EP1658502A2 - Secreted polypeptide species and use thereof - Google Patents
Secreted polypeptide species and use thereofInfo
- Publication number
- EP1658502A2 EP1658502A2 EP04764307A EP04764307A EP1658502A2 EP 1658502 A2 EP1658502 A2 EP 1658502A2 EP 04764307 A EP04764307 A EP 04764307A EP 04764307 A EP04764307 A EP 04764307A EP 1658502 A2 EP1658502 A2 EP 1658502A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- hpp
- disease
- level
- polypeptide
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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Definitions
- the invention relates to polypeptide species secreted in human plasma, isolated polynucleotides encoding such polypeptides, polymo ⁇ hic variants thereof, and the use of said nucleic acids and polypeptides or compositions thereof for detection assays and disease diagnosis and medical treatment.
- BACKGROUND The non-specific nature of many disease symptoms often makes definitive diagnosis difficult for medical practitioners. Patient descriptions are often variable or inaccurate. More quantitative diagnostic methods suffer from variability, both between individuals and between readings on a single individual. Thus, diagnostic measures must be standardized and applied to individuals with well- documented and extensive medical histories. Even with these controls, symptoms for entirely different underlying conditions can appear identical.
- neuropsychiatric diseases e.g., schizophrenia and bipolar disease, see Johnston-Wilson, et al., Int J Neuropsychopharmacol, 2001, 4:83-92
- metabolic disorders e.g., diabetes, see Gloyn and McCarthy, Best Pract Res Clin Endocrinol Metab, 2001, 15:293-308
- Current diagnostic methods often do not reveal the underlying cause(s) for a given observation or reading. Therefore, a therapeutic strategy based on a particular positive result likely will not address the causative problem and may even be harmful to the individual.
- Methods of diagnosis that rely on nucleotide detection include genetic approaches and expression profiling.
- genes that are known to be involved in a particular disorder may be screened for mutations using common genotyping techniques such as sequencing, hybridization- based techniques, or PCR.
- expression from a known gene may be tracked by standard techniques including RTPCR, various hybridization-based techniques, and sequencing. These strategies often do not enable a practitioner to detect differences in mRNA processing and splicing, translation rate, mRNA stability, and posttranslational modifications such as proteolytic processing, phosphorylation, glycosylation, and amidation.
- the invention provides new information relating to the range of polypeptides that are found in human plasma. Human blood plasma is a most useful source of proteins associated with both health and disease.
- Plasma not only contains active proteins and tell-tale disease markers; it may be obtained non-invasively in fairly large quantities from both patients and control subjects (as opposed to tissue, which is often difficult to obtain in large amounts from controls).
- blood, plasma, and serum are used quite generally for existing clinical tests (e.g., genomic approaches).
- genomic approaches many of the cells responsible for the protein content of plasma are not to be found in the blood, thus limiting genomic approaches.
- plasma is a fluid and therefore may be pooled (again, in contrast to tissue) to obtain a large and representative sample for analysis. Analysis of the plasma proteome is challenging in view of the large numbers of proteins expected to be present and the wide dynamic range of concentrations, known to span at least 11 to 12 orders of magnitude.
- HPPs Human Plasma Polypeptides
- HPPs Human Plasma Polypeptides
- HPPs Human Plasma Polypeptides
- the HPPs of the invention represent an important tool for diagnosis and drug development. HPPs are secreted factors and as such, are easy to detect and target, e.g., with a detectable molecule, protein chip, or modulator.
- compositions related to polypeptide species secreted in human plasma are designated herein "Human Plasma Polypeptides," or HPPs.
- Human Plasma Polypeptides comprise an amino acid sequence selected from the list of Table 3.
- Compositions include HPP precursors, antibodies specific for HPPs, including monoclonal antibodies and other binding compositions derived therefrom. Further included are methods of making and using these compositions.
- Precursors of the invention include unmodified precursors, proteolytic precursors of the group consisting of the sequences from Table 3, and intermediates resulting from alternative proteolytic sites in the group consisting of the sequences from Table 3.
- a preferred embodiment of the invention includes HPPs having a posttranslational modification, such as a phosphorylation, glycosylation, acetylation, amidation, or a C-, N- or O- linked carbohydrate group. Additionally preferred are HPPs with intra- or inter-molecular interactions, e.g., disulfide and hydrogen bonds that result in higher order structures. Also preferred are HPPs that result from differential mRNA processing or splicing. Preferably, the HPPs represent posttranslationally modified species, structural variants, or splice variants that are present in plasma. In another aspect, the invention includes HPPs comprising a sequence which is at least 95 percent identical to a sequence selected from the group of sequences listed in Table 3.
- the invention includes polypeptides comprising at least 97 percent, and more preferably at least 98 percent, and still more preferably at least 99 percent, identity with any one of the sequences selected from Table 3.
- the invention includes polypeptides comprising a sequence at least 99 percent identical to a sequence selected from the group of sequences listed in Table 3.
- the invention includes natural variants of HPPs having a frequency in a selected population of at least two percent. More preferably, such natural variant has a frequency in a selected population of at least five percent, and still more preferably, at least ten percent. Most preferably, such natural variant has a frequency in a selected population of at least twenty percent. The selected population may be any recognized population of study in the field of population genetics.
- the selected population is Caucasian, Negroid, or Asian. More preferably, the selected population 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, Finnish, French of Caucasian ancestry, Swiss, Singaporean of Chinese ancestry, Korean, Japanese, Quebecian, North American Pima Indians, Pennsylvanian Amish and Amish Mennonite, Newfoundlander, or Polynesian.
- a preferred aspect of the invention provides a composition comprising an isolated HPP, i.e., an HPP free from proteins or protein isoforms having a significantly different isoelectric point or a significantly different apparent molecular weight from the HPP.
- the isoelectric point and molecular weight of an HPP may be indicated by affinity and size-based separation chromatography, 2- dimensional gel analysis, and mass spectrometry.
- the invention includes modified HPPs. Such modifications include protecting/blocking groups, linkage to an antibody molecule or other cellular ligand, and detectable labels, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
- Chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, or metabolic synthesis in the presence of tunicamycin.
- chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (e.g., water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol).
- the HPPs are modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
- the invention includes polynucleotides encoding an HPP of the invention, polynucleotides encoding a polypeptide having an amino acid sequence selected from the group of sequences listed in Table 3, antisense oligonucleotides complementary to such sequences, oligonucleotides complementary to HPP gene sequences for diagnostic and analytical assays (e.g., PCR, hybridization-based techniques).
- the invention provides a vector comprising DNA encoding an HPP.
- the invention also includes host cells and transgenic nonhuman animals comprising such a vector.
- One preferred method comprises the steps of (a) providing a host cell containing an expression vector as disclosed above; (b) culturing the host cell under conditions whereby the DNA segment is expressed; and (c) recovering the protein encoded by the DNA segment.
- Another preferred method comprises the steps of: (a) providing a host cell capable of expressing an HPP; (b) culturing said host cell under conditions that allow expression of said HPP; and (c) recovering said HPP.
- the expression vector further comprises a secretory signal sequence operably linked to the DNA segment, the cell secretes the protein into a culture medium, and the protein is recovered from the medium.
- an especially preferred method of making an HPP includes chemical synthesis using standard peptide synthesis techniques, as described in the section titled "Chemical Manufacture of HPP compositions" and in Example 2.
- the invention includes isolated antibodies specific for any of the polypeptides, peptide fragments, or peptides described above.
- the antibodies of the invention are monoclonal antibodies.
- Anti-HPP antibodies have purification, detection, diagnostic and prognostic applications. Preferred anti-HPP antibodies for purification and detection are attached to a label group.
- Detection methods include, but are not limited to, those that employ antibodies or antibody-derived compositions specific for an HPP antigen.
- a preferred detection method is an enzyme-linked immunosorption assay (ELISA).
- Compositions comprising one or more antibodies described above, together with a pharmaceutically acceptable carrier are also within the scope of the invention, e.g, for in vivo detection.
- Detection methods for identifying HPPs in specific tissue samples and biological fluids (preferably plasma) form part of the invention.
- Detection methods for identifying HPP expression in cell-based samples are also included.
- the invention further provides methods that comprise detecting the level of at least one HPP in a sample of body fluid, preferably blood plasma.
- kits that may be used in the above-recited methods and that may comprise single or multiple preparations, adsorbant and substrate materials, antibodies, label groups, other reagents, if needed, and directions for use.
- kits may be used for diagnosis or for assays to identify new diagnostic agents.
- detection of increased plasma levels of at least one HPP of the invention indicates an increased risk of an HPP disorder associated with the increased HPP.
- said detection indicates that an individual has at least a 1.05-fold, 1.1 -fold, 1.15-fold, and more preferably at least a 1.2-fold increased likelihood of developing said HPP disorder.
- detection of decreased plasma levels of at least one HPP of the invention indicates that an individual has an increased risk of an HPP disorder associated with the increased HPP.
- the amount of HPP increase or decrease observed in an individual compared to a control sample will correlate with the certainty of the prediction.
- HPP is detected in a human plasma sample by the methods of the invention.
- Especially preferred techniques are mass spectrometry, retentate chromatography (including protein arrays), and immunodetection.
- a prediction or diagnosis is based on at least a 1.1-, 1.15-, 1.2-, 1.25-, and more preferably a 1.5-fold increase (resp. decrease) in the experimental HPP level as compared to the control. Further aspects of the invention are also described in the specification and in the claims.
- compositions, methods, and kits useful for screening and diagnosis of human plasma; for identifying individuals most likely to respond to a particular therapeutic treatment; and for monitoring the results of therapy.
- the invention will be described with respect to the analysis of blood plasma samples.
- the assays and techniques described below can be applied to other biological fluid samples (e.g. cerebrospinal fluid, lymph, bile, plasma, saliva or urine) or tissue samples.
- the methods and compositions of the present invention are useful for screening, diagnosis and prognosis of a living individual, but may also be used for postmortem diagnosis in an individual.
- nucleic acids and “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
- the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
- nucleotide sequence may be employed to designate indifferently a polynucleotide or a nucleic acid. More precisely, the expression “nucleotide sequence” encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e.
- nucleic acids are one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
- an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
- the isolated HPP nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
- an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
- HPP nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
- plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
- vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
- plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
- the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
- viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
- hybridizes to is intended to describe conditions for moderate stringency or high stringency hybridization, preferably where the hybridization and washing conditions permit nucleotide sequences at least 60% homologous to each other to remain hybridized to each other.
- the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85%, 90%, 95% or 98% homologous to each other typically remain hybridized to each other.
- Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
- stringent hybridization conditions for nucleic acid interactions are as follows: the hybridization step is realized at 65°C in the presence of 6 x SSC buffer, 5 x Denhardt's solution, 0,5% SDS and lOO ⁇ g/ml of salmon sperm DNA.
- the hybridization step is followed by four washing steps: - two washings during 5 min, preferably at 65°C in a 2 x SSC and 0.1%SDS buffer; - one washing during 30 min, preferably at 65°C in a 2 x SSC and 0.1% SDS buffer, - one washing during 10 min, preferably at 65°C in a 0.1 x SSC and 0.1%SDS buffer, these hybridization conditions being suitable for a nucleic acid molecule of about 20 nucleotides in length. It will be appreciated that the hybridization conditions described above are to be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art, for example be adapted according to the teachings disclosed in Hames B.D.
- Percent homology is used herein to refer to both nucleic acid sequences and amino acid sequences.
- Amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes).
- the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90% or 95% of the length of the reference sequence.
- 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 comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm.
- a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77.
- 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.
- Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
- the default parameters of the respective programs e.g., XBLAST and NBLAST
- polypeptide refers to a polymer of amino acids without regard to the length of the polymer; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide.
- polypeptides which include the covalent attachment of glycosyl, acetyl, phosphate, amide, lipid, carboxyl, acyl, or carbohydrate groups are expressly encompassed by the term polypeptide.
- polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
- protein as used herein may be used synonymously with the term “polypeptide” or may refer to, in addition, a complex of two or more polypeptides which may be linked by bonds other than peptide bonds, for example, such polypeptides making up the protein may be linked by disulfide bonds.
- protein may also comprehend a family of polypeptides having identical amino acid sequences but different post-translational modifications, particularly as may be added when such proteins are expressed in eukaryotic hosts.
- an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein of the invention (i.e., HPP or biologically active fragment thereof) is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
- the language “substantially free of cellular material” includes preparations of a protein according to the invention in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
- the language "substantially free of cellular material” includes preparations of a protein according to the invention having less than about 30% (by dry weight) of protein other than the protein of the invention (also referred to herein as a "contaminating protein”), more preferably less than about 20% of protein other than the protein according to the invention, still more preferably less than about 10% of protein other than the protein according to the invention, and most preferably less than about 5% of protein other than the protein according to the invention.
- contaminating protein protein other than the protein of the invention
- the protein according to the invention or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
- the language “substantially free of chemical precursors or other chemicals” includes preparations of a protein of the invention in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
- the language “substantially free of chemical precursors or other chemicals” includes preparations of a protein of the invention having less than about 30% (by dry weight) of chemical precursors or non- protein chemicals, more preferably less than about 20% chemical precursors or non-protein chemicals, still more preferably less than about 10% chemical precursors or non-protein chemicals, and most preferably less than about 5% chemical precursors or non-protein chemicals.
- recombinant polypeptide is used herein to refer to polypeptides that have been artificially designed and which comprise at least two polypeptide sequences that are not found as contiguous polypeptide sequences in their initial natural environment, or to refer to polypeptides which have been expressed from a recombinant polynucleotide.
- Plasma is the liquid part of the blood and lymphatic fluid, comprising about half of the volume. Plasma is devoid of cells and, unlike serum, has not clotted. Blood plasma contains antibodies and other proteins. Blood collected from an individual is often subjected to centrifugation to remove cellular components. Another method of plasma separation includes sterile filtration, as described in US Patent 6241886.
- HPP Human Plasma Polypeptide
- HPP Human Plasma Polypeptide
- Such polypeptide may be post- translationally modified as described herein.
- HPPs may also contain other structural or chemical modifications such as disulfide linkages or amino acid side chain interactions such as hydrogen and amide bonds that result in complex secondary or tertiary structures.
- HPPs also include mutant polypeptides, such as deletion, addition, swap, or truncation mutants, fusion polypeptides comprising such polypeptides, and polypeptide fragments of at least three, but preferably 8, 10, 12, 15, or 21 contiguous amino acids of the sequences of Table 3.
- the invention embodies polypeptides encoded by the nucleic acid sequences of HPP genes or HPP mRNA species, preferably human HPP genes and mRNA species, including isolated HPPs consisting of, consisting essentially of, or comprising the sequences from Table 3.
- Preferred HPPs retain at least one biological activity of HPPs from Table 3.
- HPP-related disorder or “HPP-related disease” describes any medical condition known to be associated with an HPP of the invention.
- HPP-related disorders include conditions where the presence of an abnormal level of an HPP or HPP polynucleotide is indicative that an individual has or is at risk of developing that condition.
- HPP-related disorders also include conditions where the presence of an abnormal form of an HPP or HPP polynucleotide (e.g., due to mutation, truncation, increased or decreased biological activity, abnormal posttranslational modification or processing) is indicative that an individual has or is at risk of developing that condition.
- an abnormal form of an HPP or HPP polynucleotide e.g., due to mutation, truncation, increased or decreased biological activity, abnormal posttranslational modification or processing
- Another aspect of the invention pertains to anti-HPP antibodies.
- antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site which specifically binds (immunoreacts with) an antigen, such as HPP, or a biologically active fragment or homologue thereof.
- Preferred antibodies bind to an HPP exclusively and do not recognize other polypeptides with high affinity.
- immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
- the invention provides polyclonal and monoclonal antibodies that bind an HPP, or a biologically active fragment or homologue thereof.
- a monoclonal antibody composition thus typically displays a single binding affinity for a particular HPP with which it immunoreacts.
- Preferred HPP antibodies are attached to a label group.
- a label group is any compound that, when attached to a polynucleotide or polypeptide (including antibodies), allows for detection or purification of said polynucleotide or polypeptide. Label groups may be detected or purified directly or indirectly by a secondary compound, including an antibody specific for said label group.
- Useful label groups include 32 35 3 125 radioisotopes (e.g., P, S, H, I), fluorescent compounds (e.g., 5-bromodesoxyuridin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin acetylaminofluorene, digoxigenin), luminescent compounds (e.g., luminol, GFP, luciferin, aequorin), enzymes or enzyme co-factor detectable labels (e.g., peroxidase, luciferase, alkaline phosphatase, galactosidase, or acetylcholinesterase), or compounds that are recognized by a secondary factor such as strepavidin, GST, or biotin.
- radioisotopes e.g., P, S, H, I
- a label group is attached to a polynucleotide or polypeptide in such a way as to not interfere with the biological activity of the polynucleotide or polypeptide.
- Radioisotopes may be detected by direct counting of radioemission, film exposure, or by scintillation counting, for example.
- Enzymatic labels may be detected by determination of conversion of an appropriate substrate to product, usually causing a fluorescent reaction. Fluorescent and luminescent compounds and reactions may be detected by, e.g., radioemission, fluorescent microscopy, fluorescent activated cell sorting, or a luminometer.
