WO1991018619A1 - Apo ai polypeptides, antibodies, and immunoassays - Google Patents
Apo ai polypeptides, antibodies, and immunoassays Download PDFInfo
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- WO1991018619A1 WO1991018619A1 PCT/US1991/004038 US9104038W WO9118619A1 WO 1991018619 A1 WO1991018619 A1 WO 1991018619A1 US 9104038 W US9104038 W US 9104038W WO 9118619 A1 WO9118619 A1 WO 9118619A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/775—Apolipopeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to diagnostic methods and polypeptides useful for immunologically determining the amount of Apo Al in a vascular fluid sample.
- the polypeptides are useful in therapeutic methods and compositions for increasing LCAT- ediated cholesterol esterification and the formation of cholesterol esters in a human patient.
- Lipoproteins are the primary carriers of plasma cholesterol. They are icellar lipid-protein complexes (particles) having a surface film, comprised of one or more proteins associated with polar lipids, that surrounds a cholesterol-containing core. Lipoproteins were originally classified based on their buoyant densities as measured by ultracentrifugation. Accordingly, four major density classes have been recognized: chylomicrons, very low- density lipoproteins (VLDL) , low-density lipoproteins LDL and high-density lipoproteins (HDL) .
- VLDL very low- density lipoproteins
- HDL high-density lipoproteins
- Apo Al has been described as containing a high proportion (55%) of alpha-helix, which increases to 70% when it is associated with phospholipids as in the HDL particle.
- the lipid binding properties of Apo Al appear to be a function of a series of tandemly repeated segments of 22 amino acid residues punctuated mostly by proline residues that are alpha-helical and amphophilic.
- the amino acid residue sequence of Apo Al determined by Ed an degradation of cyanogen bromide- and trypsin-fragments of intact Apo Al, has been described by Brewer et al. , Biochem. Biophvs. Res. Comm. , 80:623-630 (1978).
- cyanogen bromide (CNBr) cleavage of Apo Al produced four major fragments, designated CNBrl, CNBr2, CNBr3 and CNBr4, in order of their occurrence along the Apo Al sequence from amino-terminus to carboxy-terminus. Because it is of particular interest to the present invention, the amino acid residue sequence of the region of Apo Al from which CNBr2 and CNBr3 is produced is illustrated in Figure 1.
- epitope A An exemplary conserved native Apo Al epitope, designated epitope A, has been defined by Milthorpe et al., Arterio.. 6:285-296 (1986) as being that portion of Apo Al CNBrl that immunoreacts with MAB 4H1. This was in contrast to epitopes designated C, C* and C", all located in the CNBr2 region of Apo Al, and all of which were found to be "nonconserved" epitopes.
- Monoclonal antibodies AI-4, and AI-11, and AI-18 have been identified as anti-Apo Al pan antibodies; that is, antibodies that bind all or most species of Apo Al-containing lipoprotein particles in plasma. See Hogle et al., J. Lipid. Res. , 29:1221-1229 (1988); and Curtiss et al., in "Biotechnology of Dyslipoproteinemias: Clinical Applications in Diagnosis and Control", Lieri et al., eds, pp. 217- 226, Raven Press (New York), 1989. However, the specific epitopes on Apo Al with which antibodies AI-4 and AI-11 immunoreact have not been identified.
- Apo Al has also been shown to modulate lecithin:cholesterol acyltransferase (LCAT)-mediated cholesterol esterification.
- LCAT lecithin:cholesterol acyltransferase
- an Apo Al polypeptide comprising no more than about 60 amino acid residues and including an amino acid residue sequence represented by the formula: KVQPYLDDFQKKWQEE-.
- an Apo Al polypeptide comprising no more than about 60 amino acid residues and including an amino acid residue sequence represented by the formula: -PYLDDXQKKWQEEMEL-, wherein X is either E of F, and preferably is represented by the formula: -PYLDDXQKKWQEEMELYRQKVEP-.
- the invention contemplates a diagnostic system, in kit form, comprising, in an amount sufficient to perform at least one assay, an Apo Al polypeptide of the present invention is operatively linked to a solid matrix.
- the diagnostic system further comprises an anti-Apo Al antibody molecule that immunoreacts with the Apo Al polypeptide of the kit as disclosed herein.
- Still further contemplated is a method of assaying the amount of Apo Al in a vascular fluid sample comprising the steps of:
- step (c) determining the amount of product formed in step (b) .
- a therapeutic method for increasing the amount of esterified cholesterol in a patient comprises administering to a patient an esterified cholesterol-increasing amount of an Apo Al polypeptide of this invention.
- Figure 1 illustrates the amino acid residue sequence of Apo Al, as reported by Brewer et al. , Biochem. Biophvs. Res. Comm.. 80:623-630 (1978), from amino acid residue positions 85 through 148 using the single letter code.
- Apo Al CNBr2 which is formed by cleavage at the methionine (M) residues located at positions 86 and 112, corresponds in sequence to positions 87 through 111 with the carboxy terminal methionine being converted to homoserine lactone.
- Apo Al CNBr3 which is formed by cleavage at the methionine (M) residues located at positions 112 and 148, corresponds in sequence to positions 113 through 147 with the carboxy terminal methionine being converted to homoserine lactone.
- the conserved native epitope on Apo Al that is defined by the MAB AI-4 spans CNBr fragments 2 and 3 and is comprised of amino acid residues sequence 99- 114.
- the conserved native epitope on Apo Al that is defined by the MAB AI-11 is confined to CNBr2 from the amino acid residue sequence 96-111.
- the conserved native epitope on Apo Al that is defined by the MAB AI-18 is confined to CNBR2 from the amino acid residue sequence 95-105.
- Figure 2 illustrates the ability of Apo AI/HDL, HDL present in fresh plasma, and the polypeptide AI96-111 to competitively inhibit MAB AI-11 from immunoreacting with AI96-111.
- Protein concentrations were determined according to the method of Markwell et al., Anal. Biochem.. 87:206-220 (1978). The units on the X-axis vary linearly with respect to the three competitors used in the assay.
- the data points for Apo AI/HDL correspond to a starting concentration of 1 mg/ml followed by 5 serial 2-fold dilutions.
- the data points for HDL present in fresh plasma correspond to a starting dilution of 1:10 followed by 6 serial 2-fold dilutions.
- Polypeptide AI96-111 is diluted as above for plasma with a starting concentration of 1 mg/ml.
- Figure 3 illustrates the ability of Apo AI/HDL and the polypeptides AI94-125 and AI99-121 to competitively inhibit MAB AI-4 from immunoreacting with Apo AI/HDL.
