for the prevention and / or therapeutic treatment of cardiovascular disease. Selecting a test compound to be produced.   CPP154, 155, 181-192, 197, 198, 210, 214, 258-268, 273, 275-289, 292, 294-297, 302-402, a cardiovascular disease plasma polypeptide (CPP An isolated polypeptide having the amino acid sequence of   An isolated polynucleotide encoding the polypeptide of claim 14. A method of identifying a modulator of cardiovascular disease comprising:
(A) administering a candidate drug to a non-human test animal susceptible to or suffering from cardiovascular disease;
(B) administering the candidate agent of (a) to a corresponding control non-human animal that is unlikely to suffer from cardiovascular disease or not suffering from cardiovascular disease;
(C) in a biological sample obtained from the non-human test animal of step (a) and a biological sample obtained from the control animal of step (b),
i) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402);
ii) have at least 75% sequence identity with one or more amino acid substitutions, deletions or insertions relative to an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) And iii) detecting and / or quantifying the level of a polypeptide selected from fragments of the polypeptide as defined in i) or ii) above that are at least 10 amino acids in length; and (d) step Transferring the level of the polypeptide in a biological sample obtained from a non-human test animal to the level of the polypeptide in a biological sample obtained from a control animal, comprising comparing the level of the polypeptide of (c) Wherein the candidate agent is a modulator of cardiovascular disease. A non-human test animal susceptible to cardiovascular disease or suffering from cardiovascular disease i) a polymorph containing an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) peptide;
ii) have at least 75% sequence identity with one or more amino acid substitutions, deletions or insertions relative to an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) 17. A method according to claim 16, comprising: iii) an increase in plasma levels of a polypeptide selected from fragments of the polypeptide as defined in i) or ii) as defined in i) or ii) above, and iii) at least 10 amino acids in length. A method for monitoring the effectiveness of treatment with a drug in a subject having a cardiovascular disease or at risk of developing a cardiovascular disease comprising:
(A) obtaining a pre-dose biological sample from the subject prior to administration of the drug;
(B) in the subject biological sample,
i) a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402);
ii) have at least 75% sequence identity with one or more amino acid substitutions, deletions or insertions relative to an amino acid sequence selected from the group consisting of the amino acid sequences listed in Table 3 (CPP 149-402) And iii) detecting and / or quantifying the level of a polypeptide selected from a polypeptide as defined in i) or ii) above that is at least 10 amino acids in length;
(C) obtaining one or more post-administration biological samples from the subject;
(D) detecting the level of the polypeptide in the sample after the administration;
(E) comparing the level of the polypeptide in the pre-administration sample with the level of the polypeptide in the post-administration sample; and (f) adjusting the administration of the agent accordingly. .   A method for the treatment of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or bowel motility comprising administering to a mammal, including a human suffering from said disease, an effective amount of a CPP232 polypeptide. Method.   The intestinal motility disorder is a gastrointestinal motility disorder selected from the group consisting of irritable bowel syndrome, diabetes, gastric insufficiency, esophageal spasm, Hirschsprung's disease, chronic intestinal pseudo-obstruction, scleroderma and achalasia. The method described.   20. The method of claim 19, wherein the symptom associated with bowel movement disorder is a symptom selected from the group consisting of dysphagia, heartburn, gas, bloating, nausea, vomiting, constipation and diarrhea. A method for identifying modulators of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility disorders, comprising:
i) contacting a test compound with CPP232 under sample conditions that permit CPP232 biological activity;
ii) determining the level of said CPP232 biological activity;
iii) comparing the level to the level of a control sample not containing the test compound; and iv) prevention and / or cardiovascular disease and symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility disorders Selecting a test compound that alters said level for further testing as a CPP232 modulator for therapeutic treatment.   23. The method of claim 22, wherein the level of CPP232 biological activity is measured by determining the expression level of one or more genes shown in Table 4.   A method for the prognosis or diagnosis of gallstones or celiac disease, comprising detecting plasma levels of CPP232, wherein elevated levels of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility disorders An indicator, a method. A method for the prognosis or diagnosis of symptoms and symptoms associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility disorders, comprising:
i) in a sample of suitable tissue obtained from the subject, detecting the expression level of at least one gene shown in Table 4 to obtain a first value; and ii) obtaining the first value from a disease-free subject The level of expression in the subject sample when compared to a sample from a disease-free subject, wherein the patient is associated with biliary cirrhosis, gallstones, celiac disease or intestinal motility disorders A method of indicating that it is susceptible or afflicted with symptoms and symptoms.   26. The method of claim 25, wherein the expression level of at least 2, at least 3, at least 4, or at least 5 genes shown in Table 4 is detected. 27. The CPP232 is a variant having one or more amino acid substitutions, deletions or insertions relative to the amino acid sequence shown in SEQ ID NO: 706 and having at least 75% sequence identity. the method of.