- “Adsorbent” refers to any material capable of adsorbing a polypeptide (i.e., an HPP).
- adsorbent is used herein to refer both to a single material (“monoplex adsorbent”) (e.g., a compound or functional group) to which a polypeptide is exposed, and to a plurality of different materials (“multiplex adsorbent”) to which a sample is exposed.
- the adsorbent materials in a multiplex adsorbent are referred to as "adsorbent species.”
- an addressable location on a substrate can comprise a multiplex adsorbent characterized by many different adsorbent species (e.g., anion exchange materials, metal chelators, or antibodies), having different binding characteristics.
- the basis of attraction is generally a function of chemical or biological molecular recognition.
- Bases for attraction between an adsorbent and a polypeptide include, for example, (1) a salt-promoted interaction, e.g., hydrophobic interactions, thiophilic interactions, and immobilized dye interactions; (2) hydrogen bonding and/or van der Waals forces interactions and charge transfer interactions, such as in the case of a hydrophilic interactions; (3) electrostatic interactions, such as an ionic charge interaction, particularly positive or negative ionic charge interactions; (4) the ability of the polypeptide to form coordinate covalent bonds (i.e., coordination complex formation) with a metal ion on the adsorbent; (5) enzyme-active site binding; (6) reversible covalent interactions, for example, disulfide exchange interactions; (7) glycoprotein interactions; (8) biospecific interactions; or (9) combinations of two or more of the foregoing modes of interaction. That is, the adsorbent can exhibit two or more bases of attraction, and thus be known as a "mixed functionality" adsorbent.
- HPPs of the invention The Human Plasma Polypeptides (HPPs) of the invention are described in Table 1 and 3. HPPs comprising an amino acid sequence selected from the group consisting of the sequences from Table 3 and fragments thereof are secreted and circulate in blood plasma.
- HPPs of the invention are known polypeptides that have not previously been found in human plasma. Thus, the invention introduces a new role or function for these polypeptides.
- HPPs listed in Table 1 include polypeptide species that are not expected to be found in plasma, as described in Table 1.
- the terms "Human Plasma Polypeptide" and "HPP" are used herein to embrace any and all of the peptides, polypeptides and proteins of the present invention.
- HPPs from humans, including isolated or purified HPPs consisting of, consisting essentially of, or comprising an amino acid sequence selected from the group consisting of the sequences from Table 3. Further included are unmodified precursors, proteolytic precursors and intermediates of the sequence selected from the group consisting of the sequences from Table 3.
- the present invention embodies an isolated, purified, and recombinant polypeptide fragment comprising a contiguous span of at least 3 amino acids, preferably at least 8 to 10 amino acids, of an amino acid sequence selected from the group consisting of the sequences from Table 3, wherein said fragment has an HPP biological activity.
- the contiguous stretch of amino acids comprises the site of a mutation or functional mutation, including a deletion, addition, swap or truncation of the amino acids in the HPP sequence.
- the invention also concerns the polypeptide encoded by the HPP nucleotide sequences of the invention, or a complementary sequence thereof or a fragment thereof. Said polypeptide fragment may represent the actual peptide species that is present in human plasma.
- Said polypeptide fragment may be used, for example, to generate HPP-specific antibodies or to design another type of HPP-specific adsorbant.
- One aspect of the invention pertains to isolated HPPs, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-HPP antibodies.
- native HPP peptides can be isolated from plasma, cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
- HPPs are produced by recombinant DNA techniques.
- an HPP can be synthesized chemically using peptide synthesis techniques, as described in the section titled "Chemical Manufacture of HPP compositions" and in Example 2.
- biologically active portions comprise a domain or motif with at least one activity of an HPP.
- a biologically active HPP may, for example, comprise at least 1, 2, 3, or 5 amino acid changes from the sequence selected from the group consisting of the sequences from Table 3, or comprise at least 1%, 2%, 3%, 5%, 8%, 10% or 15% change in amino acids from the sequence selected from the group consisting of the sequences from Table 3.
- HPPs are defined by the polypeptides of 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.
- Table 1 provides: • an accession number in a public database, corresponding to the related polypeptide sequence; • the amino acid positions defining the observed polypeptide with respect to the polypeptide in the public database.
- o a proteins not previously known to be plasmatic, most probably secreted
- o b proteins not previously known to be plasmatic, most probably from cellular leakage.
- accession numbers listed in Table 1 are references for the SwissPROT/TrEMBL databases, both of which are publicly available, for example at: http://www.expasy.ch. HPPs 40-57, however, are defined as sequences appearing in published patent applications, as detailed in Table 1.
- accession numbers for HPPs 58-61 correspond to predicted protein sequences obtained by running a number of softwares on GenBank entries, as detailed in Table 1.
- the accession numbers for HPPs 1-4 represent proteins entries available from NCBI, at http://www.ncbi.nlm.nih.gov/.
- accession number for HPP 5 represents an EST, whose sequence is available from the dbEST database, for example from NCBI, at http://www.ncbi.nlm.nih.gov/
- the HPPs of the invention are all less than or around 20kD in molecular weight. Clear identification of low abundance proteins (leptin and ghrelin) and peptides (bradykinin) confirms the sensitivity of the detection methods of the invention.
- the first separation is on a cation exchange chromatography column, which is eluted with increasing salt concentration. Eighteen fractions are collected.
- the CEX column in Table 3 lists which fraction contained tryptic 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 provides an indication of the overall positive charge of a polypeptide species. Cation exchange is followed by a reverse phase HPLC separation.
- Table 3 lists in which of the 30 fractions tryptic peptides eluted for each HPP.
- Table 2 provides the elution conditions (%B), according to the protocol described in Step 4 of Example 1. Separation by reverse phase provides an indication of the overall hydrophobicity of a polypeptide species.
- Table 3 provides for each CEX fraction and for each RPl fraction, the list of tryptics detected in these fractions, along with the RP2 fractions (within parenthesis after each tryptic sequence) where these tryptics were detected.
- HPjP nucleic acids One aspect of the invention pertains to purified or isolated nucleic acid molecules that encode ⁇ PPs or biologically active portions thereof as further described herein, as well as nucleic acid fragments thereof. Said nucleic acids may be used, for example, in detection methods as further described herein.
- An object of the invention is a purified, isolated, or recombinant nucleic acid coding for an ⁇ PP, complementary sequences thereto, and fragments thereof.
- the invention also pertains to a purified or isolated nucleic acid comprising a polynucleotide having at least 95% nucleotide identity with a polynucleotide coding for an ⁇ PP, advantageously 99 % nucleotide identity, preferably 99.5% nucleotide identity and most preferably 99.8% nucleotide identity with a polynucleotide coding for an ⁇ PP, or a sequence complementary thereto or a biologically active fragment thereof.
- Another object of the invention relates to purified, isolated or recombinant nucleic acids comprising a polynucleotide that hybridizes, under the stringent hybridization conditions defined herein, with a polynucleotide coding for an ⁇ PP, or a sequence complementary thereto or a variant thereof or a biologically active fragment thereof.
- the invention pertains to purified or isolated nucleic acid molecules that encode a portion or variant of an ⁇ PP, wherein the portion or variant displays an ⁇ PP biological activity.
- said portion or variant is a portion or variant of a naturally occurring ⁇ PP or precursor thereof.
- Another object of the invention is a purified, isolated, or recombinant nucleic acid encoding an ⁇ PP comprising, consisting essentially of, or consisting of the amino acid sequence selected from the group of sequences from Table 3, or fragments thereof, wherein the isolated nucleic acid molecule encodes one or more motifs, such as a target binding site.
- a nucleic acid fragment encoding a "biologically active portion of an ⁇ PP" can be prepared by isolating a portion of a nucleotide sequence coding for an ⁇ PP, which encodes a polypeptide having an ⁇ PP biological activity, expressing the encoded portion of the ⁇ PP (e.g., by recombinant expression in vitro or in vivo) and assessing the activity of the encoded portion of the ⁇ PP.
- the invention further encompasses nucleic acid molecules that differ from the HPP nucleotide sequences of the invention due to degeneracy of the genetic code and encode the same HPPs of the invention.
- DNA sequence polymorphisms that lead to changes in the amino acid sequences of the HPPs may exist within a population (e.g., the human population). Such genetic polymorphism may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of an HPP-encoding gene or nucleic acid sequence.
- Nucleic acid molecules corresponding to natural allelic variants and homologues of the HPP nucleic acids of the invention can be isolated based on their homology to the HPP nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. It will be appreciated that the invention comprises polypeptides having an amino acid sequence encoded by any of the polynucleotides of the invention.
- HPP nucleic acids Polynucleotide sequences (or the complements thereof) encoding HPPs have various applications, including uses as hybridization probes.
- HPP-encoding nucleic acids are useful for the preparation of HPPs by recombinant techniques, as described herein.
- the polynucleotides described herein, including sequence variants thereof, can be used in detection assays. Accordingly, detecting the presence of such polynucleotides in body fluids or tissue samples is a feature of the present invention.
- nucleic acid based detection assays include, but are not limited to, hybridization assays (e.g., in situ hybridization or nucleotide arrays) and PCR-based assays.
- Polynucleotides including extended length polynucleotides, sequence variants and fragments thereof, as described herein, may be used to generate hybridization probes or PCR primers for use in such assays.
- probes and primers will be capable of detecting polynucleotide sequences, including genomic sequences that are similar, or complementary to, the HPP polynucleotides described herein.
- the invention includes primer pairs for carrying out a PCR to amplify a segment of a polynucleotide of the invention.
- Each primer of a pair is an oligonucleotide having a length of between 15 and 30 nucleotides such that i) one primer of the pair forms a perfectly matched duplex with one strand of a polynucleotide of the invention and the other primer of the pair form a perfectly match duplex with the complementary strand of the same polynucleotide, and ii) the primers of a pair form such perfectly matched duplexes at sites on the polynucleotide that separated by a distance of between 10 and 2500 nucleotides.
- the annealing temperature of each primer of a pair to its respective complementary sequence is substantially the same.
- Hybridization probes derived from polynucleotides of the invention can be used, for example, in performing in situ hybridization on tissue samples, such as fixed or frozen tissue sections prepared on microscopic slides or suspended cells. Briefly, a labeled DNA or RNA probe is allowed to bind its DNA or RNA target sample in the tissue section on a prepared microscopic, under controlled conditions.
- dsDNA probes consisting of the DNA of interest cloned into a plasmid or bacteriophage DNA vector are used for this purpose, although ssDNA or ssRNA probes may also be used.
- Probes are generally oligonucleotides between about 15 and 40 nucleotides in length.
- the probes can be polynucleotide probes generated by PCR random priming primer extension or in vitro transcription of RNA from plasmids (riboprobes). These latter probes are typically several hundred base pairs in length.
- the probes can be labeled by any of a number of label groups and the particular detection method will correspond to the type of label utilized on the probe (e.g., autoradiography, X-ray detection, fluorescent or visual microscopic analysis, as appropriate).
- the reaction can be further amplified in situ using immunocytochemical techniques directed against the label of the detector molecule used, such as an antibody directed to a fluorescein moiety present on a fluorescently labeled probe.
- immunocytochemical techniques directed against the label of the detector molecule used, such as an antibody directed to a fluorescein moiety present on a fluorescently labeled probe.
- Specific labeling and in situ detection methods can be found, for example, in Howard, G. C, Ed., Methods in Nonradioactive Detection, Appleton & Lange, Norwalk, Conn., (1993), herein incorporated by reference.
- Hybridization probes and PCR primers may also be selected from the genomic sequences corresponding to the full-length proteins identified in accordance with the present invention, including promoter, enhancer elements and introns of the gene encoding the naturally occurring polypeptide.
- Nucleotide sequences encoding an HPP can also be used to construct hybridization probes for mapping the gene encoding that HPP and for the genetic analysis of individuals. Individuals carrying variations of, or mutations in the gene encoding an HPP of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids used for diagnosis may be obtained from a patient's cells, including, for example, tissue biopsy and autopsy material. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et al. Nature 324: 163-166 (1986)) prior to analysis. RNA or cDNA may also be used for the same purpose.
- PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the
- Nucleotides of the invention may be synthesized by conventional means on a commercially available automated DNA synthesizer, e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer.
- a commercially available automated DNA synthesizer e.g. an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer.
- phosphoramidite chemistry is employed, e.g. as disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al, U.S. patent 4,980,460; Koster et al, U.S. patent
- Primers and probes of the invention can also be prepared by, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang SA et al (Methods Enzymol 1979; 68:90-98), the phosphodiester method of Brown EL et al (Methods Enzymol 1979; 68:109-151), the diethylphosphoramidite method
- Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as peptide nucleic acids which are disclosed in WO 92/20702, morpholino analogs which are described in U.S. Patents 5,185,444; 5,034,506 and 5,142,047. If desired, the probe may be rendered
- nucleic acid probes can be rendered non-extendable by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation.
- the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group.
- any of the polynucleotides of the present invention can be labeled, if desired, by incorporating any label group known in the art to be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Additional examples include non-radioactive labeling of nucleic acid fragments as described in Urdea et al. (Nucleic Acids Research. 11 :4937-4957, 1988) or Sanchez-Pescador et al. (J. Clin. Microbiol. 26(10): 1934-1938, 1988).
- the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al (Nucleic Acids Symp. Ser. 24:197-200, 1991) or in the European patent No. EP 0225807 (Chiron).
- a label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support.
- a capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin).
- a polynucleotide or a probe may be employed to capture or to detect the target DNA.
- the polynucleotides, primers or probes provided herein may, themselves, serve as the capture label.
- a solid phase reagent's binding member is a nucleic acid sequence
- it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase.
- a polynucleotide probe itself serves as the binding member those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to the target.
- a polynucleotide primer itself serves as the capture label
- at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase.
- DNA labeling techniques are well known to the skilled technician.
- the probes of the present invention are useful for a number of purposes. They can be notably used in Southern hybridization to genomic DNA. The probes can also be used to detect PCR amplification products. They may 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 the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others.
- the solid support is not critical and can be selected by one skilled in the art.
- latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples.
- Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like.
- a solid support refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
- the solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
- the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
- the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
- the receptor molecule can be any specific binding member attached to the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction.
- the receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay.
- the solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes and other configurations known to those of ordinary skill in the art.
- the nucleic acids, polynucleotides, primers and probes of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention to a single solid support.
- polynucleotides other than those of the invention may be attached to the same solid support as one or more polynucleotides of the invention. Any polynucleotide provided herein may be attached in overlapping areas or at random locations on a solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be "addressable" where the distinct locations are recorded and can be accessed as part of an assay procedure.
- Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these "addressable" arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips, and has been generally described in US Patent 5,143,854; PCT publications WO 90/15070 and 92/10092. Preferably, more than one HPP polynucleotide probe is included in such an array.
- variants including 1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the amino acid residues includes a substituent group, or 3) one in which the mutated HPP is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the HPP, such as a leader, a signal or anchor sequence, a sequence which is employed for purification of the HPP, or sequence from a precursor protein.
- HPP chimeric or fusion proteins comprises an HPP of the invention or fragment thereof, operatively linked or fused in frame to a non-HPP polypeptide sequence.
- an HPP fusion protein comprises at least one biologically active portion of an HPP.
- an HPP fusion protein comprises at least two biologically active portions of an HPP.
- the fusion protein is a GST-HPP fusion protein in which HPP domain sequences are fused to the C-terminus of the GST sequences.
- fusion proteins can facilitate the purification of recombinant HPPs.
- the fusion protein is an HPP containing a heterologous signal sequence at its N-terminus, for example, to allow for a desired cellular localization in a certain host cell.
- the fusion is an HPP biologically active fragment and an immunoglobulin molecule.
- Such fusion proteins are useful, for example, to increase the valency of HPP binding sites.
- a bivalent HPP binding site may be formed by fusing biologically active HPP fragments to an IgG Fc protein.
- HPP fusion proteins of the invention are used as immunogens to produce anti-HPP antibodies in a subject, to purify HPP, or HPP ligands and adsorbants.
- isolated fragments of HPPs can also be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
- fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
- an HPP of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
- the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments with an HPP biological activity, for example, by microinjection assays or in vitro protein binding assays.
- peptidyl portions of an HPP can be tested for HPP activity (e.g., immunogenicity) by expression as thioredoxin fusion proteins, each of which contains a discrete fragment of the HPP (see, for example, U.S. Patents 5, 270,181 and 5 5,292,646; and WO94/02502).
- HPP activity e.g., immunogenicity
- thioredoxin fusion proteins each of which contains a discrete fragment of the HPP
- Peptides of the invention are synthesized by standard techniques (e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, IL, 1984).
- standard techniques e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company, Rockford, IL, 1984.
- polypeptides of the invention may be assembled from multiple, separately synthesized and purified, peptide in a convergent synthesis approach, e.g. Kent et al, U.S. patent 6,184,344 and Dawson and Kent, Annu. Rev. Biochem., 69: 923-960 (2000).