- Polypeptides AI90-111, AI93-111 and AI96-111 are not competitive inhibitors.
- the concentration of the competitor is shown in ⁇ g protein/ml determined as described in Figure 2.
- Figure 4 illustrates the amino acid residue sequence of a portion of Apo Al as described in Figure 1.
- a conserved native epitope is defined by MAB AI-4 and the epitope is immunologically mimicked by the polypeptide AI99-121, which is shown as a bold line.
- Polypeptides AI94-125 and AI99-121 also in bold, also immunoreacts with MAB AI-4 and mimics the epitope.
- Polypeptides derived from Apo Al which do not immunoreact with MAB AI-4 nor mimic the epitope are shown as thin lines. Alignments of the thin lines with actual Apo Al sequences are approximate with respect to the amino acid residue sequence of Apo Al, and the actual span of amino acid residues which comprise the thin line polypeptides are designated numerically.
- Figure 5 illustrates the ability of Apo AI/HDL and the polypeptides AI90-111, AI93-111, and AI96-111 to competitively inhibit MAB AI-11 from immunoreacting with Apo AI/HDL.
- Polypeptide AI99-121 is not a competitive inhibitor.
- Figure 6 illustrates the amino acid residue sequence of a portion of Apo Al as described in Figure 1.
- a conserved native epitope is defined by MAB AI-11, and the epitope is immunologically mimicked by polypeptide AI96-111, which is shown as a bold line.
- Polypeptides derived from Apo Al which do not immunoreact with MAB AI-11 nor mimic the epitope are shown as thin lines. Alignments of the polypeptides which do not react with MAB AI-11 are as described in Figure 4.
- Figure 7 illustrates the ability of the anti-Apo Al monoclonal antibodies of this invention to inhibit LCAT-mediated cholesterol esterification as described in Example 11.
- Figure 8 provides a comparison of an antibody's ability to bind to proteoliposomes with its ability to inhibit LCAT-mediated cholesterol esterification as described in Example 11. Binding data (open bars) was taken from a fluid-phase immunoassay where the conditions of the LCAT assay were duplicated. Apo A- I was used at a concentration of 220 ug/ml and antibodies were added at a 4-fold molar excess. LPDP and B-mercaptoethanol were added as in the LCAT assay. Bars represent means +/- S.E. for two experiments performed in duplicate. Data for inhibition of LCAT-mediated esterification (hatched bars) was taken from Figure 7 and is expressed here as the % of inhibition.
- amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
- NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
- C00H refers to the free carboxy group present at the carboxy terminus of a polypeptide.
- amino acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates either a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to a carboxyl or hydroxyl end group.
- Apo AI/HDL Designates Apo Al when it is present on HDL particles.
- Delipidated Apo Al Refers to Apo Al that is substantially free of associated lipids.
- Homoserine Lactone refers to a derivatized carboxy terminal methionine residue on a polypeptide that is formed by conventional cyanogen bromide
- Homoserine lactone is not normally found in proteins, and is preferably not present in a polypeptide of this invention.
- Isolated Apo Al Designates Apo Al that is substantially free of both associated lipids and other proteins, such as those, like Apo All, that are typically found on HDL in addition to Apo Al.
- Polypeptide and Peptide are terms used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
- Protein is a term used herein to designate a linear series of greater than about 50 amino acid residues connected one to the other as in a polypeptide.
- Synthetic peptide refers to a chemically produced chain of amino acid residues linked together by peptide bonds that is free of naturally occurring proteins and fragments thereof.
- Al polypeptide refers to a polypeptide whose amino acid residue sequence corresponds, and preferably is identical to a portion of the Apo Al molecule.
- an Apo Al polypeptide of the present invention comprises no more than about 60 amino acid residues, preferably no more than about 32 amino acid residues, and includes an amino acid residue sequence represented by the formula: -KVQPYLDDFQKKWQEE-.
- This polypeptide defines a conserved native epitope on Apo Al that is defined by the ability of the polypeptide to immunoreact with the monoclonal antibody MAB AI-11 (i.e., the polypeptide defines a MAB AI-11 epitope) .
- the polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: QEMSKDLEEVKAKVQPYLDDFQKKWQEE, EMSKDLEEVKAKVQPYLDDFQKKWQEE, LEEVKAKVQPYLDDFQKKWQEE, VKAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEEMELYRQKVEPLRAE, and
- a related APO Al polypeptide defining a MAB AI-11 epitope comprises 26 to 60 amino acid residues, preferably 27 to 32 amino acid residues, and includes an amino acid residue sequence represented by the formula: -KVQPYLDDFQKKWQEE-.
- the polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: QEMSKDLEEVKAKVQPYLDDFQKKWQEE,
- KAKVQPYLDDFQKKWQEEMELYRQKVEPLRAE Another related Apo Al polypeptide defining a MAB AI-11 epitope comprises no more than 25 amino acid residues, preferably no more than about 22 amino acid residues, and includes an amino acid residue sequence represented by the formula: -KVQPYLDDFQKKWQEE-.
- the polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: LEEVKAKVQPYLDDFQKKWQEE, VKAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEE, AND KVQPYLDDFQKKWQEE.
- an Apo Al polypeptide of this invention comprises no more than about 60 amino acid residues and includes an amino acid residue sequence represented by the formula:
- the Apo Al polypeptide includes an amino acid residue sequence represented by the formula:
- polypeptide has an amino acid residue represented by a formula selected from the group consisting of: KAKVQPYLDDXQKKWQEEMEL,
- KAKVQPYLDDXQKKWQEEMELYRQKVEPLRAE QPYLDDXQKKWQEEMEL, QPYLDDXQKKWQEEMELYRQKVEP, PYLDDXQKKWQEEMEL, and PYLDDXQKKWQEEMELYRQKVEP.
- an Apo Al polypeptide of this invention comprises no more than about 40 amino acid residues and includes an amino acid residue sequence represented by the formula: -AKVQPYLDDFQ-.
- This polypeptide defines a conserved native epitope on Apo Al that is defined by the ability of the polypeptide to immunoreact with the monoclonal antibody MAB AI-18 (i.e., the polypeptide defines a MAB AI-18 epitope) .
- the polypeptide has an amino acid residue sequence represented a formula selected from the group consisting of:
- an Apo Al polypeptide of this invention is free of homoserine lactone.
- An "X" at amino acid residue position 104 indicates that either an E for glutamic acid or an F for phenylalanine is present at residue position 104.
