JP2007502971A - Secreted polypeptide traits and uses thereof - Google Patents

Abstract

The present invention provides polypeptide traits secreted in human plasma, isolated polynucleotides encoding the polypeptides, polymorphic variants thereof, and nucleic acids and polypeptides or detection thereof for detection assays and disease diagnosis It relates to the use of the composition.

Description

Detailed Description of the Invention

(Field of Invention)
The present invention is directed to polypeptide species secreted in human plasma, isolated polynucleotides encoding such polypeptides, polymorphic variants thereof, and detection assays and disease diagnosis and medical treatments. It relates to the use of nucleic acids and polypeptides or compositions thereof.

(background)
Many disease symptoms are non-specific in nature, and it is often difficult for healthcare professionals to make a definitive diagnosis. Patient descriptions are subject to change and are often inaccurate. Even more quantitative diagnostic methods have the disadvantage of varying both between individuals and between interpretations in a single individual. That is, the diagnostic scale must be standardized and applied to individuals whose medical history is well documented and perfect. Even with these controls, the symptoms for completely different underlying conditions may appear the same. In addition, many serious conditions result in combinations of factors such as neuropsychiatric disorders (see, eg, schizophrenia and bipolar disease, Johnston-Wilson et al., Int J Neuropsychopharmacol, 2001, 4: 83-92) and It arises from metabolic disorders (eg, see diabetes, Gloyn and McCarthy, Best Pract Res Clin Endocrinol Metab, 2001, 15: 293-308). Current diagnostic methods often do not reveal the underlying incentive (s) for an observation or interpretation. Thus, treatment strategies based on specific positive results do not appear to address the causative problem, and in some cases may be harmful to the individual.

  Diagnostic methods that rely on nucleotide detection include genetic methods and expression profiling. For example, genes known to be involved in a particular disease can be screened for mutations using common genetic analysis techniques such as sequencing, hybridization based techniques or PCR. In another example, expression from known genes can be followed by standard techniques including RTPCR, various hybridization-based techniques, and sequencing. These strategies often do not allow health professionals to detect differences in mRNA processing and splicing, translation rate, mRNA stability, and post-translational modifications such as proteolytic processing, phosphorylation, glycosylation and amidation.

  In order to address current weaknesses in the diagnostic context of the industry, the present invention provides new information regarding the range of polypeptides found in human plasma. Human plasma is the most useful source of proteins related to both health and disease. Plasma not only contains active proteins and tell tale disease markers. It can be obtained in large quantities non-invasively from both patients and control subjects (as opposed to tissues, which are often difficult to obtain in large quantities from controls). Furthermore, blood, plasma, and serum are quite commonly used in existing clinical trials (eg, genomic methods). However, genomic methods are limited because many of the cells involved in plasma protein content are not found in blood. Finally, because plasma is a fluid, it is pooled (also in contrast to tissue), resulting in a large amount of typical samples for analysis.

  Plasma proteome analysis is an interesting method in terms of a constantly changing concentration range, which is known to have a large number of proteins that are expected to exist and span at least 11-12 orders of magnitude. In order to identify and characterize polypeptides present at very low concentrations, it is necessary to start with a large amount of sample to ensure a sufficient amount for detection by mass spectrometry. For example, if 100 fmol protein is required for effective separation, digestion and mass spectrometry identification, a protein concentration of at least 1 nM is required with a sample size of 100 μl. However, if a 1 liter sample is available, this required minimum concentration drops to 100 fM.

  The invention described in the specification derives from this logic. Industrial scale (2.5 L) methods involving sample pools are detailed for small protein (molecular weight less than about 40 kD, most often less than 20 kD) analysis. Thousands of peptides arising from the polypeptide have been identified from a single pool. As proof of concept for the method of the invention, low abundance proteins such as leptin and ghrelin and peptides such as bradykinin were clearly identified. This is the first time that a small protein in human plasma has been thoroughly analyzed. That is, the present invention discloses a protein that has never been found in plasma. Providing actual plasma polypeptide traits has revealed differences in mRNA processing and splicing, translation rate, mRNA stability, and post-translational modifications. Such post-translational modifications (eg, proteolytic processing, phosphorylation, glycosylation, and amidation) can, and often do, affect the function of a particular polypeptide. Furthermore, plasma localization refers to a new and previously unknown function for the polypeptides of the invention. These polypeptides are described as “human plasma polypeptides” or HPP. These polypeptide sequences are related to polypeptides having the accession numbers listed in Table 1 and include polypeptide traits that include one or more of the amino acid sequences listed in Table 3.

  The present invention discloses “human plasma polypeptides” (HPP), fragments of HPP present in human plasma, and post-translationally modified traits. The HPP of the present invention represents an important tool for diagnosis and drug development. HPP is a secreted factor and is easy to detect and target by itself, eg, a detectable molecule, protein chip or modulator.

(Summary of the Invention)
The present invention relates to compositions related to polypeptide traits secreted in human plasma. These polypeptide traits are referred to herein as “human plasma polypeptides” or HPP. The human plasma polypeptide comprises an amino acid sequence selected from the list in Table 3. The compositions include HPP precursors, antibodies specific for HPP, such as 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, a group of proteolytic precursors consisting of sequences from Table 3, and intermediates originating from another proteolytic site in the group consisting of sequences from Table 3. .

  Preferred embodiments of the invention include HPP with post-translational modifications such as phosphorylation, glycosylation, acetylation, amidation, or C-, N- or O-linked hydrocarbon groups. Further preferred are HPPs with disulfides and hydrogen bonds that result in intramolecular or intermolecular interactions such as highly ordered structures. Also preferred are HPPs resulting from differential mRNA processing or splicing. Preferably, HPP means a post-translationally modified trait, a structural variant, or a splice variant present in plasma.

  In another aspect, the invention includes an HPP comprising a sequence that is at least 95 percent identical to a sequence selected from the group of sequences listed in Table 3. Preferably, the present invention comprises a polypeptide having at least 97 percent identity with any one of the sequences selected from Table 3, more preferably at least 98 percent, and more preferably at least 99 percent. Most preferably, the invention comprises a polypeptide comprising a sequence that is at least 99 percent identical to a sequence selected from the group of sequences listed in Table 3.

  In another aspect, the present invention includes natural variants of HPP having a frequency of at least 2 percent in a selected population. More preferably, the natural variant has a frequency of at least 5 percent, more preferably at least 10 percent in the selected population. Most preferably, the natural variant has a frequency of at least 20 percent in the selected population. The selected population can be a test population recognized in the field of population genetics. Preferably, the selected population is white, black or Asian. More preferably, the selected group is French, German, English, 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 selected population is Icelandic, Saami (lap), Finnish, Caucasian French, Swiss, Chinese Singaporean, Korean, Japanese, Quebec, North American Pima Indian, Pennsylvania Amish and Amish Mennonite, Newfoundland or Polynesian.

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

  In additional aspects, the invention includes modified HPP. Such modifications include protecting / blocking groups, linkage to antibody molecules or other cellular ligands, and detectable labels, such as enzymes, fluorescence, isotopes or affinity labels that allow detection and isolation of proteins. Chemical modifications include but are not limited to cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, specific chemical cleavage by NaBH4, acetylation, formylation, oxidation, reduction or metabolism in the presence of tunicamycin It can be performed by known techniques including synthesis.

  The present invention also provides additional advantages such as chemically modified derivatives of the polypeptides of the invention (eg, water solubility) that may result in increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity. Polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol). HPP may be modified at random positions within the molecule, or at predetermined positions within the molecule, and may include 1, 2, 3 or more attached chemical moieties.

  In another aspect, the invention relates to polynucleotides encoding the HPPs of the invention, groups of sequences listed in Table 3, for diagnostic and analytical assays (eg, PCR, hybridization based techniques). A polynucleotide encoding a polypeptide having an amino acid sequence selected from: an antisense oligonucleotide complementary to the above sequence, and an oligonucleotide complementary to the HPP gene sequence.

  In another aspect, the present invention provides a vector comprising DNA encoding HPP. The present invention also includes host cells and transgenic non-human animals comprising the above vectors.

  Also provided are methods for producing HPP or HPP precursors. One preferred method is (a) providing a host cell comprising the expression vector disclosed above, (b) culturing the host cell under conditions in which the DNA segment is expressed, and (c) encoding with the DNA segment. Recovering the purified protein. Another preferred method includes the steps of (a) providing a host cell capable of expressing HPP, (b) culturing the host cell under conditions capable of expressing HPP, and (c) recovering HPP. Within one aspect, the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, the cell secretes the protein into the culture medium, and the protein is recovered from the medium. Particularly preferred methods for producing HPP include chemical synthesis using standard peptide synthesis techniques, as described in the section entitled “Chemical Production of HPP Compositions” and in Example 2.

  In another aspect, the invention includes an isolated antibody specific for any of the above polypeptides, peptide fragments, or peptides. Preferably, the antibody of the present invention is a monoclonal antibody. Further preferred are antibodies that specifically or exclusively bind to HPP, ie antibodies that do not recognize other polypeptides with high affinity. Anti-HPP antibodies have purification, detection, diagnostic and prognostic applicability. Preferred anti-HPP antibodies for purification and detection are conjugated to a labeling group. Detection methods include, but are not limited to, those using antibodies or antibody-derived compositions specific for HPP antigen. A preferred detection method is the enzyme linked immunosorbent assay (ELISA). Compositions comprising one or more of the above antibodies together with a pharmaceutically acceptable carrier are also included within the scope of the invention, for example for in vivo detection purposes.

  Detection methods for identifying HPP in specific tissue samples and biological fluids (preferably plasma) form part of the present invention. Detection methods for identifying HPP expression from cell-based samples are also encompassed.

  The present invention further provides a method comprising detecting the level of at least one HPP in a body fluid, preferably a plasma sample. Furthermore, a method of using an HPP composition comprising a primer complementary to an HPP gene and / or messenger RNA and an anti-HPP antibody for the purpose of detecting and measuring the amount of HPP in tissues and biological fluids, preferably plasma Is also included. In addition, the present invention includes detection methods including mass spectrometry, retentate chromatography (including protein arrays), and surface enhanced laser desorption / ionization (SELDI) techniques. These methods are also appropriate for clinical screening, diagnosis, monitoring of therapeutic results, and identification of patients most likely to respond to a specific therapeutic treatment.

  The present invention provides kits that can be used in the methods listed above and can include single or multiple preparations, adsorbents and substrate materials, antibodies, labeling groups, other reagents and instructions for use if necessary. . The kit can be used in an assay to identify a diagnostic or novel diagnostic agent.

  In a preferred embodiment, the detection of at least one increased plasma level of HPP of the present invention indicates an increased risk of HPP disease associated with increased HPP. Preferably, the detection indicates that the likelihood of developing HPP disease for the individual has increased at least 1.05 times, 1.1 times, 1.15 times and more preferably at least 1.2 times. Alternatively, the detection of at least one reduced plasma level of HPP of the present invention by specific HPP indicates an increased risk of HPP disease associated with increased HPP for an individual. The amount of increase or decrease in HPP observed in an individual when compared to a control sample correlates with certainty of prediction. Individual plasma HPP levels will vary depending on family history and other risk factors and are preferably investigated individually for each. In a preferred embodiment, HPP is detected in a human plasma sample by the method of the present invention. Particularly preferred techniques are mass spectrometry, retentate chromatography (including protein arrays), and immunodetection methods. Preferably, prognosis or diagnosis is based on at least 1.1, 1.15, 1.2, 1.25 and more preferably a 1.5-fold increase (or decrease) in experimental HPP levels compared to controls. ing.

  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, provides compositions, methods, and kits useful for screening and diagnosing human plasma, for identifying individuals most likely to respond to specific therapeutic treatments, and for monitoring therapeutic results. I will provide a. For purposes of clarity of disclosure, the present invention will be described with respect to the analysis of plasma samples, without being limited thereto. However, those skilled in the art will readily appreciate that the following assays and techniques can also be applied to other biological fluid samples (eg, cerebrospinal fluid, lymph fluid, bile, plasma, saliva or urine) or tissue samples. Should be able to understand. The methods and compositions of the present invention are useful for screening, diagnosis and prognosis of living individuals, but can also be used for autopsy in individuals.

(Definition)
As used herein, the terms “nucleic acid” and “nucleic acid molecule” refer to DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and DNA produced using nucleotide analogs or It is intended to include analogs of RNA. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. Throughout this specification, the expression “nucleotide sequence” may be used generally to describe a polynucleotide or nucleic acid. More precisely, because the expression “nucleotide sequence” encompasses the nucleic acid material itself, sequence information (ie, a sequence selected between four base characters) that biochemically characterizes a particular DNA or RNA molecule. The character is not limited to. Also, the terms “nucleic acid”, “oligonucleotide” and “polynucleotide” are used interchangeably herein.

  An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is a sequence that is naturally adjacent to both ends of the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (ie, sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). Not included. For example, in various embodiments, an isolated HPP nucleic acid molecule is about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb naturally adjacent to both ends of the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived. It may contain less than a nucleotide sequence. Furthermore, an “isolated” nucleic acid molecule, eg, a cDNA molecule, is substantially free of other culture media or culture media when produced by recombinant technology, or chemically when chemically synthesized. It may be substantially free of precursors or other chemicals. By using all or part of the nucleic acid as a hybridization probe, HPP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (eg, Sambrook, J., Frish, EF and Maniatis, T. Molecular Cloning). A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

  As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. 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, where additional DNA segments can be ligated to the viral genome. Certain vectors are capable of self-replication in the 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 replicated along with the host genome by integrating into the host cell genome upon introduction into the host cell. In addition, certain vectors may direct the expression of genes that are operably linked to them. This vector is referred to herein as an “expression vector”. In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of the above-described expression vectors that perform equivalent functions, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

As used herein, the term “hybridizes with” shall describe conditions for moderate or high stringency hybridization, preferably the hybridization and wash conditions are at least 60% of each other. Nucleotide sequences that are homologous can be allowed to remain hybridized to each other. Preferably, the conditions are typically at least about 70%, more preferably at least about 80%, more preferably at least about 85%, 90%, 95% or 98% homologous to each other It is to maintain the state that is. Stringent conditions are known to those of skill in the art and are described in Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989), 6.3.1-6.3.6. In a preferred, non-limiting example, stringent hybridization conditions for nucleic acid interaction are as follows: hybridization step consists of 6 × SSC buffer, 5 × Denhart solution, 0.5% SDS and 100 μg / Performed at 65 ° C. in the presence of ml salmon semen DNA. Following the hybridization step, the following four washing steps are performed:
-Preferably 2 washes for 5 minutes at 65 ° C in 2x SSC and 0.1% SDS buffer;
-1 wash for 30 minutes, preferably at 65 ° C in 2x SSC and 0.1% SDS buffer;
-Preferably 1 wash for 10 minutes at 65 ° C in 0.1x SSC and 0.1% SDS buffer;
These hybridization conditions are appropriate for nucleic acid molecules about 20 nucleotides in length. The hybridization conditions are adapted according to the desired nucleic acid length according to techniques well known to those skilled in the art, for example Hames BD and Higgins SJ (1985) Nucleic Acid Hybridization: A Practical Approach. Edited by Hames and Higgins, IRL. It shall be adapted according to the content disclosed in Press, Oxford, and Current Protocols in Molecular Biology.

  As used herein, “percent homology” refers to both nucleic acid and amino acid sequences. Amino acid or nucleic acid “identity” is equivalent to an amino acid or nucleic acid “homology”. To measure the percent homology of two amino acid sequences or two nucleic acids, align the sequences in parallel for optimal comparison purposes (eg, gaps of the first amino acid to optimally align with the second amino acid or nucleic acid sequence). Sequence or nucleic acid sequence, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned in parallel for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, and more preferably the length of the reference sequence. At least 70%, 80%, 90% or 95%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of identical positions shared by the sequences (ie,% homology = number of identical positions / total number of positions · 100).

  Sequence comparison between two sequences and determination of percent homology can be performed using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used for sequence comparison is Karlin and Altschul (1990), modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77. Proc. Natl. Acad. Sci. USA 87: 2264-68. 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. By conducting a BLAST nucleotide search with the NBLAST program, score = 100, word length = 12, a nucleotide sequence homologous to the sequence of the present invention is obtained. By performing a BLAST protein search with the XBLAST program, score = 50, word length = 3, an amino acid sequence homologous to the polypeptide sequence of the present invention is obtained. To obtain a gap alignment for comparative purposes, gap BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Research 25 (17): 3389-3402. When using BLAST and Gap BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller, CABIOS (1989). 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 amino acid sequence comparison, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

  The term “polypeptide” encompasses amino acid polymers, regardless of the length of the polymer. That is, peptides, oligopeptides and proteins are included within the definition of polypeptide. The term is also not intended to identify or exclude post-translational modifications of the polypeptide, for example, a polypeptide containing a covalent linkage of a glycosyl, acetyl, phosphate, amide, lipid, carboxyl, acyl, or hydrocarbon group Which is expressly included in the term polypeptide. Also, polypeptides, including substitution chains, including one or more analogs of amino acids (eg, including non-natural amino acids, amino acids that are naturally occurring only in unrelated biological systems, modified amino acids from mammalian systems, etc.) Also included within the definition are other modifications known in the art, including both natural and non-naturally occurring polypeptides.

  As used herein, the term “protein” can be used synonymously with the term “polypeptide” or, in addition, two or more polypeptides that can be joined by bonds other than peptide bonds. For example, the polypeptide constituting the protein can be bound by a disulfide bond. The term “protein” can also encompass a group of polypeptides that have the same amino acid sequence but different post-translational modifications that can be added, particularly when the protein is expressed in a eukaryotic host.

  An “isolated” or “purified” protein or biologically active portion thereof contains cellular material or other contaminating protein from a cell or tissue source from which the protein of the invention (ie, HPP or biologically active fragment thereof) is derived. It shall be substantially free of chemical precursors or other chemical substances during chemical synthesis. The term “substantially free of cellular material” includes protein preparations in which the protein is separated from cellular components of cells from which the protein of the invention is isolated or genetically modified. In one embodiment, the term “substantially free of cellular material” means that the percentage of proteins other than the protein of the present invention (also referred to herein as “contaminating proteins”) is less than about 30% (in dry weight), More preferably, the proportion of proteins other than the protein of the present invention is less than about 20%, more preferably the proportion of proteins other than the protein of the present invention is less than about 10%, and most preferably the proportion of proteins other than the protein of the present invention is less than 5%. A protein preparation of the present invention. When the protein according to the invention or the biologically active part thereof is produced by genetic recombination, it is also preferably free of culture medium, ie the culture medium is less than about 20% of the volume of the protein preparation, more preferably Should be less than about 10%, and most preferably less than about 5%.

  The term “substantially free of chemical precursors or other chemicals” includes protein preparations in which the protein of the invention is separated from chemical precursors or other chemicals involved in protein synthesis. In one aspect, the term “substantially free of chemical precursors or other chemicals” is more preferably less than about 30% (in dry weight) of chemical precursors or non-protein chemicals Has a chemical precursor or non-protein chemical ratio of less than about 20%, more preferably a chemical precursor or non-protein chemical ratio of less than about 10%, and most preferably a chemical precursor or non-protein chemical. Including preparations of the protein of the present invention wherein the percentage of the substance is less than about 5%.

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

  “Cola” refers to the liquid portion of blood and lymph and constitutes about half of the volume. Serum is cell free and, unlike serum, does not clot. Plasma contains antibodies and other proteins. In many cases, cell components are removed from blood collected from an individual by centrifugation. Another plasma separation method includes sterile filtration as described in US Pat. No. 6,241,886.

  The term “human plasma polypeptide” or “HPP” is an amino acid sequence selected from the group consisting of a polypeptide comprising the sequence described by any one of the accession numbers listed in Table 1 or the sequence of Table 3 Say. The polypeptide can be post-translationally modified as described herein. HPP may also include 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 or tertiary structures. HPP is also a mutant polypeptide, such as a deletion, addition, substitution (swap) or truncation mutant, a fusion polypeptide comprising the above polypeptide, and at least three of the sequences in Table 3, preferably 8, 10 , 12, 15 or 21 contiguous amino acid polypeptide fragments. Further included are HPP proteolytic precursors and intermediates of a sequence selected from the group consisting of the sequences from Table 3. The present invention relates to an HPP gene or HPP mRNA trait, preferably a human HPP gene and mRNA trait, comprising an isolated HPP composed by, consisting essentially of, or comprising them from Table 3. Includes polypeptides encoded by nucleic acid sequences. Preferred HPPs retain at least one biological activity of HPP from Table 3.

  As used herein, the term “biological activity” refers to a function performed by HPP. This includes (1) circulation throughout the bloodstream of a human individual, (2) the ability to bind antigenic or anti-HPP specific antibodies, (3) the ability to produce immunogenicity or anti-HPP specific antibodies, and (4) There is an interaction with the HPP target molecule or adsorbent, but it is not limited.

  “HPP-related disorder” or “HPP-related disease” refers to a medical condition known to be related to the HPP of the present invention. HPP-related disorders include conditions in which the presence of an abnormal level of HPP or HPP polynucleotide indicates that the individual is afflicted with or at risk for developing the condition. An HPP-related disorder is also caused by the presence of an abnormal form of HPP or HPP polynucleotide (eg, due to mutation, truncation, increased or decreased biological activity, abnormal post-translational modification or processing) Or a condition that indicates that there is a risk of developing or developing it.

Another aspect of the invention relates to anti-HPP antibodies. As used herein, the term “antibody” specifically binds to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie antigens such as HPP, or biologically active fragments or homologues thereof (immune reactions). Refers to a molecule containing an antigen binding site. Preferred antibodies bind exclusively to HPP 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 HPP or a biologically active fragment or homologue thereof. As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to a population of antibody molecules that contain a unique trait of an antigen binding site that can immunoreact with a particular epitope of HPP. That is, a monoclonal antibody composition typically displays a single binding affinity for a particular HPP with which it immunoreacts. Preferred HPP antibodies are conjugated to a labeling group.

As used herein, a “labeling group” is a compound that, when attached to a polynucleotide or polypeptide (including antibodies), allows for detection or purification of the polynucleotide or polypeptide. The labeling group can be detected or purified directly or 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-bromodesoxyuridine, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, Dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin acetylaminofluorene, digoxigenin), luminescent compounds (eg, luminol, GFP, luciferin, aequorin), enzymes or enzyme cofactor detectable labels (eg, peroxidase, luciferase, alkaline) Phosphatase, galactosidase, or acetylcholinesterase), or compounds recognized by secondary factors such as streptavidin, GST, or biotin. Preferably, the labeling group is attached to the polynucleotide or polypeptide in a manner that does not interfere with the biological activity of the polynucleotide or polypeptide.