- Peptides of the invention may be assembled by solid phase synthesis on a cross-linked polystyrene support starting from the carboxyl
- polypeptides of the invention is carried out by the assembly of peptide fragments by native chemical ligation, as described by Dawson et al, Science, 266: 776-779 (1994) and Kent el al, U.S. patent 6,184,344. Briefly, in the approach a first peptide fragment is
- N-terminal cysteine having an unoxidized sulfhydryl side chain and a second peptide fragment is provided with a C-terminal thioester.
- the unoxidized sulfhydryl side chain of the N-terminal cysteine is then condensed with the C-terminal thioester to produce an intermediate peptide fragment which links the first and second peptide fragments with a ⁇ -aminothioester bond.
- the ⁇ -aminothioester bond of the intermediate peptide fragment then undergoes an intramolecular
- the N-terminal cysteine of the internal fragments is protected from undesired cyclization and/or concatenation reactions by a cyclic thiazolidine protecting group as described below.
- a cyclic thiazolidine protecting group is a thioprolinyl group.
- Peptide fragments having a C-terminal thioester may be produced as described in the following references, which are incorporated by reference: Kent et al, U.S. patent 6,184,344; Tam et al, Proc. Natl. Acad.
- peptide fragments are synthesized on a solid phase support (described below) typically on a 0.25 mmol scale by using the in situ neutralization/HBTU activation procedure for Boc chemistry disclosed by Schnolzer et al, Int. J. Peptide Protein Res, 40: 180-193 (1992), which reference is incorporated herein by reference.
- HBTU is 2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate and Boc is tert- butoxycarbonyl).
- Each synthetic cycle consists of N°-Boc removal by a 1- to 2- minute treatment with neat TFA, a 1 -minute DMF flow wash, a 10- to 20-minute coupling time with 1.0 mmol of preactivated Boc-a ino acid in the presence of DIEA, and a second DMF flow wash.
- TFA trifluoroacetic acid
- DMF is N,N-dimethylformamide
- DIEA is N,N-diisopropylethylamine
- N°- Boc-amino acids (1.1 mmol) are preactivated for 3 minutes with 1.0 mmol of HBTU (0.5 M in DMF) in the presence of excess DIEA (3 mmol).
- yields are determined by measuring residual free amine with a conventional quantitative ninhydrin assay, e.g. as disclosed in Sarin et al, Anal. Biochem, 117: 147-157 (1981).
- a DCM flow wash is used before and after deprotection by using TFA, to prevent possible high-temperature (TFA/DMF)-catalyzed pyrrolidone formation.
- TFA high-temperature
- the imidazole side-chain 2,4-dinitrophenyl (dnp) protecting groups remain on the His residues because the dnp-removal procedure is incompatible with C- terminal thioester groups. However, dnp is gradually removed by thiols during the ligation reaction. After cleavage, peptide fragments are precipitated with ice-cold diethylether, dissolved in aqueous acetonitrile, and lyophihzed. Thioester peptide fragments described above are preferably synthesized on a trityl-associated mercaptopropionic acid-leucine (TAMPAL) resin, made as disclosed by hackeng et al (1999), or comparable protocol.
- TAMPAL trityl-associated mercaptopropionic acid-leucine
- N"-Boc-Leu (4 mmol) is activated with 3.6 mmol of HBTU in the presence of 6 mmol of DIEA and coupled for 16 minutes to 2 mmol of p-methylbenzhydrylamine (MBHA) resin, or the equivalent.
- MBHA p-methylbenzhydrylamine
- 3 mmol of S-trityl mercaptopropionic acid is activated with 2.7 mmol of HBTU in the presence of 6 mmol of DIEA and coupled for 16 minutes to Leu-MBHA resin.
- the resulting TAMPAL resin can be used as a starting resin for polypeptide-chain assembly after removal of the trityl protecting group with two 1 -minute treatments with 3.5% triisopropylsilane and 2.5% H 2 0 in TFA.
- the thioester bond can be formed with any desired amino acid by using standard in situ-neutralization peptide coupling protocols for 1 hour, as disclosed in Schnolzer et al (cited above). Treatment of the final peptide fragment with anhydrous HF yields the C-terminal activated mercaptopropionic acid-leucine (MPAL) thioester peptide fragments.
- MPAL C-terminal activated mercaptopropionic acid-leucine
- thiazolidine-protected thioester peptide fragment intermediates are used in native chemical ligation under conditions as described by Hackeng et al (1999), or like conditions. Briefly, 0.1 M phosphate buffer (pH 8.5) containing 6 M guanidine, 4% (vol/vol) benzylmercaptan, and 4% (vol/vol) thiophenol is added to dry peptides to be ligated, to give a final peptide concentration of 1-3 mM at about pH 7, lowered because of the addition of thiols and TFA from the lyophihzed peptide.
- the ligation reaction is performed in a heating block at 37°C and is periodically vortexed to equilibrate the thiol additives.
- the reaction may be monitored for degree of completion by MALDI-MS or HPLC and electrospray ionization MS.
- the N-terminal thiazolidine ring of the product is opened by treatment with a cysteine deprotecting agent, such as O- methylhydroxylamine (0.5 M) at pH 3.5-4.5 for 2 hours at 37° C, after which a 10-fold excess of Tris- (2-carboxyethyl)-phosphine is added to the reaction mixture to completely reduce any oxidizing reaction constituents prior to purification of the product by conventional preparative HPLC.
- a cysteine deprotecting agent such as O- methylhydroxylamine (0.5 M) at pH 3.5-4.5 for 2 hours at 37° C
- Tris- (2-carboxyethyl)-phosphine is added to the reaction mixture to completely reduce any oxidizing reaction constituents prior to purification of the product by conventional preparative HPLC.
- fractions containing the ligation product are identified by electrospray MS, are pooled, and lyophihzed.
- the final polypeptide product may be refolded by conventional techniques, e.g. Creighton, Meth. Enzymol, 107: 305-329 (1984); White, Meth. Enzymol, 11 : 481-484 (1967); Wetlaufer, Meth. Enzymol, 107: 301-304 (1984); and the like.
- a final product is refolded by air oxidation by the following, or like:
- the reduced lyophihzed product is dissolved (at about 0.1 mg/mL) in 1 M guanidine hydrochloride (or like chaotropic agent) with 100 mM Tris, 10 mM methionine, at pH 8.6. After gentle overnight stirring, the re-folded product is isolated by reverse phase HPLC with conventional protocols.
- the polynucleotide sequences described herein can be used in recombinant DNA molecules that direct the expression of the corresponding polypeptides in appropriate host cells. Because of the degeneracy in the genetic code, other DNA sequences may encode the equivalent amino acid sequence, and may be used to clone and express the HPPs. Codons preferred by a particular host cell may be selected and substituted into the naturally occurring nucleotide sequences, to increase the rate and or efficiency of expression.
- the nucleic acid (e.g, cDNA or genomic DNA) encoding the desired HPP may be inserted into a replicable vector for cloning (amplification of the DNA), or for expression.
- the polypeptide can be expressed recombinantly in any of a number of expression systems according to methods known in the art (Ausubel, et al, editors, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1990).
- Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells, for example primary cells, including stem cells, including, but not limited to bone marrow stem cells. More specifically, these include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, and yeast transformed with yeast expression vectors.
- insect cells infected with a recombinant insect virus such as baculovirus
- mammalian expression systems The nucleic acid sequence to be expressed may 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. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.
- the HPPs of the present invention are produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding an HPP, under the appropriate conditions to induce or cause expression of the protein.
- the conditions appropriate for HPP expression will vary with the choice of the expression vector and the host cell, as ascertained by one skilled in the art.
- the use of constitutive promoters in the expression vector may require routine optimization of host cell growth and proliferation, while the use of an inducible promoter requires the appropriate growth conditions for induction.
- the timing of the harvest is important.
- the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
- a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
- modifications of the protein include, but are not limited to, glycosyl, acetyl, phosphate, amide, lipid, carboxyl, acyl, or carbohydrate groups.
- Post-translational processing which cleaves a "prepro" form of the protein, may also be important for correct insertion, folding and/or function.
- host cells such as CHO, HeLa, BHK, MDCK, 293, W138, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
- 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, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, human cells and other primary cells.
- the nucleic acid encoding an HPP must be "operably linked" by placing it into a functional relationship with another nucleic acid sequence.
- DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
- "operably linked" DNA sequences are contiguous, and, in the case of a secretory leader or other polypeptide sequence, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites.
- Promoter sequences encode either constitutive or inducible promoters.
- the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
- the expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
- 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 the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.
- the expression vector contains at least one sequence homologous to the host cell genome, and preferably, two homologous sequences which flank the expression construct.
- the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
- a heterologous expression control element may be operably linked with the endogenous gene in the host cell by homologous recombination (described in US Patents 6410266 and 6361972). This technique allows one to regulate expression to a desired level with a chosen control element while ensuring proper processing and modification of HPP endogenously expressed by the host cell.
- Useful heterologous expression control elements include but are not limited to CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), and metallothionein promoters.
- the expression vector contains a selectable marker gene to allow the 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 will typically contain a selection gene, also termed a selectable marker.
- Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g, ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available for from complex media, e.g, the gene encoding D-alanine racemase for Bacilli.
- Host cells transformed with a nucleotide sequence encoding an HPP may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
- the protein produced by a recombinant cell may be secreted, membrane-bound, or contained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides encoding the HPP can be designed with signal sequences which direct secretion of the HPP through a prokaryotic or eukaryotic cell membrane.
- the desired HPP may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
- the signal sequence may be a component of the vector, or it may be a part of the HPP-encoding DNA that is inserted into the vector.
- the signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.
- the signal sequence may be, e.g, the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), or the signal described in WO 90113646 published Nov. 15, 1990.
- mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.
- the coding sequence is inserted into an appropriate vector, which in turn may require the presence of certain characteristic "control elements” or "regulatory sequences.”
- Appropriate constructs are known generally in the art (Ausubel, et al, 1990) and, in many cases, are available from commercial suppliers such as Invitrogen (San Diego, Calif), Stratagene (La Jolla, Calif), Gibco BRL (Rockville, Md.) or Clontech (Palo Alto, Calif).
- Transformation of bacterial cells may be achieved using an inducible promoter such as the hybrid lacZ promoter of the "BLUESCPJPT" Phagemid (Stratagene) or "pSPORTl” (Gibco BRL).
- an inducible promoter such as the hybrid lacZ promoter of the "BLUESCPJPT" Phagemid (Stratagene) or "pSPORTl” (Gibco BRL).
- a number of expression vectors may be selected for use in bacterial cells to produce cleavable fusion proteins that can be easily detected and/or purified, including, but not limited to "BLUESCRJPT” (a-galactosidase; Stratagene) or pGEX (glutathione S-transferase; Promega, Madison, Wis.).
- a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the 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. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art.
- tat promoter is a hybrid of the tip and lac promoter sequences.
- a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. An efficient ribosome-binding site is also desirable.
- the expression vector may also include a signal peptide sequence that provides for secretion of the HPP in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art.
- the protein is either secreted into the growth media (gram- positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
- the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. 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. When large quantities of HPPs are needed, e.g., for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
- Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCPJPT (Stratagene), in which the HPP coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; PIN vectors (Van Heeke & Schuster JBiol Chem 264:5503-5509 1989)); PET vectors (Novagen, Madison Wis.); and the like.
- Expression vectors for bacteria include the various components set forth above, and are well known in the art. Examples include vectors for Bacillus subtilis, E.
- Bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride mediated transfection, electroporation, and others.
- Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillermondii and Ppastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
- suitable promoters for use in yeast hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman et al, J. Biol Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg.
- yeast promoters which are inducible have the additional advantage of transcription controlled by growth conditions, include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors andpromoters for use in yeast expression are further described in EP 73,657. Yeast selectable markers include ADE2. HIS4. LEU2. TRP1.
- Yeast expression vectors can be constructed for intracellular production or secretion of an HPP from the DNA encoding the HPP of interest.
- a selected signal peptide and the appropriate constitutive or inducible promoter may be inserted into suitable restriction sites in the selected plasmid for direct intracellular expression of the HPP.
- DNA encoding the HPP can be cloned into the selected plasmid, together with DNA encoding the promoter, the yeast alpha-factor secretory signal/leader sequence, and linker sequences (as needed), for 5 expression of the HPP.
- Yeast cells can then be transformed with the expression plasmids described above, and cultured in an appropriate fermentation media.
- the protein produced by such transformed yeast can then be concentrated by precipitation with 10% trichloroacetic acid and analyzed following separation by SDS-PAGE and staining of the gels with Coomassie Blue stain.
- the recombinant HPP can subsequently be isolated and purified from the fermentation medium by techniques known to
- the HPP may be expressed in mammalian cells.
- Mammalian expression systems are known in the art, and include retroviral vector mediated expression systems.
- Mammalian host cells may be
- a preferred expression vector system is a retroviral vector system such as is generally
- Suitable mammalian expression vectors contain a mammalian promoter which is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for HPP into mRNA.
- a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the
- a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
- An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
- mammalian promoters are the promoters from mammalian viral lO genes, since the viral genes are often highly expressed and have a broad host range. Examples include promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211, 504 published Jul.
- adenovirus such as Adenovirus 2
- bovine papilloma virus such as Adenovirus 2
- bovine papilloma virus such as avian sarcoma virus
- cytomegalovirus a retrovirus
- hepatitis-B virus and Simian Virus 40 SV40
- heterologous mammalian promoters e.g, the actin promoter or an immunoglobulin promoter
- heat-shock promoters provided such promoters are compatible with the host cell systems.
- Transcription of DNA encoding an HPP by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.
- Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription.
- enhancer sequences are now known 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 on the late side of the replication origin, and adenovirus enhancers. The enhancer is preferably located at a site 5' from the promoter. In general, the transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
- the 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
- transcription terminator and polyadenylation signals include those derived from SV40.
- Long term, high-yield production of recombinant proteins can be effected in a stable expression system.
- Expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene may be used for this purpose. Appropriate vectors containing selectable markers for use in mammalian cells are readily available commercially and are known to persons skilled in the art.
- selectable markers include, but are not limited to herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase for use in tk- or hprt-cells, respectively.
- the methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well 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 the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
- HPPs can be purified from culture supernatants of mammalian cells transiently transfected or stably transformed by an expression vector carrying an HPP-encoding sequence.
- HPP is purified from culture supernatants of COS 7 cells transiently transfected by the pcD expression vector.
- Transfection of COS 7 cells with pcD proceeds.as follows: One day prior to transfection, approximately 10 ⁇ COS 7 monkey cells are seeded onto individual 100 mm plates in Dulbecco's modified Eagle medium (DME) containing 10% fetal calf serum and 2 mM glutamine.
- DME Dulbecco's modified Eagle medium
- the medium is aspirated from each plate and replaced with 4 ml of DME containing 50 mM Tris.HCl pH 7.4, 400 mg/ml DEAE-Dextran and 50 ⁇ g of plasmid DNA.
- the plates are incubated for four hours at 37°C, then the DNA-containing medium is removed, and the plates are washed twice with 5 ml of serum-free DME.
- DME is added back to the plates which are then incubated for an additional 3 hrs at 37°C.
- the plates are washed once with DME, after which DME containing fetal calf serum, 2 mM glutamine, penicillin (100 U/L) and streptomycin (100 ⁇ gL) at standard concentrations is added.
- Plasmid DNA for the transfections is obtained by growing pcD(SR ⁇ ), or like expression vector, containing the HPP-encoding cDNA insert in E. coli MCI 061 (described by Casadaban and Cohen, J. Mol. Biol, Vol. 138, pgs. 179-207 (1980)), or like organism.
- the plasmid DNA is isolated from the cultures by standard techniques, e.g. Sambrook et al. Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, New York, 1989) or Ausubel et al (1990, cited above).
- HPPs may also be produced in insect cells.
- Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
- the HPP-encoding DNA is fused upstream of an epitope tag contained within a baculovirus expression vector.
- Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda Sf9 cells or in Trichoplusia larvae.
- the HPP- encoding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
- telomere sequence Successful insertion of an HPP-encoding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat.
- the recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the HPP is expressed (Smith et al, J. Wol. 46:584 (1994); Engelhard E K et al, Proc. Nat. Acad. Sci. 91:3224-3227 (1994)).
- Suitable epitope tags for fusion to the HPP-encoding DNA include poly-his tags and immunoglobulin tags (like Fc regions of IgG).
- a variety of plasmids may be employed, including commercially available plasmids such as pVL1393 (Novagen).
- the HPP-encoding DNA or the desired portion of the HPP-encoding DNA is amplified by PCR with primers complementary to the 5' and 3' regions.
- the 5' primer may incorporate flanking restriction sites.
- the PCR product is then digested with the selected restriction enzymes and subcloned into an expression vector. Recombinant baculovirus is generated by co-transfecting the above plasmid and
- BaculoGoldTM virus DNA (Pharmingen) into Spodoptera frugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL), or other methods known to those of skill in the art.
- Virus is produced by day 4-5 of culture in Sf9 cells at 28°C, and used for further amplifications. Procedures are performed as further described in O'Reilley et al, BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, Oxford University Press (1994). Extracts may be prepared from recombinant virus-infected Sf9 cells as described in Rupert et al. Nature 362:175- 179 (1993).