- an Apo Al polypeptide of this invention is further characterized by its ability to immunologically mimic an epitope (antigenic determinant) expressed by Apo Al on substantially all HDL.
- an Apo Al polypeptide of this invention is further characterized by its ability to immunologically mimic an epitope (antigenic determinant) expressed by Apo Al on substantially all HDL.
- the phrase "immunologically mimic” in its various grammatical forms refers to the ability of an Apo Al polypeptide of this invention to immunoreact with a antibody of the present invention that recognizes a conserved native epitope of Apo Al as defined herein. It should be understood that a subject polypeptide nee not be identical to the amino acid residue sequence of Apo Al, so long as it includes the required sequence and is able to immunoreact with antibodies of the present invention.
- a subject polypeptide includes any analog, fragment or chemical derivative of a polypeptide whose amino acid residue sequence is shown herein so long as the polypeptide is capable of immunologically mimicking an Apo Al native conserved epitope. Therefore, a present polypeptide can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use.
- analog includes any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the ability to mimic Apo Al as described herein.
- conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
- “Chemical derivative” refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group.
- Such derivatized molecules include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
- Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
- Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
- the imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.
- chemical derivatives those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For examples: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
- Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions or residues relative to the sequence of a polypeptide whos sequence is shown herein, so long as the requisite activit is maintained.
- a polypeptide is free of homoserine lactone when ther is no detectable homoserine lactone present in the polypeptide when subjected to conventional amino acid analysis able to indicate the presence of homoserine lactone or other amino acids.
- Amino acid analysis method suitable to detect homoserine lactone are generally well known in the art.
- fragment refers to any subject polypeptide having an amino acid residue sequence shorter than that o a polypeptide whose amino acid residue sequence is shown herein.
- a polypeptide of the present invention has a sequence that is not identical to the sequence of a Apo Al because one or more conservative or non-conservative substitutions have been made, usually no more than about 3 number percent, more usually no more than 20 number percent, and preferably no more than 10 number percent of the amino acid residues are substituted, except that the proline residue at position 99 cannot be substituted or deleted where additional residues have been added at eithe terminus for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently affixed to a label or solid matrix, or carrier, the linker residues do not form Apo Al epitopes, i.e., are not simila is structure to the Apo Al. Labels, solid matrices and carriers that can be used with the polypeptides of this invention are described hereinbelow.
- Amino acid residue linkers are usually at least one residue and can be 40 or more residues, more often 1 to 10 residues, but do not form Apo Al epitopes.
- Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.
- a subject polypeptide can differ, unless otherwise specified, from the natural sequence of Apo Al b the sequence being modified by terminal-NH 2 acylation, e.g., acetylation, or thi ⁇ glycolic acid amidation, by terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like.
- an Apo Al polypeptide of the present invention When coupled to a carrier to form what is known in the art as a carrier-hapten conjugate, an Apo Al polypeptide of the present invention is capable of inducing antibodies that immunoreact with Apo Al, preferably Apo Al when it is part of an HDL particle (Apo AI/HDL) .
- Apo AI/HDL an HDL particle
- the present invention therefore contemplates antigenically related variants of the polypeptides shown in Table 1.
- An "antigenically related variant” is a subject polypeptide that is capable of inducing antibody molecules that immunoreact with a polypeptide from Table 1 and Apo Al.
- Any peptide of the present invention may be used in the form of a pharmaceutically acceptable salt.
- Suitable acids which are capable of forming salts with the peptides of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like.
- Suitable bases capable of forming salts with the peptides of the present invention include inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like) .
- inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like
- organic bases such as mono-, di- and tri-alkyl and aryl amines (e.g. triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g. ethanolamine, diethanolamine and the like) .
- An Apo Al polypeptide of the present invention also referred to herein as a subject polypeptide, can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, are preferred for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production and the like. An excellent summary of the many techniques available can be found in J.M. Steward and J.D. Young, “Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969; M. Bodanszky, et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976 and J.
- either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group.
- a different, selectively removable protecting group is utilized for amino acids containing a reactive side group such as lysine.
- the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
- the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted under conditions suitable for forming the amide linkage with the residue already attached to the solid support.
- the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to afford the final polypeptide.
- An Apo Al polypeptide can be used, inter alia, in the diagnostic methods and systems of the present invention to detect Apo Al present in a body sample, or can be used to prepare an inoculum as described herein for the preparation of antibodies that immunoreact with conserved epitopes on Apo Al.
- an Apo Al polypeptide of this invention can be used in the therapeutic methods of the present invention to increase esterification of cholesterol in a patient.
- antibody in its various grammatical forms is used herein as a collective noun that refers to a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.
- An "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
- antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
- Exemplary antibody molecules for use in the diagnostic methods and systems of the present invention are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab » ) 2 and F(v) .
- Fab and F(ab') 2 portions of antibodies are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibodies by methods that are well known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous and Dixon.
- Fab' antibody portions are also well known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoaceta ide.
- An antibody containing intact antibody molecules are preferred, and are utilized as illustrative herein.
- An antibody of the present invention i.e., an anti-Apo Al antibody
- an anti-Apo Al antibody in one embodiment is characterized as being capable of immunoreacting with 1) Apo Al present on HDL particles (Apo AI/HDL), 2) isolated Apo Al, 3) Apo Al (CNBr2-CNBr3) , and 4) the polypeptide PYLDDFQKKWQEEMEL, and being substantially free of antibody molecules that immunoreact with 1) Apo Al CNBrl, and 2 ) the polypeptides: SKDLEEVKAKVQPYLDDFQKKWQEE,
- an anti-Apo Al antibody is characterized as being capable of immunoreacting with 1) Apo AI/HDL, 2) isolated Apo Al, 3) Apo Al CNBr2, and 4) the polypeptide KVQPYLDDFQKKWQEE, and being substantially free of antibody molecules that immunoreact with 1) Apo Al CNBrl, 2) Apo Al CNBr3, and 3) the polypeptides:
- Antibody immunoreactivity with Apo Al-containing antigens can be measured by a variety of immunological assays known in the art. Exemplary immunoreaction of an anti-Apo Al antibody with a CNBr fragment is described in
- Example 8 Direct binding with Apo AI/HDL, isolated Apo Al (prepared as described in Example 4) , and with Apo Al polypeptides can be assayed at least by the methods described in Example 9.
- An antibody of the present invention is typically produced by immunizing a mammal with an inoculum containing an Apo Al polypeptide of this invention and thereby induce in the mammal antibody molecules having immunospecificity for Apo Al polypeptide. The antibody molecules are then collected from the mammal and isolated to the extent desired by well known techniques such as, for example, by using DEAE Sephadex to obtain the IgG fraction.