  The radioisotope can be detected by direct counting of radiation, film exposure, or, for example, by a scintillation counter. Enzymatic labels can be detected by measuring the conversion of an appropriate substrate into a product that normally induces a fluorescent reaction. Fluorescent and luminescent compounds and reactions can be detected by, for example, radiation, fluorescence microscopy, fluorescence activated cell sorting, or luminometer.

  “Adsorbent” refers to a substance capable of adsorbing a polypeptide (ie, HPP). As used herein, the term “adsorbent” refers to a single substance to which a polypeptide is exposed (“monoplex adsorbent”) (eg, a compound or functional group), and a plurality of samples to which a sample is exposed. Includes both different materials ("multiplex adsorbents"). The adsorbed material in the multiplex adsorbent is referred to as “adsorption trait”. For example, an accessible position in the substrate may be characterized by many different adsorption traits (eg, anion exchange materials, metal chelators or antibodies) and include multiplex adsorbents with different binding properties. The basis of attraction is generally the function of chemical or biological molecular recognition. The basis for the attraction between the adsorbent and the polypeptide includes, for example, (1) salt-promoted interactions, such as hydrophobic interactions, thiophilic interactions, and immobilized dye interactions, (2) hydrogen bonds and / or van der In the case of Waals force interactions and charge transfer interactions, eg hydrophilic interactions, (3) electrostatic interactions, eg ionic charge interactions, in particular positive or negative ionic charge interactions, (4) in polypeptide adsorbents Coordination covalent bond formation (ie, coordination complex formation) ability with metal ions, (5) Enzyme active site binding, (6) Reversible covalent interaction, eg, disulfide exchange interaction, (7) Glycoprotein interaction ( There are 8) biospecific interactions, or (9) a combination of two or more of the above interaction types. That is, the adsorbent can be known as a “mixed-functional” adsorbent because it can exhibit two or more attractive bases.

HPP of the present invention
The human plasma polypeptides (HPP) of the present invention are listed in Tables 1 and 3. HPP and fragments thereof comprising an amino acid sequence selected from the group consisting of the sequences from Table 3 are secreted and circulated in plasma.

  The HPP of the present invention is a known polypeptide that has never been found in human plasma. That is, the present invention introduces a new role or function for these polypeptides. The HPPs listed in Table 1 include polypeptide traits that are not expected to be found in plasma, as described in Table 1.

  As used herein, the terms “human plasma polypeptide” and “HPP” encompass all peptides, polypeptides and proteins of the invention. Polypeptides encoded by the polynucleotides of the present invention and fusion polypeptides comprising the above polypeptides also form part of the present invention. The present invention relates to a human-derived HPP comprising an isolated or purified HPP composed of, consisting essentially of, or comprising an amino acid sequence selected from the group consisting of the sequences of Table 3. Include. Further included are unmodified precursors, proteolytic hydrolysis precursors and intermediates of a sequence selected from the group consisting of the sequences in Table 3.

  The present invention provides an isolated, purified, and recombinant polypeptide fragment comprising a contiguous range of at least 3 amino acids, preferably at least 8-10 amino acids of an amino acid sequence selected from the group consisting of the sequences of Table 3. And the fragment should have HPP biological activity. In an encompassed embodiment, the continuous stretch of amino acids comprises a site of single or functional mutation including amino acid deletion, addition, substitution (swap) or truncation in the HPP sequence. The present invention also relates to a polypeptide encoded by the HPP nucleotide sequence of the present invention, or a complementary sequence thereof, or a fragment thereof. The polypeptide fragment may represent the actual peptide trait present in human plasma. The polypeptide fragments can be used, for example, for the production of HPP specific antibodies or the design of another type of HPP specific adsorbent.

  One aspect of the invention relates to isolated HPP, and biologically active portions thereof, as well as polypeptide fragments suitable for use as an immunogen to produce anti-HPP antibodies. In one aspect, native HPP peptides can be isolated from plasma, cell or tissue sources by a suitable purification scheme using standard protein purification techniques. In another embodiment, HPP is produced by recombinant DNA technology. As an alternative to recombinant expression, HPP can be chemically synthesized using peptide synthesis techniques as described in the section entitled “Chemical Production of HPP Compositions” and in Example 2.

  Typically, the biologically active portion includes a domain or motif having at least one activity of HPP. The biologically active HPP comprises, for example, at least 1, 2, 3, or 5 amino acid changes from a sequence selected from the group consisting of the sequences from Table 3, or a sequence selected from the group consisting of the sequences from Table 3 From at least 1%, 2%, 3%, 5%, 8%, 10% or 15% of amino acids from

Characterization of HPP The polypeptide of the present invention, HPP, is identified by the polypeptides in Table 3 and the accession numbers listed in Table 1. These peptides were isolated from human plasma and characterized according to the MicroProt® method as described in Example 1. For each HPP, Table 1 provides the following:
The accession number in the public database corresponding to the relevant polypeptide sequence;
An amino acid position that identifies the observed polypeptide for the polypeptide in a public database;
・ Classification into 3 categories according to the following keys ・ a: Protein that was not previously known to be plasma, seems to be secreted.
B: Protein that was previously unknown to be plasma, probably due to cell leakage.
C: unclassified proteins-most of them are novel proteins, eg predicted from DNA.

  Most of the accession numbers listed in Table 1 are references to the SwissPROT / TrEMBL database, both of which are publicly available, eg http://www.expasy.ch. However, HPP 40-57 are identified as sequences appearing in published patent applications, as detailed in Table 1. Furthermore, the accession numbers for HPP accession numbers 58-61 correspond to the predicted protein sequences obtained by using some software at the GenBank entry, as detailed in Table 1. The accession numbers for HPP 1-4 indicate protein entries available from NCBI at http://www.ncbi.nlm.nih.gov/. Finally, the accession number for HPP indicates EST and its sequence is available from the dbEST database, eg NCBI, at http://www.ncbi.nlm.nih.gov/.

  All HPPs of the present invention have a molecular weight of less than or around 20 kD. The unambiguous identification of low abundance proteins (leptin and ghrelin) and peptides (bradykinin) confirms the sensitivity of the detection method of the present invention.

  The initial separation is on a cation exchange chromatography column and is eluted with increasing salt concentration. Collect 18 fractions. The CEX column in Table 3 lists which fractions contained trypsin peptides for each HPP. Table 2 provides the NaCl concentration at which each fraction was eluted according to the protocol described in Step 3 of Example 1 herein. Separation by cation exchange gives an indication of the overall positive charge of the polypeptide trait. Subsequent to cation exchange, reverse phase HPLC separation is performed. The RP1 column in Table 3 lists which tryptic peptides out of 30 fractions were eluted for each HPP. Table 2 provides elution conditions (% B) according to the protocol described in Example 1, Step 4. Separation by reverse phase provides an indication of the overall hydrophobicity of the polypeptide trait. Finally, for each HPP, Table 3 shows, for each CEX fraction and each RP1 fraction, the trypsin detected from these fractions along with the RP2 fraction in which these trypsins were detected (in parentheses after each trypsin sequence). Provide a list of

Table 3

HPP Nucleic Acid One aspect of the invention relates to purified or isolated nucleic acid molecules that encode HPP or biologically active portions thereof, as further detailed herein, as well as nucleic acid fragments thereof. The nucleic acid can be used, for example, in the detection methods described in further detail herein.

  One object of the present invention is a purified, isolated or recombinant nucleic acid encoding HPP, its complementary sequence, and fragments thereof. The present invention also provides a polynucleotide encoding at least 95% nucleotide identity, advantageously 99% nucleotide identity, preferably 99.5% nucleotide identity and most preferably HPP encoding with a polynucleotide encoding HPP, or It relates to a purified or isolated nucleic acid comprising a polynucleotide having 99.8% nucleotide identity with its complementary sequence or biologically active fragment thereof. Another object of the present invention is to provide a polynucleotide that hybridizes under stringent hybridization conditions described herein with a polynucleotide encoding HPP, or a sequence complementary thereto, or a variant thereof or a biologically active fragment thereof. It relates to purified, isolated or recombinant nucleic acids containing nucleotides.

  In another preferred aspect, the invention relates to a purified or isolated nucleic acid molecule that encodes a portion or variant of HPP, wherein the portion or variant exhibits HPP biological activity. Preferably, the portion or variant is a portion or variant of natural HPP or a precursor thereof.

  Another object of the present invention is a purification, simple substance encoding an HPP comprising, consisting essentially of, or constituted by an amino acid sequence selected from the group consisting of the sequences from Table 3. Isolated or recombinant nucleic acid, an isolated nucleic acid molecule shall encode one or more motifs, eg, target binding sites.

  A nucleic acid fragment that encodes a “biologically active portion of HPP” is a portion of a nucleotide sequence that encodes HPP, wherein the portion encoding a polypeptide having HPP biological activity is isolated and encoded by HPP. Can be produced by expressing (eg, by in vitro or in vivo recombinant expression) and assessing the activity of the coding portion of HPP.

  The invention further encompasses nucleic acid molecules that encode the same HPP of the invention, although differing from the HPP nucleotide sequences of the invention due to the degeneracy of the genetic code.

  In addition to the above HPP nucleotide sequences, one of ordinary skill in the art should readily understand that DNA sequence polymorphisms that cause changes in the amino acid sequence of HPP can exist within a population (eg, the human population). The genetic polymorphism may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1-5% variation in the nucleotide or nucleic acid sequence of the HPP-encoded gene.

  Nucleic acid molecules corresponding to naturally occurring allelic variants and homologues of the HPP nucleic acids of the invention are cDNAs or portions thereof disclosed herein as hybridization probes by standard hybridization techniques under stringent hybridization conditions. Can be isolated based on their homology with the HPP nucleic acids disclosed herein.

  The present invention is intended to encompass polypeptides having an amino acid sequence encoded by any of the polynucleotides of the present invention.

Use of HPP Nucleic Acids Polynucleotide sequences encoding HPP (or their complements) have a variety of applications, including use as hybridization probes. Furthermore, HPP-encoded nucleic acids are useful for the production of HPP by genetic recombination techniques as described herein. The polynucleotides described herein, including their sequence variants, can be used in detection assays. Accordingly, detection of the presence of the polynucleotide in a body fluid or tissue sample is a feature of the invention. Examples of nucleic acid based detection assays according to the present invention include, but are not limited to, hybridization assays (eg, in situ hybridization or nucleotide arrays) and PCR based assays. The polynucleotides, including extended polynucleotides, sequence variants and fragments thereof described herein can be used to generate hybridization probes or PCR primers used in the above assays. The probes and primers can detect polynucleotide sequences, including genomic sequences that are similar or complementary to the HPP polynucleotides described herein.

  The present invention includes primer pairs that amplify a segment of the polynucleotide of the present invention by performing PCR. Each pair of primers is an oligonucleotide having a length of 15 to 30 nucleotides, i) one primer of the pair forms a double helix that perfectly matches one strand of the polynucleotide of the present invention, and the other of the pair Ii) a pair of primers that form a perfect match duplex with a complementary strand of the same polynucleotide, and ii) a pair of primers that are separated by a distance between 10 and 2500 nucleotides at the site in the polynucleotide Take the form to form. Preferably, the annealing temperature of each primer in the pair to its respective complementary sequence is substantially the same.

  Hybridization probes derived from the polynucleotides of the invention can be used, for example, when performing in situ hybridization on fixed or frozen tissue pieces or suspension cells prepared on a tissue sample, such as a microscope slide. Briefly, a labeled DNA or RNA probe can bind its DNA or RNA target sample in a piece of tissue on a microscope slide under controlled conditions. Generally, dsDNA probes consisting of the DNA of interest cloned into a plasmid or bacteriophage DNA vector are used for this purpose, but ssDNA or ssDNA probes can also be used. Probes are oligonucleotides generally between about 15 and 40 nucleotides in length. Alternatively, the probe can be a polynucleotide probe (riboprobe) generated by PCR random priming primer extension or in vitro transcription of RNA from a plasmid. These latter probes are typically several hundred base pairs in length. The probe can be labeled with any of a number of labeling groups, and the particular detection method corresponds to the type of label used in the probe (eg, autoradiography, X-ray detection, fluorescence or visual as appropriate). Microscopic analysis). The reactants can be further amplified in situ by using immunocytochemical techniques directed against the label of the detection molecule used, eg, antibodies directed against the fluorescein moiety present in the fluorescently labeled probe. Specific labeling and in situ detection methods are described, for example, in Howard, GC, Ed., Methods in Nonradioactive Detection, Appleton & Lange, Norwalk, Connecticut (1993) (incorporated 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. The nucleotide sequence encoding an HPP can also be used to map a gene encoding that HPP and to construct a hybridization probe used for genetic analysis of the individual. Individuals with a variant or mutation of the gene encoding the HPP of the invention can be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis are obtained from patient cells, including, for example, tissue biopsy and autopsy material. Genomic DNA can be used directly for detection or can be amplified enzymatically by 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 acid of the invention can be used for the identification and analysis of mutations in the gene of the invention. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to a radiolabeled RNA of the present invention or alternatively to a radiolabeled antisense DNA sequence of the present invention. Sequence changes at specific positions can also be performed using nuclease protection assays, such as ribonuclease and S1 protection or chemical cleavage methods (eg, Cotton, et al., Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985). ) Or a difference in melting temperature. “Molecular beacons” (Kostrikis LG et al., Science 279: 1228-1229 (1998)), hairpin-shaped, single-stranded synthetic oligonucleotides comprising probe sequences that are complementary to the nucleic acids of the invention are also point mutations or It can be used to detect other sequence changes as well as monitor HPP expression levels.

  The nucleotides of the present invention, including PCR primers and probes, are synthesized by conventional means in commercially available automated DNA synthesizers, such as the Applied Biosystems (Foster City, Calif.) Model 380B, 392 or 394 DNA / RNA synthesizer. Can be done. Preferably, phosphoramidite chemistry is used and is disclosed, for example, in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al., US Pat. No. 4,980,460. Koster et al., U.S. Pat. No. 4,725,677; Caruthers et al., U.S. Pat. Nos. 4,415,732, 4458066, and 4,973,679.

  The primers and probes of the present invention can also be used, for example, for cloning and restriction of appropriate sequences and for example the phosphodiester method of Narang SA et al (Methods Enzymol 1979; 68: 90-98), Brown EL et al (Methods Enzymol 1979; 68 : 109-151), the diethylphosphoramidite method of Beaucage et al (Tetrahedron Lett 1981, 22: 1859-1862) and the solid support method described in European Patent No. 0707592 by direct chemical synthesis. Can be manufactured.

  Detection probes are generally described in nucleic acid sequences or uncharged nucleic acid analogs, eg, peptide nucleic acids disclosed in WO 92/20702, US Pat. Nos. 5,185,444, 5,034,506, and 5,142,047. Is a morpholino analogue. If desired, the probe can be made “non-extensible” in that no additional dNTPs can be added to the probe. In essence and as such, analogs are usually non-extensible, and nucleic acid probes are rendered non-extendable by modifying the 3 ′ end of the probe so that the hydroxyl group cannot participate in further extension. obtain. For example, hydroxyl groups can be consumed or otherwise blocked by functionalizing the 3 ′ end of the probe with a capture or detection label.

  The polynucleotides of the invention can be labeled by incorporating a labeling group known in the art to be detectable by microscopic, photochemical, biochemical, immunochemical or chemical means, if desired. Additional examples include in Urdea et al. (Nucleic Acids Research, 11: 4937-4957, 1988) or Sanchez-Pescador et al. (J. Clin. Microbiol. 26 (10): 1934-1938, 1988). There are non-radioactive labels of the nucleic acid fragments described. In addition, the probes according to the invention may have structural features such that they allow signal amplification, such as the Urdea et al (Nucleic Acids Symp. Ser, 24: 197-200, 1991) or European Patent No. EP 0225807 (Chiron).

  Also, the use of a label for primer capture can facilitate the immobilization of a primer or primer extension product, such as amplified DNA, on a solid support. The capture label can be a specific binding member that is bound to a primer or probe and forms a binding pair with a solid phase reagent specific binding member (eg, biotin and streptavidin). Thus, depending on the type of label carried by the polynucleotide or probe, it can be used to capture or detect target DNA. Furthermore, it can be seen that the polynucleotides, primers or probes provided in the present invention can themselves serve as capture labels. For example, if the solid phase reagent binding member is a nucleic acid sequence, it can be selected to immobilize the primer or probe to the solid phase by binding to the complementary portion of the primer or probe. If the polynucleotide probe itself serves as a binding member, one skilled in the art will readily recognize that the probe includes a sequence or “tail” that is not complementary to the target. If the polynucleotide primer itself serves as a capture label, it will hybridize to the nucleic acid on the solid phase due to the release of at least a portion of the primer. DNA labeling techniques are well known to those skilled in the art.

  The probe of the present invention is useful for some purposes. They can be used in particular in Southern hybridization with genomic DNA. The probe can also be used to detect PCR amplification products. They can also be used to detect mismatches in HPP-encoding genes or mRNA 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 supports are known to those skilled in the art and include wells of reaction trays, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, dura Site (duracyte). The solid support is not critical and can be selected by one skilled in the art. That is, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, microtiter well walls, glass or microchips, sheep (or other suitable animal) erythrocytes and durasite are all suitable examples. Suitable methods for immobilizing nucleic acids on the solid phase include ionic, hydrophobic, and covalent interactions. The solid support used in the present invention can be any material that is insoluble or can be rendered insoluble by subsequent reactions. The solid support can be selected for its inherent ability to attract and immobilize the capture reagent. Alternatively, the solid phase may hold additional receptors that have the ability to attract and immobilize capture reagents. The additional receptor may comprise a charged substance that is oppositely charged with respect to the capture reagent itself or a charged substance conjugated to the capture reagent. As yet another method, the receptor molecule can be a specific binding member that is bound to a solid support and has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule can indirectly bind the capture reagent to the solid support material prior to or during the performance of the assay. That is, 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, dura Other configurations known to the site and those skilled in the art can be used. The nucleic acids, polynucleotides, primers and probes of the present invention can be present on at least 2, 5, 8, 10, 12, 15, 20 or 25 different polys of the present invention individually on a solid support or to a single solid support. It can be bound or immobilized in a group consisting of nucleotides. In addition, polynucleotides other than those of the present invention can be bound to the same solid support as one or more polynucleotides of the present invention.

  Any of the polynucleotides provided in the present invention can be bound to overlapping regions or random locations on a solid support. Alternatively, the polynucleotides of the invention can be bound to an ordered array, with each polynucleotide bound to a different region of the solid support that does not overlap with the binding site of the other polynucleotide. Preferably, the regularly arranged array of polynucleotides is designed to be “addressable” so that well-defined locations can be recorded and evaluated as part of the assay procedure. Addressable polynucleotide arrays typically include a plurality of different oligonucleotide probes coupled to the surface of a substrate at different known locations. The precise location information for each polynucleotide position makes these “addressable” arrays particularly useful in hybridization assays. Addressable array technology known in the art can be used with the polynucleotides of the invention. One embodiment of these polynucleotide arrays, known as gene chips, is generally described in US Pat. No. 5,143,854, PCT Publications WO 90/15070 and 92/10092. Preferably, a plurality of HPP polynucleotide probes are included in the array.

Methods for Obtaining Variant Nucleic Acids and Polypeptides In addition to naturally occurring allelic variants of HPP sequences that may be present in a population, those skilled in the art introduce changes by mutations to nucleotide sequences encoding HPP. It should be readily understood that this leads to changes in the amino acid sequence of the encoded HPP with or without modification of the functional capabilities of HPP.

  Some types of variants are: 1) one or more of the amino acid residues are replaced by conserved or non-conserved amino acid residues, and the substituted amino acid residues are encoded by the genetic code May or may not be, or 2) one or more of the amino acid residues contain a substituent, or 3) the mutant HPP increases the half-life of another compound, eg, a polypeptide Fused to a compound (eg, polyethylene glycol), or 4) additional amino acids such as leader, signal or anchor sequences, sequences used to purify HPP, or sequences from precursor proteins fused to HPP It is thought to include what is. The above variants are also considered to be within the research area of those skilled in the art.

  The present invention provides an HPP chimera or fusion protein. As used herein, an HPP “chimeric protein” or “fusion protein” encompasses an HPP of the invention, or fragment thereof, operably linked or fused to a non-HPP polypeptide sequence. . In preferred embodiments, the HPP fusion protein comprises at least one biologically active portion of HPP. In another preferred embodiment, the HPP fusion protein comprises at least two biologically active portions of HPP. For example, in one embodiment, the fusion protein is a GST-HPP fusion protein in which the HPP domain sequence is fused to the C-terminus of the GST sequence. The fusion protein can facilitate the purification of recombinant HPP. In another embodiment, the fusion protein is an HPP that includes a heterologous signal sequence at its N-terminus, which can provide the desired cellular localization in certain host cells, for example. In yet another aspect, the fusion is an HPP biologically active fragment and an immunoglobulin molecule. The fusion protein is useful, for example, to increase the valency of the HPP binding site. For example, a bivalent HPP binding site can be formed by fusing a biologically active HPP fragment to an IgG Fc protein.

  In a preferred embodiment, the HPP fusion proteins of the invention are used as an immunogen to produce anti-HPP antibodies in a subject, to purify HPP, or as an HPP ligand and adsorbent.

  In addition, isolated fragments of HPP can also be obtained by screening peptides produced by genetic recombination from corresponding fragments of nucleic acids encoding the 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 HPPs of the present invention can be arbitrarily divided into fragments of the desired length without overlapping fragments, or preferably can be divided into overlapping fragments of the desired length. By producing and testing the fragments (by recombinant or chemical synthesis), peptidyl fragments with HPP biological activity can be identified, for example, by microinjection assays or in vitro protein binding assays. In an illustrative embodiment, the peptidyl portion of HPP, eg, the HPP target binding region, can be tested for HPP activity (eg, immunogenicity) by expression as a thioredoxin fusion protein, each containing a separate fragment of HPP (eg, the US Patents 5270181 and 5292646 and WO94 / 02502).