- expressed epitope-tagged HPP can be purified by affinity chromatography, or for example, purification of an IgG tagged (or Fc tagged) HPP can be performed using chromatography techniques, including Protein A or protein G column chromatography.
- Gene expression may be evaluated in a sample directly, for example, by standard techniques known to those of skill in the art, e.g. Northern blotting to determine the transcription of mRNA, dot blotting (DNA or RNA), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein.
- antibodies may be used in assays for detection of polypeptides, nucleic acids, such as specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes.
- Such antibodies may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
- Gene expression alternatively, may be measured by immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to directly evaluate the expression of an HPP polypeptide or polynucleotide.
- Antibodies useful for such immunological assays may be either monoclonal or polyclonal, and may be prepared against a native sequence HPP. Protein levels may also be detected by mass spectrometry.
- a further method of protein detection is with retentate chromatography (including protein arrays) and surface enhanced laser desorption/ionization (SELDI) techniques.
- HPP Purification of Expressed Protein Expressed HPP may be purified or isolated after expression, using any of a variety of methods known to those skilled in the art. The appropriate technique will vary depending upon what other components are present in the sample. Contaminant components that are removed by isolation or purification are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other solutes.
- the purification step(s) selected will depend, for example, on the nature of the production process used and the particular HPP produced. As HPPs are secreted, they may be recovered from culture medium. Alternatively, the HPP may be recovered from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g.
- Triton-X 100 or by enzymatic cleavage.
- cells employed in expression of HPP can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or by use of cell lysing agents.
- Exemplary purification methods include, but are not limited to, ion-exchange column chromatography; chromatography using silica gel or a cation-exchange resin such as DEAE; gel
- an isolated HPP will be prepared by at least one purification step.
- the HPP may be purified using a standard anti-HPP antibody column.
- the invention further provides methods of testing the activity of or obtaining functional fragments and variants of HPPs and HPP sequences. Such methods involve providing a variant or modified HPP-encoding nucleic acid and assessing whether the encoded
- polypeptide displays an HPP biological activity.
- a method of assessing the function of an HPP comprising: (a) providing an HPP, or a biologically active fragment or homologue thereof; and (b) testing said HPP, or a biologically active fragment or homologue thereof for an HPP biological activity under conditions suitable for HPP activity.
- Cell free, cell-based and in vivo assays may be used to test HPP activity.
- said assay may comprise expressing an HPP nucleic
- HPP biological activities include: (1) circulating through the bloodstream of human individuals; (2) antigenicity, or the ability to bind an anti-HPP specific antibody; (3) immunogenicity,
- HPP biological activity can be assayed by any suitable method known in the art.
- Antigenicity and immunogenicity may be detected, for example, as described in the sections titled "Anti HPP antibodies” and “Uses of HPP antibodies”.
- Interaction of a test compound with an HPP can be detected, for example, by coupling the HPP or biologically active portion thereof with a label group such that binding of the HPP or biologically active portion thereof to its cognate target molecule can be determined by detecting the labeled HPP or biologically active portion thereof in a complex.
- the extent of complex formation may be measured by immunoprecipitating the complex or by performing gel electrophoresis. Determining the ability of the HPP to bind to an HPP target molecule may also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA).
- BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g, BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. Protein array methods are useful for detecting interaction. For example, one member of a receptor/ ligand pair is docked to an adsorbent, and its ability to bind the binding partner is determined in the presence of the 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 the interaction.
- SPR surface plasmon resonance
- HPPs are docked to the adsorbent.
- Binding partners are preferably labeled, thus enabling detection of the interaction.
- a test substance is docked to the adsorbent.
- the polypeptides of the invention are exposed to the test substance and binding detected.
- Anti-HPP Antibodies The present invention provides antibodies and binding compositions specific for HPPs.
- Such antibodies and binding compositions include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv fragments thereof, bispecific antibodies, heteroconjugates, and humanized antibodies.
- Such antibodies and binding compositions may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. There is abundant guidance in the literature for selecting a particular production methodology, e.g. Chadd and Chamow, Curr. Opin. Biotechnol, 12: 188-194 (2001).
- antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost.
- Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species.
- Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system.
- Single chain Fv fragments are highly selective for in vivo tumors, show good tumor penetration and low immunogenicity, and are cleared rapidly from the blood, e.g. Freyre et al, J. Biotechnol, 76: 157-163 (2000). Thus, such molecules are desirable for radioimmunodetection.
- the anti-HPP antibodies of the present invention may be polyclonal antibodies.
- Such polyclonal antibodies can be produced in a mammal, for example, following one or more injections of an immunizing agent, and preferably, an adjuvant.
- the immunizing agent and/or adjuvant will be injected into the mammal by a series of subcutaneous or intraperitoneal injections.
- the immunizing agent may include HPPs 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.
- immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), methylated bovine serum albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
- Adjuvants include, for example, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicoryno-mycolate).
- the immunization protocol may be determined by one skilled in the art based on standard protocols or by routine experimentation. Alternatively, a crude protein preparation which has been enriched for an HPP or a portion thereof can be used to generate antibodies.
- Such proteins, fragments or preparations are introduced into the non-human mammal in the presence of an appropriate adjuvant.
- the serum contains polyclonal antibodies to undesired epitopes, the polyclonal antibodies are purified by immunoaffinity chromatography. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. Also, host animals vary in response to site of inoculations and dose, with both inadequate and excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appear to be most reliable. Techniques for producing and processing polyclonal antisera are known in the art, see for example, Mayer and Walker (1987).
- Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al. Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum.
- Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D, Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol, Washington, D. C. (1980).
- the anti-HPP antibodies may be monoclonal antibodies.
- Monoclonal antibodies may be produced by hybridomas, wherein a mouse, hamster, or other appropriate host animal, is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent, e.g. Kohler and Milstein, Nature 256:495 (1975).
- the immunizing agent will typically include the HPP or a fusion protein thereof and optionally a carrier.
- the lymphocytes may be immunized in vitro.
- lymph node cells are used if non-human mammalian sources are desired, or peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired.
- the lymphocytes are fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to produce a hybridoma cell, e.g. Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE, Academic Press, pp. 59-103 (1986); Liddell and Cryer, A Practical Guide to Monoclonal Antibodies (John Wiley & Sons, New York, 1991); Malik and Lillenoj, Editors, Antibody Techniques (Academic Press, New York, 1994).
- a suitable fusing agent such as polyethylene glycol
- immortalized cell lines are transformed mammalian cells, for example, myeloma cells of rat, mouse, bovine or human origin.
- the hybridoma cells are cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells.
- a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of unfused, immortalized cells.
- the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT)
- HGPRT hypoxanthine guanine phosphoribosyl transferase
- the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT), substances which prevent the growth of HGPRT-deficient cells.
- Preferred immortalized cell lines are those that fuse efficiently, support stable high level production of antibody, and are sensitive to a medium such as HAT medium.
- More preferred immortalized cell lines are murine or human myeloma lines, which can be obtained, for example, from the American Type Culture Collection (ATCC), Rockville, MD. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies, e.g. Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al. Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987). The culture medium (supernatant) in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against an HPP.
- the binding specificity of monoclonal antibodies present in the hybridoma supernatant is determined by immunoprecipitation or by an in vitro binding assay, such as radio-immunoassay (RIA) or Enzyme-Linked Immuno Sorbent Assay (ELISA). Appropriate techniques and assays are known in the art.
- the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem. 107:220 (1980). After the desired antibody-producing hybridoma cells are identified, the cells may 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.
- the hybridoma cells may be grown in vivo as ascites in a mammal.
- the monoclonal antibodies secreted by selected clones may be isolated or purified from the culture medium or ascites fluid by immunoglobulin purification procedures routinely used by those of skill in the art such as, for example, protein A-Sepharose, hydroxyl-apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
- the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567.
- DNA encoding the monoclonal antibodies of the invention can be isolated from the HPP-specific hybridoma cells and sequenced, e.g, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies.
- the ' DNA may be inserted into an expression vector, which is then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
- the DNA also may be modified, for example, by substituting the coding sequence for the murine heavy and light chain constant domains for the 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 by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
- the non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
- the antibodies may also be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, in vitro methods are suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. Antibodies and antibody fragments characteristic of hybridomas of the invention can also be produced by recombinant means by extracting messenger RNA, constructing a cDNA library, and selecting clones which encode segments of the antibody molecule.
- such techniques can be used to produce interspecific monoclonal antibodies, wherein the binding region of one species is combined with non-binding region of the antibody of another species to reduce immunogenicity, e.g. Liu et al, Proc. Natl. Acad. Sci, Vol. 84, pgs. 3439-3443 (1987), and patents 6,054,297 and 5,530,101.
- recombinantly produced Fab and Fv fragments are expressed in bacterial host systems.
- full-length antibodies are produced by mammalian cell culture techniques. More preferably, full-length antibodies are expressed in Chinese Hamster Ovary (CHO) cells or NSO cells.
- Both polyclonal and monoclonal antibodies can be screened by ELISA.
- the test is based on the tendency of macromolecules to adsorb nonspecif ⁇ cally to plastic. The irreversibility of this reaction, without loss of immunological activity, allows the formation of antigen-antibody complexes with a simple separation of such complexes from unbound material.
- peptide conjugated to a carrier different from that used in immunization is adsorbed to the wells of a 96-well microtiter plate. The adsorbed antigen is then allowed to react in the wells with dilutions of anti-peptide serum.
- HPP peptide carriers The invention includes immunogens derived from HPPs, and immunogens comprising conjugates between carriers and peptides of the invention.
- immunogen refers to a substance which is capable of causing an immune response.
- carrier refers to any substance which when chemically conjugated to a peptide of the invention permits a host organism immunized with the resulting conjugate to generate antibodies specific for the conjugated peptide. Carriers include red blood cells, bacteriophages, proteins, or synthetic particles such as agarose beads.
- carriers are proteins, such as serum albumin, gamma-globulin, keyhole limpet hemocyanin (KLH), thyroglobulin, ovalbumin, or fibrinogen.
- proteins such as serum albumin, gamma-globulin, keyhole limpet hemocyanin (KLH), thyroglobulin, ovalbumin, or fibrinogen.
- KLH keyhole limpet hemocyanin
- thyroglobulin thyroglobulin
- ovalbumin ovalbumin
- fibrinogen fibrinogen
- a general rule for selecting an appropriate method for coupling a given peptide to a protein carrier can be stated as follows: the group involved in attachment should occur only once in the sequence, preferably at the appropriate end of the segment. For example, BDB should not be used if a tyrosine residue occurs in the main part of a sequence chosen for its potentially antigenic character. Similarly, centrally located lysines rule out the glutaraldehyde method, and the occurrences of aspartic and glutamic acids frequently exclude the carbodiimide approach. On the other hand, suitable residues can be positioned at either end of chosen sequence segment as attachment sites, whether or not they occur in the "native" protein sequence.
- tyrosine can be introduced as a terminal residue if it is not part of the peptide sequence defined by the native polypeptide.
- Preferred carriers are proteins, and preferred protein carriers include bovine serum albumin, myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or the like.
- Peptides can be linked to KLH through cysteines by MBS as disclosed by Liu et al. Biochemistry, Vol. 18, pgs. 690- 697 (1979). The peptides are 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 the dissolution of the peptide is chosen to optimize peptide solubility.
- the content of free cysteine for soluble peptides is determined by Ellman's method, Ellman, Arch. Biochem. Biophys, Vol. 82, pg. 7077 (1959).
- 4 mg KLH in 0.25 ml of 10 mM sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg MBS
- KLH-MBS (dissolved in dimethyl formamide) and stirred for 30 min at room temperature.
- the MBS is added dropwise to ensure that the local concentration of formamide is not too high, as KLH is insoluble in >30% formamide.
- the reaction product, KLH-MBS is then passed through Sephadex G-25 equilibrated with 50 mM sodium phosphate buffer (pH 6.0) to remove free MBS, KLH recovery from peak fractions of the column eluate (monitored by OD280) is estimated to be approximately 80%.
- KLH-MBS is then reacted with 5 mg peptide dissolved in 1 ml of the chosen buffer. The pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at room temperature.
- Coupling efficiency is monitored with radioactive peptide by dialysis of a sample of the conjugate against phosphate- buffered saline, and may range from 8% to 60%.
- polyclonal or monoclonal antibodies are produced by standard techniques, e.g. as disclosed by
- the anti-HPP antibodies of the invention may further comprise humanized antibodies or human antibodies.
- humanized antibody refers to humanized forms of non-human (e.g, murine) antibodies that are chimeric antibodies, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding partial sequences of antibodies) which contain some portion of the sequence derived from non-human antibody.
- Humanized antibodies include human immunoglobulins in which residues from a complementary determining region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired binding specificity, affinity and capacity.
- CDR complementary determining region
- the humanized antibody will comprise substantially all of at least one, and generally two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
- the humanized antibody optimally also will 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)).
- Fc immunoglobulin constant region
- a humanized antibody has one or more amino acids introduced into it from a source which is non-human in order to more closely resemble a human antibody, while still retaining the original binding activity of the antibody.
- Methods for humanization of antibodies are further detailed in Jones et al. Nature 321:522-525 (1986); Riechmann et al. Nature 332:323-327 (1988); and Verhoeyen et al. Science 239:1534-1536 (1988).
- Such "humanized" antibodies are chimeric antibodies in that substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
- Heteroconjugate antibodies which comprise two covalently joined antibodies, are also within the scope of the present invention.
- Heteroconjugate antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
- immunotoxins may be prepared using a disulfide exchange reaction or by forming a thioether bond.
- Bispecific antibodies have binding specificities for at least two different antigens. Such antibodies are monoclonal, and preferably human or humanized. One of the binding specificities of a bispecific antibody of the present invention is for an HPP, and the other one is preferably for a cell- surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art, and in general, the recombinant production of bispecific antibodies is based on the co- expression of two immunoglobulin heavy-chain/light-chain pairs in hybridoma cells, where the two heavy chains have different specificities, e.g. Milstein and Cuello, Nature 305:537-539 (1983). Given that the random assortment of immunoglobulin heavy and light chains results in production of potentially ten different antibody molecules by the hybridomas, purification of the correct molecule usually requires some sort of affinity purification, e.g. affinity chromatography.
- HPP antibodies are preferably specific for the HPPs of the invention and, as such, do not bind peptides derived from other proteins with high affinity.
- the term "heavy chain variable region” means a polypeptide (1) which is from 110 to 125 amino acids in length, and (2) whose amino acid sequence corresponds to that of a heavy chain of an antibody of the invention, starting from the heavy chain's N-terminal amino acid.
- the term "light chain variable region” means a polypeptide (1) which is from 95 to 115 amino acids in length, and (2) whose amino acid sequence corresponds to that of a light chain of an antibody of the invention, starting from the light chain's N-terminal amino acid.
- the term "monoclonal antibody” refers to homogeneous populations of immunoglobulins which are capable of specifically binding to HPPs.
- the use of antibody fragments is also well known, e.g. Fab fragments: Tijssen, Practice and Theory of Enzyme Immunoassays (Elsevier, Amsterdam, 1985); and Fv fragments: Hochman et al. Biochemistry, Vol. 12, pgs. 1 130-1135 (1973), Sharon et al. Biochemistry, Vol. 15, pgs. 1591-1594 (1976) and Ehrlich et al, U.S. Patent 4,355,023; and antibody half molecules: Auditore- Hargreaves, U.S. Patent 4,470,925.
- monoclonal antibodies, Fv fragments, Fab fragments, or other binding compositions derived from monoclonal antibodies of the invention have a high affinity to HPPs.
- the affinity of monoclonal antibodies and related molecules to HPPs may be measured by conventional techniques including plasmon resonance, ELISA, or equilibrium dialysis. Affinity measurement by plasmon resonance techniques may be carried out, for example, using a BIAcore 2000 instrument (Biacore AB, Uppsala, Sweden) in accordance with the manufacturer's recommended protocol.
- affinity is measured by ELISA, as described in U.S. patent 6,235,883, for example.
- the dissociation constant between HPPs and monoclonal antibodies of the invention is less than 10 "5 molar. More preferably, such dissociation constant is less than 10 "8 molar; still more preferably, such dissociation constant is less than 10 "9 molar; and most preferably, such dissociation constant is in the range of 10 "9 to 10 "11 molar.
- the antibodies of the present invention are useful for detecting HPPs. Such detection methods are advantageously applied to diagnosis of HPP-related disorders.
- the antibodies of the invention may be used in most assays involving antigen-antibody reactions.
- the assays may be homogeneous or heterogeneous.
- the sample can be a biological sample or fluid such as serum, urine, whole blood, lymphatic fluid, plasma, saliva, cells, tissue, and material secreted by cells or tissues cultured in vitro.
- the sample can be pretreated if necessary to remove unwanted materials.
- the immunological reaction usually involves the specific antibody, a labeled analyte, and the sample suspected of containing the antigen.
- the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
- Immunochemical labels which may be employed include free radicals, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
- the reagents are usually the sample, the specific antibody, and means for producing a detectable signal.
- the specimen is generally placed on a support, such as a plate or a slide, and contacted with the antibody in a 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 employing means for producing such signal or signal producing system.