- the antibodies may be purified by immunoaffinity chromatography using solid phase-affixed immunizing polypeptide.
- the antibody is contacted with the solid phase-affixed immunizing polypeptide for a period of time sufficient for the polypeptide to immunoreact with the antibody molecules to form a solid phase-affixed immunocomplex.
- the bound antibodies are separated from the complex by standard techniques.
- the antibody so produced can be used, inter alia, in the diagnostic methods and systems of the present invention to detect Apo Al present in a body sample. See, for example, the method described in Example 9.
- the word "inoculum” in its various grammatical forms is used herein to describe a composition containing an Apo Al polypeptide of this invention as an active ingredient used for the preparation of antibodies against an Apo Al polypeptide.
- the polypeptide can be used in various embodiments, e.g., alone or linked to a carrier as a conjugate, or as a polypeptide polymer.
- the various embodiments of the polypeptides of this invention are collectively referred to herein by the term "polypeptide", and its various grammatical forms.
- polypeptide that contains fewer than about 35 amino acid residues
- One or more additional amino acid residues can be added to the amino- or carboxy-termini of the polypeptide to assist in binding the polypeptide to a carrier.
- Cysteine residues added at the amino- or carboxy-termini of the polypeptide have been found to be particularly useful for forming conjugates via disulfide bonds.
- Exemplary additional linking procedures include the use of Michael addition reaction products, di- aldehydes such as glutaraldehyde, Klipstein, et al., J. Infect.
- Useful carriers are well known in the art, and are generally proteins themselves. Exemplary of such carriers are keyhole limpet hemocyanin (KLH) , edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) or human serum albumin (HSA) , red blood cells such as sheep erythrocytes (SRBC) , tetanus toxoid, cholera toxoid as well as polyamino acids such as poly (D-lysine: D-glutamic acid) , and the like.
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- HSA human serum albumin
- red blood cells such as sheep erythrocytes (SRBC)
- tetanus toxoid cholera toxoid
- polyamino acids such as poly (D-lysine: D-glutamic acid) , and the like.
- carrier is more dependent upon the ultimate use of the inoculum and is based upon criteria not particularly involved in the present invention. For example, a carrier that does not generate an untoward reaction in the particular animal to be inoculated should be selected.
- the present inoculum contains an effective, immunogenic amount of a polypeptide of this invention, typically as a conjugate linked to a carrier.
- the effective amount of polypeptide per unit dose sufficient to induce an immune response to the immunizing polypeptide depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen inoculation regimen as is well known in the art.
- Inocula typically contain polypeptide concentrations of about 10 micrograms to about 500 milligrams per inoculation (dose) , preferably about 50 micrograms to about 50 milligrams per dose.
- unit dose refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent; i.e., carrier, or vehicle.
- the specifications for the novel unit dose of an inoculum of this invention are dictated by and are directly dependent on (a) the unique characteristics of the active material and the particular immunologic effect to be achieved, and (b) the limitations inherent in the art of compounding such active material for immunologic use in animals, as disclosed in detail herein, these being features of the present invention.
- Inocula are typically prepared from the dried solid polypeptide-conjugate by dispersing the polypeptide- conjugate in a physiologically tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline to form an aqueous composition.
- a physiologically tolerable (acceptable) diluent such as water, saline or phosphate-buffered saline to form an aqueous composition.
- Inocula can also include an adjuvant as part of the diluent.
- Adjuvants such as complete Freund's adjuvant (CFA) , incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and are available commercially from several sources.
- One or more additional amino acid residues may be added to the amino- or carboxy-termini of the polypeptide to assist in binding the polypeptide to form a conjugate.
- Cysteine residues usually added at the carboxy-terminus of the polypeptide, have been found to be particularly useful for forming conjugates via disulfide bonds, but other methods well-known in the art for preparing conjugates may be used.
- a preferred anti-Apo Al antibody is a monoclonal antibody and is used herein as exemplary of an anti-Apo Al antibody.
- the phrase "monoclonal antibody” in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with a particular epitope.
- a monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts.
- a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, e.g., a bispecific monoclonal antibody.
- a monoclonal antibody in one embodiment, is characterized as being capable of immunoreacting with 1) Apo AI/HDL, 2) isolated Apo Al, 3) Apo Al CNBr2-CNBr3, and 4) the polypeptide PYLDDFQKKWQEEMEL, and being substantially free of antibody molecules that immunoreact with 1) Apo Al CNBrl, and 2) the polypeptides:
- YRQKVEPLRAEL Particularly preferred is the monoclonal antibody produced by the hybridoma AI-4, having an ATCC accession number HB8744.
- a monoclonal antibody is characterized as being capable of immunoreacting with 1) Apo AI/HDL, 2) isolated Apo Al, 3) Apo Al CNBr2, and 4) the polypeptide KVQPYLDDFQKKWQEE, and being substantially free of antibody molecules that immunoreact with 1) Apo Al CNBrl, 2) Apo Al CNBr3, and 3) the polypeptides:
- PYLDDFQKKWQEEMELYRQKVEP Particularly preferred in this embodiment is the monoclonal antibody produced by the hybridoma AI-11, having an ATCC accession number HB9201.
- a monoclonal antibody is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) but one kind of antibody molecule.
- the hybridoma cell is formed by fusing an antibody-producing cell and a myeloma or other self- perpetuating cell line.
- the preparation of such antibodies was first described by Kohler and Milstein, Nature 256:495- 497 (1975) , which description is incorporated by reference.
- the hybridoma supernates so prepared can be screened for the presence of antibody molecules that immunoreact with an Apo Al polypeptide, or for inhibition of binding to HDL by the Apo Al polypeptides of this invention.
- a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an Apo Al antigen, such as is present in an Apo Al-containing lipoprotein particles, or with an Apo Al polypeptide of this invention.
- Apo Al antigen such as is present in an Apo Al-containing lipoprotein particles, or with an Apo Al polypeptide of this invention.
- the polypeptide-induced hybridoma technology is described by Niman et al. , Proc. Natl. Sci. , U.S.A.. 80:4949-4953 (1983), which description is incorporated herein by reference.
- the myeloma cell line used to prepare a hybridoma be from the same species as the lymphocytes.
- a mouse of the strain 129 G1X + is the preferred mammal.