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

  Preferably, the chemical synthesis of the polypeptides of the present invention is by natural chemical ligation as described by Dawson et al., Science, 266: 776-779 (1994) and Kent et al., US Pat. No. 6,184,344. Performed by construction of peptide fragments. Briefly, this method adds an N-terminal cysteine with a non-oxidized sulfhydryl side chain to the first peptide fragment and a C-terminal thioester to the second peptide fragment. The non-oxidized sulfhydryl side chain of the N-terminal cysteine is then condensed with a C-terminal thioester to produce an intermediate peptide fragment that joins the first and second peptide fragments with a β-aminothioester bond. A peptide fragment product is then produced that joins the first and second peptide fragments with an amide bond by intramolecular rearrangement to the β-aminothioester bond of the intermediate peptide fragment. Preferably, the N-terminal cysteine of the internal fragment is protected from unwanted ring closure and / or chain formation reactions by a cyclic thiazolidine protecting group as described below. Preferably, the cyclic thiazolidine protecting group is a thioprolinyl group.

Peptide fragments with C-terminal thioesters can be prepared 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: 12485-12589 (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. Briefly, in situ neutralization / HBTU activation for the Boc chemistry disclosed by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992), incorporated by reference. Peptide fragments are typically synthesized on a solid support (below) on a 0.25 mmol scale by using the procedure. (HBTU is 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate and Boc is tert-butoxycarbonyl). Each synthesis cycle consists of 1-2 min treatment with pure TFA, 1 min DMF flow wash, 10-20 min coupling time with 1.0 mmol pre-activated Boc-amino acid in the presence of DIEA, and second DMF flow. It is comprised by N ( ^alpha) -Boc removal by washing | cleaning. (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 with 1.0 mmol HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol) for 3 min. After each coupling step, the yield is measured by measuring the residual free amine with a conventional quantitative ninhydrin assay, for example as disclosed in Sarin et al, Anal. Biochem., 117: 147-157 (1981). After coupling of the Gln residue, DCM flow washing is used before and after deprotection by using TFA to prevent potential high temperature (TFA / DMF) -catalyzed pyrrolidone formation. After chain assembly 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 incompatible with the C-terminal thioester group, the imidazole side chain 2,4-dinitrophenyl (dnp) protecting group remains in the His residue. However, dnp is gradually removed by thiols during the ligation reaction. After cleavage, the peptide fragment is precipitated with ice-cold diethyl ether, dissolved in aqueous acetonitrile and lyophilized.

Preferably, the thioester peptide fragment is synthesized in a trityl-associated mercaptopropionate leucine (TAMPAL) resin made according to the protocol disclosed by Hackeng et al (1999) or an equivalent protocol. Briefly, the ^{N α -Boc-Leu (4mmol)} , were activated by HBTU in 3.6mmol the presence of DIEA in 6 mmol, in 16 minutes 2 mmol p-methylbenzhydrylamine (MBHA) resin or equivalent content, Coupling with things. Next, 3 mmol S-trityl mercaptopropionic acid is activated with 2.7 mmol HBTU in the presence of 6 mmol DIEA and coupled with Leu-MBHA resin for 16 minutes. The resulting TAMPAL resin is used as a starting resin for polypeptide-chain construction after removal of the trityl protecting group by two 1 minute treatments with 3.5% triisopropylsilane and 2.5% H ₂ O in TFA. Can be done. A thioester bond can be formed with the desired amino acid by using a 1 hour standard in situ neutralizing peptide coupling protocol as disclosed by Schnolzer et al (supra). Treatment of the final peptide fragment with anhydrous HF yields a C-terminal activated mercaptopropionic acid-leucine (MPAL) thioester peptide fragment.

  Preferably, the thiazolidine protected thioester peptide fragment intermediate is used in a natural chemical ligation reaction under conditions described by Hackeng et al (1999) or equivalent conditions. Briefly, 0.1M phosphate buffer (pH 8.5) containing 6M guanidine, 4% (v / v) benzyl mercaptan, and 4% (v / v) thiophenol was added to the dried peptide and ligated. To obtain a final peptide concentration of 1-3 mM at about pH 7, which is reduced due to the addition of thiol and TFA from the lyophilized peptide. Preferably, the ligation reaction is carried out at 37 ° C. in a heating block and equilibrates the thiol adduct by periodic vortexing. The reaction can be monitored for the degree of completion 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 subjected to a cysteine deprotecting agent such as O-methylhydroxylamine (0. 5M) followed by the addition of a 10-fold excess of tris- (2-carboxyethyl) -phosphine to the reaction mixture to remove any oxidation reaction components prior to purification of the product by conventional preparative HPLC. Reduce them completely. Preferably, ligation product containing fractions are identified by electrospray MS, pooled and lyophilized.

  After synthesis is complete and the final product is purified, the final polypeptide product can be obtained, for example, from 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 regenerated by air oxidation or the like according to the following procedure. The reduced lyophilized product is dissolved (at about 0.1 mg / mL) in 1M guanidine hydrochloride (or similar chaotropic agent) containing 100 mM Tris, 10 mM methionine at pH 8.6. After gentle stirring overnight, the regenerated product is isolated by reverse phase HPLC with conventional protocols.

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 can encode equivalent amino acid sequences and can be used to clone and express HPP. By selecting codons preferred by a particular host cell and substituting the native nucleotide sequence, the rate and / or effectiveness of expression can be increased. Nucleic acid (eg, cDNA or genomic DNA) encoding the desired HPP can be inserted into a replicable vector for cloning (amplification of DNA) or expression. Polypeptides are expressed recombinantly in any of several expression systems according to methods known in the art (Ausubel et al., Ed., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1990). obtain. Suitable host cells include yeast, bacteria, archaea, fungi and insects, and animal cells, including mammalian cells such as primary cells, but stem cells including, but not limited to bone marrow stem cells. More specifically, these examples include, but are not limited to, microorganisms such as recombinant bacteriophage, bacteria transformed with plasmid or cosmid DNA expression vectors, and yeast expression vectors. There is yeast. Also included are insect cells infected with recombinant insect viruses (eg, baculovirus), and mammalian expression systems. The nucleic acid sequence to be expressed can be inserted into the vector by a variety of procedures. 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 of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Do not mean. Construction of suitable vectors containing one or more of these components uses standard ligation techniques known to those skilled in the art.

  The HPP of the present invention is produced by culturing host cells transformed with an expression vector comprising a nucleic acid encoding HPP under conditions suitable to induce or induce protein expression. Appropriate conditions for HPP expression will vary with the choice of the expression vector and the host cell, as confirmed by those skilled in the art. For example, the use of a constitutive promoter in an expression vector may require routine optimization procedures for host cell growth and proliferation, and the use of an inducible promoter requires appropriate growth conditions for induction. Furthermore, the timing of collection may be important in some embodiments. For example, since the baculovirus system used in insect cell expression is a lytic virus, selection of harvest time can be critical to product yield.

  Host cell lines can be selected based on their 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, acetyl, phosphate, amide, lipid, carboxyl, acyl or carbohydrate groups. Post-translational processing also cleaves the “prepro” form of the protein and may be important for correct insertion, folding and / or function. As an example, host cells such as CHO, Healer, BHK, MDCK, 293, W138, etc. have specific tissue and characteristic mechanisms for the above-mentioned post-translational activities, and correct modification of introduced foreign proteins. And can be selected to ensure processing. Of particular interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129. Cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, schwannoma cell lines, immortalized mammalian bone marrow and lymphocyte cell lines, Jurkat cells, human cells and others Primary cell.

  A nucleic acid encoding HPP must be “operably linked” by placing it in a functional relationship with another nucleic acid sequence. For example, DNA for a precursor sequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide. A promoter or enhancer is operably linked to a sequence if it affects the transcription of the coding sequence. Or, a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. In general, DNA sequences that are “operably linked” are contiguous and, in the case of secretory leaders or other polypeptide sequences, are contiguous in the reading phase. However, enhancers do not have to be contiguous. Linkage is achieved by ligation reactions at conventional restriction sites. In the absence of such sites, synthetic oligonucleotide adapters or linkers are used according to conventional practice. A promoter sequence encodes a constitutive or inducible promoter. The promoter can be a naturally occurring promoter or a hybrid promoter. A hybrid promoter is a combination of elements of a plurality of promoters, and is known in the art, and is useful in the present invention. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, so in two organisms, such as a mammalian or insect cell for expression and a prokaryotic host for cloning and amplification. Can be maintained. 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 are well known for a variety of bacteria, yeast, and viruses. The origin of replication from plasmid pBR322 is appropriate for most gram-negative bacteria; 2: the plasmid origin is appropriate for yeast and the various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are mammalian Useful for cloning vectors in cells. In addition, for integrating an expression vector, the expression vector comprises at least one sequence homologous to the host cell genome, and preferably two homologous sequences flanking the expression construct. The integrating vector can be directed to a specific location in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrative vectors are well known in the art. In additional embodiments, the heterologous expression control element can be operably linked to an endogenous gene in the host cell by homologous recombination (described in US Pat. Nos. 6,410,266 and 6,636,1972). This technique makes it possible to regulate expression to a desired level with selected control elements while ensuring proper processing and modification of HPP endogenously expressed by the host cell. Useful heterologous expression control elements include CMV immediate promoters, HSV thymidine kinase promoters, early and late SV40 promoters, retroviral LTR promoters such as the Rous sarcoma virus (RSV) promoter, and metallothionein promoters. It is not limited.

  Preferably, the expression vector contains a selectable marker gene that allows for 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 typically 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 auxotrophic gene deletions, or (c) complex media There is a gene that encodes a protein that supplies critical nutrients that are not available from, for example, D-alanine racemase for Baccilli.

  Host cells transformed with a nucleotide sequence encoding HPP can be cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. Proteins produced by recombinant cells can be secreted by the sequence and / or the vector used, membrane bound, or contained within the cell. As will be readily appreciated by those skilled in the art, an expression vector comprising a polynucleotide encoding HPP can be designed with a signal sequence that directs secretion of HPP through prokaryotic or eukaryotic cell membranes. . The desired HPP is as a fusion polypeptide with a heterologous polypeptide, which can be directly by genetic recombination and also a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Can also be manufactured. In general, the signal sequence can be a component of the vector, or it can be a portion of the HPP-encoded DNA inserted into the vector. The signal sequence can be, for example, a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, lpp or a thermostable enterotoxin II leader sequence. For yeast secretion, signal sequences include, for example, yeast invertase leader sequences, alpha factor leader sequences (Saccharomyces and Kluyveromyces a-factor leader sequences, the latter in US Pat. No. 5,010,182. Described), or an acid phosphatase leading sequence, a C. albicans glucoamylase leading sequence (European Patent No. 362179 published April 4, 1990), or International Publication No. 90 published November 15, 1990 / 113646 may be the signal described. In mammalian cell expression, mammalian signal sequences can be used to direct protein secretion, including signal sequences from secreted polypeptides of the same or related species, and viral secretion leader sequences. Depending on the chosen expression system, the coding sequence is inserted into an appropriate vector, which may require the presence of certain characteristic “control elements” or “regulatory sequences”. Appropriate constructs are known in the art (Ausubel et al., 1990) and are often vendors such as Invitrogen (San Diego, CA), Stratagene (La Jolla, CA), Gibco BRL (Rock Bill, Maryland) or Clontech (Palo Alto, California).

Expression in bacterial systems Transformation of bacterial cells can be accomplished using inducible promoters such as the hybrid lacZ promoter of "BLUESCRIPT" phagemid (Stratagene) or "pSPORT1" (Gibco BRL). In addition, some expression vectors can be selected that, when used in bacterial cells, produce a cleavable fusion protein that can be easily detected and / or purified, including but not limited to “BLUESCRIPT” (a -Galactosidase; Stratagene) or pGEX (Glutathione S-transferase; Promega, Madison, Wisconsin). Suitable bacterial promoters are nucleic acid sequences that can bind bacterial RNA polymerase and initiate downstream (3 ′) transcription of the coding sequence of the HPP gene into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5 ′ end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. A sequence that encodes a metabolic pathway enzyme provides a particularly useful promoter sequence. Examples include promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophages 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 include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. Effective ribosome-binding sites are also desirable. The expression vector may also include a signal peptide sequence that causes secretion of HPP in bacteria. The signal sequence typically encodes a signal peptide composed of hydrophobic amino acids that direct the secretion of the protein from the cell, as is well recognized in the art. Proteins are secreted into the peripheral space between the inner and outer membranes of growth media (Gram positive bacteria) or cells (Gram negative bacteria). Bacterial expression vectors can also include a selectable marker gene that allows 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 in the histidine, tryptophan and leucine biosynthetic pathways. For example, if large amounts of HPP are required for antibody induction, vectors that direct high level expression of easily purified fusion proteins may be desirable. Such vectors include multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), where the HPP coding sequence is the sequence for the amino terminal Met and the subsequent 7 residues of beta-galactosidase. And resulting in hybrid protein production; PIN vectors (Van Heeke & Schuster J Biol Chem 264: 5503-5509, 1989); PET vectors (Novagen, Madison, Wisconsin) etc. However, it is not limited to these. Bacterial expression vectors contain the various components described above and are well known in the art. Examples are in particular vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans. Bacterial expression vectors are transformed into bacterial host cells using techniques known in the art such as calcium chloride mediated transfection, electroporation and the like.

Yeast Expression Yeast expression systems are well known in the art and include Saccharomyces, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillermondii and P pastoris, Schizosaccharomyces pombe and Expression vectors for Yarrowia lipolytica are included. Examples of suitable promoters for use in yeast hosts include 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073 (1980)) or other glycolytic enzymes (Hess et al. J. Adv. Enzyme Reg. 7: 149 (1968); Holland, Biochemistry 17: 4900 (1978)), eg enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase , Glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, promoter for alpha factor, ADH2IGAPDH promoter, glucokinase alcohol oxidase, and PGH. See, for example, Ausubel et al., 1990; Grant et al., Methods in Enzymology 153: 516-544 (1987). Other yeast promoters that are inducible have the added advantage of transcription controlled by growth conditions, such as alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde-3 -Contains promoter regions for phosphate dehydrogenase and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73657. Yeast selectable markers include ADE2. HIS4. LEU2. TRP1. And ALG7 (confers resistance to tunicamycin), the neomycin phosphotransferase gene that confers resistance to G418, and the CUP1 gene that can grow yeast in the presence of copper ions. Yeast expression vectors can be constructed for intracellular production and secretion of HPP from DNA encoding the HPP of interest. For example, the selected signal peptide and an appropriate constitutive or inducible promoter can be inserted into an appropriate restriction site in the selected plasmid for direct intracellular expression of HPP. In order to secrete HPP, the DNA encoding HPP is combined with DNA encoding promoter, yeast alpha-factor secretion signal / leading sequence, and linker sequence (optional) for the purpose of expression of HPP. Can be cloned into a selected plasmid. Yeast cells can then be transformed with the expression plasmid and cultured in a suitable fermentation medium. The protein produced by the transformed yeast can then be concentrated by precipitation with 10% trichloroacetic acid and analyzed after separation by SDS-PAGE and gel staining with Coomassie blue dye. Recombinant HPP can be continuously isolated and purified from the fermentation medium by techniques known to those skilled in the art.

Expression in mammalian systems HPP can be expressed in mammalian cells. Mammalian expression systems are known in the art and include retroviral vector-mediated expression systems. Mammalian host cells can be transformed with any of a number of different viral-based expression systems, such as adenovirus, in which case the coding sequence is linked to an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. obtain. Insertion in the non-essential E1 or E3 region of the viral genome results in a viable virus that can express the polypeptide of interest in the infected host cell. A preferred expression vector system is a retroviral vector system, itself as described generally in PCT / US97 / 01019 and PCT / US97 / 101048. Suitable mammalian expression vectors include a mammalian promoter, which is a DNA sequence that can bind to mammalian RNA polymerase and initiate downstream (3 ′) transcription of the coding sequence for HPP into mRNA. A promoter usually has a TATA box, with a transcription initiation region located proximal to the 5 'end of the coding sequence and 25-30 base pairs located upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to initiate RNA synthesis at the correct site. Mammalian promoters also contain an upstream promoter element (enhancer element), typically located within 100 to 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 orientation. Since viral genes are often highly expressed and have a broad host range, promoters from mammalian viral genes are particularly useful as mammalian promoters. Examples include viruses such as polyoma virus, fowlpox virus (UK Patent No. 2211504 published July 5, 1989), adenovirus (eg adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus. Promoters derived from the genomes of retroviruses, hepatitis B virus and simian virus 40 (SV40), heterologous mammalian promoters, such as actin promoters or immunoglobulin promoters, and heat shock promoters, which promoters are host cells It shall be compatible with the system. Transcription of DNA encoding HPP by higher eukaryotes can be enhanced by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10-300 bp, that acts on a promoter to increase its transcription. Many enhancer sequences are known to be derived from mammalian genes (globin, elastase, albumin, a-fetoprotein and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer late in the origin of replication, and the adenovirus enhancer. The enhancer is preferably located 5 'to the promoter. In general, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 'to the translation stop codon and are therefore adjacent to the coding sequence together with the promoter element. 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 derived from SV40. Long-term, high-yield production of recombinant proteins can be performed in a stable expression system. Expression vectors containing a viral origin of replication or endogenous expression element and a selectable marker gene can be used for this purpose. Appropriate vectors containing selectable markers for use in mammalian cells are readily available 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 used in tk- or hprt- cells, respectively. Methods for introducing exogenous nucleic acid into mammalian hosts and other hosts are known in the art and will vary with the host cell used. Techniques include dextran mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of polynucleotide (s) in liposomes, and direct microinjection of DNA into the nucleus.

HPP can be purified from the culture supernatant of mammalian cells transiently transfected or stably transformed with an expression vector having the HPP coding sequence. Preferably, HPP is purified from the culture supernatant of COS7 cells transiently transfected with a pcD expression vector. Transfection of COS7 cells with pcD proceeds as follows: 1 day prior to transfection, about 10 ⁶ COS7 monkey cells were transferred to individual 100 mm in Dulbecco's Modified Eagle Medium (DME) containing 10% fetal bovine serum and 2 mM glutathione. Seed the plate. To perform the transfection, the medium is aspirated from each plate and replaced with 4 ml DME containing 50 mM Tris.HCl pH 7.4, 400 mg / ml DEAE-dextran and 50 μg plasmid DNA. The plate is incubated for 4 hours at 37 ° C., then the DNA-containing medium is removed and the plate is washed twice with 5 ml of serum free DME. DME is added back to the plate and then incubated for an additional 3 hours at 37 ° C. After washing the plate once with DME, a standard concentration of DME containing 4% fetal bovine serum, 2 mM glutathione, penicillin (100 U / L) and streptomycin (100 μg / L) is added. The cells are then incubated at 37 ° C. for 72 hours, after which the growth medium is collected to purify HPP. E. coli MC1061 (reported by Casadaban and Cohen, J. Mol. Biol., 138, 179-207 (1980)) or a similar organism comprising an HPP-encoded cDNA insert, Plasmid DNA for transfection is obtained by growing pcD (SRα) or similar expression vector. Plasmid DNA is isolated from culture by standard techniques, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory, New York, 1989) or Ausubel et al (1990, supra). .

Expression in insect cells HPP can also be produced in insect cells. Expression vectors for transformation of insect cells and in particular expression vectors based on baculovirus are known in the art. In one of the above systems, the HPP encoded DNA is fused upstream of an epitope tag contained within a baculovirus expression vector. Foreign genes are expressed in Spodoptera frugiperda Sf9 cells or Trichoplusia larvae by using Autographa californica nuclear multifaceted disease virus (AcNPV) as a vector. The HPP coding sequence is cloned into a nonessential region of the virus, such as a polyhedron protein gene, and placed under the control of a polyhedron protein promoter. Effective insertion of the HPP coding sequence renders the polyhedron protein gene inactive and produces a recombinant virus lacking the coat protein coat. The recombinant virus is then used to infect Spodoptera frugiperda cells or Trichoplusia larvae in which HPP is expressed (Smith et al., J. Wol. 46: 584 (1994)). Engelhard EK et al., Proc. Natl. Acad. Sci. 91: 3224-3227 (1994)). Suitable epitope tags for fusion with HPP-encoded DNA include poly-his tags and immunoglobulin tags (also IgG Fc regions). Various plasmids can be used, including commercially available plasmids such as pVL1393 (Novagen). Briefly, HPP-encoded DNA or a desired portion of HPP-encoded DNA is amplified by PCR with primers complementary to the 5 ′ and 3 ′ regions. The 5 ′ primer can incorporate flanking restriction sites. The PCR product is then digested with the selected restriction enzyme and subcloned into an expression vector. Recombinant baculovirus can be obtained using the above plasmid and BaculoGold ™ viral DNA (Pharmingen) using Lipofectin (commercially available from Gibco-BRL), or other methods known to those skilled in the art. Spodoptera frugiperda ("Sf9") produced by co-transfection into cells (ATCC CRL 1711). Virus is produced by day 4-5 of culture in Sf9 cells at 28 ° C. and used for further amplification. Further, the procedure described in O'Reilley et al., BACULOVIRUD EXPRESSION VECTORS: A LABORATORY MANUAL, Oxford University Press (1944) is performed. Extracts can be produced from recombinant virus-infected Sf9 cells as described in Rupert et al., Nature 362: 175-179 (1993). Alternatively, the expressed epitope-tagged HPP can be purified by affinity chromatography or, for example, purification of IgG-tagged (or Fc-tagged) HPP can be accomplished by chromatographic techniques, including protein A or protein G column chromatography. Can be implemented.

Assessment of gene expression Gene expression is measured directly in the sample, for example by measuring the transcription of mRNA by standard techniques known to those skilled in the art, eg Northern blotting, dot blotting (DNA or RNA) or provided in the present invention It can be assessed by in situ hybridization using an appropriate labeled probe based on the sequence. Alternatively, antibodies can be used in assays to detect polypeptides, nucleic acids, such as specific duplexes such as DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. An assay can be performed so that the presence of antibody bound to the duplex can be detected when the duplex is formed on the surface by labeling the antibody and binding the duplex to the surface. Alternatively, expression of HPP polypeptides or polynucleotides can be directly assessed by measuring gene expression using immunohistochemical staining of cells or tissue pieces and cell culture or body fluid assays. Antibodies useful in the immunoassay can be monoclonal or polyclonal and can be produced against native sequence HPP. Protein levels can also be detected by mass spectrometry. Further protein detection methods are by retention material chromatography (including protein arrays) and surface enhanced laser desorption / ionization (SELDI) techniques.