- the signal is related to the presence of the antigen in the sample.
- Means for producing a detectable signal includes the use of radioactive labels, fluorescent compounds, enzymes, and so forth.
- Exemplary heterogeneous immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
- an assay employing an antibody of the present invention involves the use of a surface to which the monoclonal antibody of the invention is attached.
- the underlying structure of the surface may take different forms, have different compositions and may be a mixture of compositions or laminates or combinations thereof.
- the surface may assume a variety of shapes and forms and may have varied dimensions, depending on the manner of use and measurement. Illustrative surfaces may be pads, beads, discs, or strips which may be flat, concave or convex.
- Thickness is not critical, generally being from about 0.1 to 2 mm thick and of any convenient diameter or other dimensions.
- the surface typically will be supported on a rod, tube, capillary, fiber, strip, disc, plate, cuvette and will typically be porous and polyfunctional or capable of being polyfunctionalized so as to permit covalent binding of an antibody and permit bonding of other compounds which form a part of a means for producing a detectable signal.
- a wide variety of organic and inorganic polymers, both natural and synthetic, and combinations thereof, may be employed as the material for the solid surface.
- Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethracrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and latex.
- Other surfaces include paper, glasses, ceramics, metals, metaloids, semiconductor materials, cements, silicates or the like.
- substrates that form gels, gelatins, lipopolysaccharides, silicates, agarose and polyacrylamides or polymers which form several aqueous phases such as dextrans, polyalkylene glycols (alkylene of 2 to 3 carbon atoms) or surfactants such as phospholipids.
- dextrans polyalkylene glycols (alkylene of 2 to 3 carbon atoms)
- surfactants such as phospholipids.
- the binding of the antibody to the surface may be accomplished by well known techniques, commonly available in the literature (see, for example, “Immobilized Enzymes,” Ichiro Chibata, Press, New York (1978) and Cuatrecasas, J. Bio. Chem, 245: 3059 (1970)).
- the sample is mixed with aqueous medium and the medium is contacted with the surface having an antibody bound thereto.
- Labels may be included in the aqueous medium, either concurrently or added subsequently so as to provide a detectable signal associated with the surface.
- the means for producing the detectable signal can involve the incorporation of a labeled analyte or it may involve the use of a second monoclonal antibody having a label conjugated thereto. Separation and washing steps will be carried out as needed.
- the signal detected is related to the presence of HPP in the sample. It is within the scope of the present invention to include a calibration on the same support.
- a particular embodiment of an assay in accordance with the present invention involves the use of a support such as a slide or a well of a petri dish.
- the technique involves fixing the sample to be analyzed on the support with an appropriate fixing material and incubating the sample on the slide with a monoclonal antibody. After washing with an appropriate buffer such as, for example, phosphate buffered saline, the support is contacted with a labeled specific binding partner for the antibody. After incubation as desired, the slide is washed a second time with an aqueous buffer and the determination is made of the binding of the labeled monoclonal antibody to the antigen.
- an appropriate buffer such as, for example, phosphate buffered saline
- the slide may be covered with a fluorescent antibody mounting fluid on a cover slip and then examined with a fluorescent microscope to determine the extent of binding.
- the label can be an enzyme conjugated to the monoclonal antibody and the extent of binding can be determined by examining the slide for the presence of enzyme activity, which may be indicated by the formation of a precipitate, color, etc.
- a particular example of an assay utilizing the present antibodies is a double determinant ELISA assay.
- a support such as, e.g, a glass or vinyl plate, is coated with an antibody specific for HPP by conventional techniques. The support is contacted with the sample suspected of containing HPP, usually in aqueous medium.
- the support is separated from the medium, washed to remove unbound HPP with, for example, water or an aqueous buffered medium, and contacted with an antibody specific for HPP, again usually in aqueous medium.
- the antibody is labeled with an enzyme directly or indirectly such as, e.g, horseradish peroxidase or alkaline phosphatase.
- the support is separated from the medium, and washed as above. The enzyme activity of the support or the aqueous medium is determined. This enzyme activity is related to the amount of HPP in the sample.
- Another antibody-utilizing method of detection includes retentate chromotography methods, as described herein.
- the adsorbant is an antibody.
- more than one antibody specific for more than one HPP of the invention is included on a surface or substrate.
- the invention also includes kits, e.g, diagnostic assay kits, for carrying out the methods disclosed above.
- the kit comprises in packaged combination (a) a monoclonal antibody more specifically defined above and (b) a conjugate of a specific binding partner for the above monoclonal antibody and a label capable of producing a detectable signal.
- the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g, polysaccharides and the like.
- the kit may further include, where necessary, other members of the signal producing system of which system the label is a member, agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
- the diagnostic kit comprises a conjugate of monoclonal antibody of the invention and a label capable of producing a detectable signal.
- Ancillary agents as mentioned above may also be present.
- an anti-HPP antibody e.g, monoclonal antibody
- an anti- HPP antibody can be used to isolate HPPs by standard techniques, such as affinity chromatography or immunoprecipitation.
- an anti- HPP antibody can facilitate the purification of natural HPPs from cells and of recombinantly produced HPP expressed in host cells.
- an anti-HPP antibody can be used to isolate HPP to aid in detection of low concentrations of HPP (e.g, in plasma, cellular lysate or cell supernatant) or in order to evaluate the abundance and pattern of expression of the HPP.
- Anti-HPP antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a label group.
- Detection and purification of the polypeptides of the invention may be accomplished using retentate chromatography (preferably, protein arrays or chips), as described by U.S. Patent 6225027 and U.S. Patent Application 20010014461. Briefly, retentate chromatography describes methods in which polypeptides (and/ or other sample components) are retained on an adsorbent (e.g, array or chip) and subsequently detected.
- retentate chromatography preferably, protein arrays or chips
- Such methods involve (1) selectively adsorbing polypeptides from a sample to a substrate under a plurality of different adsorbent/eluant combinations ("selectivity conditions") and (2) detecting the retention of adsorbed polypeptides by desorption spectrometry (e.g, by mass spectrometry).
- desorption spectrometry refers to a method of detecting a substance (e.g, polypeptide) in which the substance is exposed to energy which desorbs it from a stationary phase into a gas phase, and the desorbed substance or a distinguishable portion of it is directly detected by a detector, without an intermediate capture on a second stationary phase.
- polypeptides are eluted off of the adsorbent prior to detection.
- the coupling of adsorption chromatography with detection by desorption spectrometry provides extraordinary sensitivity, the ability to rapidly analyze retained components with a variety of different selectivity conditions, and parallel processing of components adsorbed to different sites (i.e., "affinity sites” or “spots") on the array under different elution conditions.
- a preferred embodiment provides adsorbents, either chemical (e.g, ion-affinity or hydrophobic substances) or biospecific (e.g, antibodies or antigen-binding fragments thereof), developed to detect a specific polypeptide, preferably an HPP.
- a substrate has an array of adsorbent spots selected for a combination of polypeptides, e.g. as diagnostic markers. As few as two and as many as 10, 100, 1000, or more adsorbents can be coupled to a single substrate.
- the size of the adsorbent site may be varied, depending on experimental design and purpose. However, it does not need to be larger than the diameter of the impinging energy source (e.g, laser spot diameter).
- the spots can be made of the same or different adsorbents.
- adsorbent at multiple locations on the substrate to permit evaluation against a plurality of different eluants or so that the bound polypeptide can be preserved for future use or reference, perhaps in secondary processing.
- a substrate with a plurality of different adsorbents, it is possible to utilize the plurality of binding characteristics provided by the combination of different adsorbents with respect to a single sample (e.g, plasma sample) and thereby bind and detect a wider variety of different polypeptides (preferably HPPs).
- the use of a plurality of different adsorbents on a substrate for evaluation of a single sample is essentially equivalent to concurrently conducting multiple chromatographic experiments, each with a different chromatography column, but the present method has the advantage of requiring only a single system.
- the substrate includes a plurality of adsorbents, it is particularly useful to provide the adsorbents in predetermined addressable locations. By providing the adsorbents in predetermined addressable locations, it is possible to wash an adsorbent at a first predetermined addressable location with a first eluant and to wash an adsorbent at a second predetermined addressable location with a second eluant.
- Retentate chromotography may be used in a combinatorial separation method that includes separation and detection of multiple polypeptides in parallel.
- the method comprises the steps of a) exposing a sample (e.g, a biological fluid such as plasma) to at least two different selectivity conditions, each selectivity condition defined by the combination of an adsorbent and an eluant, to allow retention of a polypeptide by the adsorbent; and b) detecting retained polypeptide under the different selectivity conditions by desorption spectrometry.
- a sample e.g, a biological fluid such as plasma
- Each different selectivity condition is defined at a different predetermined, addressable location for parallel processing.
- the method comprises the steps of i) exposing a sample to a first selectivity condition at a defined location to allow retention of a polypeptide by an adsorbent; ii) detecting retained polypeptide under the first selectivity condition by desorption spectrometry; iii) washing the adsorbent under a second, different selectivity condition at the defined location to allow retention of a polypeptide to the adsorbent at that location; and iv) detecting retained polypeptides under the second selectivity condition by desorption spectrometry.
- Polypeptides may be detected by gas phase ion spectrometry, SELDI, or mass spectrometry techniques detailed herein.
- Gas phase ion spectrometry refers to a method of employing an ionization source to generate gas phase ions from a substance presented on a surface and detecting the gas phase ions with a gas phase ion spectrometer.
- Surface-enhanced laser desorption/ionisation or “SELDI” is a method of gas phase ion spectrometry in which the substance-presenting surface plays an active role in desorption and ionization process.
- SELDI technology is described in, e.g., U.S. Patents 5,719,060 and 6,225,047 (Hutchens and Yip).
- HPPs of the invention or HPP-binding substances are preferably attached to a label group, and thus directly detected, enabling simultaneous transmission of two or more signals from the same "circuit" (i.e., addressable "chip” location) during a single unit operation.
- a preferred embodiment of the invention encompasses use of retentate chromotography to identify at least one HPP in a sample, preferably plasma.
- a variation of microchip-based detection utilizes electrophoresis, as described by Chen, et al. (Anal Chem 2002; 74:5146-53).
- a flow-through sampling chip is applied to immunoseparation, protein purification, concentration, and detection purposes.
- This device uses hydrodynamic pressure to drive the sample flow, and a gating voltage is applied to the electrophoretic channel on the microchip.
- the wash/elution step can be integrated on-line with electrophoretic separation and detection on the microchip.
- the electrical field-free bed ensures that protein-adsorbant interaction will not be affected by the electric field during the wash/elution step.
- any instrument, method, process, etc. can be utilized to determine the identity and abundance of proteins in a sample.
- a preferred method of obtaining identity is by mass spectrometry, where protein molecules in a sample are ionized and then the resultant mass and charge of the protein ions are detected and determined.
- mass spectrometry it is preferred that the protein be converted to a gas-ion phase.
- Various methods of protein ionization are useful, including, e.g, fast ion bombardment (FAB), plasma desorption, laser desorption, thermal desorption, preferably, electrospray ionization
- mass analyzers are available for peptide and protein analysis, including, but not limited to, Time-of-Flight (TOF), ion trap (ITMS), Fourier transform ion cyclotron (FTMS), quadrupole ion trap, and sector (electric and/or magnetic) spectrometers. See, e.g, U.S. Pat. No. 5,572,025 for an ion trap MS.
- TOF Time-of-Flight
- IMS ion trap
- FTMS Fourier transform ion cyclotron
- quadrupole ion trap and sector (electric and/or magnetic) spectrometers. See, e.g, U.S. Pat. No. 5,572,025 for an ion trap MS.
- Mass analyzers can be used alone, or in combination with other mass analyzers in tandem mass spectrometers.
- a first mass analyzer can be use to separate the protein ions (precursor ion) from each other and determine the molecular weights of the various protein constituents in the sample.
- a second mass analyzer can be used to analyze each separated constituents, e.g, by fragmenting the precursor ions into product ions by using, e.g. an inert gas. Any desired combination of mass analyzers can be used, including, e.g, triple quadrupoles, tandem time-of-flights, ion traps, and/or combinations thereof.
- Different kinds of detectors can be used to detect the protein ions. For example, destructive detectors can be utilized, such as ion electron multipliers or cryogenic detectors (e.g, U.S.
- Non-destructive detectors can be used, such as ion traps which are used as ion current pick-up devices in quadrupole ion trap mass analyzers or FTMS.
- ion traps which are used as ion current pick-up devices in quadrupole ion trap mass analyzers or FTMS.
- FTMS quadrupole ion trap mass analyzers
- MALDI-TOF a number of sample preparation methods can be utilized including, dried droplet (Karasand Hillenkamp, Anal. Chem, 60:2299-2301, 1988), vacuum-drying (Winberger et al. In Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics, San Francisco, May 31-June 4, 1993, pp. 775a-b), crush crystals (Xiang et al. Rapid Comm. Mass
- samples are prepared as solid-state co-crystals or thin films by mixing them with an energy absorbing compound or colloid (the matrix) in the liquid phase, and ultimately drying the solution to the solid state upon the surface of an inert probe.
- an energy absorbing compound or colloid the matrix
- EAM energy absorbing molecule
- the probe contents are allowed to dry to the solid state prior to introduction into the laser desorption/ionization time-of-flight mass spectrometer (LDIMS).
- Ion detection in TOF mass spectrometry is typically achieved with the use of electro-emissive detectors such as electron multipliers (EMP) or microchannel plates (MCP).
- EMP electron multipliers
- MCP microchannel plates
- Both of these devices function by converting primary incident charged particles into a cascade of secondary, tertiary, quaternary, etc. electrons.
- the probability of secondary electrons being generated by the impact of a single incident charged particle can be taken to be the ion-to-electron conversion efficiency of this charged particle (or more simply, the conversion efficiency).
- the total electron yield for cascading events when compared to the total number of incident charged particles is typically described as the detector gain. Because generally the overall response time of MCPs is far superior to that of EMPs, MCPs are the preferred electro-emissive detector for enhancing mass/charge resolving power. However, EMPs function well for detecting ion populations of disbursed kinetic energies, where rapid response time and broad frequency bandwidth are not necessary.
- LC-TMS liquid-chromatography tandem mass spectrometer
- ESI Electrospray ionization
- MALDI MALDI
- CE capillary electrophoresis
- MS Moini, M, Anal Bioanal Chem, 2002; 373:466-80
- a small portion of intact proteins eluting from RP2 may be diverted to online detection using LC-ESI MS.
- the proteins are aliquoted on a number of plates allowing digestion or not with trypsin, preparation for MALDI-MS as well as for ESI-MS, as well as preparation of the MALDI plates with different matrices.
- the methods thus allow, in addition to information on intact mass, to conduct an analysis by both peptide mass fingerprinting and MS-MS techniques.
- the methods described herein of separating and fractionating proteins provide individual proteins or fractions containing small numbers of distinct proteins. These proteins can be identified by mass spectral determination of the molecular masses of the protein and peptides resulting from the fragmentation thereof.
- Data collected from a mass spectrometer typically comprises the intensity and mass to charge ratio for each detected event.
- Spectral data can be recorded in any suitable form, including, e.g, in graphical, numerical, or electronic formats, either in digital or analog form.
- Spectra are preferably recorded in a storage medium, including, e.g, magnetic, such as floppy disk, tape, or hard disk; optical, such as CD-ROM or laser-disc; or, ROM-CHJPS.
- the mass spectrum of a given sample typically provides information on protein intensity, mass to charge ratio, and molecular weight.
- the molecular weights of proteins in the sample are used as a matching criterion to query a database.
- the molecular weights are calculated conventionally, e.g, by subtracting the mass of the ionizing proton for singly- charged protonated molecular ions, by multiplying the measured mass/charge ratio by the number of charges for multiply-charged ions and subtracting the number of ionizing protons.
- Various databases are useful in accordance with the present invention. Useful databases include, databases containing genomic sequences, expressed gene sequences, and/or expressed protein sequences. Preferred databases contain nucleotide sequence-derived molecular masses of proteins present in a known organism, organ, tissue, or cell-type.
- ORF open reading frames
- Several publicly accessible databases are available, including, the SwissPROT/TrEMBL database (http://www.expasy.ch).
- a mass spectrometer is equipped with commercial software that identifies peaks above a certain threshold level, calculates mass, charge, and intensity of detected ions. Correlating molecular weight with a given output peak can be accomplished directly from the spectral data, i.e., where the charge on an ion is one and the molecular weight is therefore equal to the numerator value minus the mass of the ionizing proton.
- protein ions can be complexed with various counter- ions and adducts, such as N, C, and K.
- a given protein ion would exhibit multiple peaks, such as a triplet, representing different ionic states (or species) of the same protein.
- This analysis can be carried out conventionally, e.g, as described by Mann et al, anal. Chem, 61:1702-1708 (1989).
- post-translation processing may have to be considered.
- processing events which modify protein structure, including, proteolytic processing, removal of N-terminal methionine, acetylation, methylation, glycosylation, phosphorylation, etc.
- a database can be queried for a range of proteins matching the molecular mass of the unknown.