- Suitable mouse myelomas for use in the present invention include the hypoxanthine-aminopterin- thymidine-sensitive (HAT) cell lines P3X63-Ag8.653, and Sp2/0-Agl4 that are available from the American Type
- Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 1500. Fused hybrids are selected by their sensitivity to HAT. Hybridomas producing a monoclonal antibody of this invention are identified using the radioimmunoassay (RIA) and the enzyme linked immunosorbent assay (ELISA) described in Examples 10 and 9, respectively.
- a monoclonal antibody of the present invention can also be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate polypeptide specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well known techniques.
- DMEM Dulbecco's minimal essential medium
- fetal calf serum An exemplary inbred mouse strain is the Balb/c.
- the monoclonal antibodies of this invention can be used in the same manner as disclosed herein for antibodies of the present invention.
- the monoclonal antibody can be used in the diagnostic methods and systems disclosed herein where formation of an Apo Al-containing immunoreaction product is desired.
- a hybridoma useful in producing a subject monoclonal antibody i.e., MAB AI-4 and MAB AI-11, are hybridomas 611AV63C2.111 and H10308.1011, said hybridomas being deposited pursuant to Budapest Treaty Requirements with the American Type Culture Collection (ATCC) , Rockville, MD 20852 U.S.A. on March 5, 1985 and September 16, 1986, respectively, and given the ATCC designations HB8744 and HB9201, respectively.
- ATCC American Type Culture Collection
- hybridoma ATCC 1580 can be used, as is well known in the art, to produce other immortal cell lines that produce a subject monoclonal antibody, and thus production of a subject monoclonal antibody is not dependent on culturing hybridomas by ATCC HB8744 and HB9201 per se.
- the present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of Apo Al or an Apo Al polypeptide in a fluid sample.
- a diagnostic system includes, in an amount sufficient for at least one assay, a subject Apo Al polypeptide and/or a subject antibody or monoclonal antibody, as a separately packaged immunochemical reagent. Instructions for use of the packaged reagent are also typically included.
- a package refers to a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody or monoclonal antibody of the present invention.
- a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or it can be a microtiter plate well to which icrogram quantities of a contemplated polypeptide have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody.
- Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.
- a diagnostic system for assaying for the presence of or to quantitate Apo Al in a sample comprises a package containing at least one Apo Al polypeptide of this invention.
- a diagnostic system of the present invention for assaying for the presence or amount of Apo Al or an Apo Al polypeptide in a sample further includes an anti-Apo Al antibody composition of this invention.
- An exemplary diagnostic system is described in Example 9.
- a diagnostic system of the present invention further includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.
- complex refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction.
- exemplary complexes are immunoreaction products.
- label and indicating means in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents.
- labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
- the labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
- Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5- dimethylamine-1-naphthalenesulfonyl chloride (DANSC) , tetramethylrhodamine isothiocyanate (TRITC) , lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
- fluorochromes such as fluorescein isocyanate (FIC) , fluorescein isothiocyante (FITC) , 5- dimethylamine-1-naphthalenesulfonyl chloride (DANSC) , tetramethylrho
- the indicating group is an enzyme, such as horseradish peroxidase (HRP) , glucose oxidase, or the like.
- HRP horseradish peroxidase
- glucose oxidase or the like.
- additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
- additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
- An additional reagent useful with glucose oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G- sulfonic acid) (ABTS) .
- Radioactive elements are also useful labeling agents and are used illustratively herein.
- An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 12A I, 125 I, 128 I, 132 I and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 125 I.
- Another group of useful labeling means are those elements such as - ⁇ C, 18 F, 15 0 and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such as - 11 indium of 3 H.
- labeling of, polypeptides and proteins is well known in the art.
- antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth. Enzvmol.. 73:3-46 (1981).
- the techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas, et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech. , 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
- the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
- a "specific binding agent” is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention.
- Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like.
- the specific binding agent binds the reagent species when that species is present as part of a complex.
- the specific binding agent is labeled.
- the agent is typically used as an amplifying means or reagent.
- the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
- the diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of Apo Al in a vascular fluid sample such as blood, serum, or plasma.
- ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample.
- an Apo Al polypeptide or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
- a reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides well known to those skilled in the art, can be used.
- Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross- linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ) ; agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.
- the reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
- the indicating means is an enzyme
- the enzyme's substrate can also be provided in a separate package of a system.
- a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
- packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
- a package refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene and polycarbonate) , paper, foil and the like capable of holding within fixed limits a diagnostic reagent such as a polypeptide, antibody or monoclonal antibody of the present invention.
- a package can be a bottle, vial, plastic and plastic-foil laminated envelope or the like container used to contain a contemplated diagnostic reagent or it can be a microtiter plate well to which microgram quantities of a contemplated diagnostic reagent have been operatively affixed, i.e., linked so as to be capable of being immunologically bound by an antibody or polypeptide to be detected.
- the present invention contemplates various immunoassay methods for determining the amount of Apo Al in a biological fluid sample using a polypeptide, polyclonal antibody or monoclonal antibody of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of Apo Al in the sample.
- a polypeptide, polyclonal antibody or monoclonal antibody of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of Apo Al in the sample.
- an immunochemical reagent of this invention can be used to form an immunoreaction product whose amount relates to the amount of Apo Al present in a body sample.
- exemplary assay methods are described herein, the invention is not so limited.
- the present invention contemplates a competitive method for assaying the amount of Apo Al in a vascular fluid sample which comprises the steps of:
- an antibody of the present invention preferably a monoclonal antibody
- an Apo Al polypeptide of the present invention that is able to immunoreact with the antibody added in step (i) .
- the vascular fluid sample is provided as a known amount of blood, or a blood derived product such as serum or plasma. Regardless of the type of sample used, it is preferably obtained from a person who has fasted at least about 12 hours as is known in the art. Such a sample is referred to as a "fasting" sample. It is also noted that where serum or plasma is used as the sample, that sample need not be subjected to treatment with a denaturing or chaotropic agent for purposes of altering the expression of the Apo Al epitope being assayed.
- the diagnostic method includes forming an immunoreaction admixture by admixing a vascular fluid sample with:
- an Apo Al polypeptide of the present invention that includes an amino acid residue sequence represented by the formula: -PYLDDXQKKWQEEMEL-, and preferably includes an amino acid residue sequence represented by the formula: -PYLDDXQKKWQEEMELYRQKVEP-.
- the antibody is the monoclonal antibody produced by the hybridoma cell line having the ATCC designation HB 8744, and the polypeptide is selected from the group polypeptides shown in Table 1 consisting of AI99- 121, AI94-125, AI94-114, AI98-114, AI98-121, and AI99-114.