Purification of Expressed Protein Expressed HPP can be purified or isolated after expression by using 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 substances that typically interfere with diagnostic or therapeutic use for polypeptides and may include enzymes, hormones, and other solutes. The selected purification step (s) will depend on, for example, the manufacturing method used and the nature of the specific HPP produced. Since HPPs are secreted, they can be recovered from the culture medium. Alternatively, HPP can be recovered from the host cell lysate. In the case of membrane binding, it can be dissociated from the membrane using an appropriate detergent solution (eg, Triton-X100) or by enzymatic cleavage. Alternatively, the cells used for HPP expression can be disrupted by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical disruption, or the use of cytolytic agents. Examples of purification methods include ion exchange column chromatography, chromatography on silica gel or cation exchange resin, eg DEAE, eg gel filtration using Sephadex G-75, contaminants on protein A Sepharose column, eg IgG. Removal, chromatography using metal chelating columns to bind epitope-tagged forms of HPP, ethanol precipitation, reverse phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation methods, but are not limited to these. Usually, isolated HPP is produced by at least one purification step. For example, HPP can be purified using a standard anti-HPP antibody column. Ultrafiltration and dialysis techniques in conjunction with protein concentration are also useful (see, eg, Scopes, R., PROTEIN PURIFICATION, Springer-Fellark, New York, New York, 1982). The required degree of purification depends on the use of HPP. In some cases, purification may not be necessary. The HPPs and nucleic acids of the invention, expressed and purified as needed, are useful in some applications detailed herein.

Evaluation of HPP activity Furthermore, the present invention is intended to provide a method for testing or obtaining the activity of functional fragments and variants of HPP and HPP sequences. In the above method, a mutant or modified HPP-encoding nucleic acid is provided to assess whether the encoded polypeptide exhibits HPP biological activity. A method for evaluating the function of HPP, comprising: (a) providing HPP, or a biologically active fragment or homologue thereof, and (b) for HPP biological activity under conditions suitable for HPP activity. Also encompassed are methods comprising testing HPP or biologically active fragments or homologues thereof. Cell-free assays, cell-based assays and in vivo assays can also be used to test for HPP activity. For example, the assay can include expressing an HPP nucleic acid in a host cell and observing HPP activity in the cell and other diseased cells. In another example, HPP or a biologically active fragment or homologue thereof is contacted with a cell and the HPP biological activity is observed.

  HPP biological activity is (1) circulating throughout the bloodstream of a human individual, (2) ability to bind antigenic or anti-HPP specific antibodies, (3) immunogenicity, or anti-HPP specific antibody production. Capacity, and (4) interaction with HPP target molecules or adsorbents.

  HPP biological activity can be assayed by any suitable method known in the art. Antigenicity and immunogenicity can be deleted, for example, as described in the sections entitled “Anti-HPP antibodies” and “Use of HPP antibodies”. Circulation in plasma can be detected as described in “Diagnostic and Prognostic Use”.

  Measuring the ability of HPP to bind to or interact with an HPP target molecule can be performed by direct or indirect binding measurement methods common in the art. The method can be cell-based (eg, take the form in which binding to membrane-bound HPP is detected) or cell-free. The interaction of the test compound with HPP can be detected, for example, by coupling HPP or its biologically active moiety with a labeling group, which binds HPP or its biologically active moiety and its cognate target molecule. Is a method that can be measured by detecting the labeled HPP or biologically active portion thereof in the complex. For example, the extent of complex formation can be measured by performing immunoprecipitation of the complex or gel electrophoresis. Further, the measurement of the ability of HPP to bind to an HPP target molecule can be performed using 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. Struct. Biol. 5: 699-705. As used herein, “BIA” is a technique for testing biospecific interactions in real time without labeling any reactants (eg, BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indicator of real-time reactions between biomolecules. Protein array methods are useful for detecting interactions. For example, a member of a receptor / ligand pair is docked to an adsorbent and its binding ability with a binding partner is measured in the presence of a test substance. Because of the rapidity with which adsorption can be tested, combinatorial libraries of test substances can be easily screened for their ability to modulate interactions. In a preferred method, HPP is docked to the adsorbent. The interaction can be detected, preferably by labeling the binding partner. Alternatively, in certain embodiments, the test substance is docked to the adsorbent. The polypeptide of the present invention is exposed to a test substance and binding is detected.

Anti-HPP Antibodies The present invention provides antibodies and binding compositions specific for HPP. Such antibodies and binding compositions include polyclonal antibodies, monoclonal antibodies, Fab and side chain Fv fragments thereof, bispecific antibodies, heteroconjugates, and humanized antibodies. The antibodies and binding compositions can be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. Extensive guidance is given in the literature for the selection of specific production methods: eg, Chadd and Chamow, Curr. Opin. Biotechnol., 12: 188-194 (2001).

  The choice of manufacturing method depends on several factors including the desired antibody structure, the importance of the carbohydrate moiety for the antibody, ease of culture and purification, and cost. Many different antibody structures can be generated using standard expression techniques, including full length antibodies, antibody fragments, such as Fab and Fv fragments, and chimeric antibodies comprising components from different species. Small size antibody fragments, such as Fab and Fv fragments, that have no effector function and limited pharmokinetic activity can be generated in bacterial expression systems. Single chain Fv fragments are highly selective for in vivo tumors, exhibit good tumor penetration and low immunogenicity, and are rapidly cleared from the blood, eg, Freyre et al., J. Biotechnol. 76: 157-163 (2000). That is, the molecule is desirable for radioimmunodetection.

Polyclonal antibody The anti-HPP antibody of the present invention may be a polyclonal antibody. Such polyclonal antibodies can be produced in mammals after, for example, one or more injections of an immunizing agent and preferably an adjuvant. Typically, the immunizing agent and / or adjuvant is injected into the mammal by a series of subcutaneous or intraperitoneal injections. The immunizing agent can comprise HPP or a fusion protein thereof. It may be useful to conjugate the antigen to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include keyhole limpet hemocyanin (KLH), methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), hepatitis B surface antigen, serum albumin, bovine thyroglobulin, and soybean There are, but are not limited to, trypsin inhibitors. Adjuvants include, for example, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicoryno-mycolate). The immunization protocol can be determined by those skilled in the art based on standard protocols or by routine experimentation.

  Alternatively, crude protein preparations enriched for HPP or a portion thereof can be used for the production of antibodies. The protein, fragment or preparation is introduced into a non-human mammal 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 the antigen and the host species. Host animals will also vary depending on the site and dose of inoculation, resulting in low titer antisera both when the antigen dose is insufficient and excessive. Small doses (ng level) of antigen administered at many intradermal sites appear to be most reliable. Polyclonal antisera production and processing techniques are known in the art, see, for example, Mayer and Walker (1987). An effective immunization protocol for rabbits is described in Vaitukaitis, J. et al. J, Clin. Endocrinol. Metab. 33: 988-991 (1971). Antiserum can be collected when booster injections are made at regular intervals and when the antibody titer begins to fall semi-quantitatively, eg, by double immunodiffusion in agar against a known concentration of antigen. See, for example, Ouchterlony, O. et al., Chapter 19, 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. Antisera affinity for antigen is described, for example, in Fisher, D., Chapter 42, Manual of Clinical Immunology, 2nd edition (Edited by Rose and Friedman) Amer. Soc. For Microbiol., Washington, D.C. (1980). Measured by creating competitive binding curves according to the procedure described in.

Monoclonal Antibodies Alternatively, the anti-HPP antibody can be a monoclonal antibody. Monoclonal antibodies can be produced by hybridomas, in which case immunizing a mouse, hamster or other suitable host animal with an immunizing agent produces or produces an antibody that specifically binds to the immunizing agent. Inducing resulting lymphocytes, eg, Kohler and Milstein, Nature 256: 495 (1975). The immunizing agent typically comprises HPP or a fusion protein thereof and an optional carrier. Alternatively, lymphocytes can be immunized in vitro. In general, spleen cells or lymph node cells are used if a non-human mammalian source is desired, or peripheral blood lymphocytes ("PBL") are used if human-derived cells are desired. Hybridoma cells are produced by fusing lymphocytes with an immortal cell line using a suitable fusing agent such as polyethylene glycol, eg, Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, 59-103 ( 1986); Liddell and Cryer, A Practical Guide to Monoclonal Antibodies (John Wiley and Sons, New York, 1991); edited by Malik and Lillenoj, Antibody Techniques (Academic Press, New York, 1994). In general, an immortalized cell line is a myeloma cell derived from a transformed mammalian cell, eg, a rat, mouse, bovine or human. Preferably, the hybridoma cells are 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 lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT), the culture medium for hybridomas typically inhibits the growth of hypoxanthine, aminopterin and thymidine (HAT), HGPRT-deficient cells. Contains substances. Preferred immortal cell lines are those that fuse effectively, support stable high level production of antibodies, and are sensitive to media such as HAT media. A more preferred immortal cell line is the mouse or human myeloma line, which can be obtained, for example, from the American Type Culture Collection (ATCC, Rockville, MD). Human myeloma and mouse-human heteromyeloma cell lines have also been reported for the production of human monoclonal antibodies, eg, Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Decker, Incorporated, New York, 51-63 (1987).

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

  Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibody of the present invention is isolated from HPP-specific hybridoma cells, for example, by using an oligonucleotide probe that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody. Can be sequenced. After isolation, the DNA can be inserted into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. By doing so, the synthesis of the monoclonal antibody in the recombinant host cell is achieved. DNA also uses coding sequences for mouse heavy and light chain constant domains in place 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 immunizing all or part of the coding sequence for a non-immunoglobulin polypeptide. It can be modified by covalent attachment to a globulin coding sequence. By using a non-immunoglobulin polypeptide in place of the constant domain of the antibody of the present invention or in place of the variable domain of one antigen-binding site of the antibody of the present invention, a chimeric bivalent antibody can be produced. The antibody can also be a monovalent antibody. Methods for producing monovalent antibodies are well known in the art. For example, in vitro methods are suitable for the production of monovalent antibodies. Production of the fragments, particularly Fab fragments, by digestion of the antibody can be performed using conventional techniques known in the art.

  Antibodies and antibody fragments characteristic of the hybridomas of the invention can also be produced by recombinant techniques by extracting messenger RNA, constructing a cDNA library, and selecting clones that encode segments of the antibody molecule. Examples of references disclosing genetic recombination techniques for antibody production include the following: Wall et al., Nucleic Acids Research, Vol. 5, 313-3128 (1978); Zakut et al., Nucleic Acids Research, 8, 3591-3601 (1980); Cabilly et al., Proc. Natl. Acad. Sci., 81, 3273-3277 (1984); Boss et al., Nucleic Acids Research, 12, 3791-3806 (1984); Amster et al., Nucleic Acids Research, 8, 2055-2065 (1980); Moore et al., US Pat. No. 4,642,334; Skerra et al., Science, 240, 1038-1041 (1988); Huse et al., Science, 246, 1275-1281 (1989); and US Pat. Nos. 6,054,297, 5530101, 4816567, 5750105. Beauty No. 5648237, for these are incorporated by reference. In particular, the techniques described above can be used in the production of interspecies monoclonal antibodies, reducing the immunogenicity by combining one binding region with the non-binding region of another antibody, eg, Liu et al , Proc. Natl. Acad. Sci., 84, 3439-3443 (1987), and patents 6054297 and 5530101. 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, the full-length antibody is expressed in Chinese hamster ovary (CHO) cells or NSO cells.

Both polyclonal and monoclonal antibodies can be screened by ELISA. As with other solid phase immunoassays, this test is based on the tendency of macromolecules to be non-specifically adsorbed to plastic. Since this reaction is irreversible and does not involve a loss of immunological activity, an antigen-antibody complex can be formed, simply separating the complex from unbound material. To titrate anti-peptide sera, peptides conjugated to a different carrier than that used for immunization are adsorbed to the wells of a 96-well microtiter plate. The adsorbed antigen is then reacted in a well with a dilution of anti-peptide serum. Unbound antibody is washed and the remaining antigen-antibody complex is reacted with an antibody specific for IgG of the immunized animal. This secondary antibody is conjugated to an enzyme such as alkaline phosphatase. The visible color reaction that occurs when the enzyme substrate is added indicates which wells have bound anti-peptide antibodies. By using spectrometer readings, the amount of bound peptide-specific antibody can be more accurately quantified. A high titer antiserum gives a linear titration curve with a dilution rate between 10 ^{−3 and} 10 ⁻⁵ .

HPP Peptide Carriers The present invention includes immunogens derived from HPP and immunogens including conjugates between the carrier and the peptides of the present invention. As used herein, the term immunogen refers to a substance that can elicit an immune response. As used herein, the term carrier refers to the production of antibodies specific for a conjugated peptide in a host organism immunized with the resulting conjugate when chemically conjugated to the peptide of the invention. Refers to a substance that can be Carriers include red blood cells, bacteriophages, proteins, or synthetic particles such as agarose beads. Preferably, the carrier is a protein such as serum albumin, gamma-globulin, keyhole limpet hemocyanin (KLH), thyroglobulin, ovalbumin or fibrinogen.

  General techniques for attaching synthetic peptides to carriers are described in several references: eg, Walter and Doolittle, “Antibodies Against Synthetic Peptides”, Setlow et al., Genetic Engineering, Vol. 5, 61. -91 (Plenum Press, New York, 1983); Green et al. Cell, 28, 477-487 (1982); Lerner et al., Proc. Natl. Acad. Sci., 78, 3403. -3407 (1981); Shimizu et al., U.S. Pat. No. 4,474,754 and Ganfield et al., U.S. Pat. No. 4,311,639. Therefore, these references are incorporated by reference. Also, the technique used to bind the hapten to the carrier is essentially the same as the technique according to the above references, eg Tijssen, Practice and Theory of Enzyme Immunoassays, Chapter 20 (Elsevier, New York, 1985). The four most commonly used schemes for conjugating peptides to carriers are (1) eg Kagan and Glick, edited by Jaffe and Behrman, Methods of Hormone Radioimmunoassay, pages 328-329 (Academic Press, New York, 1979). ), And glutaraldehyde for amino coupling disclosed by Walter et al., Proc. Natl. Acad. Sci., 77, 5197-5200 (1980); (2) For example, Hoare et al. , J. Biol. Chem., 242, 2447-2453 (1967), amino-coupled for water-soluble carbodiimides; (3) For example, Bassiri et al., 46-47, Jaffe And tyrosine side chain coupling for tyrosine as disclosed by Behrman (supra) and Walter et al. (Supra). Bis-diazobenzidine (BDB); and (4) disclosed, for example, by Kitagawa et al., J. Biochem. (Tokyo), 79, 233-239 (1976), and Lerner et al. (Supra). Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), which couples cysteine (or other sulfhydryl) to an amino group. The general rules for selecting an appropriate method for coupling a given peptide to a protein carrier are as follows. The group involved in the binding should be found only once in the sequence, preferably at the appropriate end of the segment. For example, BDB should not be used if tyrosine residues are present in the major portion of the sequence selected for its potential antigenic properties. Similarly, the glutaraldehyde method is excluded in the case of centrally located lysine, and the carbodiimide method is often excluded when aspartic acid and glutamic acid are found. On the other hand, suitable residues may be placed at either end of the sequence segment selected as the binding site, whether or not they are found from the “native” protein sequence. The internal segment differs from the amino and carboxy termini in that it differs significantly from the "non-binding end" from the same sequence found in a native protein with a continuous polypeptide backbone. By acetylating the α-amino group and then attaching the peptide via its carboxy terminus, this problem can be ameliorated to some extent. Coupling efficiency to the carrier protein is conveniently achieved by using a radiolabeled peptide produced by using a radioactive amino acid for one step of the synthesis or by labeling the finished peptide with iodination of tyrosine residues. Measured. Also, the presence of tyrosine in the peptide allows for the setting up of a sensitive radioimmunoassay if desired. Thus, tyrosine can be introduced as a terminal residue if it is not part of the peptide sequence specified by the native polypeptide.

  Preferred carriers are proteins, and preferred protein carriers include bovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH) and the like. As disclosed by Liu et al., Biochemistry, Vol. 18, 690-697 (1979), peptides can be linked to KLH through cysteine by MBS. 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 pH for peptide dissolution is selected to optimize peptide solubility. The free cysteine content for soluble peptides is determined by the Ellman method of Ellman, Arch. Biochem. Biophys., 82, 7077 (1959). For each peptide, 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg MBS (dissolved in dimethylformamide) and stirred at room temperature for 30 minutes. Since KLH is insoluble in> 30% formamide, dropping MBS ensures that the local concentration of formamide does not become too high. Free MBS was removed by passing the reaction product, KLH-MBS, through Sephadex G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0), and KLH was recovered from the peak fraction of the column eluate. When evaluated (monitored by OD280), it is about 80%. KLH-MBS is then reacted with 5 mg of 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 efficiencies can range from 8% to 60% when monitored with radioactive peptides by dialysis of conjugate samples against phosphate buffered saline. When peptide-carrier conjugates are available, for example, Campbell, Monoclonal Antibody Technology (Elsevier, New York, 1984); Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications (CRC Press, Boca Raton, Florida, 1982); Schreier et al. , Hybridoma Techniques (Cold Spring Harbor Laboratory, New York, 1980); polyclonal or monoclonal antibodies are produced by standard techniques as disclosed in US Pat. No. 4,456,2003.

Humanized antibodies Furthermore, the anti-HPP antibodies of the invention may comprise humanized antibodies or human antibodies. The term “humanized antibody” refers to a chimeric antibody, immunoglobulin chain or fragment thereof (eg, Fv, Fab, Fab ′, F (ab ′), or other antibody that contains a portion of a sequence derived from a non-human antibody. The humanized form of a non-human (eg, mouse) antibody. Humanized antibodies have residues from complementarity-determining regions (CDRs) of human immunoglobulins from CDRs of non-human species, such as mouse, rat or rabbit, that have the desired binding specificity, affinity and acceptability. Includes human immunoglobulins substituted by groups. In general, a humanized antibody corresponds to the case where all or substantially all of the CDR regions are non-human immunoglobulin, and all or substantially all of the FR regions are at least the same region of a human immunoglobulin consensus sequence. It includes substantially all of one and generally two variable domains. Humanized antibodies also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically 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, a humanized antibody has been introduced into it from a source other than human, such that it retains the original binding activity of the antibody, but more closely resembles a human antibody. Has more amino acids. Methods for humanizing antibodies are further 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). The “humanized” antibody is a chimeric antibody in that substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species.

Heteroconjugate antibodies Heteroconjugate antibodies comprising an antibody linked by two covalent bonds are also included within the scope of the present invention. Heteroconjugate antibodies can be produced in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be produced using a disulfide exchange reaction or by forming a thioether bond.

Bispecific antibodies Bispecific antibodies have binding specificities for at least two different antigens. The antibody is monoclonal, preferably a human or humanized antibody. One of the binding properties of the bispecific antibody of the invention is for HPP and the other is preferably for cell surface proteins or receptors or receptor subunits. Methods for producing bispecific antibodies are known in the art, and in general, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs in hybridoma cells. The two heavy chains have different specificities, eg, Milstein and Cuello, Nature 305: 537-539 (1983). When 10 different antibody molecules are produced as possible by hybridomas by randomly combining immunoglobulin heavy and light chains, certain types of affinity purification, such as affinity chromatography, are usually used for accurate molecular purification. A graphic is required.

Use of HPP antibodies HPP antibodies are preferably specific for the HPP of the invention and as such do not bind with high affinity to peptides derived from other proteins. As used herein, the term “heavy chain variable region” refers to (1) 110-125 amino acids long and (2) the amino acid sequence of the antibody of the present invention beginning with the heavy chain N-terminal amino acid. It means a polypeptide corresponding to the heavy chain sequence. Similarly, the term “light chain variable region” refers to the sequence of the light chain of an antibody of the invention, which is (1) 95-115 amino acids long and (2) the amino acid sequence begins with the light chain N-terminal amino acid. The corresponding polypeptide is meant. As used herein, the term “monoclonal antibody” refers to a heterogeneous population of immunoglobulins that can specifically bind to HPP.

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

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

  The antibody of the present invention is useful for detection of HPP. The detection method is advantageously applied to diagnosis of HPP-related diseases. The antibodies of the invention can be used in most assays involving antigen-antibody reactions. This assay can be homogeneous or heterogeneous. In a homogeneous assay method, the sample can be a biological sample or fluid, such as serum, urine, whole blood, lymph, plasma, saliva, cells, tissues, and substances secreted by cells or tissues cultured in vitro. . If necessary, the sample can be pretreated to remove unwanted material. Immunological reactions usually involve specific antibodies, labeled analyte samples, and samples suspected of containing antigens. The signal resulting from the label is modified directly or indirectly when the antibody binds to the labeled analyte. Both the immunological reaction and the detection of its range are performed in a homogeneous solution. Immunological labels that can be used include free radicals, fluorescent dyes, enzymes, bacteriophages, coenzymes and the like.

  In the heterogeneous assay method, the reagents are usually a sample, a specific antibody, and a detectable signal generating means. Generally, 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 separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal by using the signal generating means or signal generating system. The signal is associated with 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, Incorporated, Boca Raton, Florida, 1980). See also, for example, U.S. Pat. Nos. 3,690,837, 3791932, 3,817,837, 3,850,578, 3853987, 3867517, 3901654, 3935074, 3984533, 3966345, and 4098876 (not all of which are covered in this list). Methods for conjugating labels to antibodies and antibody fragments are well known in the art. Such methods are described in U.S. Pat. Nos. 4,220,450, 4,235,869, 3935974 and 3966345. Another example of a technique in which the antibodies of the invention can be used is immunoperoxidase labeling (Sternberger, Immunocytochemistry (1979) 104-169).