- the range window can be determined by the accuracy of the instrument, the method by which the sample was prepared, etc. Based on the number of hits (where a hit is match) in the spectrum, the unknown protein or peptide is identified or classified.
- Methods of identifying one or more HPP by mass spectrometry are useful for diagnosis and prognosis.
- such methods are used to detect one or more HPP present in human plasma.
- Exemplary techniques are described in U.S. Patent Applications 02/0060290, 02/0137106, 02/0138208, 02/0142343, 02/0155509.
- the nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics as further described herein.
- the invention provides diagnostic and prognostic assays for detecting HPP nucleic acids and proteins, as further described. Also provided are diagnostic and prognostic assays for detecting interactions between HPPs 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 quantity or quality of a polypeptide between samples.
- differences could result at any stage of protein expression from transcription through post-translational modification.
- two samples are bound to affinity spots on different sets of adsorbents (e.g, chips) and recognition maps are compared to identify polypeptides that are differentially retained by the two sets of adsorbents. Differential retention includes quantitative retention as well as qualitative differences in the polypeptide.
- differences in post-translational modification of a protein can result in differences in recognition maps detectable as differences in binding characteristics (e.g, glycosylated proteins bind differently to lectin adsorbents) or differences in mass (e.g, post- translational cleavage products).
- an adsorbent can have an array of affinity spots selected for a combination of markers diagnostic for a disease or syndrome. Differences in polypeptide levels between samples (e.g, differentially expressed HPPs in plasma samples) can be identified by exposing the samples to a variety of conditions for analysis by desorption spectrometry (e.g, mass spectrometry). Proteins can be identified by detecting physicochemical characteristics (e.g, molecular mass), and this information can be used to search databases for proteins having similar profiles. Preferred methods of detecting an HPP utilize mass spectrometry techniques. Such methods provide information about the size and character of the particular HPP isoform that is present in a sample, e.g, a biological sample submitted for diagnosis or prognosis.
- desorption spectrometry e.g, mass spectrometry
- Example 1 outlines a preferred detection scheme, wherein a biological sample is separated by chromatography before characterization by mass spectrometry.
- the invention provides a method of detecting an HPP in a biological sample comprising the steps of: fractionating a biological sample (e.g, plasma, serum, lymph, cerebrospinal fluid, cell lysate of a particular tissue) by at least one chromatographic step; subjecting a fraction to mass spectrometry; and comparing the characteristics of polypeptide species observed in mass spectrometry with known characteristics of HPP polypeptides.
- a biological sample e.g, plasma, serum, lymph, cerebrospinal fluid, cell lysate of a particular tissue
- An especially preferred method includes detection of at least one HPP using retentate chromotography methods, including, for example, protein arrays or chips. Such methods are described in the section titled "Detection Using Protein Arrays.”
- said more than one HPP is detected in a biological sample, preferably plasma.
- a favored embodiment provides a protein chip capable of detecting an HPP on an addressable array representing proteins present in human plasma.
- one embodiment of the present invention involves a method of use (e.g, a diagnostic or prognostic assay) wherein a molecule of the present invention (e.g, an HPP, HPP nucleic acid, or antibody) is used to diagnose or prognose an HPP-related disorder or one in which any of the aforementioned HPP activities is indicated.
- a molecule of the present invention e.g, an HPP, HPP nucleic acid, or antibody
- the present invention involves a method of use wherein a molecule of the present invention is used, for example, for the diagnosis or prognosis of subjects, preferably a human subject, in which any of the aforementioned activities is pathologically perturbed.
- the invention encompasses a method of determining whether an HPP is expressed within a biological sample comprising: a) contacting said biological sample with: i) a polynucleotide that hybridizes under stringent conditions to an HPP nucleic acid; or ii) a detectable polypeptide (e.g. antibody) that selectively binds to an HPP; and b) detecting the presence or absence of hybridization between said polynucleotide and an RNA species within said sample, or the presence or absence of binding of said detectable polypeptide to a polypeptide within said sample. Detection of said hybridization or of said binding indicates that said HPP is expressed within said sample.
- a detectable polypeptide e.g. antibody
- the polynucleotide is a primer, wherein said hybridization is detected by detecting the presence of an amplification product comprising said primer sequence, or the detectable polypeptide is an antibody.
- detection involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g, U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g, Landegren et al. (1988) Science 241:1077-1080; and Nakazawa et al.
- PCR polymerase chain reaction
- LCR ligation chain reaction
- a method of determining whether a mammal, preferably human, has an elevated or reduced level of expression of an HPP comprising: a) providing a biological sample from said mammal; and b) comparing the amount of an HPP or of an HPP RNA species encoding an HPP within said biological sample with a level detected in a control sample.
- An increased amount of said HPP or said HPP RNA species within said biological sample compared to said level detected in or expected from said control sample indicates that said mammal has an elevated level of HPP • expression
- a decreased amount of said HPP or said HPP RNA species within said biological sample compared to said level detected in or expected from said control sample indicates that said mammal has a reduced level of expression of an HPP.
- the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic purposes.
- one aspect of the present invention relates to diagnostic assays for determining HPP and/or nucleic acid expression as well as HPP activity, in the context of a biological sample (e.g, blood, plasma, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant HPP expression or activity.
- the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with an HPP, nucleic acid expression or activity. For example, mutations in an HPP-encoding gene can be assayed in a biological sample.
- Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with HPP expression or activity.
- biological sample is intended to include tissues, cells and biological fluids isolated from an individual, as well as tissues, cells and fluids present within an individual. That is, the detection methods of the invention can be used to detect an HPP mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
- Prefered biological samples are biological fluids such as cell lysate, lymph, cerebrospinal fluid, blood, and especially blood plasma.
- in vitro techniques for detection of an HPP mRNA include Northern hybridizations and in situ hybridizations.
- In vitro techniques for detection of an HPP include mass spectrometry, Enzyme Linked Immuno Sorbent Assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
- In vitro techniques for detection of an HPP-encoding genomic DNA include Southern hybridizations.
- in vivo techniques for detection of an HPP include introducing into an individual a labeled anti- HPP antibody.
- compositions suitable for administration comprises one or more HPP polypeptides, in conjunction with a pharmaceutically acceptable carrier.
- the pharmaceutical composition comprises compounds capable of detecting or modulating a HPP or a HPP biological activity including small molecules, peptides, HPP nucleic acid molecules, and anti- HPP antibodies of the invention.
- Such compositions typically comprise a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
- a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
- routes of administration include parenteral, e.g, intravenous, intradermal, subcutaneous, oral (e.g, inhalation), transdermal (topical), transmucosal, and rectal administration.
- Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
- a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
- antibacterial agents such as benzyl alcohol or methyl parabens
- antioxidants
- 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.
- suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, N.J.) 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 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 the action microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and other required ingredients from those enumerated above.
- the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
- Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
- the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
- the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g, a gas such as carbon dioxide, or a nebulizer.
- a suitable propellant e.g, a gas such as carbon dioxide, or a nebulizer.
- Systemic administration can also be by transmucosal or transdermal means.
- 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.
- the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Most preferably, active compound is delivered to a subject by intravenous injection.
- the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to 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 U.S. Pat. No. 4,522,811, the disclosure of which is incorporated herein by reference in its entirety.
- the active compound may be coated on a microchip drug delivery device.
- a microchip drug delivery device Such devices are useful for controlled delivery of proteinaceous compositions into the bloodstream, cerebrospinal fluid, lymph, or tissue of an individual without subjecting such compositions to digestion or subjecting the individual to injection. Methods of using microchip drug delivery devices are described in US Patents 6123861, 5797898 and US Patent application 20020119176A1, disclosures of which are hereby incorporated in their entireties. It is especially advantageous to formulate oral or preferably parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
- Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
- Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
- the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
- the therapeutically effective dose can be estimated initially from cell culture assays.
- a dose may be formulated in animal models to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
- the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
- HPP polypeptides of the present invention as well as HPP modulators and HPP-related compositions of the invention can be used in the treatment or prevention of HPP-related disorders.
- Such diseases and disorders include, but are not limited to the diseases and disorders set forth below.
- Example 4 provides a method suitable for determining diseases and disorders in which HPP polypeptides of the present invention could be used as therapeutics, prognostics and/or diagnostics.
- HPP-38 polypeptide is provided for the treatment of a cancer disease or a disease or condition associated with hyperplasia or for the prognosis or diagnosis of said diseases.
- Polypeptide HPP-38 which had been detected in human plasma in accordance with the present invention was found to have an amino acid sequence which is part of Esophageal cancer related gene 2 (ECRG2) Swiss-Prot P58062.
- ECRG2 Esophageal cancer related gene 2
- HPP-38 polypeptide includes polypeptides of the sequence as set forth in SEQ ED No: 391 and 392.
- variants and derivatives wherein the variants and derivatives have a sequence identical to the sequences forth in SEQ ID No: 391 and 392 of desirably at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
- the variants and derivatives may have one, two or three amino acid substitutions, deletions or insertions relative to an amino acid sequence set forth in SEQ ID No: 391 and 392.
- the variants and derivatives may also be chemically modified as described herein.
- HPP-38 polypeptide was synthezised by GeneProt, Inc (Geneva, Switzerland) and, in an approach as described in Example 4, the peptide was injected into mice and gene expression profiling on many organs performed.
- Table 4 shows that HPP-38 polypeptide affects expression of genes which are involved in the positive regulation of apoptosis, genes with indication in breast cancer, stress response genes and tumor suppressor genes.
- RNAs encoding tight junction proteins were downregulated in a concerted manner in blood, heart, kidney, liver and spleen. All of those gene groups can be linked to regulation of cell growth. To confirm our finding ECRG2 was tested for its growth regulatory activity in a cell growth assay at
- 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 was able to significantly inhibit the growth ( ⁇ 50% of cell proliferation) of 11 of the 15 cell lines tested, including MCF-7 (breast), DLD-1 (colon), A-498 (kidney), HepG2 (Liver), A549 (lung), SK-MEL-5 (melanoma), SK-N-MC (neuroepithelioma), PANC-1 (pancreas), PC-3 (prostate), A431 (skin), MES- SA (uterus).
- MCF-7 breast
- DLD-1 colon
- A-498 kidney
- HepG2 Liver
- A549 lung
- SK-MEL-5 melanoma
- SK-N-MC neuroepithelioma
- PANC-1 pancreas
- PC-3 prostate
- A431 skin
- the present invention now provides HPP-38 polypeptide as suitable therapeutic for the treatment of a cancer disease or a disease or condition associated with hyperplasia comprising administering an effective amount of an HPP-38 polypeptide to a mammal, including a human, suffering from said disease.
- the cancer disease is selected from the group consisting of neuroepithelioma, prostate cancer, basal cell carcinoma, squamous cell carcinoma, melanomas, and cervical intraepithelial neoplasia.
- the disease or condition associated with hyperplasia is a disease or condition selected from the group consisting of fibrosis, prostatic hyperplasia, adrenal hyperplasia, endometrial hyperplasia, psoriasis, hyperplasia due to inflammation.
- the use of HPP-38 polypeptide in medicine is provided.
- the present invention provides a method for the prognosis or diagnosis of a cancer disease or a disease or condition associated with hyperplasia comprising detecting the plasma level of a HPP-38 polypeptide, wherein an increased level is indicative of a cancer disease or a disease or condition associated with hyperplasia.
- An increased HPP-38 polypeptide plasma level within the context of the present invention is relative to the HPP-38 polypeptide plasma level as found in individuals which do not suffer of a cancer disease or a disease or condition associated with hyperplasia.
- the increase is preferably at least 1.2 fold, more preferably at least 1.5 fold, 2 fold, 3 fold, 5 fold or 10 fold.
- the present invention provides a method for the prognosis or diagnosis of a cancer disease or a disease or condition associated with hyperplasia comprising i) detecting a level of expression of at least one gene identified in Table 4 in a sample of a suitable tissue obtained from the subject to provide a first value; and ii) comparing the first value with a level of expression of said gene from a disease-free subject, wherein a greater or smaller expression level in the subject sample as compared to the sample from the disease-free subject is indicative of the subject being predisposed to or having a cancer disease or a disease or condition associated with hyperplasia.
- Gene expression may be detected on mRNA or protein level.
- Suitable tissues include, but are not limited to skin, prostate, pancreas, uterus, lung, liver, intestines, kidney.
- the mRNA expression level may be detected by any suitable technique, such as for instance Microarray analysis, Northern blot analysis, reverse transcription PCR and real time quantitative PCR.
- the protein level may be detected by any suitable technique, such as for instance through western blotting by utilizing a labeled probe specific for the protein.
- the gene(s) are selected from the gene(s) set forth in Table 4 which are upregulated or downregulated.
- the gene(s) are selected from the gene(s) set forth in Table 4 which are upregulated 1.2 fold or more, 1.3 fold or more, or 1.5 fold or more; or from the gene(s) selected from the genes set forth in Table 4 are downregulated 0.8 fold or less, 0.7 fold or less.
- the expression of at least 1, 2, 3, 4 or 5 genes is measured.
- the present invention provides a method of identifying a modulator of a cancer disease or a disease or condition associated with hyperplasia comprising i) contacting a test compound with a HPP-38 polypeptide under sample conditions permissive for HPP- 38 biological activity; ii) determining the level of said at least one HPP-38 biological activity; iii) comparing said level to that of a control sample lacking said test compound.
- said test compound, which causes said level to change is selected for further testing as a HPP-38 modulator for the prophylactic and/or therapeutic treatment of a cancer disease or a disease or condition associated with hyperplasia.
- the level of HPP-38 biological activity is measured by detecting the level of expression of one or more genes set forth in Table 4.
- HPP-13 which had been detected in human plasma in accordance with the present invention, was found to have an amino acid sequence which is part of human thymosin beta 4 (Swiss-Prot P01253).
- HPP-13 polypeptides includes polypeptides of the sequence as set forth in SEQ ID No: 393 (human form and short mouse isoform) and SEQ ID No: 394 (long mouse isoform).
- variants and derivatives wherein the variants and derivatives have a sequence identical to the sequences forth in SEQ ID No: 393 and 394 of desirably at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
- the variants and derivatives may have one, two or three amino acid substitutions, deletions or insertions relative to an amino acid sequence set forth in SEQ ID No: 393 and 394.
- the variants and derivatives may also be chemically modified as described herein.
- HPP-13 polypeptides were synthezised by GeneProt, Inc (Geneva, Switzerland) and, in an approach as described in Example 4, the peptides were injected into mice and gene expression profiling on many organs performed. Table 6, 7, 8, 9 and/or 10 shows that HPP-13 polypeptides affect expression of genes which are involved in heme biosynthesis and metabolism, porphyrin biosynthesis and metabolism and erythropoiesis. Additionally, genes involved in dietary iron balance, iron transport are also differentially regulated. Accordingly, the present invention now provides HPP-13 polypeptides for the treatment of conditions related to diseases or conditions where body iron balance is altered, leading to iron overload diseases, anemia or deficient red blood cell production.
- HPP-13 polypeptides are provided as suitable therapeutics for the treatment of a disease or condition associated with iron balance or iron transport comprising administering an effective amount of an HPP-13 polypeptide to a mammal, including a human, suffering from said disease.
- the disease or condition associated with iron balance or iron transport is selected from the group consisting of hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron-overload, anemia, sickle cell anemia.
- the use of HPP-13 polypeptides in medicine is provided.
- the present invention provides a method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising detecting the plasma level of an HPP-13 polypeptide, wherein an increased level is indicative of a disease or condition associated with iron balance or iron transport.
- An increased HPP-13 polypeptides plasma level within the context of the present invention is relative to the HPP-13 polypeptide plasma level as found in individuals which do not suffer of a disease or condition associated with iron balance or iron transport.
- the increase is preferably at least 1.2 fold, more preferably at least 1.5 fold, 2 fold, 3 fold, 5 fold or 10 fold.
- the present invention provides a method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising i) detecting a level of expression of at least one gene identified in Table 6, 7, 8, 9 and/or 10 in a sample of a suitable tissue obtained from the subject to provide a first value; and ii) comparing the first value with a level of expression of said gene from a disease-free subject, wherein a greater or smaller expression level in the subject sample as compared to the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease or condition associated with iron balance or iron transport.
- Gene expression may be detected on mRNA or protein level. Suitable tissues include, but are not limited to liver, heart, intestines such as duodenum, spleen, bone marrow.
- the mRNA expression level may be detected by any suitable technique, such as for instance
- the gene(s) are selected from the gene(s) set forth in Table 6, 7, 8, 9 and/or 10 which are upregulated or downregulated.
- the gene(s) are selected from the gene(s) set forth in Table 6, 7, 8, 9 and/or 10 which are upregulated 1.2 fold or more, 1.3 fold or more, or 1.5 fold or more; or from the gene(s) selected from the genes set forth in Table 6, 7, 8, 9 and/or 10 are downregulated 0.8 fold or less, 0.7 fold or less, or 0.6 fold or less.
- the expression of at least 1, 2, 3, 4 or 5 genes is measured.
- the present invention provides a method of identifying a modulator of a disease or condition associated with iron balance or iron transport comprising i) contacting a test compound with a HPP-13 polypeptide under sample conditions permissive for HPP- 13 biological activity; ii) determining the level of said at least one HPP-13 biological activity; iii) comparing said level to that of a control sample lacking said test compound.