- the immunoreaction admixture is formed by admixing a vascular fluid sample with:
- the antibody is the monoclonal antibody produced by the hybridoma cell line having the ATCC designation HB9201, and the polypeptide is selected from the group of polypeptides shown in Table 1 consisting Of AI96-111, AI93-111, AI84-111, AI85-111, AI87-111, AI94- 111, AI94-125, and AI90-111.
- the amount of antibody that is admixed is known. Further preferred are embodiments where the antibody is labeled, i.e., operatively linked to an indicating means such as an enzyme, radionuclide and the like.
- the Apo Al polypeptide is present as part of a solid support, i.e., operatively linked to a solid matrix, so that the immunoreaction admixture formed has a solid and a liquid phase.
- the amount of polypeptide present in the immunoreaction admixture is an amount sufficient to form an excess of epitopes relative to the number of antibody combining sites present in the immunoreaction admixture capable of immunoreacting with those epitopes.
- the immunoreaction admixture is maintained under biological assay conditions for a predetermined time perio such as about 10 minutes to about 16-20 hours at a temperature of about 4 degrees C to about 45 degrees C that, such time being sufficient for the Apo Al present in the sample to immunoreact with (immunologically bind) a portion of the anti-Apo Al antibody combining sites present in the monoclonal antibody to form an Apo Al-containing immunoreaction product (immunocomplex) .
- the immunocomplex formed is also present in the solid phase.
- Biological assay conditions are those that maintain the biological activity of the immunochemical reagents of this invention and the Apo Al sought to be assayed. Those conditions include a temperature range of about 4 degrees C to about 45 degrees C, a pH value range of about 5 to about 9 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride. Methods for optimizing such conditions are well known in the art.
- step (c) The amount of Apo Al-containing immunoreaction product that formed in step (b) is determined, thereby determining the amount of Apo Al present in the sample. Determining the amount of the Apo Al-containing immunoreaction product, either directly or indirectly, can be accomplished by assay techniques well known in the art, and typically depend on the type of indicating means used. In preferred competitive assay methods, the amount of product determined in step (c) is related to the amount of immunoreaction product similarly formed and determined using a control sample in place of the vascular fluid sample, wherein the control sample contains a known amount of a subject polypeptide from which a standard curve is determined.
- the present invention contemplates a double antibody or "sandwich" immunoassay comprising the steps of:
- a first immunoreaction admixture by admixing a vascular fluid sample with a first antibody, preferably a monoclonal antibody, wherein the antibody and Apo AI/HDL present in the sample are capable of forming a first immunoreaction product that can immunoreact with a subject monoclonal antibody.
- the first antibody is operatively linked to a solid matrix.
- the first immunoreaction product is then separated from the sample.
- the subject monoclonal antibody of step (c) is labeled, preferably with an enzyme, and therefore the second immunoreaction product formed is a labeled product.
- the detection of Apo Al polypeptides in a body sample is utilized as a means to monitor the fate of therapeuticallly administered Apo Al polypeptides according to the therapeutic methods disclosed herein.
- immunological assays capable of detecting the presence of immunoreaction product formation without the use of a label. Such methods employ a "detection means", which means are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel polypeptides, methods and systems.
- Exemplary detection means include methods known as biosensors and include biosensing methods based on detecting changes in the reflectivity of a surface, changes in the absorption of an evanescent wave by optical fibers or changes in the propagation of surface acoustical waves.
- F. Therapeutic Compositions include methods known as biosensors and include biosensing methods based on detecting changes in the reflectivity of a surface, changes in the absorption of an evanescent wave by optical fibers or changes in the propagation of surface acoustical waves.
- compositions of the present invention contemplates therapeutic compositions useful for practicing the therapeutic methods described herein.
- Therapeutic compositions of the present invention contain a physiologically tolerable carrier together with an Apo Al polypeptide, as described herein, dissolved or dispersed therein as an active ingredient.
- the therapeutic composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes.
- compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
- physiological effects such as nausea, dizziness, gastric upset and the like.
- compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.
- the active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein.
- Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.
- the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.
- the therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein.
- Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethy1amine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art.
- liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
- the Apo Al polypeptides of the present invention have the capacity to modulate the enzymatic activity of lecithin:cholesterol acyltransferase (LCAT) and thereby increase the level of esterified cholesterol in a human patient.
- LCAT lecithin:cholesterol acyltransferase
- the esterification of cholesterol, where it occurs by the activity of LCAT, is referred to herein as "LCAT-mediated cholesterol esterification”.
- the present invention provides for a method for increasing esterified cholesterol in a patient comprising administering to the patient a therapeutically effective amount of a physiologically tolerable composition containing an Apo Al polypeptide of the present invention.
- a therapeutically effective amount is a predetermined amount calculated to achieve the desired effect, i.e., to increase the amount of esterified cholesterol in a patient.
- a therapeutically effective amount is typically an amount of an Apo Al polypeptide of the present invention that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about 0.1 ⁇ g/ml to about 100 ⁇ g/ l, preferably from about 1.0 ⁇ g/ml to about 50 ⁇ l, more preferably at least about 2 ⁇ g/ml and usually 5 to 10 ⁇ g/ml.
- the level of esterified cholesterol present in a patient can be readily determined by routine clinical analysis. Exemplary assays to monitor the level of esterified cholesterol are described in Example 11. In addition, changes in esterified cholesterol can be monitored during a treatment regimen to determine the effectiveness of the administered Apo Al polypeptide over time.
- the present therapeutic method provides a means for in vivo increasing esterified cholesterol in a human patient displaying symptoms of elevated serum cholesterol, or is otherwise at medical risk by the presence of serum cholesterol, wherein it is beneficial to reduce the levels of cholesterol by cholesterol esterification.
- the therapeutic compositions containing an Apo Al polypeptide of this invention are conventionally administered intravenously, as by injection of a unit dose, for example.
- unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
- compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
- quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual.
- suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
- a diagnostic method of this invention for detecting an APO Al polypeptide in the subject's blood is useful to characterize the fate of the administered therapeutic composition.
- Polypeptides AI84-111, AI85-111, AI87-111, AI90-111, AI93-111, AI94-111, AI94-114, AI94-125, AI96-111, AI98-114, AI98-121, AI99-114, AI99-121, AI90-105, AI87-105, and AI95- 105 were synthesized using the classical solid-phase technique described by Merrifield, Adv. Enzvmol.. 32:221-96 (1969) as adapted for use with a Model 430A automated peptide synthesizer (Applied Biosystems, Foster City, CA) .