  One aspect of the assay using the antibodies of the invention involves the use of a surface to which the monoclonal antibodies of the invention are bound. The surface substructure may take various forms, have different compositions, and may be a composition or a mixture of laminates or combinations thereof. The surface can take on various shapes and forms and can have different dimensions depending on the method of use and measurement. Examples of surfaces can be pads, beads, disks, or strips that can be flat, concave or convex. The thickness is not critical and is generally about 0.1-2 mm thick and has a convenient diameter or other dimensions. Typically, the surface is supported on rods, tubes, capillaries, fibers, strips, discs, plates, cuvettes and is typically porous, allowing covalent binding of antibodies and detection It can be multifunctional or can be multifunctional to allow attachment of other compounds that form part of the possible signal generating means. A wide variety of organic and inorganic polymers and combinations thereof, including both natural and synthetic, can be used as materials for solid surfaces. Examples of polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), rayon, nylon, poly (vinyl butyrate), silicone, polyformaldehyde, cellulose, cellulose acetate, nitro There are cellulose and latex. Other surfaces include paper, glass, ceramics, metals, metalloids, semiconductor materials, cement, silicates and the like. Also polymers or interfaces that form substrates, forming several gels, gelatins, lipopolysaccharides, silicates, agarose and polyacrylamides or several aqueous phases, eg dextran, polyalkylene glycols (alkylenes of 2 to 3 carbon atoms) Active agents such as phospholipids are also included. Binding of the antibody to the surface can be accomplished by known techniques commonly available in the literature (eg, “Immobilized Enzymes”, 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 surface to which the antibody is bound is contacted with the medium. The label can be included in the aqueous medium by being added simultaneously or subsequently to provide a detectable signal associated with the surface. The means for generating a detectable signal may involve the incorporation of a labeled analyte sample, or it may involve the use of a second monoclonal antibody conjugated to the label. Separation and washing steps are performed as needed. The detected signal is associated with the presence of HPP in the sample. It is within the scope of the present invention to define a scale on the same support. One particular embodiment of the assay method according to the present invention is intended to be illustrative but not limiting, and includes the use of a support, such as a slide or well of a petri dish. In this 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 a monoclonal antibody. After washing with a suitable buffer, such as phosphate buffered saline, the support is contacted with a label specific binding partner for the antibody. After incubation as desired, the slide is washed again with aqueous buffer and a measurement consisting of binding of labeled monoclonal antibody to the antigen is performed. If the label is fluorescent, the extent of binding can be measured by covering the slide with a fluorescent antibody mounting solution on a cover glass and then examining with a fluorescent microscope. On the other hand, the label can be an enzyme conjugated to a monoclonal antibody and the extent of binding can be measured by examining the slide for the presence of enzyme activity, which can be indicated by precipitation tail formation, coloration, and the like.

  A specific example of an assay using the antibody of the present invention is a double determinant ELISA assay. A support, such as a glass or vinyl plate, is coated with antibodies specific for HPP by conventional techniques. A sample suspected of containing HPP is usually brought into contact with the support in an aqueous medium. After an incubation period of 30 seconds to 12 hours, the support is separated from the medium, unbound HPP is removed, for example by washing with water or an aqueous buffer medium, and again an antibody specific for HPP usually in aqueous medium. Contact with. The antibody is directly or indirectly labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase. After incubation, the support is separated from the medium and washed as above. The enzyme activity of the support or aqueous medium is measured. This enzyme activity is related to the amount of HPP in the sample.

  Another antibody-based detection method is the retentate chromatography method described herein. In this case, the adsorbent is an antibody. Preferably, a plurality of antibodies specific for a plurality of HPPs of the invention are included on the surface or substrate.

  The present invention also includes kits for carrying out the methods disclosed above, such as diagnostic assay kits. In one aspect, the kit is a packaged set of (a) a monoclonal antibody more specifically identified above and (b) a specific binding partner for the monoclonal antibody and a conjugate of a label capable of generating a detectable signal. Including together. Reagents can also include adjuvants such as buffers and protein stabilizers such as polysaccharides. The kit may further include other members of the signal generation system of which the label is a member, agents that reduce background interference in the test, control reagents, equipment for performing the test, etc., if necessary. In another embodiment, a diagnostic kit comprises a conjugate of a monoclonal antibody of the invention and a label capable of generating a detectable signal. Such auxiliaries may also be present.

  Furthermore, anti-HPP antibodies (eg, monoclonal antibodies) can be used for isolation of HPP by standard techniques such as affinity chromatography or immunoprecipitation. For example, anti-HPP antibodies can simplify the purification of native HPP from cells and the purification of HPP produced by genetic recombination expressed in host cells. In addition, isolating HPP using anti-HPP antibodies may aid in the detection of low concentrations of HPP (eg, in plasma, cell lysate or cell supernatant) or assess HPP abundance and expression patterns . Anti-HPP antibodies can be used in diagnosis to monitor protein levels in tissues as part of a clinical trial procedure, for example to determine the efficacy of a given therapy. Detection can be facilitated by coupling (ie, physically linking) the antibody to a labeling group.

Detection using protein array Detection and purification of the polypeptide of the present invention is carried out using retention substance chromatography (preferably, protein array or chip) as described in US Pat. No. 6,225,027 and US Patent Application No. 2001014461. Can be implemented. Briefly, retention material chromatography refers to a method in which a polypeptide (and / or other sample component) is retained on an adsorbent (eg, an array or chip) and subsequently detected. In the above method, (1) a polypeptide from a sample is selectively adsorbed to a substrate under a plurality of different adsorbent / eluent combinations (“selective conditions”), and (2) desorption spectroscopy. Retention of the adsorbed polypeptide is detected by a measurement method (eg, by mass spectrometry). “Desorption spectroscopy” is a method for detecting a substance (eg, polypeptide), exposing the substance to energy that desorbs the substance from the stationary phase to the gas phase, and intermediates in the secondary stationary phase. A method in which a desorbed substance or an identifiable part thereof is directly detected by a detector without capturing. In conventional chromatographic methods, the polypeptide is eluted from the adsorbent prior to detection. The combination of adsorption chromatography and detection by desorption spectroscopy provides surprising sensitivity, the ability to quickly analyze retained components with a variety of different selectivity conditions, and different sites on the array under different elution conditions Parallel processing of the components adsorbed on (ie, “affinity sites” or “spots”) is achieved.

  Preferred embodiments are either chemically (eg, ion-affinity or hydrophobic substances) or biospecific (eg, antibodies or antigen-binding fragments thereof) developed to detect specific polypeptides, preferably HPP. Provide an adsorbent that is In certain embodiments, the substrate has an array of adsorption spots selected for a combination of polypeptides, eg, as a diagnostic marker. As few as 2, and 10, 100, 1000 or more adsorbents can be coupled to a single substrate. The size of the adsorption site can vary depending on the experimental design and purpose. However, it need not be larger than the impact energy source diameter (eg, laser spot diameter). The spots can be composed of the same or different adsorbents. In some cases, the same adsorbent can be placed in multiple locations on the substrate, as multiple different eluents can be evaluated or the bound polypeptide can be stored for future use or control, possibly in secondary processing. It may be advantageous to do so. By placing multiple different adsorbents on the substrate, it takes advantage of the multiple binding properties provided by the combination of different adsorbents for a single sample (eg, a plasma sample), thereby allowing a wide variety of different polypeptides. (Preferably HPP) can be bound and detected. The use of multiple different adsorbents on a substrate for single sample evaluation is essentially equivalent to performing multiple chromatographic experiments simultaneously, each with a different chromatography column, but the method of the present invention is single The advantage is that only the system is required.

  When the substrate includes multiple adsorbents, it is particularly useful to place the adsorbent at a predetermined addressable location. By adsorbing the adsorbent at a predetermined addressable location, the adsorbent at the first predetermined addressable location is washed with the first eluent and the second predetermined with the second eluent. It is possible to clean the adsorbent at the designated addressable location. In this way, the binding properties of a single adsorbent for that particular polypeptide can be evaluated in the presence of a number of eluents that each selectively modify each set of adsorbent-polypeptide binding properties.

  Retention material chromatography can be used in combinatorial separation methods involving parallel separation and detection of multiple polypeptides. The method comprises: a) exposing a sample (eg, biological fluid, eg plasma) to at least two different selection conditions, each of which is a selection condition specified by a combination of adsorbent and eluent. And b) detecting the retained polypeptide under different selection conditions by desorption spectroscopy. Each different selection condition is specified at a different addressable location that is predetermined for parallel processing. In this method, i) a polypeptide is held by an adsorbent by exposing the sample to a first selection condition at a specific location, and ii) a polypeptide held under the first selection condition by desorption spectroscopy. Iii) washing the adsorbent under a second different selection condition at a specific location, thereby allowing the adsorbent at that location to retain the polypeptide, and iv) second by desorption spectroscopy Detecting a polypeptide retained under the selection conditions.

  Polypeptides can be detected by gas phase ion spectrometry, SELDI or mass spectrometry techniques detailed herein. “Gas phase ion spectrometry” refers to a method in which gas phase ions are generated from a substance presented on the surface by using an ionization source, and gas phase ions are detected by a gas phase ion spectrometer. “Surface-enhanced laser desorption / ionization” or “SELDI” is a gas phase ion spectrometry in which the material-presenting surface plays an active role in the desorption and ionization processes. SELDI technology is described, for example, in US Pat. Nos. 5,719,060 and 6225047 (Hutchens and Yip).

  These methods are useful for biochemical separation and purification of HPP combinations (combinatorial), testing differential gene expression, and detecting differences in protein levels between samples (eg, diagnostic). WO 03/018193 (Ciphergen) reports on the use of the above detection method for specific kidney disease markers in fluid samples. Yip and Lomas (Technol Cancer Res Treat, 2002, 1: 273-80) employ a similar method to detect cancer-related polypeptides. The HPP or HPP binding material of the present invention is preferably bound to a labeling group so that it is detected directly and two or more from the same “circuit” (ie, addressable “chip” location) during single unit operation. The above signals can be transmitted simultaneously. A preferred embodiment of the present invention involves the use of retentate chromatography to identify at least one HPP from a sample, preferably plasma.

  As reported by Chen et al. (Anal Chem 2002; 74: 5146-53), a variation of the microchip-based detection method utilizes electrophoresis. In this method, a flow-through sampling chip is applied for immunoseparation, protein purification, concentration and detection purposes. This device uses hydrodynamic pressure to drive the sample flow and applies a gating voltage to the electrophoresis channel on the microchip. With this device, the wash / elution step can be integrated on-line with electrophoretic separation and detection on the microchip. Furthermore, the electroless bed ensures that the protein-adsorbent interaction is not affected by the electric field during the wash / elution phase.

Detection of HPP by Mass Spectrometry According to the present invention, any instrument, method, process, etc. can be used to measure protein identity and abundance in a sample. A preferred method of obtaining identity is by mass spectrometry, where 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 analyze the protein using mass spectrometry, it is preferable to convert the protein into a gas-ion phase. Various protein ionization methods are useful, such as fast ion bombardment (FAB), plasma desorption, laser desorption, thermal desorption, preferably electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). Can be mentioned. Many different mass spectrometers are available for peptide and protein analysis, such as time-of-flight (TOF), ion trap (ITMS), Fourier transform ion cyclotron (FTMS), quadrupole ion trap, and sector (electric And / or magnetic) spectrometers, but is not limited to these. For example, see US Pat. No. 5,572,025 for ion trap MS. The mass spectrometer can be used alone or in combination with other mass spectrometers in a tandem mass spectrometer. In the latter case, the first mass spectrometer can be used to separate protein ions (precursor ions) from each other and measure the molecular weight of various protein components in the sample. The second mass spectrometer can be used to analyze each separated component, for example, by fragmenting the precursor ions into product ions, for example by using an inert gas. Any desired combination of mass spectrometers can be used, including, for example, triple quadrupole, tandem time of flight, ion trap, and / or combinations thereof.

  Various types of detection devices can be used to detect protein ions. For example, destructive detection devices such as ion electron multipliers or cryogenic detection devices can be used (eg, US Pat. No. 5,641,010). In addition, non-destructive detection devices such as ion traps used as ion current pick-up devices in quadrupole ion trap mass spectrometers or FTMS can also be used.

  For MALDI-TOF, some sample preparation methods can be used: dry droplets (Karasand Hillenkamp, Anal. Chem. 60: 2299-2301, 1988), vacuum drying (Winberger et al., Proceedings of the 41st ASMS Conference). on Mass Spectrometry and Allied Topics, San Francisco, May 31-June 4, 1993, pages 775a-b), ground crystals (Xiang et al., Rapid Comm. Mass Spectrom., 8: 199-204, 1994). , Slow crystal growth (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), pneumatic spray (Kochling et al., Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, Georgia, 19 (May 21-26, 1995, p. 1225), electrospray (Hensel et al., Proceedings of the 43rd ASMS Conference on Mass Spectrometry and Allied Topics, Atlanta, Georgia, May 21, 1995, p. 947) , Fast solvent concentration (Vorm et al., Anal. Chem., 66: 3281-3287, 1994), sandwich (Li et al., J. Am. Chem. Soc., 118: 11662-11663, 1996), And two-layer methods (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).

  For MALDI analysis, the sample is mixed as an energy absorbing compound or colloid (matrix) in the liquid phase, and finally dried as a solid on the surface of the inert probe, thereby producing a sample as a solid co-crystal or thin film. To prepare. In some cases, energy absorbing molecules (EAM) may be an essential component of the sample presentation surface. Regardless of the EAM application strategy, the probe contents are dried to a solid state prior to introduction into a laser desorption / ionization time-of-flight mass spectrometer (LDIMS).

  Ion detection in TOF mass spectrometry is typically accomplished by the use of an electron emission detector, such as an electron multiplier (EMP) or microchannel plate (MCP). Both of these devices work by converting primary incident charged particles into secondary, tertiary, quaternary, etc. cascades of electrons. The probability of secondary electrons generated by the impact of a single incident charged particle can be considered as the ion-to-electron conversion efficiency (or more simply conversion efficiency) of this charged particle. The electron yield for cascade events when compared to the total number of incident charged particles is typically reported as detector gain. MCP is a preferred electron emission detector for mass / charge resolution enhancement because the overall response time of MCP is generally much better than that of EMP. However, EMP works well for the detection of ion populations with distributed kinetic energy when rapid response times and wide frequency bandwidths are not required.

  In a preferred aspect for digested protein analysis, a liquid chromatography tandem mass spectrometer (LC-TMS) is used. This system provides an additional stage of sample separation through the use of liquid chromatography followed by tandem mass spectrometry.

  Electrospray ionization (ESI) and MALDI are routine ionization techniques that couple capillary electrophoresis (CE) to MS (Moini, M., Anal Bioanal Chem, 2002; 373: 466-80). CE has the advantages of high sensitivity and separation efficiency. The high concentration detection limit of CE has been addressed by the development of sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis are currently coupled to the MS. Deterding et al. (Electrophoresis 2002; 23: 2296-305) report on the effective application of CE-MS technology for the identification of apolipoprotein species from plasma samples. That is, these methods are also considered to be applied to the detection of HPP particularly in human plasma samples.

  In a preferred aspect, the protein eluted from the column by the system described in Example 1 is analyzed using both MS and MS-MS analysis. For example, a small portion of intact protein eluting from RP2 can be converted to on-line detection using LC-ESI MS. Divide the protein into several plates that allow trypsin digestion or non-trypsin digestion, preparation for MALDI-MS and ESI-MS, and preparation of MALDI plates with different matrices. That is, by these methods, in addition to information regarding intact mass, analysis can be performed both by peptide mass fingerprinting and MS-MS techniques.

  The protein separation and fractionation methods described herein provide fractions containing individual proteins or a small number of different proteins. These proteins can be identified by mass spectral measurements of the molecular mass of the protein and the peptides resulting from its fragmentation. By utilizing the information available in the protein sequence database, a comparison can be made between the proteolyzed peptide mass pattern generated in silico and the experimentally observed peptide mass pattern. Based on the number of matches between theoretical and experimental proteolytic fragments (among other criteria), a “hit list” ranking candidate proteins in the database can be created by the compiler. Several websites providing online protein identification software based on peptide mapping and sequence database search strategies 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 US Pat. No. 5,696,376. See also Yates, J. Mass Spec., 33: 1 (1998).

  The data collected from the mass spectrometer typically includes the intensity and mass to charge ratio for each detected event. Spectral data may be recorded in any suitable form including, for example, digital or analog form in graph, numerical or electronic format. The spectrum is preferably recorded on a storage medium including, for example, magnetic, eg floppy disk, tape, or hard disk, optical, eg CD-ROM or laser disk, or ROM-CHIPS.

  The mass spectrum of a given sample typically provides information regarding protein strength, mass to charge ratio, and molecular weight. In a preferred embodiment of the invention, the database is queried by using the molecular weight of the protein in the sample as a matching criterion. Conventionally, for example, the molecular weight is calculated by subtracting the mass of ionized protons for single charged protonated molecular ions, multiplying the measured mass / charge ratio by the number of charges for multicharged ions, and subtracting the number of ionized protons. To do.

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

  Typically, mass spectrometers are equipped with commercially available software that identifies peaks that exceed certain threshold levels and calculates the mass, charge and intensity of detected ions. The correlation between a given output peak and molecular weight can be revealed directly from the spectral data, i.e., since the charge on the ion is one, the molecular weight is equal to the value of the calculator-the mass of the ionized proton. However, protein ions can form complexes with various counter ions and adducts such as N, C and K. In such a case, it is prognostic that a given protein ion will exhibit multiple peaks, such as triplets, representing various ionic states (or species) of the same protein. That is, it may be necessary to measure groups of peaks originating from the same protein by analyzing and processing the spectral data. This analysis can be performed conventionally, for example as described by Mann et al., Anal. Chem., 61: 1702-1708 (1989).

  In matching the molecular weight calculated from the mass spectrometer with a molecular weight prognostic from a database, such as a genome or database of expressed genes, post-translational processing must be taken into account. There are a variety of processing events that modify protein structure, including proteolytic processing, N-terminal methionine removal, acetylation, methylation, glycosylation, phosphorylation, and the like.

  The database can be queried for a set of proteins that match an unknown molecular weight. The range window may be determined by instrument accuracy, the method by which the sample was prepared, and the like. Identify or classify unknown proteins or peptides based on the number of hits in the spectrum (if the hit is a match).

  Methods for identifying one or more HPPs by mass spectrometry are useful for diagnosis and prognosis. Preferably, one or more HPPs present in human plasma are detected by using the above method. Examples of techniques are described in US patent applications 02/0060290, 02/0137106, 02/0138208, 02/0142343, 02/0155509.

Diagnostic and Prognostic Applications The nucleic acid molecules, proteins, protein homologs, and antibodies described herein can be used in one or more of the following ways: diagnostic assays, prognostics, clinical trials Monitoring, and the pharmacogenetic methods detailed herein.

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

  The present invention provides methods for identifying polypeptides that are differentially expressed between two or more samples. “Differential expression” refers to differences in the amount or quality of a polypeptide between samples. The difference can occur at any stage of protein expression from transcription to post-translational modification. For example, using the protein array method, two sets of adsorbents are differentially retained by binding two samples to affinity spots in different sets of adsorbents (eg, chips) and comparing the recognition maps. Identify the polypeptide. Differential retention includes quantitative and qualitative retention in the polypeptide. For example, differences in protein post-translational modifications can be detected as differences in binding properties (eg, glycosylated proteins bind differentially to lectin adsorbents) or mass differences (eg, post-translational cleavage products). Can lead to map differences. In certain embodiments, the adsorbent can have an array of affinity spots selected for a combination of markers for diagnosing disease or syndrome.

  Differences in polypeptide levels between samples (eg, differentially expressed HPP in plasma samples) can be identified by desorption spectroscopy (eg, mass spectrometry) by exposing the sample to various analytical conditions. Proteins can be identified by detecting physicochemical properties (eg, molecular weight), and this information can be used to search a database for proteins with similar profiles.

  A preferred method for detecting HPP utilizes mass spectrometry techniques. The method provides information about the size and characteristics of a particular HPP isoform present in a sample that has been subjected to diagnosis or prognosis, eg, a biological sample. Mass spectrometry techniques are detailed in the section entitled “Detection of HPP by mass spectrometry”. Example 1 outlines a preferred detection scheme in which biological samples are separated by chromatography prior to characterization by mass spectrometry. The present invention relates to a method for detecting HPP in a biological sample, wherein the biological sample (eg, plasma, serum, lymph fluid, cerebrospinal fluid, cell lysate of a specific tissue) is fractionated by at least one chromatography step. And subjecting the fraction to mass spectrometry and comparing the characteristics of the polypeptide trait observed with mass spectrometry to the known characteristics of the HPP polypeptide.

  Particularly preferred methods include the detection of at least one HPP using retention material chromatography methods, including, for example, protein arrays or chips. The above method is described in the section entitled “Detection using protein arrays”. Preferably, the plurality of HPPs are detected from a biological sample, preferably plasma. An advantageous embodiment provides a protein chip capable of detecting HPP in an addressable array showing proteins present in human plasma.

  Accordingly, one aspect of the present invention is a method for diagnosing or prognosing an HPP-related disease or a disease showing any of the aforementioned HPP activities by using the molecule of the present invention (eg, HPP, HPP nucleic acid, or antibody). (Eg, diagnostic assays or prognostics). In another aspect, the invention includes a method of using the molecules of the invention for the diagnosis or prognosis of a subject, preferably a human subject, whose pathology is perturbed, for example, any of the aforementioned activities.

  For example, the invention relates to a method for determining whether HPP is expressed in a biological sample, comprising a) i) a polynucleotide that hybridizes with a HPP nucleic acid under stringent conditions, or ii) HPP Contacting a biological sample with a detectable polypeptide (eg, antibody) that selectively binds, and b) the presence or absence of hybridization between the polynucleotide and an RNA trait in the sample, or detectable A method comprising detecting the presence or absence of binding between the active polypeptide and a polypeptide in the sample. The detection of hybridization or binding as described above indicates that HPP is expressed in the sample and tails. Preferably, the polynucleotide is a primer, in which hybridization is detected by detecting the presence of an amplification product comprising the primer sequence, or the detectable polypeptide is an antibody.

  In certain embodiments, detection is performed by polymerase chain reaction (PCR) (eg, see US Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or alternatively ligation chain reaction (LCR) (eg, Landegren et al. al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) PNAS 91: 360-364), with the latter in particular the point mutations in the HPP encoding gene. (See Abravaya et al. (1995) Nucleic Acids Res. 23: 675-682).

  There is also a method for determining whether a mammal, preferably a human, exhibits an increase or decrease in the level of HPP expression, comprising: a) obtaining a biological sample from the mammal, and b) HPP in the biological sample. Alternatively, a method comprising comparing the amount of HPP RNA trait encoding HPP to the level detected in a control sample is also contemplated. A high amount of HPP or HPP RNA trait in the biological sample relative to the level detected or predicted from the control sample indicates an increased level of HPP expression in the mammal and is detected or predicted from the control sample A low amount of HPP or HPP RNA trait in the biological sample relative to the level indicates a decrease in HPP expression level in the mammal.

  The present invention also relates to the field of predictive medicine using diagnostic assays, prognostics, and clinical trial monitors for prognostic purposes. Accordingly, one aspect of the present invention is that an individual can detect harmful HPP expression or in a biological sample (eg, blood, plasma, cell, tissue) by measuring HPP and / or nucleic acid expression and HPP activity. The present invention relates to a diagnostic assay for measuring whether or not a disease or disorder associated with activity is present or at risk of developing a disease. The present invention also provides a prognostic (or predictive) assay that measures whether an individual is at risk of developing a disease associated with HPP, nucleic acid expression or activity. For example, mutations in the HPP encoding gene can be assayed in a biological sample. By using the above assay for prognostic or predictive purposes, an individual can be prophylactically treated before a disease characterized by or associated with HPP expression or activity begins.