- said test compound which causes said level to change, is selected for further testing as a HPP-13 modulator for the prophylactic and/or therapeutic treatment of a disease or condition associated with iron balance or iron transport.
- the level of HPP-13 biological activity is measured by detecting the level of expression of one or more genes set forth in Table 6, 7, 8, 9 and/or 10.
- mice with HPP-13 polypeptide resulted in gene expression profiles showing that these polypeptides would be useful in the treatment of conditions of neurodegeneration and peripheral neuropathies, such as multiple sclerosis, demyelinating diseases, Guillian-Barre disease, diabetic neuropathy, chemotherapy induced neuropathy, autoimmune related neuropathy, CNS-related neuropathies, such as Alzheimer's disease and neuronal injury related for example to stoke (nervous system ischemia).
- GPAIOI polypeptide is provided in accordance with the present invention to be useful for the treatment of the above conditions.
- GAP101 is a polypeptide with a randomized sequence of HPP-13.
- GPAIOI polypeptide includes polypeptides of the sequence as set forth in SEQ ID No: 397 as well as variants and derivatives as described above.
- the present invention provides a polypeptide comprising the amino acid sequence set forth in SEQ ID No: 397
- the present invention provides a polypeptide consisting of the sequence set forth in SEQ ID No: 397. Accordingly, the present invention now provides HPP-13 and GPAIOI polypeptides as suitable therapeutics for the treatment of a disease or condition associated with neurodegeneration comprising administering an effective amount of an HPP-13 or GPAIOI polypeptides to a mammal, including a human, suffering from said disease.
- the disease or condition associated with neurodegeneration is selected from the group consisting of spinal cord injuries or CNS injuries, , Alzheimer's, Parkinson's, multiple sclerosis, , ALS (amyotrophic lateral sclerosis), peripheral neuropathy, Guillian-Barre disease, diabetic neuropathy, demyelinating neuropathies.
- the use of HPP-13 or GPAIOI polypeptide in medicine is provided.
- the present invention provides a method for the prognosis or diagnosis of a disease or condition associated with neurodegeneration comprising detecting the plasma level of a HPP-13 polypeptide, wherein an increased level is indicative of a disease or condition associated with neurodegeneration.
- An increased HPP-13 polypeptide plasma level within the context of the present invention is relative to the HPP-13 polypeptide plasma level as found in individuals which do not suffer of a disease or condition associated with neurodegeneration.
- the increase is preferably at least 1.2 fold, more preferably at least 1.5 fold, 2 fold, 3 fold, 5 fold or 10 fold.
- the present invention provides a method for the prognosis or diagnosis of a disease or condition associated with neurodegeneration comprising i) detecting a level of expression of at least one gene identified in Table 11 in a sample of a suitable tissue obtained from the subject to provide a first value; and ii) comparing the first value with a level of expression of said gene from a disease-free subject, wherein a greater or smaller expression level in the subject sample as compared to the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease or condition associated with neurodegeneration.
- Gene expression may be detected on mRNA or protein level.
- Suitable tissues include, but are not limited to brain, spinal cord, neuromotor tissue, peripheral nerves, central and peripheral nervous tissues.
- the mRNA expression level may be detected by any suitable technique, such as for instance Microarray analysis, Northern blot analysis, reverse transcription PCR and real time quantitative PCR.
- the protein level may be detected by any suitable technique, such as for instance through western blotting by utilizing a labeled probe specific for the protein.
- the gene(s) are selected from the gene(s) set forth in Table 11 which are upregulated or downregulated.
- the gene(s) are selected from the gene(s) set forth in Table 11 which are upregulated 1.2 fold or more, 1.3 fold or more, or 1.5 fold or more; or from the gene(s) selected from the genes set forth in Table 11 are downregulated 0.8 fold or less, 0.7 fold or less, or 0.6 fold or less.
- the expression of at least 1, 2, 3, 4 or 5 genes is measured.
- the present invention provides a method of identifying a modulator of a disease or condition associated with neurodegeneration comprising i) contacting a test compound with a HPP-13 or GPAIOI polypeptide under sample conditions permissive for HPP-13 or GPAIOI biological activity; ii) determining the level of said at least one HPP-13 or GPAIOI biological activity; iii) comparing said level to that of a control sample lacking said test compound.
- said test compound, which causes said level to change is selected for further testing as a HPP-13 or GPAIOI modulator for the prophylactic and/or therapeutic treatment of a disease or condition associated with neurodegeneration.
- the level of HPP- 13 biological activity is measured and the expression of one or more genes set forth in Table 11 is detected.
- Disease Therapies Related to Defects in Glucose Metabolism Polypeptide HPP-23 which had been detected in human plasma in accordance with the present invention, was found to have an amino acid sequence which is part of Pancreastatin (Swiss-Prot P10645).
- HPP-23 polypeptide includes polypeptides of the sequence as set forth in SEQ ID No: 395 and SEQ ID No: 396. In a particularly preferred embodiment the polypeptide is not amidated.
- variants and derivatives wherein the variants and derivatives have a sequence identical to the sequences forth in SEQ ID No: 395 and SEQ ID No: 396 of desirably at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%.
- the variants and derivatives may have one, two or three amino acid substitutions, deletions or insertions relative to an amino acid sequence set forth in SEQ ID No: 395 and SEQ ID No: 396.
- the variants and derivatives may also be chemically modified as described herein.
- HPP-23 polypeptide was synthezised by GeneProt, Inc (Geneva, Switzerland) and, in an approach as described in Example 4, the peptide was injected into mice and gene expression profiling on many organs performed. In the brain and other organs, HPP-23 polypeptide injection revealed a strong effect the RNA expression levels of genes involved in glucose metabolism, other carbohydrate catabolism pathways, vasculogenesis and inflammatory responses (Table 12). In addition the major steps of the signalling pathway of pancreastatin could be reconstituted. HPP-23 was tested for its modulatory effect on glucose levels in three mouse models at MDS
- the present invention now provides HPP-23 polypeptide as suitable therapeutic for the treatment of a diseases with dysregulated serum glucose comprising administering an effective amount of an HPP-23 polypeptide to a mammal, including a human, suffering from said disease.
- the diseases with dysregulated serum glucose is diabetes or other hyperglycemic conditions.
- HPP-23 reduces blood glucose levels when administered to a mammal, including a human.
- the use of HPP-23 in medicine is provided.
- the present invention provides a method for the prognosis or diagnosis of a diseases with dysregulated serum glucose comprising detecting the plasma level of a HPP-23 polypeptide, wherein an increased level is indicative of a disease with dysregulated serum glucose.
- An increased HPP-23 polypeptide plasma level within the context of the present invention is relative to the HPP-23 polypeptide plasma level as found in individuals which do not suffer of a disease with dysregulated serum glucose.
- the increase is preferably at least 1.2 fold, more preferably at least 1.5 fold, 2 fold, 3 fold, 5 fold or 10 fold.
- the present invention provides a method for the prognosis or diagnosis of a disease with dysregulated serum glucose comprising i) detecting a level of expression of at least one gene identified in Table 12 in a sample of a suitable tissue obtained from the subject to provide a first value; and ii) comparing the first value with a level of expression of said gene from a disease-free subject, wherein a greater or smaller expression level in the subject sample as compared to the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease with dysregulated serum glucose.
- Gene expression may be detected on mRNA or protein level.
- Suitable tissues include, but are not limited to adipose (fat) tissue, heart, pancreas, muscle, brain.
- the mRNA expression level may be detected by any suitable technique, such as for instance Microarray analysis, Northern blot analysis, reverse transcription PCR and real time quantitative PCR.
- the protein level may be detected by any suitable technique, such as for instance through western blotting by utilizing a labeled probe specific for the protein.
- the gene(s) are selected from the gene(s) set forth in Table 12 which are upregulated or downregulated.
- the gene(s) are selected from the gene(s) set forth in Table 12 which are upregulated 1.2 fold or more, 1.3 fold or more, or 1.5 fold or more; or from the gene(s) selected from the genes set forth in Table 12 are downregulated 0.8 fold or less, 0.7 fold or less, or 0.6 fold or less.
- the expression of at least 1, 2, 3, 4 or 5 genes is measured.
- the present invention provides a method of identifying a modulator of a disease with dysregulated serum glucose comprising i) contacting a test compound with a HPP-23 polypeptide under sample conditions permissive for HPP-23 biological activity; ii) determining the level of said at least one HPP-23 biological activity; iii) comparing said level to that of a control sample lacking said test compound.
- said test compound which causes said level to change, is selected for further testing as a HPP-23 modulator for the prophylactic and/or therapeutic treatment of a disease with dysregulated serum glucose.
- the level of HPP-23 biological activity is measured by detecting the level of expression of one or more genes set forth in Table 12.
- HPP-23 polypeptide is provided as suitable therapeutic for the treatment of a metabolic disorder, in particular of amyloidosis.
- HPP-23 polypeptide is also provided as part of the invention as suitable treatment for arrhythmia, in particular bradycardia, tachycardia, sick sinus syndrome, and angiogenesis, in particular, stroke, macular regeneration, cancer.
- Amyloidosis is a group of incompletely understood metabolic disorders resulting from the deposition of protein-containing fibrils (amyloid) in tissues. The disease may cause localized or widespread organ failure. Amyloid may infiltrate many organs, including heart and blood vessels, brain and peripheral nerves.
- amyloidosis the clinical manifestations of amyloidosis are enormously varied, and the disease may mimic other conditions ranging from nephritic syndrome to dementias or congestive heart failure.
- Pathways of oxidative stress seem to be causative for the development of amyloid fibrils. Oxidative stress precipitates both nuclear DNA degradation and membrane phosphatidylserine exposure in neuronal and vascular cells to promote loss of cellular integrity, microglial phagocytosis, and thrombotic destruction.
- Critical in the ability to foster cell survival during oxidative stress is the modulation of the metabotropic glutamate system, cell cycle regulation in post-mitotic neurons, and control of GSK-3beta activity and presenilin integrity.
- pancreastatin in brain tissue In brain of HPP-23 polypeptide treated mice it has been found in accordance with the present invention that amyloidosis-diseases are affected by HPP-23 polypeptide.
- HPP-23 polypeptide upregulates presenilinl, a large panel of peroxiredoxin genes, mitochondrial NADH-dehydrogenases, endothelial nitric oxide, cytochrome c-oxidases (15 probe sets). (All those groups were not affected by a control).
- heme oxygenase (HO-1) expression is stimulated under pro-oxidant stimuli such as dopamine, hydrogen peroxide, beta-amyloid and others, and that HO-1 is found upregulated in CNS tissues affected by Alzheimer's disease, Parkinson's disease, multiple sclerosis and other degenerative and non-degenerative CNS diseases.
- HPP-23 polypeptide leads to an upregulation of HO-1 in brain.
- Pancreastatin in heart tissue Ryanodine receptor per se is not affected in the hearts of pancreastatin treated animals, However, many ryr binding proteins are downregulated, such as FKBP12, calmodulin, sorcin, triadin, and phosphatase 1. Sorcin binds to presenilinl, which is also downregulated in heart. Presenilin is part of the Notch pathway, consisting, among others, of ADAM 17, notch, and the targets snail,hesl, and the homeobox gene msx. While snail and hesl were upregulated, all other members were downregulated. An inhibitor of notch, numb, was upregulated, pointing at a common inhibition of the pathway. Another presenilin binding protein is restin.
- Example 1 Characterization of HPP levels Portions of plasma from more than 50 healthy male volunteers were pooled in order to dilute phenotypic differences and to provide a large pool volume. Blood (100-450 ml) was obtained using standard veinous puncture procedures in a medical centre setting after informed written consent. Samples were barcoded and plasma was prepared by centrifugation and removal of white cells on filters by standard techniques. Times, temperatures and centrifugation conditions and subsequent procedures were rigorously controlled to ensure similar treatment of all samples. Protease inhibitor (Complete, Roche) was added according to the manufacturer's instructions and mixed gently to ensure dissolution. Plasma samples were then frozen and stored at -80°C. After careful consideration of medical history and clinical chemistry parameters, portions of at least 50 samples were pooled. A volume of 2.5L (of the 6 L total) was subjected to separation by multiple chromatography steps according to the Microprot.TM process as follows:
- Step 1 HSA/IgG depletion 125 ml frozen plasma were defrost and filtered on 0.45 ⁇ m sterile filter in a sterile hood. Filtrate was injected on two inline columns of respectively 300 ml of HSA ligand Sepharose fast Flow column (Amersham, Upsala, Sweden), 5cm ID, 15 cm length; and 100 ml Protein G Sepharose fast Flow column (Amersham, Upsala, Sweden), 5 cm ID, 5 cm length. Columns were equilibrated and washed with 50 mM P04 buffer, pH 7.1, 0.15M NaCl. Flow rate was 5 ml/min. Non-retained fraction (53g of depleted polypeptides) was frozen until second step. Twenty runs were performed.
- Step 2 Gel Filtration / Reverse Phase Capture step
- Sample from step 1 was defrosted and filtered on 0.45 ⁇ m sterile filter in a sterile hood. Filtrate was injected on two in line gel filtration columns: 2 X 9.5 litres Superdex 75 (Amersham, UK) column, 14 cm ID, 62 cm length. Column was equilibrated with 50mM P04 buffer pH 7.4, 0.1 M NaCl, 8M urea. Hydrophobic impurities were retained on a reverse phase precolumn: 150 ml PLRPS (Polymer Labs, UK). Precolumn was switched for sample injection. Gel filtration was performed at a flow rate of 40 ml/min.
- Low molecular weight proteins ( ⁇ 20 kDa) were oriented to in line reverse phase capture column: 50 ml PLRPS 100 angstroms (Polymer labs, UK). The three-way valve controlling injection on PLRPS column was switched at a cut-off of 33 mAU (280 nm) to send gel filtration eluate into reverse phase capture column. This cut-off value was established by first using SDS-PAGE to provide an estimated range of OD values and by subsequently evaluating three cut-off values (high, median and low values of OD range). The final cut-off value was chosen to maximize the low molecular weight protein obtained, with a low molecular protein proportion of at least 85%.
- Step 3 Cation Exchange Sample from step 2 (147 ml) was defrosted and mixed with an equal volume of cation exchange buffer A (Gly/HCl buffer 50 mM, pH 2.7, urea 8M). Sample was injected on a 100 ml Source 15S column (Amersham, Upsala, Sweden), 35 mm ID, 100 mm length. Column was equilibrated and washed with buffer A. Flow rate was 10 ml min. Proteins and peptides were eluted with step gradient from 100% buffer A until 100 % buffer B (buffer A containing IM NaCl):
- Step 4 Reduction/Alkylation and Reverse Phase HPLC Fractionation 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 alkylated with iodoacetamide (120 mM, 1 hour 25°C in the dark). The latter reaction was stopped with the addition of DTE (30 mM) followed by acidification (TFA, 0.1 %). The fractions were then injected on an Uptispher C8, 5 um, 300 angstroms column (Interchim, France), 21 mm ID, 150 mm length. Injection was performed with a 10 ml min flow rate.
- DTE dithioerythritol
- TFA acidification
- Step 5 Reverse Phase HPLC Fractionation 2 Dried samples from step 4 were resuspended in 1 ml of solution A (0.03% TFA in water) and injected on a Vydac LCMS C4 column, 5 micrometers, 300 angstroms (Vydac, USA), 4.6 mm ID, 150 mm length. Flow rate was 0.8 ml/min. C4 column was equilibrated and washed with solution A and proteins and peptides were eluted with a biphasic gradient adapted to elution position of the sample in Reverse Phase HPLC Fractionation 1. Intact mass data were acquired using Electrospray Ion Trap Mass spectrometry.
- Step 6 Mass detection 12,960 (18 x 30 x 24) fractions were collected following reverse phase HPLC fractionation 2 into 96-well deep well plates (DWP). A small proportion (2.5%) of the volume was diverted to online analysis using LC-ESI-MS (Bruker Esquire). Aliquots of undigested proteins were mixed with MALDI matrices, and spotted on MALDI plates together with mass calibration standards and sensitivity standards. Automated spotting devices (Bruker MALDI sample prep. Robots) were used.
- SA sinapic acid
- HCCA alpha-cyano-4-hydroxycinnamic acid
- MALDI plates were subjected to mass detection using Bruker Reflex HI MALDI MS apparati. The 96-well plates were stored at +4 C. 96-well plates (DWP) were recovered and subjected to two sequential concentration steps. Volumes were concentrated from 0.8 ml to about 50 microl per well by drying with a SpeedVac, and then resolubilized to ca. 200 microl and reconcentrated to about 50 microl per well, and stored at +4 C.
- Proteins were then digested by re-buffering, adding trypsin to the wells, sealing and incubating the plates at 37 C for 12 hours, followed by quenching (addition of formic acid to bring the pH down to 2.0).
- concentration of trypsin to be added to the wells was adjusted based on the OD at 280 nm recorded for each particular fraction. This ensured an optimal use of trypsin and a complete digestion of the most concentrated fractions.