- Polypeptide resins were cleaved by hydrogen fluoride, extracted and analyzed for purity by high-performance liquid chromatograph using a reverse-phase C18 column. (Waters Associates, Milford, MA) .
- the amino acid residue sequence of the polypeptides named above is shown in Table 1.
- HDL was isolated from plasma obtained by plasmaphoresis of normal fasting-donor blood at the local blood bank (San Diego Plasma Center, San Diego, CA) .
- plasma so obtained was adjusted to contain a final concentration of 2 millimolar (mM) benzamidine, 14 mM ethylenediaminetetraacetic acid (disodium salt) (EDTA) , 20 micrograms per milliliter ( ⁇ g/ml) soybean trypsin inhibitor, 10,000 units per ml aprotinin, 20 ⁇ g/ml lima bean trypsin inhibitor, 25 ⁇ g/ml polybrene, and l ⁇ M D- phenylalanyl-1-prolyl-l-arginine chloromethyl ketone (PPACK) .
- the HDL was then isolated from this adjusted plasma by sequential ultracentrifugation using solid potassium bromide (KBr) for density adjustment.
- the adjusted plasma was centrifuged at 186,000 x g for 18 to 24 hours at 4 degrees centigrade (4°C) .
- the top layer of the resulting supernatant containing Apo-VLDL was removed and retained.
- the bottom layer of the supernatant was recovered and admixed with solid KBr layer until the density was greater than 1.063 grams per milliliter (g/ml) .
- the resulting admixture was then layered under a 0.1% EDTA solution containing KBr at density of 1.063 g/ml and centrifuged at 186,000 x g for 18 to 24 hours.
- the bottom layer was again recovered and admixed with solid KBr until the density was adjusted to greater than 1.21 g/ml. That adjusted layer was layered under a 0.1% EDTA solution containing KBr at a density of 1.21 g/ml, and was centrifuged at 186,000 x g for more about 48 hours at 4°C.
- the resulting top layer was then recovered and admixed with solid KBr until the density was greater than 1.063 g/ml. That adjusted top layer was layered under a 0.1% EDTA solution containing KBr at a density of 1.063 g/ml, and still further centrifuged at 186,000 x g for 18 to 24 hours at 4°C.
- the middle layer was recovered and admixed with solid KBr until the density was adjusted to greater than 1.21 g/ml. That adjusted middle layer was layered under a 0.1% EDTA solution containing KBr at a density of 1.21 g/ml and centrifuged at 300,000 x g for 18 to 24 hours at 4°C. The resulting HDL-containing top layer, having a density equal to 1.063 to 1.21 g/ml, was recovered.
- the recovered HDL was dialyzed against lipoprotein buffer (LLB; water containing 150 mM NaCl, 0.3 mM EDTA, 0.005% alpha- tocopherol, and 5 mM benzamidine) and the resulting Apo AI/HDL was stored under sterile conditions for no more than 21 days.
- the protein concentration of Apo AI/HDL was determined to be between 15 and 25 mg/ml by a modification of the Lowry method [Lowry et al., J. Biol. Chem. , 193, 265-275 (1951) ] when conducted in the presence of SDS using a bovine serum albumin standard.
- Delipidated Apo Al was prepared by organically extracting the lipids from Apo AI/HDL.
- a sample of the Apo AI/HDL prepared in Example IB was first dialyzed overnight (approximately 18 hours) against 0.01 percent EDTA having a pH value of 7.5, then dialyzed against 0.003 percent EDTA for approximately 6 hours, and subsequently lyophilized at 10 to 20 milligrams of protein per tube.
- delipidated Apo Al contains not only Apo Al, but also other proteins associated with the HDL, such as Apo All.
- Apo Al was isolated from delipidated Apo Al by size fractionation using high pressure liquid chromatography (HPLC) following the procedures of Kinoshita et al., J. Biochem. , 94:615-617 (1983). About 300 ⁇ g of delipidated Apo Al prepared in Example 3 was dissolved in 200 microliters ( ⁇ l) of 0.1% sodium dodecyl sulfate (SDS), 0.1 M sodium phosphate (pH 7.0) and size fractionated on Spherogel - TSK 3000 SW HPLC columns (Beckman Instruments Inc., Fullerton, CA) . Fractions containing the isolated Apo Al were stored at -20°C. 5.
- top layer is then recovered, and solid KBr is admixed until the density is greater than 1.063 g/ml. That adjusted top layer is layered under a 0.1% EDTA solution containing KBr at a density of 1.063 g/ml, and still further centrifuged at 250,000 x g for 18 to 24 hours at 4"C. The top layer containing concentrated LDL is recovered and dialyzed against PBS (phosphate-buffered saline, pH 7.2) and stored at -70°C.
- PBS phosphate-buffered saline, pH 7.2
- polypeptides AI84-111, AI85-111, AI90-111, AI93- 111, AI194-111, AI94-114, AI94-125, AI96-111, A198-114, AI198-121, Al AI99-114, and AI99-121 are synthesized as described in Example 1.
- Each polypeptide is individually dissolved in 1.5 M sodium acetate, pH 7.8, to a final concentration of 6 mg/ml with a total volume of 5 mis.
- a dissolved polypeptide is admixed with 2.5 is each of a 2 mg/ml LDL solution and a 3 M sodium acetate solution, pH 7.8, for a peptide:LDL ratio of 1000:1.
- Added to the polypeptide and LDL reaction mixture is a 500 mM glutaraldehyde solution using a 2-7 molar excess of glutaraldehyde to peptide.
- the admixture is maintained at room temperature for 10 minutes, after which a 40 mM solution of sodium borohydride is added for a final concentration of 0.2 mM.
- the admixture is thereafter maintained at 37°C for 5 to 8 hours, which is followed by dialysis against PBS for 5 days with two buffer changes per day using dialysis tubing having a 12,000 to 14,000 molecular weight cut-off.
- the dialysed solution is centrifuged at 2500 x g for 10 minutes, and the resulting pellet is resuspended in 5 mis PBS to form a peptide-LDL immunogen.
- a peptide-LDL immunogen is prepared for using each of the above-described peptides in the above immunogen preparation protocol.
- Example 5A The peptide-LDL immunogen prepared in Example 5A is emulsified using the Ribi Adjuvant System (Ribi Immunochem Research, Inc. , Hamilton, Montana) according to the manufacturer's instructions, and the peptide-LDL antigens are incorporated into the emulsion at a concentration of 300 ⁇ g/ml. Two rabbits are injected with 1 ml of a prepared emulsion after pre-immune serum samples are collected.