  The term “biological sample” is intended to encompass tissues, cells and biological fluids isolated from an individual, as well as tissues, cells and fluids present within an individual. That is, by using the detection method of the present invention, HPP mRNA, protein or genomic DNA can be detected from biological samples in vitro and in vivo. Preferred biological samples are biological fluids such as cell lysates, lymph fluid, cerebrospinal fluid, blood and especially plasma. For example, in vitro detection techniques for HPP mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for HPP include mass spectrometry, enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. Southern hybridization is a technique for in vitro detection of HPP-encoded genomic DNA. Further, in vivo detection techniques for HPP include a method for introducing a labeled anti-HPP antibody into an individual.

Pharmaceutical Compositions One aspect of the present invention relates to pharmaceutical compositions suitable for administration. In one aspect, the pharmaceutical composition comprises one or more HPP polypeptides with a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises a compound capable of detecting or modulating HPP or HPP biological activity, including small molecules, peptides, HPP nucleic acid molecules and anti-HPP antibodies of the invention. The composition typically includes a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” refers to any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption delaying agent, and the like that is compatible with the pharmaceutical composition. All are included. The use of such media and agents for pharmaceutically active substances is well known in the art. Except where the conventional medium or agent 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, eg intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application may contain the following components: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycol, glycerin, propylene glycol or others Synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetic acid, citric acid or phosphate buffers, and isotonic Sex regulators 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 volume 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, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should 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 dispersion and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, 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, 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, such as an anti-HPP antibody, a sterile injectable solution may incorporate the requisite amount of active compound in a suitable solvent, optionally with one or a combination of the ingredients listed above, and then filter. It can be manufactured by sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other necessary ingredients from those enumerated above. In the case of sterile powders for the production of sterile injectable solutions, the preferred production methods are vacuum drying and lyophilization, where the active ingredient plus the desired additional ingredients are produced from the solution that has been previously sterile filtered. The

  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 that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as known in the art. Most preferably, the active compound is delivered to the subject by intravenous injection.

  In one embodiment, the active compounds are manufactured as carriers that protect the compound against rapid elimination from the body, for example as a controlled release formulation, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polybutyric acid. The method of manufacturing the above formulation can be easily understood by those skilled in the art. The material can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to cells infected with monoclonal antibodies against viral antigens) can also be used as pharmaceutically 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 (which is incorporated by reference).

  In a further aspect, the active compound can be coated in a microchip drug delivery device. The device is useful for controlled delivery of the composition to the bloodstream, cerebrospinal fluid, lymph or tissue of an individual without subjecting the protein composition to digestion or injection into the individual. Methods of using the microchip drug delivery device are described in US Pat. Nos. 6,123,861, 597898 and US Patent Application 200201119176A1, which are hereby incorporated by reference.

  It is especially advantageous to formulate oral or preferably parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physical discrete unit suitable as a unit dosage for the subject being treated, each unit with the pharmaceutical carrier required to provide the desired therapeutic effect. Containing a predetermined amount of active compound calculated in Specializations for dosage unit forms of the present invention are directly defined by the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art in formulating the active compound for individual therapy. Is done.

  Toxicity and therapeutic efficacy of the compounds are measured, for example, by standard pharmaceutical procedures in cell culture or laboratory animals for the determination of LD50 (lethal dose for 50% of the population) and ED50 (therapeutically effective dose in 50% of the population). obtain. 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. When compounds that exhibit toxic side effects can be used, pay attention to the design of delivery systems that target the compounds to affected tissue sites to reduce side effects by minimizing potential damage to uninfected cells. Should do.

  Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in the human body. The dosage of the compound is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For compounds used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. By formulating the dose in an animal model, a useful dose in the human body can be more accurately determined. Levels in plasma can be measured, for example, by high performance liquid chromatography.

  The pharmaceutical composition can be enclosed in a container, pack or dispenser together with instructions for administration.

Disease Treatment The HPP polypeptides of the invention and the HPP modulators and HPP-related compositions of the invention can be used in the treatment or prevention of HPP-related disorders. The above diseases and disorders include, but are not limited to, the following diseases and disorders. Example 4 provides a suitable method for determining diseases and disorders in which the HPP polypeptides of the invention can be used for therapy, prognosis and / or diagnosis. However, there are other suitable methods known in the art that can be used for this purpose.

Treatment of Diseases Associated with Cell Proliferation and Cancer According to one aspect of the present invention, HPP-38 polypeptides are provided for the treatment of diseases or conditions associated with cancer diseases or hyperplasia or for the prognosis or diagnosis of the diseases. .

  The polypeptide HPP-38 detected from human plasma according to the present invention was found to have an amino acid sequence that is part of the esophageal cancer associated gene 2 (ECRG2) Swiss-Prot P58062. In the present specification, the term HPP-38 polypeptide encompasses the polypeptides of the sequences set forth in SEQ ID NOs: 391 and 392. In addition, variants and derivatives are desirably represented by SEQ ID NOs: 391 and 392 in a proportion of at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 99%. Those variants and derivatives are also encompassed if they have a sequence that is identical to the rendered sequence. Variants and derivatives may have 1, 2 or 3 amino acid substitutions, deletions or insertions relative to the amino acid sequence shown in SEQ ID NOs: 391 and 392. Variants and derivatives can also be chemically modified as described herein. The HPP-38 polypeptide was synthesized by Gene Plot, Inc. (Geneva, Switzerland). The peptide was injected into mice by the method described in Example 4, and gene expression profiling was performed in many organs. . Table 4 shows that HPP-38 polypeptides affect the expression of genes involved in the positive regulation of apoptosis, genes that are indicative of breast cancer, stress response genes, and tumor suppressor genes. In addition, many RNAs encoding tight junction proteins were down-regulated in a coordinated manner in blood, heart, kidney, liver and spleen. All of these genes can be associated with the regulation of cell growth. To confirm our findings, ECRG2 was tested for its growth regulatory activity in a cell growth assay at the MDS Pharma Service. ECRG2 was added to the supernatant of 15 cancer cell lines at concentrations ranging from 0.01 to 100 micromolar and cell growth was measured over time. ECRG2 is MCF-7 (chest), DLD-1 (colon), A-498 (kidney), HepG2 (liver), A549 (lung), SK-MEL-5 (melanoma), SK-N-MC ( Significantly inhibits the growth of 11 of 15 cell lines tested (including neuroepithelioma), PANC-1 (pancreas), PC-3 (prostate), A431 (skin), MES-SA (uterus) <50%). IC-50 was less than 20 micromolar for neuroepithelioma, prostate, skin and uterine cell lines.

  Accordingly, the present invention provides an HPP-38 polypeptide as a therapeutic method suitable for the treatment of cancer diseases or diseases or conditions associated with hyperplasia, the methods comprising humans suffering from the above diseases Administering an effective amount of an HPP-38 polypeptide to the mammal. Preferably, the cancer disease is selected from the group consisting of neuroepithelioma, prostate cancer, basal cell carcinoma, squamous cell carcinoma, melanoma and cervical carcinoma in situ. In another preferred embodiment, the disease or condition associated with hyperplasia is a disease selected from the group consisting of fibrosis, prostate hyperplasia, adrenal hyperplasia, endometrial hyperplasia, psoriasis, hyperplasia resulting from inflammation or State. In a preferred aspect, the use of an HPP-38 polypeptide in medicine is provided.

  According to another aspect, the present invention provides a method for prognosis or diagnosis of a disease or condition associated with cancer disease or hyperplasia comprising detecting plasma levels of HPP-38 polypeptide, wherein the level is An increase is indicative of a disease or condition associated with cancer disease or hyperplasia. Within the scope of the present invention, an increase in HPP-38 polypeptide plasma levels is relative to HPP-38 polypeptide plasma levels found in individuals who are not afflicted with a disease or condition associated with cancer disease or hyperplasia. It is. 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 another aspect, the present invention is a method for prognosis or diagnosis of a disease or condition associated with a cancer disease or hyperplasia, i) at least as shown in Table 4 in a sample of a suitable tissue obtained from a subject. Detecting the expression level of a gene provides a first value, and ii) comparing the first value with the expression level of the gene from a disease free subject and comparing with a sample from a disease free subject Provided is a method wherein the level of expression in the subject sample is an indication that the subject is susceptible or afflicted with a disease or condition associated with cancer disease or hyperplasia. Gene expression can be detected at the mRNA or protein level. Suitable tissues include but are not limited to skin, prostate, pancreas, uterus, lung, liver, intestine, kidney. mRNA expression levels can be detected by appropriate techniques such as microarray analysis, Northern blot analysis, reverse transcription PCR and real-time quantitative PCR. Similarly, protein levels can be detected by appropriate techniques, eg, through Western blotting using a labeled probe specific for the protein.

  In a preferred embodiment of the above aspect, the gene (s) are selected from the gene (s) shown in Table 4 that are up-regulated or down-regulated. Preferably, the gene (s) are 1.2-fold or higher, 1.3-fold or higher, or 1.5-fold or higher upregulated gene (s) shown in Table 4 Selected from the genes shown in Table 4 that are down-regulated 0.8 times or less, 0.7 times or less. In yet another preferred embodiment of the above aspect, the expression of at least 1, 2, 3, 4 or 5 genes is measured.

  According to another aspect, the present invention is a method for identifying a modulator of a disease or condition associated with cancer disease or hyperplasia, i) HPP-38 under sample conditions acceptable for HPP-38 biological activity. Contacting the polypeptide with the test compound, ii) measuring the level of at least one HPP-38 biological activity, and iii) comparing the level with a level of a control sample lacking the test compound to the level. provide. In a preferred embodiment, the test compound that induces said level of change is selected for further testing as an HPP-38 modulator for the prevention and / or therapeutic treatment of a disease or condition associated with cancer disease or hyperplasia. In a preferred embodiment, the HPP-38 biological activity level is determined by detecting the expression level of one or more genes shown in Table 4.

Treatment of diseases associated with iron transport or iron balance deficiency The polypeptide HPP-13 detected in human plasma according to the present invention may have an amino acid sequence that is part of human thymosin beta 4 (Swiss-Prot P01253). It was found. In the present specification, the term HPP-13 polypeptide encompasses the polypeptide of the sequence shown in SEQ ID NO: 393 (human form and short mouse isoform) and SEQ ID NO: 394 (long mouse isoform). Also, variants and derivatives are desirably shown in SEQ ID NOs: 393 and 394 in a proportion of at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 99%. Those variants and derivatives are also encompassed if they have a sequence that is identical to the rendered sequence. Variants and derivatives may have 1, 2 or 3 amino acid substitutions, deletions or insertions relative to the amino acid sequence set forth in SEQ ID NOs: 393 and 394. Variants and derivatives can also be chemically modified as described herein. The HPP-13 polypeptide was synthesized by Gene Plot, Inc. (Geneva, Switzerland). The peptide was injected into mice by the method described in Example 4, and gene expression profiling was performed in many organs. . Tables 6, 7, 8, 9 and / or 10 show that HPP-13 polypeptides affect the expression of genes involved in heme biosynthesis and metabolism, porphyrin biosynthesis and metabolism and erythropoiesis. . Furthermore, the iron balance of the diet and genes involved in iron transport are differentially regulated.

  Accordingly, the present invention provides HPP-13 polypeptides for the treatment of conditions associated with diseases or conditions in which the body's iron balance is altered, leading to iron overload disease, anemia, or insufficient red blood cell production. In one aspect, the HPP-13 polypeptide is provided as a therapeutic suitable for treating a disease or condition associated with iron balance or iron transport, and an effective amount of the HPP-13 polypeptide is afflicted with the disease. It is administered to mammals including humans. Preferably, the disease or condition associated with iron balance or iron transport comprises hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions associated with iron overload, anemia, sickle cell anemia Selected from the group. In a preferred embodiment, the use of HPP-13 in medicine is provided.

  According to another aspect, the present invention is a method for prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising the detection of plasma levels of HPP-13 polypeptide, wherein the level increase is iron balance or iron transport. A method is provided that is indicative of a disease or condition associated with. Within the scope of this specification, an increase in HPP-13 polypeptide plasma levels is relative to HPP-13 polypeptide plasma levels found in individuals not afflicted with a disease or condition associated with iron balance or iron transport. is there. 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 another aspect, the present invention is a method of prognosis or diagnosis of a disease or condition related to iron balance or iron transport, i) in a sample of a suitable tissue obtained from a subject, as shown in Tables 6, 7, 8, 9 And / or obtaining the first value by detecting the expression level of at least one gene identified in 10, and ii) comparing the first value with the expression level of the gene from the disease-free subject, Provide a method wherein the level of expression in a subject sample relative to a sample from a subject is an indication that the subject is susceptible to or suffering from a disease or condition associated with iron balance or iron transport . Gene expression can be detected at the mRNA or protein level. Suitable tissues include, but are not limited to, liver, heart, intestine such as duodenum, spleen, bone marrow. mRNA expression levels can be detected by appropriate techniques such as microarray analysis, Northern blot analysis, reverse transcription PCR and real-time quantitative PCR. Similarly, protein levels can be detected by suitable techniques, eg, through Western blotting by using a protein-specific labeled probe.

  In preferred embodiments of the above aspects, the gene (s) are selected from the gene (s) shown in Tables 6, 7, 8, 9 and / or 10 that are up-regulated or down-regulated. . Preferably, the gene (s) are up-regulated 1.2 fold or more, 1.3 fold or more, or 1.5 fold or more, and Tables 6, 7, 8, 9 and / or Or Table 6,7,8 down-regulated from the gene (s) shown in 10, or 0.8-fold or less, 0.7-fold or less, or 0.6-fold or less , 9 and / or 10 selected from the gene (s) selected from the genes shown. In yet another preferred embodiment of the above aspect, the expression of at least 1, 2, 3, 4 or 5 genes is measured.

  According to another aspect, the present invention is a method for identifying a modulator of a disease or condition associated with iron balance or iron transport, comprising i) HPP-13 under sample conditions acceptable for HPP-13 biological activity. Contacting the polypeptide with the test compound, ii) measuring the level of at least one HPP-13 biological activity, and iii) comparing said level to that of a control sample lacking the test compound. I will provide a. In a preferred embodiment, a test compound that induces said level of change is selected for further testing as an HPP-13 modulator for prevention and / or therapeutic treatment of diseases or conditions associated with iron balance or iron transport. . In a preferred embodiment, the level of HPP-13 biological activity is determined by detecting the expression level of one or more genes shown in Tables 6, 7, 8, 9 and / or 10.

Treatment of Diseases Related to Neurodegeneration Table 11 shows that treatment of mice with HPP-13 polypeptides resulted in these polypeptides being neurodegenerative and peripheral neuropathies such as multiple sclerosis, demyelinating diseases, Guillain- Genes that are useful for the treatment of pathologies of Valley disease, diabetic neuropathy, chemotherapy-induced neuropathy, autoimmunity-related neuropathy, CNS-related neuropathy such as Alzheimer's disease and eg stroke-related nerve damage (nervous ischemia) It shows that an expression profile was obtained.

  Further provided by the present invention is a GPA101 polypeptide that is useful in the treatment of the above pathologies. GAP101 is a polypeptide having a randomized sequence of HPP-13. In the specification of the present invention, the term GPA101 polypeptide encompasses the polypeptide of the sequence shown in SEQ ID NO: 397 and the variants and derivatives described above. That is, in one embodiment, the present invention provides a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 397, and in a preferred embodiment, the present invention provides a polypeptide consisting of the sequence shown in SEQ ID NO: 397. .

  Accordingly, the present invention provides HPP-13 and GPA101 polypeptides as suitable therapeutics for treating diseases or conditions associated with neurodegeneration, in which mammals including humans suffering from the diseases Is administered an effective amount of HPP-13 or GPA101 polypeptide. Preferably, the disease or condition associated with neurodegeneration is spinal or CNS injury, Alzheimer's disease, Parkinson's disease, multiple sclerosis, ALS (amyotrophic lateral sclerosis), peripheral neuropathy, Guillain-Barre disease, Selected from the group consisting of diabetic neuropathy and demyelinating neuropathy. In a preferred aspect, the use of HPP-13 or GPA101 polypeptide in medicine is provided.

  According to another aspect, the present invention is a method for prognosis or diagnosis of a disease or condition associated with neurodegeneration comprising the detection of plasma levels of HPP-13 polypeptide, wherein the increased level is associated with neurodegeneration. Provide a method that is indicative of Within the scope of the present invention, an increase in HPP-13 polypeptide plasma levels is relative to HPP-13 polypeptide plasma levels found in individuals not afflicted with a disease or condition associated with neurodegeneration. 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 another aspect, the present invention is a method for prognosis or diagnosis of a disease or condition associated with neurodegeneration, i) expression of at least one gene identified in Table 11 in a sample of a suitable tissue obtained from a subject. A first value is obtained by detecting the level, and ii) expression in the subject sample when compared to a sample from the disease free subject, comprising comparing the first value with the expression level of the gene from the disease free subject Provide a method wherein the level of level is an indication that a subject is susceptible to or suffering from a disease or condition associated with neurodegeneration. Gene expression can be detected at the mRNA or protein level. Suitable tissues include, but are not limited to, brain, spinal cord, motor innervation tissues, peripheral nerves, central and peripheral nerve tissues. mRNA expression levels can be detected by appropriate techniques such as microarray analysis, Northern blot analysis, reverse transcription PCR and real-time quantitative PCR. Similarly, protein levels can be detected by appropriate techniques, eg, through Western blotting by using a protein-specific labeled probe.

  In a preferred embodiment of the above aspect, the gene (s) is selected from the gene (s) shown in Table 11 that are up-regulated or down-regulated. Preferably, the gene (s) is 1.2-fold or higher, 1.3-fold or higher, or 1.5-fold or higher upregulated gene (s) shown in Table 11 Or a gene selected from the genes shown in Table 11 that are down-regulated from 0.8-fold or less, 0.7-fold or less, or 0.6-fold or less ) Is selected. In yet another preferred embodiment of the above aspect, the expression of at least 1, 2, 3, 4 or 5 genes is measured.

  According to another aspect, the present invention provides a method for identifying a modulator of a disease or condition associated with neurodegeneration, i) under conditions of sample acceptable for HPP-13 or GPA101 biological activity, Contacting GPA101 polypeptide with a test compound, ii) measuring the level of at least one HPP-13 or GPA101 biological activity, and iii) comparing said level to that of a control sample lacking the test compound. A method comprising: In a preferred embodiment, test compounds that induce the above level of change are selected for further testing as HPP-13 or GPA101 modulators for the prevention and / or therapeutic treatment of diseases or conditions associated with neurodegeneration. In a preferred embodiment, the level of HPP-13 biological activity is measured and the expression of one or more genes shown in Table 11 is detected.

Treatment of Diseases Associated with a Deficiency in Glucose Metabolism Polypeptide HPP-23 detected in human plasma according to the present invention was found to have an amino acid sequence that is part of pancreatatin (Swiss-Prot P10645). . In the present specification, the term HPP-23 polypeptide encompasses the polypeptides of the sequences set forth in SEQ ID NO: 395 and SEQ ID NO: 396. In particularly preferred embodiments, the polypeptide is not amidated. Also, variants and derivatives are desirably shown in SEQ ID NOs: 395 and 396 in a proportion of at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 99%. Those variants and derivatives are also encompassed if they have a sequence that is identical to the rendered sequence. Variants and derivatives may have 1, 2 or 3 amino acid substitutions, deletions or insertions relative to the amino acid sequence shown in SEQ ID NOs: 395 and 396. Variants and derivatives can also be chemically modified as described herein. The HPP-23 polypeptide was synthesized by Gene Plot, Incorporated (Geneva, Switzerland). The peptide was injected into mice by the method described in Example 4, and gene expression profiling was performed in many organs. . In the brain and other organs, HPP-23 polypeptide injection had a strong effect on RNA expression levels of genes involved in glucose metabolism, other carbohydrate catabolic pathways, angiogenesis and inflammatory responses (Table 12). . Furthermore, the major steps of the pancreatin statin signaling pathway can be reconstituted.

  HPP-23 in three mouse models at MDS Pharma Service: fasted ICR mice, glucose-loaded ICR mice, and db / db (NIDDM, non-insulin dependent diabetes mellitus) mice, a model of type 2 diabetes It was tested for its modulator effect on glucose levels. At a dose of 10 mg / kg, non-amidated pancreatinstatin significantly reduced serum glucose levels in glucose-loaded mice by as much as 20% compared to vehicle. This is the first record of the biological activity of the non-amidated form of pancreatatin. Biological activity is the regulation of serum glucose levels.

  Accordingly, the present invention provides HPP-23 polypeptides as suitable therapeutics for treating diseases associated with impaired serum glucose regulation, in which mammals, including humans suffering from the above diseases, are treated with HPP-23. An effective amount of -23 polypeptide is administered. Preferably, the disease associated with impaired serum glucose regulation is diabetes or other hyperglycemic conditions. In a particularly preferred embodiment, HPP-23 reduces blood glucose levels when administered to a mammal, such as a human. In one preferred aspect, the use of HPP-23 in medicine is provided.

  According to another aspect, the present invention is a method for prognosis or diagnosis of a disease associated with impaired serum glucose regulation comprising detection of plasma levels of HPP-23 polypeptide, wherein increased levels indicate a disease associated with impaired serum glucose regulation Provide a way that is. Within the scope of the present invention, an increase in HPP-23 polypeptide plasma levels is relative to HPP-23 polypeptide plasma levels found in individuals who are not afflicted with a disease associated with impaired serum glucose regulation. 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 another aspect, the present invention is a method of prognosis or diagnosis of a disease associated with impaired serum glucose regulation, i) the expression level of at least one gene identified in Table 12 in a sample of a suitable tissue obtained from a subject. And ii) comparing the first value with the expression level of the gene from the disease free subject, and comparing the expression value to the sample from the disease free subject. Provides a method wherein the subject is susceptible to or afflicted with a disease associated with impaired serum glucose control. Gene expression can be detected at the mRNA or protein level. Suitable tissues include, but are not limited to, adipose (adipose) tissue, heart, pancreas, muscle, and brain. mRNA expression levels can be detected by appropriate techniques such as microarray analysis, Northern blot analysis, reverse transcription PCR and real-time quantitative PCR. Similarly, protein levels can be detected by appropriate techniques, eg, through Western blotting by using a protein-specific labeled probe.