- Automated spotting devices (Bruker MALDI sample prep. Robots) were used to deposit a volume from each well, pre-mixed with a HCCA matrix onto a
- MALDI plate together with sensitivity and mass calibration standards.
- MALDI plates were analyzed using a Bruker Reflex in MALDI MS device. Contents from each well of the 96 well plates were analyzed with LC-ESI-MS-MS Bruker Esquire ESI Ion-Trap MS devices.
- Step 7 Detection and Identification of Low Abundance Peptides in Human Plasma Separated fractions are further subjected to mass spectrometry (both MALDI and LC-ESI- MS-MS) for separation and detection. More than 1.5 million MS/MS spectra were generated and 330 000 used for manual testing and validation of the algorithms developed for automated identifications. Intact mass data, Peptide Mass Fingerprints and peptide sequence data were integrated for protein identification and characterization.
- Proteins were identified using Mascot software (Matrix Science Ltd, London, UK), and results from peptide identification were checked by manual analysis of the spectra. MS/MS spectra were interpreted with a custom identification engine (Olav) which accessed a set of 6 different databases (SwissProt, public EST and Genomic sequence data, and Patent databases). An integration step confirmed the consistency of the global identification across the different databases. Annotation (automatic and manual) was performed on the validated identifications to further characterize important features of the observed polypeptides.
- the methods of protein separation and identification according to the invention are extremely sensitive. The Microprot.TM process is able to detect very low abundance proteins with a plasma concentration in the range of 50 pM. The accuracy was confirmed while carrying out the presently described methods. In particular, proteins with a well-characterized role in human plasma were detected.
- Example 2 Chemical Synthesis of HPPs
- an HPP of the invention is synthesized. Peptide fragment intermediates are first synthesized and then assembled into the desired polypeptide.
- An HPP can initially be prepared in, e.g. 5 fragments, selected to have a Cys residue at the N- terminus of the fragment to be coupled.
- Fragment 1 is initially coupled to fragment 2 to give a first product, then after preparative HPLC purification, the first product is coupled to fragment 3 to give a second product. After preparative HPLC purification, the second product is coupled to fragment 4 to give a third product. Finally, after preparative HPLC purification, the third product is coupled to fragment 5 to give the desired polypeptide, which is purified and refolded. Thioester formation
- Fragments 2, 3, 4, and 5 are synthesized on a thioester generating resin, as described above.
- the following resin is prepared: S-acetylthioglycolic acid pentafluorophenylester is coupled to a Leu-PAM resin under conditions essentially as described by Hackeng et al (1999).
- the resulting resin is used as a starting resin for peptide chain elongation on a 0.2 mmol scale after removal of the acetyl protecting group with a 30 min treatment with 10% mercaptoethanol, 10% piperidine in DMF.
- N-terminal Cys residues of fragments 2 through 5 are protected by coupling a Boc-thioproline (Boc-SPr, i.e. Boc-L-thioproline) to the terminus of the respective chains instead of a Cys having conventional N" or S ⁇ protection, e.g. Brik et al, J. Org. Chem, 65 : 3829-3835 (2000).
- Boc-SPr Boc-L-thioproline
- Each synthetic cycle consists of N"-Boc -removal by a 1 to 2 min treatment with neat TFA, a 1-min DMF flow wash, a 10-min coupling time with 2.0 mmol of preactivated Boc-amino acid in the presence of excess DIEA and a second DMF flow wash.
- N ⁇ -Boc-amino acids (2 mmol) are preactivated for 3min with 1.8mmol HBTU (0.5M in DMF) in the presence of excess DIEA (6 mol).
- a dichloromethane flow wash is used before and after deprotection using TFA, to prevent possible high temperature (TFA DMF)-catalyzed pyrrolidone carboxylic acid formation.
- Side-chain protected amino acids are Boc-Arg(p-toluenesulfonyl)-OH, Boc-Asn(xanthyl)- OH, Boc-Asp(0-cyclohexyl)-OH, Boc-Cys(4-methylbenzyl)-OH, Boc-Glu(0-cyclohexyl)-OH, Boc- His(dinitrophenylbenzyl)-OH, Boc-Lys(2-Cl-Z)-OH, Boc-Ser(benzyl)-OH, Boc-Thr(benzyl)-OH, Boc-Trp(cyclohexylcarbonyl)-OH and Boc-Tyr(2-Br-Z)-OH (Orpagen Pharma, Heidelberg, Germany).
- C-terminal Fragment 1 is synthesized on Boc-Leu-0-CH 2 -Pam resin (0.71mmol/g of loaded resin), while for Fragments 2 through 5 machine-assisted synthesis is started on the Boc-Xaa-S-CH 2 -CO-Leu-Pam resin.
- This resin is obtained by the coupling of S-acetylthioglycolic acid pentafluorophenylester to a Leu-PAM resin under standard conditions.
- the resulting resin is used as a starting resin for peptide chain elongation on a 0.2 mmol scale after removal of the acetyl protecting group with a 30min treatment with 10% mercaptoethanol, 10% piperidine in DMF.
- the peptide fragments are deprotected and cleaved from the resin by treatment with anhydrous hydrogen fluoride for lhr at 0°C with 5% p-cresol as a scavenger.
- anhydrous hydrogen fluoride for lhr at 0°C with 5% p-cresol as a scavenger.
- the imidazole side chain 2,4-dinitrophenyl (DNP) protecting groups remain on His residues because the DNP-removal procedure is incompatible with C-terminal thioester groups.
- peptide fragments are precipitated with ice-cold diethylether, dissolved in aqueous acetonitrile and lyophihzed.
- the peptide fragments are purified by RP-HPLC with a C18 column from Waters by using linear gradients of buffer B (acetonitile/0.1% trifluoroacetic acid) in buffer A (H 2 O/0.1% trifluoroacetic acid) and UV detection at 214nm. Samples are analyzed by electrospray mass spectrometry (ESMS) using an Esquire instrument (Briicker, Bremen , Germany), or like instrument.
- ESMS electrospray mass spectrometry
- the ligation of unprotected fragments is performed as follows: the dry peptides are dissolved in equimolar amounts in 6M guanidine hydrochloride (GuHCl), 0.2M phosphate, pH 7.5 in order to get a final peptide concentration of 1-8 mM at a pH around 7, and 1% benzylmercaptan, 1% thiophenol is added. Usually, the reaction is carried out overnight and is monitored by HPLC and electrospray mass spectrometry. The ligation product is subsequently treated to remove protecting groups still present.
- GuHCl 6M guanidine hydrochloride
- 0.2M phosphate pH 7.5
- pH 7.5 a final peptide concentration of 1-8 mM at a pH around 7, and 1% benzylmercaptan, 1% thiophenol is added.
- the reaction is carried out overnight and is monitored by HPLC and electrospray mass spectrometry.
- the ligation product is subsequently treated to remove
- Opening of the N-terminal thiazolidine ring further required the addition of solid methoxamine to a 0.5M final concentration at pH3.5 and a further incubation for 2h at 37°C.
- a 10-fold excess of Tris(2-carboxyethyl)phosphine is added before preparative HPLC purification.
- Fractions containing the polypeptide chain are identified by ESMS, pooled and lyophihzed.
- the ligation of fragments 4 and 5 is performed at pH7.0 in 6 M GuHCl.
- the concentration of each reactant is 8mM, and 1% benzylmercaptan and 1% thiophenol were added to create a reducing environment and to facilitate the ligation reaction.
- Monoclonal or polyclonal antibodies to the protein are then prepared as described in the sections titled "Monoclonal antibodies” and "Polyclonal antibodies.” Briefly, to produce an anti-HPP monoclonal antibody, a mouse is repetitively inoculated with a few micrograms of the HPP or a portion thereof over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
- HAT media aminopterin
- the successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
- Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, E, Meth. Enzymol. 70: 419 (1980). Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2.
- polyclonal antiserum containing antibodies to heterogeneous epitopes in the HPP or a portion thereof are prepared by immunizing a mouse with the HPP or a portion thereof, which can be unmodified or modified to enhance immunogenicity.
- Any suitable nonhuman animal preferably a non-human mammal, may be selected including rat, rabbit, goat, or horse.
- Antibody preparations prepared according to either the monoclonal or the polyclonal protocol are useful in quantitative immunoassays which determine concentrations of HPP in biological samples; or they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
- the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
- Example 4 Characterisation of HPP polypeptides in vivo
- the mouse homologous/orthologuous proteins of the HPP polypeptides are administered subcutaneously to male C57BL/6 mice for 7 to 14 days at a dose of 300, 600 or 1000 microg/day.
- samples from all organs are subjected to snap freezing at necropsy and are analyzed with GeneChip® expression profiling.
- Total RNA is extracted from these frozen tissues using TRIzol reagent (Life Technologies) according to the manufacturer's instructions.
- RNA is stored at -80°C until analysis. Good quality total RNA is used to synthesize double-stranded cDNA using the Superscript Choice System (Life Technologies). The cDNA is then in vitro transcribed (MEGAscriptTM T7 Kit, Ambion) to form biotin labeled cRNA. Next, 12 to 15 mg of labeled cRNA is hybridized to the Affymetrix Mouse MOE430A expression probe arrays for 16 hours at 45°C. Arrays are then washed according to the EukGE-WS2 protocol (Affymetrix), and stained with 10 mg/ml of streptavidin-phycoerythrin conjugate (Molecular Probes).
- the signal is antibody-amplified with 2 mg/ml acetylated BSA (Life Technologies), 100 mM MES, 1 M [Na+], 0.05 % Tween 20, 0.005 % Antiofoam (Sigma), O.lmg/ml goat IgG and 0.5 mg/ml biotinylated antibody and re-stained with the streptavidin solution.
- the arrays are scanned twice with the Gene Array® scanner (Affymetrix).
- the expression level is estimated by averaging the differences in signal intensity measured by oligonucleotide pairs of a given probe (AvgDiff value).
- the image acquisition and numerical translation software used for this study is the Affymetrix Microarray Suite version 5 (MAS5).
- the dataset is initially filtered to exclude in a first wave of analysis genes whose values are systematically in the lower expression ranges where the experimental noise is high (at least an AvgDiff value of 50 in a number of experiments corresponding to the smallest number of replicas of any experimental point).
- a threshold t-test p-value (0.05) identifies genes with different values between treated and non-treated based on a two component error model (Global Error Model) and, where possible, with a stepdown correction for multi-hypothesis testing (Benjamini and Hochberg false discovery rate).
- the selected genelists are then compared with established genelists for pathways and cellular components using Fisher's exact test.
- Venn diagrams are used to identify the gene changes that are in common between the different organs. Expression profiles of highly relevant genes are used to find genes with correlated changes at individual experimental points, using several distance metrics (standard, Pearson). The decision to consider a specific gene relevant is based on a conjunction of numerical changes identified by exploratory filtering and statistical algorithms as described above and the relationship to other modulated genes that point to a common biological theme. Tables 4 and 6 to 11 shows that at the RNA level HPP polypeptides affect genes that are related to apoptosis pathway, proteosome, ubiquitin pathways and ribosomal RNAs and proteins. Most genes are up-regulated more than at least 1.2 fold or down-regulated at least less than 0.8 fold.
- the assay used to detect changes in cell proliferation was based on the ability of viable cells to cause alamarBlue to change from its oxidized non-fluorescent blue form to a reduced fluorescent red form. With the results obtained from the alamarBlue reaction, cell proliferation can be quantified and metabolic activity of viable cells examined.
- the test compound was dissolved and diluted with sterile distilled water to obtain an initial working solutions of 10000, 1000, 100, 10 and 1 micromolar. A 100-fold dilution was further made in culture media to generate the final assay concentrations of 100, 10, 1, 0.1 and 0.01 micromolar.
- IC 50 50% Inhibition Concentration: Test compound concentration where the increase from time 0 in the number or mass of treated cells was only 50% as much as the corresponding increase in the vehicle-control at the end of experiment.
- b TGI Total Growth Inhibition: Test compound concentration where the number or mass of treated cells at the end of experiment was equal to that at time 0 .
- c LC 5 o Test compound concentration where the number or mass of treated cells at the end of experiment was half that at timeo.
- a semi-quantitative determination of IC 50 , TGI and LC 50 was carried out by nonlinear regression analysis using GraphPad Prism (GraphPad Software, USA).
- CD cluster differentiation
- Neurotrophins such as NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin 3), NT-4/5 (neurotrophin 4/5) or GDNF (glial cell line-derived neurotrophic factor) open a large and promising investigation field.
- Neurotrophic factors have been used for the treatment of several neurodegenerative diseases. However, their use is limited by their inability to cross the blood-brain barrier, their short half life and their side effects. So, the development of new compounds which could mimic neurotrophins effects (promoting neuron survival and neurite outgrowth) without their limitation appears to be a good strategy for the development of new therapeutics in neurodegenerative diseases.
- the purpose of the present in-vitro study is to investigate the putative neurotrophic effects of HPP-13 and GPAIOI on neurite outgrowth from spinal motor neuron.
- the neurotrophic effect of test compounds are investigated 3 days after treatment by measuring the length of the major neurite. Two cultures are done and two Petri dishes per conditions are performed.
- Rat spinal motor neurons are prepared according to the method described by Martinou et al. (1992) Neuron, 8, 737-744.. Briefly, pregnant female rats of 15 days gestation are killed by cervical dislocation (Rats Wistar; Janvier, Le Genest-St-Isle, France) and the fetuses are removed from the uterus. Their spinal cords are removed and placed in ice-cold medium of Leibovitz (L15, Gibco, Invitrogen, Cergy-Pontoise, France). Meninges are carefully removed.
- the pellet of dissociated cells is resuspended in LI 5 medium and the resulting suspension is first enriched in motor neurons by centrifugation for 10 min at 180 x g for 10 min at room temperature on a layer of 3.5 % solution of bovine serum albumin (BSA) in LI 5 medium. The supernatant is discarded, the pellet is homogenized in LI 5 supplemented with DNAase I, the suspension is layered over a cushion of Optiprep (d: 1.06 g.mL-1; Abcys, Paris, France) and centrifuged at 335 x g for 15 min at room temperature.
- BSA bovine serum albumin
- the upper phase containing the purified motor neurons, is collected, resuspended in L15, centrifuged at 800 x g for 10 min at room temperature.
- the cell pellet is finally resuspended in a defined culture medium consisting of Neurobasal (Gibco) supplemented with B27 supplement (2%; Gibco) and 0.5 mM of L-Glutamine (Gibco).
- This culture medium does not contain any serum: Neurobasal is a serum-free medium for long-term viability neurons but formulation is confidential (official data sheet is available on invitrogen website); B27 is a supplement added to give the optimal growth condition (B27 supplement contains following constituents: d-Biotin, BSA, fatty acid free Fraction V, Catalase, L-Carnitine HCl, Corticosterone, Ethanolamine HCl, D-Galactose (Anhyd.), 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-Poor), Vitamin A Acetate).
- B27 supplement contains following constituents: d-Biotin, BSA,
- Viable cells are counted in a Neubauer cytometer using the trypan blue exclusion test (Sigma). Then cells are plated at 30 000 cells per Petri dish (Nunc, Dutscher, pre-coated with poly-1-lysine) and cultured at 37°C in a humidified air (95%) - C02 (5%) atmosphere. Two hours after plating (time needed for cell adhesion), tested compounds are added on culture.
- a global analysis of the data is done using a one way analysis of variance (ANOVA). Where applicable, Fisher's PLSD test is used for multiple pairwise comparisons. The level of significance was set at p ⁇ 0.05.
- BDNF tested at 50 ng/ml induced a neuritic outgrowth on spinal motor neuron.
- the principal neurite length was about 111.74 ⁇ m ⁇ 4.032 whereas with BDNF, the principal neurite reached 199.56 ⁇ m ⁇ 7.122. An increase of 77% was observed.
- HPP-13 and GPAIOI also significantly increase neurite outgrowth, the neurite outgrowth is similar to BDNF condition.
- the best concentration is found to be 10 nM.
- the mean neurite length reached 193.65.
- the mean neuritic length reaches 150% of control condition.
- Example 4.5 Characterisation of HPP-23 polypeptide in vivo HPP-23 was administered to mice, the expression level was estimated and a genelist was selected as described above (Table 14). Genes affected are related to sugar amyloidosis, arrhythmia or angiogenesis (e.g. stroke, macular regeneration, cancer). Table 14:
- hCP38826 SEAIAVDGAGKPGAEEAQDPEGKGEQEHSQQKEE-EEEMAVVPQGLFR-GGKSGELEQ-- mCP7064 SDSQAVDGDGKTEASEALPSEGKGELEHSQQEEDGEEAMVGTPQGLFPQGGKGRELEHKQ * .. **** ** * ** ***** ***** . * . ** * ***** *** . hCP38826 EEERLSKEWEDSKRWSK DQLAKELTAEKRLEGQEEEEDNRDSSMKLSFRARAYG mCP7064 EEEEEEEERLSRE ED-KR SRMDQLAKELTAEKRLEG EDDPDRSMKLSFRTRAYG ****** . **** **** **** . ********** ** . ****** . **** **** * ** ** . ****** . **** ***** ** . ****** . **** ***** ** . ****** . **** ***** ** . ******
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