  In a preferred embodiment of the above aspect, the gene (s) is selected from the gene (s) shown in Table 12 that are up-regulated or down-regulated. Preferably, the gene (s) are 1.2-fold or higher, 1.3-fold or higher, or 1.5-fold or higher upregulated gene (s) shown in Table 12 Or a gene selected from the genes shown in Table 12 that are down-regulated 0.8 times or less, 0.7 times or less, or 0.6 times or less. ) Is selected. In yet another preferred embodiment of the above aspect, the expression of at least 1, 2, 3, 4 or 5 genes is measured.

  According to another aspect, the present invention relates to a method of identifying a modulator of a disease associated with impaired serum glucose regulation, i) testing with HPP-23 polypeptides under acceptable sample conditions for HPP-23 biological activity. Contacting the compound, ii) measuring the level of at least one HPP-23 biological activity, and iii) comparing said level to that of a control sample lacking the test compound. In a preferred embodiment, test compounds that induce the above level of change are selected for further testing as HPP-23 modulators for the prevention and / or therapeutic treatment of diseases associated with impaired serum glucose regulation. In a preferred embodiment, the level of HPP-23 biological activity is determined by detecting the expression level of one or more genes shown in Table 12.

Treatment of Diseases Associated with Metabolic Disorders In a further aspect of the invention, HPP-23 polypeptides are provided as suitable therapeutics for the treatment of metabolic disorders, particularly amyloidosis. Also, HPP-23 polypeptide is provided as part of the present invention as a suitable treatment for arrhythmias, particularly bradycardia, tachycardia, sinus insufficiency syndrome, and angiogenesis, particularly stroke, macular regeneration, cancer. .

  Amyloidosis is a group of metabolic disorders that have not been fully elucidated, resulting from the deposition of protein-containing fibrils (amyloid) in tissues. The disease can cause localized or widespread organ failure. Amyloid can infiltrate many organs including the heart and blood vessels, brain and peripheral nerves. Thus, the clinical manifestations of amyloidosis are so varied that the disease can mimic other conditions ranging from nephrotic syndrome to dementia or congestive heart failure. The pathway of oxidative stress appears to be responsible for the development of amyloid fibrils. Oxidative stress promotes loss of cell integrity, microglial phagocytosis and thrombus destruction by accelerating both nuclear DNA degradation and membrane phosphatidylserine exposure in nerve and vascular cells. Critical in the ability to promote cell survival during oxidative stress is the modulation of the metabotropic glutamate system, cell cycle regulation in postmitotic neurons, and control of GSK-3beta activity and presenilin integrity. These cellular pathways ultimately converge to more central cellular mechanisms including maintenance of mitochondrial membrane permeability through Bcl-2 family members, trophic factors, and mitochondrial energy storage.

  It was found by the present invention that HPP-23 polypeptide affects amyloidosis disease in the brain of pancreatinatin: HPP-23 polypeptide treated mice in brain tissue. HPP-23 polypeptide upregulates presenilin 1, a large cluster of peroxiredoxin genes, mitochondrial NADH-dehydrogenase, endothelial nitric oxide, cytochrome c-oxidase (15 probe set). (All of these groups were unaffected by the controls). These genes represent a major change in the redox system and refer to oxidative stress. In addition, the expression of many cell cycle genes (cyclin and cyclin dependent kinases) is altered.

  Another indicator of the involvement of pancreatin and HPP-23 polypeptides in the expression of amyloidosis is a strong upregulation of the tubulin gene (tubulin alpha 1,2,4 beta, gamma, cofactor alpha). Tubulin-amyloid deposits are found in familial cerebral amyloid angiopathy (Baumann et al., Biochem Biophys Res Comm, 1996, 219: 238-242). (And those changes are not found in the control brain).

  Strong expression of presenilin and apolipoprotein E (apo E) involved in Notch- and amyloid precursor protein (APP) processing has been shown to be part of the mechanism for familial Alzheimer's disease progression (Tezapsidis et al. FASEB 2003, 17: 1322-1324). Both presenilin and apoE were upregulated by HPP-23 polypeptide. The amyloid precursor gene (APP) and various APP binding proteins are also upregulated by HPP-23. Schipper (Ann NY Acad Sci 2004, 1012: 84-93) shows that heme oxygenase (HO-1) expression is stimulated under pro-oxidant stimulators such as dopamine, hydrogen peroxide, beta-amyloid, etc. And HO-1 have been found to be up-regulated in diseased CNS tissues due to Alzheimer's disease, Parkinson's disease, multiple sclerosis and other degenerative and non-degenerative CNS diseases. HPP-23 polypeptide leads to up-regulation of HO-1 in the brain.

  Ferreiro et al (J Neurosci Res, 2004, 76: 872-880) show that in vitro cultured neurons treated with amyloid-beta peptide are at least partially due to perturbation of intracellular Ca (2+) homeostasis. Announced to show neuronal loss. Inhibition of Ca (2+) release from the endoplasmic reticulum, mediated by the ryanodine receptor (Ryr) and IP (3) R, eliminated this neurotoxic effect. Ryr is down-regulated approximately 2-fold in HPP-23 polypeptide treated brains.

  Pancreatastatin in heart tissue: The ryanodine receptor itself is not affected in the heart of pancreasatin treated animals. However, many ryr binding proteins such as FKBP12, calmodulin, sorcin, triazine, and phosphatase 1 are down-regulated. Sorcin binds to presenilin 1, which is also down-regulated in the heart. Presenilin is in particular part of the Notch pathway consisting of ADAM17, notch (notch), and target snail, hes1 and homeobox gene msx. snail and hes1 were up-regulated and all other personnel were down-regulated. The notch inhibitor, numb, was up-regulated and pointed to general inhibition of the pathway. Another presenilin binding protein is restin. The disruption of this complex is associated with both reduced secretion of endogenous amyloid beta peptide and reduced uptake of exogenous amyloid beta from cultured cell media (Tezapsidis et al, FASEB 2003, 17: 1322-1324). Restin was upregulated in the heart of HPP-23 polypeptide treated mice.

  The Notch signaling pathway is essential for heart development (eg, Timmerman et al, Genes Dev 2004 18: 99-115). Furthermore, it cross-talks with VEGF (vascular endothelial growth factor), for example notch4 is an inhibitor of VEGF signaling, and VEGF and notch1 regulate germination (Liu et al. Mol Cell Biol 2003, 23: 14-). 25). In addition to changes in VEGF expression in both the heart and brain, we have detected changes in angiomotin and angiopoietin and other angiogenesis-related genes. Nakajima et al announced that abnormal blood vessels develop in mice lacking presenilin-1 (Mech dev 2003, 120: 657-667). From our data, this may be due to the participation of presenilin in the notch pathway.

  Taken together, these gene expression changes in the heart are associated with HPP-23 in conditions such as arrhythmias (bradycardia, tachycardia, sinus syndrome) and angiogenesis (eg, stroke, macular regeneration, cancer). Polypeptide participation is indicated. Accordingly, the present invention provides HPP-23 polypeptides for the therapeutic, prognostic and diagnostic methods and screening methods (eg, modulator identification methods) described above for these indications.

  The disclosures of the references cited throughout this specification are hereby incorporated by reference. Although the invention has been described generally, it is for illustrative purposes only and is not intended to be limiting unless otherwise specified for further understanding by reference to certain embodiments described herein. Should be able to deepen.

Example 1: Characterization of HPP levels Plasma fractions from over 50 healthy male volunteers were pooled to dilute phenotypic differences and provide a large pool capacity. Blood (100-450 ml) was obtained by using a standard venipuncture procedure in a setting after obtaining informed consent in writing at a medical center. Samples were barcoded and plasma was prepared by centrifugation and filter removal of white blood cells by standard techniques. The same treatment was ensured for all samples by tightly controlling the number of times, temperature and centrifugation conditions and subsequent procedures. Protease inhibitors (complete, roche) were added according to the manufacturer's instructions and mixed to ensure dissolution. The plasma samples were then frozen and stored at -80 ° C. After careful consideration of medical history and clinical chemistry parameters, aliquots of at least 50 samples were pooled. According to the microplot, a volume of 2.5 L (out of a total 6 L) was subjected to separation by multiple chromatography steps. The TM process is 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.

  The filtrates were each 300 ml HSA ligand Sepharose fast flow column (Amersham, Uppsala, Sweden), 5 cm ID, 15 cm long, and 100 ml Protein G Sepharose fast flow column (Amersham, Uppsala, Sweden), 5 cm ID, 5 cm length 2 It was injected into the inline column of the book.

  The column was equilibrated with 50 mM PO4 buffer, pH 7.1, 0.15 M NaCl and washed. The flow rate was 5 ml / min.

  The non-retained fraction (53 g of depleted polypeptide) was frozen until the second stage. Work was performed 20 times.

Step 2: Gel filtration / reverse phase capture step Samples from Step 1 were 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: a 2 × 9.5 liter Superdex 75 (Amersham, UK) column, 14 cm ID, 62 cm long. The column was equilibrated with 50 mM PO4 buffer pH 7.4, 0.1 M NaCl, 8 M urea. Hydrophobic impurities were retained in the reverse phase precolumn: 150 ml of PLRPS (Polymer Labs, UK). The precolumn was replaced for sample injection. Gel filtration was performed at a flow rate of 40 ml / min.

  Low molecular weight protein (<20 kDa) was directed to an in-line reverse phase capture column: 50 ml of PLRPS 100 angstrom (Polymer Labs, UK). The gel filtration eluent was sent to the reverse phase capture column by switching the three-way valve controlled injection on the PLRPS column with a cutoff of 33 mAU (280 nm). This cut-off value was established by first giving an estimated range of OD values using SDS-PAGE followed by evaluation of three cut-off values (high value, median and low value of the OD range). . The final cutoff value was chosen to maximize the low molecular weight protein obtained with a low molecular weight protein ratio of at least 85%. Low molecular weight proteins and peptides were eluted from the reverse phase capture PLRPS column with a one column volume gradient of 0.1% TFA, 80% CH3CN in water.

  About 1.5 g of small protein was obtained, as measured by analytical gel filtration HPLC monitored at 210 nm according to BSA standard, of which 1.3 g was <20 kDa. The elution fraction (50 ml) was frozen to the next stage. Work was performed 20 times. At the end of this stage, all reverse phase eluents were thawed, pooled (1 liter) and divided into 7 polypeptide containers (143 ml). The container was kept at −20 ° C. until used in the next step.

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

  Samples were injected into a 100 ml source 15S column (Amersham, Uppsala, Sweden), 35 mm ID, 100 mm length. The column was equilibrated with buffer A and washed. The flow rate was 10 ml / min.

Proteins and peptides were eluted with a step gradient from 100% buffer A to 100% buffer B (buffer A containing 1 M NaCl):
3 column volume 7.5% B (75 mM NaCl).
3 column capacity 10% B (100 mM NaCl)
3 column volume 17.5% B (175 mM NaCl)
2 column capacity 22.5% B (225 mM NaCl)
2 column volume 27.5% B (275 mM NaCl)
2 column volume 100% B (1M NaCl).

  45-60 fractions were collected based on peak. The work was carried out 7 times. After 7 runs, 18 fractions were obtained by pooling fractions during and between operations. The fraction was kept at −20 ° C. until used in 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 the 18 cation exchange fractions was reduced with dithioerythritol (DTE, 30 mM, 3 hours at 37 ° C.) and iodoacetamide (120 mM, 1 hour 25 hours in the dark). Alkylation). The subsequent reaction was stopped by adding DTE (30 mM) followed by acidification (TFA, 0.1%). Fractions were then injected on an Uptiper C8, 5 μm, 300 Angstrom column (Interkim, France), 21 mm ID, 150 mm length. Injection was performed at a flow rate of 10 ml / min.

  The C8 column was equilibrated with 0.1% TFA in water (solution A) and washed. Proteins and peptides were eluted with a biphasic gradient from 100% A to 100% B (0.1% TFA, 80% CH3CN, in water) in 60 minutes. The flow rate was 20 ml / min. 30 fractions of 40 ml were collected.

  Based on the optical density (OD) measured at 280 nm of each fraction, reflecting the protein concentration in that fraction, an aliquot of similar protein content was made for each fraction.

  To prevent over-drying, 500 μl of 10% glycerin in water was added in each fraction, and then all aliquots were frozen and stored until further use, but one fraction was obtained by Speed Vac (Savant, Fischer, Geneva). Dried. The dried fraction was stored at −20 ° C. until use in the next step.

Step 5: Reversed phase HPLC fraction 2
The dried sample from step 4 was resuspended in 1 ml of solution A (0.03% TFA in water), Vydac LCMS C4 column, 5 micrometer, 300 angstrom (Vydac, USA), 4.6 mm ID, 150 mm length Injected in. 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 position of the sample in reverse phase HPLC fraction 1. As-is mass data was obtained using electrospray ion trap mass spectrometry. Sixteen different gradients were used with CH3CN concentration range-and + 5% CH3CN for RP1 fraction corresponding solvent concentrations. For proteins that eluted with RP1 with a solvent concentration equal to or greater than 30% CH3CN, the starting elution conditions for the RP2 gradient were set at CH3CN percentage at RP1 elution concentration-30%. Twenty-four elution fractions were collected in a deep well plate employing an optimized SpeedVac enrichment and different optimized collection configurations designed for further robotic processing.

Step 6: Mass Detection After reverse phase HPLC fraction 2 to 96 well deep well plates (DWP), 12960 (18 × 30 × 24) fractions were collected. For small capacity (2.5%) capacity, we switched to on-line analysis using LC-ESI-MS (Bruker Esquire). An aliquot of undigested protein was mixed with a MALDI matrix and spotted on a MALDI plate combined with a mass measurement standard and a sensitivity standard. An automatic spot device (Bruker MALDI sample preparation robot) was used. Two different MALDI matrices were used: sinapic acid (SA), also known as sinapinic acid, trans-3,5-dimethoxy-4-hydroxycinnamic acid, and alpha-cyano- 4-hydroxycinnamic acid (HCCA). The MALDI plate was subjected to mass detection using a Bruker Reflex III MALDI MS instrument. 96 well plates were stored at + 4 ° C.

  A 96-well plate (DWP) was collected and subjected to two successive concentration steps. By drying with SpeedVac, the volume was concentrated from 0.8 ml per well to about 50 microliters, then redissolved per well to about 200 microliters and reconcentrated to about 50 microliters at + 4 ° C. Stored in The protein was then digested by rebuffering, adding trypsin to the wells, sealing, and incubating the plate for 12 hours at 37 ° C. before quenching (adding formic acid to lower the pH 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 that trypsin was optimally used and that most concentrated fractions were completely digested. An automated spot device (Bruker MALDI sample preparation robot) was used to deposit an aliquot from each well that had been premixed with the HCCA matrix onto a MALDI plate with sensitivity and mass measurement standards. MALDI plates were analyzed using a Bruker Reflex III MALDI MS instrument. The contents from each well of the 96 well plate were analyzed by LC-ESI-MS-MS Bruker Esquire ESI ion-trap MS instrument.

Step 7: Detection and identification of low abundance peptides in human plasma The separated fractions were subjected to mass spectrometry (both MALDI and LC-ESI-MS-MS) for further separation and detection.

  Over 1.5 million MS / MS spectra were generated, and 330,000 were used for validation of algorithms developed for manual testing and automated identification. Intact mass data, peptide mass fingerprint and peptide sequence data were integrated for protein identification and characterization. Proteins were identified using mascot software (Matrix Science Limited, London, UK) and the results obtained from peptide identification were checked by manual analysis of the spectra. MS / MS spectra were translated by a custom identification engine (Olav) accessing a set of six different databases (SwissProt, published EST and genomic sequence data, and patent databases). The integration phase confirmed the consistency of the comprehensive identification results across different databases. Annotations (automatic and manual) of the identified identification results further characterize the important features of the observed polypeptides.

  The protein separation and identification method according to the invention is very sensitive. The Micropurot.TM process can detect even very low abundance proteins with plasma concentrations in the range of 50 pM. The accuracy was confirmed while carrying out the method described herein. In particular, proteins with well-characterized roles were detected in human plasma.

Example 2: Chemical synthesis of HPP In this example, the HPP of the present invention is synthesized. The peptide fragment intermediate is first synthesized and then assembled into the desired polypeptide.

  HPP can be made initially, for example, in 5 fragments selected to have a Cys residue at the N-terminus of the coupled fragment. Fragment 1 is first coupled to fragment 2 to give a first product, and after preparative HPLC purification, the first product is coupled to fragment 3 to give a second product. After preparative HPLC purification, the third product is obtained by coupling the second product to fragment 4. Finally, after preparative HPLC purification, the desired product is obtained by coupling the third product to fragment 5, which is purified and regenerated.

Thioester formation Fragments 2, 3, 4 and 5 are synthesized in a thioester-generating resin as described above. For this purpose, the following resin is prepared: S-acetylthioglycolic acid pentafluorophenyl ester is coupled to Leu-PAM resin essentially under the conditions described in Hackeng et al (1999). In the first case, the resulting resin is used as a 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. The N ^alpha of N- terminal Cys residues from fragment 2 5, at the end of each strand in place of Cys with conventional N ^alpha or S ^beta protective Boc- thioproline (Boc-SPr, i.e. Boc-L-thioproline ) By coupling, eg, Brik et al., J. Org. Chem., 65: 3829-3835 (2000).

Peptide synthesis
In situ neutralization / 2- (1H-benzotriazol-1-yl) -1,1,1,3 as described by Schnolzer et al, Int. J. Peptide Protein Res., 40: 180-193 (1992). Solid phase synthesis is performed on a custom modified 433A peptide synthesizer manufactured by Applied Biosystems using the 3,3-tetramethyluronium hexafluoro-phosphate (HBTU) activation protocol. Each synthesis cycle is 1-2 minutes N ^alpha -Boc-removal by treatment, 1 minute DMF flow wash, 10 min coupling with preactivated Boc- amino acids 2.0mmol in the presence of excess DIEA in neat TFA Consists of time and second DMF flow wash. 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 coupling of the Gln residue, a high temperature (TFA / DMF) -catalyzed pyrrolidone carboxylic acid formation is prevented by using a dichloromethane flow wash before and after deprotection with TFA. 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 (Olpergen Pharma, Heidelberg, Germany). Other amino acids are used without side chain protection. C-terminal fragment 1 was synthesized with Boc-Leu-O—CH ₂ -Pam resin (loaded resin, 0.71 mmol / g), and fragment 2 was Boc-Xaa-S—CH ₂ through 5 machine-assisted synthesis. Start with -CO-Leu-Pam resin. This resin is obtained by coupling S-acetylthioglycolic acid pentafluorophenyl ester to Leu-PAM resin under standard conditions. After removal of the acetyl protecting group by treatment with 10% mercaptoethanol, 10% piperidine in DMF for 30 minutes, the resulting resin is used as the starting resin for peptide chain extension on the 0.2 mmol scale.

After chain construction is complete, the peptide fragment is deprotected and cleaved from the resin by treatment with anhydrous hydrogen fluoride at 0 ° C. for 1 hour using 5% p-cresol as a scavenger. In all cases except fragment 1, the DNP-removal procedure cannot be adapted to the C-terminal thioester group, so the imidazole side chain 2,4-dinitrophenyl (DNP) protecting group remains in the His residue. Yes. However, during the ligation reaction, DNP is gradually removed by thiols, resulting in unprotected His. After cleavage, the peptide fragment is precipitated with ice cold diethyl ether, dissolved in aqueous acetonitrile and lyophilized. Peptide fragments were collected on a C18 column 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). Purify by RP-HPLC and UV detect at 214 nm. Samples are analyzed by electrospray mass spectrometry (ESMS) using an Esquire instrument (Bruker, Bremen, Germany) or similar instrument.

  As described in further detail below, the ligation reaction of the unprotected fragment is performed as follows: The dried peptide is equimolarly 6M guanidine hydrochloride (GuHCl), 0.2M phosphoric acid, pH 7.5. To achieve a final peptide concentration of 1-8 mM at a pH around 7, and add 1% benzyl mercaptan, 1% thiophenol. Typically, the reaction is performed overnight and monitored by HPLC and electrospray mass spectrometry. Subsequent treatment of the ligation product removes protecting groups still present. Opening of the N-terminal thiazolidine ring requires a further incubation at 37 ° C. for 2 hours with a final concentration of 0.5 M at pH 3.5 by the addition of solid methoxamine. 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 carried out at pH 7.0 in 6M GuHCl. The concentration of each reactant is 8 mM and 1% benzyl mercaptan and 1% thiophenol are added to create a reducing environment and facilitate the ligation reaction. After stirring overnight at 37 ° C., a nearly quantitative ligation reaction is observed. At this point in the reaction, the N-terminal thiazolidine ring is opened by adding CH ₃ —O—NH ₂ .HCl to the solution to achieve a final concentration of 0.5M and adjusting the pH to 3.5. After 2 hours incubation at 37 ° C., the completion of the reaction is confirmed using ESMS. Subsequently, the reaction mixture was treated with a 10-fold excess of tris (2-carboxyethylphosphine) over the peptide fragment and after 15 minutes preparative HPLC (eg C4, 20-60% CH ₃ CN, 1 minute). The ligation product is purified using 0.5% per liter), lyophilized and stored at -20 ° C.

  Repeat the same procedure with minor modifications for the remaining ligation reactions.

Peptide folding Full-length peptides are regenerated 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 gentle stirring overnight, the protein solution is purified by RP-HPLC as described above.

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

  Briefly, mice are repeatedly inoculated with several micrograms of HPP or a portion thereof over a period of several weeks to produce anti-HPP monoclonal antibodies. The mice are then sacrificed and spleen antibody-producing cells are isolated. Spleen cells are fused with mouse myeloma cells by polyethylene glycol means, and excess non-fused cells are destroyed by growing the system in selective medium containing aminopterin (HAT medium). Effectively fused cells are diluted and an aliquot of the dilution is placed in a well of a microtiter plate where the culture is allowed to continue to grow. First, Engvall, E., Meth. Antibody-producing clones are identified by detection of antibodies from well supernatants by immunoassay procedures, eg, ELISA, as described by Enzymol. 70: 419 (1980). Selected positive clones can be expanded and picked to use their monoclonal antibody products. Detailed procedures for monoclonal antibody production are described in Davis, L. et al., Basic Methods in Molecular Biology Elsevier, New York, Sections 21-2.

  For polyclonal antibody production by immunization, polyclonal antisera comprising antibodies to heterologous epitopes in HPP or a portion thereof by immunizing mice with HPP or a portion thereof, which may be unmodified or modified to increase immunogenicity To prepare. Appropriate non-human animals, preferably non-human mammals such as rats, rabbits, goats or horses may be selected.

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

Example 4: Characterization of HPP Polypeptide In Vivo Mouse homolog / orthologous protein of HPP polypeptide is administered subcutaneously to male C57BL / 6 mice at doses of 300, 600 or 1000 micrograms / day for 7-14 days. . At the end of the treatment period, samples from all organs are snap frozen at necropsy and analyzed by 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 assessed by the A260 nm / A280 nm ratio. Check integrity by denaturing gel electrophoresis. RNA is stored at −80 ° C. until analysis. Use good quality total RNA to synthesize double stranded cDNA using Superscript selection 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 at 45 ° C. for 16 hours. The array is then washed and stained with 10 mg / ml streptavidin-phycoerythrin conjugate (Molecular Probes) according to the EukGE-WS2 protocol (Affymetrix). 2 mg / ml acetylated BSA (Life Technologies), 100 mM MES, 1 M [Na ⁺ ], 0.05% Tween 20, 0.005% anthioform (Sigma), 0.1 mg / ml goat IgG and 0 Amplify the signal with .5 mg / ml biotinylated antibody and restain with streptavidin solution. After washing, the array is scanned twice with a Gene Array® scanner (Affymetrix).

  Expression levels are assessed by averaging the difference in signal intensity measured by an oligonucleotide pair of a given probe (AvgDiff value). The image capture and numerical translation software used for this test is Affymetrix Microarray Suite Version 5 (MAS5). To identify the genes affected by the treatment, the data set is first filtered so that the values are systematically low in the expression range when the experimental noise is high (slightly corresponding to the minimum number of replicas of the experimental points). In the first experiment, at least 50 AvgDiff values) are excluded in the first wave of analysis. In the second round of selection, the threshold t-test p-value (0.05) is based on a two-component error model (global error model) and, if possible, a gradual reduction correction (Benjamini And Hochberg false discovery rate) identify genes that differ in value between treated and untreated.

  A Fisher exact test is then used to compare the selected gene list with the established gene list for pathways and cellular components. By using Venn diagrams, genetic changes that are common between different organs are identified. By using highly relevant gene expression profiles, we find genes that correlate with changes at individual experimental points using several distance metrics (standard, Pearson).

  The decision to consider that a particular gene is relevant is based on the linkage of numerical changes confirmed by the above-described exploratory filtering and statistical algorithms and relationships with other modulated genes directed at a common biological theme. ing. Tables 4 and 6-11 show that at the RNA level, HPP polypeptides affect genes associated with the apoptotic pathway, proteosome, ubiquitin pathway and ribosomal RNA and proteins. Most genes are up-regulated at a rate of at least over 1.2 fold or down-regulated at a rate of at least less than 0.8 fold.

4.1: Characterization of HPP-38 polypeptides in vivo As described above, HPP-38 polypeptides were administered to mice, expression levels were evaluated, and gene lists were selected (Table 4).

  In vitro experiments were performed with 15 tumor cell lines. The assay used to detect changes in cell proliferation was based on the ability of viable cells to change Alamar Blue from its oxidized non-fluorescent blue form to a reduced fluorescent red form. Results obtained from the Alamar Blue reaction can quantify cell proliferation and assess the metabolic activity of viable cells. 10000, 1000, 100, 10 and 1 micromolar initial working solutions were obtained by dissolving and diluting the test compound with sterile distilled water. Further, 100-fold dilutions in culture medium were made to final assay concentrations of 100, 10, 1, 0.1 and 0.01 micromolar. A 100 microliter aliquot of cell suspension (approximately 1500-5000 cells per well) was placed in a 96-well microtiter plate at 37 degrees Celsius in a 5% carbon dioxide atmosphere. After 24 hours, 100 microliters of growth medium and 2 microliters of test solution, mitomycin (positive control material) or vehicle (distilled water) were added in duplicate per well and incubated for a further 72 hours. Test substances were evaluated at 100, 10, 1, 0.1 and 0.01 micromolar concentrations for possible inhibitory effects on cell proliferation. At the end of the incubation, 20 microliters of 90% Alamar Blue reagent was added to each well and incubated for another 6 hours before detecting cell viability by fluorescence intensity. Fluorescence intensity was measured using a Spectafluor Plus plate reader with absorbance at 530 nm and emission at 590 nm. A 50% or greater decrease in fluorescence intensity relative to the vehicle-treated control culture is indicative of significant cell growth inhibition, quiescence or cytotoxicity, and then semi-quantitative IC50, TBI by non-linear regression analysis And LC50 were measured. The results indicated that at least some tumor cell lines were more sensitive to HPP-38 induced cytotoxicity or cell growth inhibition than other tumor cell lines. Therefore, HPP-38 has a certain degree of selectivity. These results demonstrate the potential of HPP-38 as an anticancer agent.

^a IC ₅₀ (50% inhibitory concentration): Test compound concentration where the increase from time 0 in the number or mass of treated cells was only 50% of the corresponding increase in vehicle control at the end of the experiment.
^b TGI (total growth inhibition): test compound concentration at which the number or mass of treated cells at the end of the experiment was equal to the value at time zero.
^c LC ₅₀ (50% lethal concentration): test compound concentration at which the number or mass of treated cells at the end of the experiment was half of the value at time zero.
Semi-quantitative measurements of IC ₅₀ , TGI and LC ₅₀ were performed by non-linear regression analysis using GraphPad Prism (GraphPad software, USA).

4.2: Characterization of HPP-13 polypeptide in vivo In the same manner as described above, HPP-13 polypeptide was administered to mice, expression levels were evaluated, and gene lists were selected (Tables 6, 7, 8). 9 and 10). Affected genes are associated with iron transport, iron metabolism, heme biosynthesis, erythropoiesis and immunomodulation, or with neurodegeneration (Table 11). The rate of change is greater or less than 1.2.

4.3: Characterization of HPP-23 polypeptide in vivo As described above, HPP-23 was administered to mice, expression levels were evaluated, and a gene list was selected (Table 12). Affected genes are related to glucose metabolism (eg, glucose), angiogenesis, angiogenesis, blood pressure.

4.4: Characterization of HPP-13 and GPA101 polypeptide in vivo 4.4.1 Introduction Progressive cell loss in neuronal populations is a prominent pathological feature of neurodegenerative diseases. Neurotrophic, eg NGF (nerve growth factor), BDNF (brain derived neurotrophic factor), NT-3 (neurotrophin 3), NT-4 / 5 (neurotrophin 4/5) or GDNF (derived from glial cell line) The discovery of neurotrophic factors) has become a breakthrough in a large and promising research field. Neurotrophic factors are already used in the treatment of several neurodegenerative diseases. However, this application is limited by their inability to cross the blood brain barrier, their short half-life and their side effects. Therefore, the development of new compounds that can mimic neurotrophic effects (promoting neuronal survival and neurite outgrowth) without these limitations seems to be an excellent strategy for the development of new therapies in neurodegenerative diseases It is.

  The purpose of this in vitro test is to investigate the putative neurotrophic effects of HPP-13 and GPA101 on neurite outgrowth from spinal motor neurons. The neurotrophic effect of the test compound is examined after 3 days of treatment by measuring the length of the major neurites. Two cultures are performed and performed in two petri dishes per condition.

4.4.2: Materials and methods 4.4.2.1: Motor neuron culture
Martinou et al. (1992) Neurons, 8, 737-744, rat rat spinal motor neurons are prepared. Briefly, 15-day gestation (pregnant) female rats are killed by cervical dislocation (Latz Wister; Jeanvia, Lugenu-Saint-Isle, France) and the fetus is removed from the uterus. The spinal cord is removed and placed in Leibovitz's ice-cold medium (L15, Gibco, Invitrogen, Serge-Pontois, France). Carefully remove the meninges. Spinal cord tissue is dissociated by trypsinization (trypsin, Gibco) for 30 minutes at 37 ° C. in the presence of deoxyribonuclease I (Roche Meyrin). The reaction is stopped by adding DMEM containing 10% fetal bovine serum (Gibco). The suspension is ground with a 10 ml pipette and the cells are then mechanically dissociated several times through a 21 gauge needle of a syringe. The cells are then centrifuged at 580 × g for 10 minutes at room temperature. The pellet of dissociated cells was resuspended in L15 medium and the resulting suspension was first prepared at 180 × g for 10 minutes at room temperature in a layer of a 3.5% solution of bovine serum albumin (BSA) in L15 medium. Concentrate on motor neurons by centrifugation for 10 min. The supernatant is discarded, the pellet is homogenized in L15 supplemented with deoxyribonuclease I, and the suspension is placed on a cushion of Optiprep (d: 1.06 g.mL-1; Absiy, Paris, France). Layer in and centrifuge at 335 × g for 15 minutes at room temperature. The upper phase containing purified motor neurons is collected, resuspended in L15, and centrifuged at 800 × g for 10 minutes at room temperature. The cell pellet is finally resuspended in defined culture medium consisting of Neurobasal (Neurobasal, Gibco) supplemented with B27 supplement (2%, Gibco) and 0.5 mM L-glutamine (Gibco). This culture medium does not contain any serum. Neurobasal is a serum-free medium that allows neurons to survive for a long time, but the formulation is confidential (official data sheets are published on the Invitrogen website). B27 is a supplement added to give optimal growth conditions (B27 supplement contains the following components: d-biotin, BSA, fatty acid free fraction V, catalase, L-carnitine HCl, corticosterone, ethanolamine HCl , D-galactosidase (anhydride), glutathione (reduced), insulin (human, recombinant), linoleic acid, linolenic acid, progesterone, putrescine.2HCl, sodium selenite (1000X), superoxide dismutase, T- 3 / albumin complex, DL alpha-tocopherol, DL alpha tocopherol acetate, transferrin (human, iron deficient), vitamin A acetate). Viable cells are counted on a Neubauer hemocytometer using the trypan blue exclusion test (Sigma). Cells are then cultured at 30,000 cells per Petri dish (Nunk, Ducher, pre-coated with poly-1-lysine) and cultured at 37 ° C. in a humidified air (95%)-CO 2 (5%) atmosphere. After 2 hours of plating (time required for cell adhesion), test compounds are added to the culture.

This test used the following conditions:
・ Control ・ + BDNF, 50ng / ml
-+ HPP-13 (SEQ ID NO: 393), 1 nM
+ HPP-13 (SEQ ID NO: 393), 10 nM
+ HPP-13 (SEQ ID NO: 393), 30 nM
・ + GPA101, 1nM
・ + GPA101, 10nM
・ + GPA101, 30nM

4.4.2.2 Evaluation of neurite outgrowth After 3 days of treatment, cultures are washed with phosphate buffered saline (PBS, Gibco) and fixed in 2.5% glutaraldehyde in PBS. Under one condition (about half of each dish), 80-100 photographs of cells with unbranched neurites were taken with a camera (Coolpix 995, Nikon) fixed to a microscope (Nikon, objective lens 20x). The length is measured by analyzing the photos with software (Image-Pro Plus, France).

4.4.2.3 Data analysis Data analysis is performed using one-way analysis of variance (ANOVA). If applicable, the Fisher PLSD test is used in the many-pair comparison method. The level of significance was set at p <0.05.

4.4.3 Results 4.4.3.1 Effects of BDNF BDNF tested at 50 ng / ml induced neurite outgrowth in spinal motor neurons. In the control condition after 3 days of culture, the main neurite length was about 111.74 μm ± 4.032, and according to BDNF, the main neurite reached 199.56 μm ± 7.122. A 77% increase was observed.

4.4.3.2 Effects of HPP-13 and GPA101 HPP-13 and GPA101 also significantly increased neurite outgrowth, which is similar to BDNF. For each compound, the best concentration was found to be 10 nM. For this concentration, the average neurite length reached 193.65 μm ± 8.737 for HPP-13 and 179.74 μm ± 7.545 for GPA101. For all test concentrations, the average neurite length reaches 150% of the control condition.

4.4.4 Discussion In this study, a large neurotrophic effect of HPP-13 and GPA101 was observed.

Example 4.5: In vivo characterization of HPP-23 polypeptide In the same manner as above, HPP-23 was administered to mice, expression levels were evaluated, and a gene list was selected (Table 14). Affected genes are associated with sugar amyloidosis, arrhythmia or angiogenesis (eg, stroke, macular regeneration, cancer).

Example 5: Alignment of human and mouse ortholog peptides In the manner described in Example 4, proteins detected according to the present invention to be present in human plasma were searched for homologous mouse sequences suitable for injection into mice. .

The underline indicates the peptide sequence used for synthesis.
GPA056 (HPP-38)
Alignment type for Gene Plot ID: GP_1727089: Esophageal cancer related gene 2
Gene symbol: ECG2
Identity: 26/74 = 35%
Orthology: Celera synthetic ortholog

GPA065 (HPP-13)
Alignment Type for Gene Plot ID: GP_909415: Thymosin Paralog, Beta 4, X Chromosome Gene Symbol:
Identity: 43/43 = 100%
Orthology: Novartis Mutual Optimal Match Ortholog, SwissProt Ortholog

GPA066
Alignment Type for Gene Plot ID: GP_909415: Thymosin Paralog, Beta 4, X Chromosome Gene Symbol:
Identity: 44/44 = 100%
Orthology: Novartis Mutual Optimal Match Ortholog, SwissProt Ortholog

GPA068 (HPP-23)
Alignment type for Gene Plot ID: GP_805366: Chromogranin A (parathyroid secretion protein 1)
Gene symbol: CHGA
Identity: 28/52 = 54%
Orthology: Celera Synthetic Ortholog

Claims (40)

A method for detecting human plasma polypeptide (HPP) comprising:
i) contacting the biological sample with an HPP-bound adsorbent; and
ii) A method comprising detecting and / or quantifying the binding of HPP to the HPP-bound adsorbent.   The method of claim 1, wherein the biological sample is a plasma sample.   2. The method of claim 1, wherein the HPP binding adsorbent is an HPP specific antibody.   The method of claim 1, wherein the HPP bound adsorbent is bound to a substrate.   Claims wherein the detection and / or quantification step comprises a method selected from the group consisting of radioimmunoassay, enzyme-linked immunosorbent assay, retentate chromatography, protein array, surface enhanced laser desorption / ionization, and mass spectrometry. Item 2. The method according to Item 1.   A protein array comprising an adsorbent specific for at least one human plasma polypeptide (HPP). A method for detecting an abnormal concentration of at least one human plasma polypeptide (HPP) in an individual comprising:
i) obtaining a sample from an individual;
ii) measuring a first level of at least one HPP in the sample; and
iii) A method comprising the step of comparing the level with that of a control sample, wherein the difference between the first level and the control level is diagnostically determinative of an abnormal concentration of HPP.   A pharmaceutical composition comprising an effective amount of an HPP-38 polypeptide and a pharmaceutically acceptable carrier.   A method of treating a disease or condition associated with a cancer disease or hyperplasia, comprising administering an effective amount of an HPP-38 polypeptide to a mammal, including a human suffering from said disease.   The disease or condition associated with hyperplasia is a disease or condition selected from the group consisting of fibrosis, prostate hyperplasia, adrenal hyperplasia, endometrial hyperplasia, psoriasis, hyperplasia resulting from inflammation. 8. The method according to 8. A method of identifying a disease or condition associated with a cancer disease or hyperplasia, comprising:
i) contacting the HPP-38 polypeptide with the test compound under sample conditions acceptable for HPP-38 biological activity;
ii) measuring the level of at least one HPP-38 biological activity;
iii) comparing the level to that of a control sample lacking the test compound, and
iv) to select a test compound that induces said level of change for the purpose of further testing as a HPP-38 modulator for the prevention and / or therapeutic treatment of a disease or condition associated with cancer disease or hyperplasia. Including methods.   12. The method of claim 11, wherein the HPP-38 bioactivity level is determined by measuring the expression level of one or more genes shown in Table 4.   A method of prognosis or diagnosis of a disease or condition associated with cancer disease or hyperplasia comprising detection of plasma levels of HPP-38, wherein increased levels indicate a disease or condition associated with cancer disease or hyperplasia . A method for prognosis or diagnosis of a disease or condition associated with cancer disease or hyperplasia, comprising:
i) obtaining a first value by detecting the expression level of at least one gene identified in Table 4 in a sample of the appropriate tissue obtained from the subject; and
ii) Comparing the first level with the expression level of the gene from the disease-free subject, the magnitude of the expression level in the subject sample when compared to the sample from the disease-free subject is A method that is predisposed to or is an indicator of being afflicted with a related disease or condition.   A polypeptide comprising the one shown in SEQ ID NO: 397.   A non-amidated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 395 or SEQ ID NO: 396.   A pharmaceutical composition comprising an effective amount of an HPP-13 polypeptide or GPA101 polypeptide or a combination thereof and a pharmaceutically acceptable carrier.   A method of treating a disease or condition associated with neurodegeneration, comprising administering an effective amount of HPP-13 or GPA101 polypeptide or a combination thereof to a mammal suffering from said disease, for example a human.   Diseases or conditions associated with neurodegeneration include spinal cord injury or CNS injury, Alzheimer's disease, Parkinson's disease, multiple sclerosis, ALS (amyotrophic lateral sclerosis), peripheral neuropathy, Guillain-Barre disease, diabetic neuropathy 19. The method of claim 18, wherein the disease or condition is selected from the group consisting of a demyelinating neuropathy. A method for identifying a modulator of a disease or condition associated with neurodegeneration comprising:
i) contacting an HPP-13 or GPA101 polypeptide with a test compound under sample conditions acceptable for HPP-13 or GPA101 biological activity;
ii) measuring the level of at least one HPP-13 or GPA101 biological activity;
iii) comparing the level to that of a control sample lacking the test compound, and
iv) selecting test compounds that induce the above level of change for the purpose of further testing as HPP-13 or GPA101 polypeptide modulators for the prevention and / or therapeutic treatment of diseases or conditions associated with neurodegeneration. Including methods.   21. The method of claim 20, wherein the HPP-13 bioactivity level is measured by measuring the expression level of one or more genes shown in Table 11.   A method of prognosis or diagnosis of a disease or condition associated with neurodegeneration comprising the detection of plasma levels of HPP-13 polypeptide, wherein increased levels are indicative of a disease or condition associated with neurodegeneration. A prognosis or diagnostic method for a disease or condition associated with neurodegeneration, comprising:
i) obtaining a first value by detecting the expression level of at least one gene identified in Table 11 in a sample of the appropriate tissue obtained from the subject; and
ii) Comparing the first level with the expression level of a gene from a disease-free subject, the magnitude of the expression level in the subject sample when compared to the sample from the disease-free subject is a disease in which the subject is associated with neurodegeneration Or a method that is susceptible to or an indication of being afflicted with a medical condition.   A method of treating a disease or condition associated with iron balance or iron transport, comprising administering an effective amount of an HPP-13 polypeptide to a mammal, such as a human, suffering from said disease.   A disease or condition related to iron balance or iron transport is selected from the group consisting of hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron overload, anemia, and sickle cell anemia 25. The method of claim 24, wherein: A method of identifying a modulator of a disease or condition associated with iron balance or iron transport comprising:
i) contacting the HPP-13 polypeptide with a test compound under sample conditions acceptable for HPP-13 biological activity;
ii) measuring the level of at least one HPP-13 biological activity;
iii) comparing the level to that of a control sample lacking the test compound, and
iv) to select test compounds that induce changes in the above levels for the purpose of further testing as HPP-13 modulators for the prevention and / or therapeutic treatment of diseases or conditions associated with iron balance or iron transport. Including methods.   27. The method of claim 26, wherein the HPP-13 bioactivity level is measured by measuring the expression level of one or more genes shown in Tables 6, 7, 8, 9 and / or 10.   A method for prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising detection of plasma levels of HPP-13 polypeptide, wherein increased levels indicate a disease or condition associated with iron balance or iron transport There is a way. A method for prognosis or diagnosis of a disease or condition associated with iron balance or iron transport, comprising:
i) obtaining a first value by detecting the expression level of at least one gene identified in Tables 6, 7, 8, 9 and / or 10 in a sample of the appropriate tissue obtained from the subject; and
ii) Comparing the expression level of the gene from the disease-free subject with the first value, the magnitude of the expression level in the subject sample when compared to the sample from the disease-free subject is A method that is predisposed to or is an indicator of being afflicted with a related disease or condition.   A pharmaceutical composition comprising an effective amount of an HPP-23 polypeptide and a pharmaceutically acceptable carrier.   A method of treating a disease associated with impaired serum glucose regulation, comprising administering an effective amount of an HPP-23 polypeptide to a mammal, such as a human, suffering from said disease.   32. The method of claim 31, wherein the disease associated with impaired serum glucose regulation is diabetes.   A method of treating a metabolic disorder, comprising administering an effective amount of an HPP-23 polypeptide to a mammal, such as a human, suffering from said disorder.   34. The method of claim 33, wherein the metabolic disorder is amyloidosis.   A method of reducing blood glucose levels in a mammal, eg, a human, comprising administering to the mammal an HPP-23 polypeptide. A method for identifying a modulator of a disease or metabolic disorder associated with impaired serum glucose regulation comprising:
i) contacting an HPP-23 polypeptide with a test compound under sample conditions acceptable for HPP-23 biological activity;
ii) measuring the level of at least one HPP-23 biological activity;
iii) comparing the level to that of a control sample lacking the test compound, and
iv) selecting test compounds that induce the above-mentioned changes for the purpose of further testing as HPP-23 modulators for the prevention and / or therapeutic treatment of diseases or metabolic disorders associated with impaired serum glucose regulation. Method.   38. The method of claim 36, wherein the HPP-23 bioactivity level is measured by measuring the expression level of one or more genes shown in Table 12 or 14.   A method for the prognosis or diagnosis of a disease or metabolic disorder associated with impaired serum glucose regulation, comprising detection of plasma levels of HPP-23, wherein increased levels indicate a disease or metabolic disorder associated with impaired serum glucose regulation. A method for prognosis or diagnosis of a disease or metabolic disease associated with impaired serum glucose regulation, comprising:
i) obtaining a first value by detecting the expression level of at least one gene identified in Table 12 or 14 in a sample of the appropriate tissue obtained from the subject; and
ii) Comparing the first level with the expression level of the gene from the disease-free subject, the magnitude of the expression level in the subject sample when compared to the sample from the disease-free subject is that the subject is associated with impaired serum glucose regulation A method that is susceptible to or is indicative of being afflicted with a disease or metabolic disorder. 40. The method of claims 31-39, wherein the HPP-23 polypeptide is not amidated.