- Ribi Adjuvant System Ribi Immunochem Research, Inc. , Hamilton, Montana
- the 1 ml emulsion dose is administered as follows: 0.30 ml intradermal (0.05 ml in each of 6 sites); 0.40 ml intramuscular (0.2 ml into each hind leg); 0.10 ml subcutaneous (neck region) ; and 0.20 ml intraperitoneal.
- the rabbits are injected 6 times at three-week intervals following the injection protocol as detailed.
- blood samples are collected to check antibody titer against the specific peptide used as an immunogen by the SPRIA assay described below.
- the collected blood samples are stirred in a 37°C oven for 1 hour, after which the samples are centrifuged at 3000 x g for 20 minutes.
- the interface is collected and spun in a microfuge at 12,000 x g for 5 minutes.
- the supernatant containing anti-peptide antibodies is collected and stored at -20°C.
- the peptide antibody titers are determined by solid phase radioimmunoassay (SPRIA) essentially as described in Curtiss and Edgington, J. Biol. Chem. , 257:15213-15221 (1982) . Briefly, 50 ⁇ l of PBS containing 5 ⁇ g/ml synthetic peptides are admixed into the wells of microtiter plates.
- the plates are maintained overnight (about 16 hours) at 4°C to permit the peptides to adhere to well walls.
- SPRIA buffer (2.68 mM KCL, 1.47 mM KH 2 P0 4 , 137 mM NaCl, 8.03 mM Na 2 HP0 4f 0.05% Tween-20, 0.1 KlU/ml Traysol, 0.1% BSA, 0.015% NaN 3 )
- 200 ⁇ l of SPRIA buffer containing 3% normal goat serum (NGS) and 3% bovine serum albumin (BSA) are admixed to each well to block excess protein binding sites.
- NGS normal goat serum
- BSA bovine serum albumin
- the plates are maintained for 30 minutes at 20°C, the wells emptied by shaking, and blotted dry to form a solid-support, i.e., a solid matrix to which Apo AI/HDL was operatively affixed.
- a solid-support i.e., a solid matrix to which Apo AI/HDL was operatively affixed.
- 50 ⁇ l of serum sample to form a solid-liquid phase immunoreaction admixture.
- the admixture is maintained for 2 hours at 37°C to permit formation of solid-phase immunoreaction products.
- 50 ⁇ l of 125 I- labeled goat anti-mouse IgG at 0.25 ⁇ g protein per ml are admixed to each well to form a labeling reaction admixture.
- That admixture is maintained for 1 hour at 37°C to permit formation of 15 I-labeled solid-phase immunoreaction products.
- the amount of 125 I-labeled product bound to each well is determined by gamma scintillation.
- Specific anti-peptide antibody titers in collected serum samples from immunized rabbits are determined in comparison to pre-immunized normal rabbit serum samples which are a measure of non ⁇ specific background. Serum samples are considered to contain anti-peptide polyclonal antibodies if the radioactive signal is 5 times over that seen with normal rabbit serum. 6.
- A. Anti-peptide The polypeptides designated AI84-111, AI85-111, AI90- 111, AI93-111, AI94-111, AI94-114, AI94-125, AI96-111, AI98-114, AI98-121, AI99-114, and AI99-121 are individually prepared as immunogens according to Example 5A.
- Balb/c ByJ mice (Scripps Clinic and Research Foundation Vivarium, La Jolla, CA) are immunized intraperitoneally (i.p.) with 50 ⁇ g of prepared peptide-LDL immunogens in complete Freund's adjuvant (CFA) followed by a second and third immunization using the same peptide-LDL immunogen, each about three weeks apart, in incomplete Freund's adjuvant (IFA) .
- CFA complete Freund's adjuvant
- IFA incomplete Freund's adjuvant
- the mice receive a boost of 50 ⁇ g of prepared peptides intravenously (i.v.) in normal saline 4 days prior to fusion and a second similar perfusion boost one day later.
- the animals so treated are sacrificed and the spleen of each mouse is harvested.
- a spleen cell suspension is then prepared.
- Spleen cells are then extracted from the spleen cell suspension by centrifugation for about 10 minutes at 1000 r.p.m., at 23°C. Following removal of supernatant, the cell pellet is resuspended in 5 ml cold NH 4 C1 lysing buffer, and was incubated for about 10 minutes.
- DMEM Dulbecco's Modified Eagle Medium
- HEPES [4-(2-hydroxyethyl)-l-piperidineethanesulfonic acid] buffer
- the supernatant is decanted, the pellet is resuspended in 15 ml of DMEM and HEPES, and is centrifuged for about 10 minutes at 1000 r.p.m. at 23°C. The above procedure is repeated.
- the pellet is then resuspended in 5 ml DMEM and HEPES. An aliquot of the spleen cell suspension is then removed for counting. Fusions are accomplished in the following manner using the non-secreting mouse myeloma cell line P3X63Ag8.653.1, a subclone of line P3x63Ag 8.653 (ATCC 1580) .
- a myeloma to spleen cell ratio of about 1 to 10 or about 1 to 5
- a sufficient quantity of myeloma cells are centrifuged into a pellet, washed twice in 15 ml DMEM and HEPES, and centrifuged for 10 minutes at 1000 r.p.m. at 23°C.
- Spleen cells and myeloma cells are combined in round bottom 15 ml tubes.
- the cell mixture is centrifuged for 10 minutes at 1000 r.p.m. at 23°C, and the supernatant is removed by aspiration. Thereafter, 200 ⁇ l of 50 percent (weight per volume) aqueous polyethylene glycol 4000 molecular weight (PEG; ATCC Baltimore, MD) at about 37°C are admixed using a 1 ml pipette with vigorous stirring to disrupt the pellet, and the cells are gently mixed for between 15 and 30 seconds. The cell mixture is centrifuged 4 minutes at 700 r.p.m.
- PEG polyethylene glycol 4000 molecular weight
- DMEM plus HEPES buffer are admixed slowly to the pellet, without disturbing the cells.
- the resulting admixture is broken up with a 1 ml pipette, and is incubated for an additional 4 minutes. This mixture is centrifuged for 7 minutes at 1000 r.p.m. The supernatant is decanted, 5 ml of HT (hypoxanthine/thymidine) medium are slowly admixed to the pellet, and the admixture is maintained undisturbed for 5 minutes.
- HT hyperxanthine/thymidine
- the pellet is then broken into large chunks, and the final cell suspension is placed into T75 flasks (2.5 ml per flask) into which 7.5 ml HT medium have been placed previously.
- the resulting cell suspension is incubated at 37°C to grow the fused cells.
- 10 ml of HT medium are admixed to the flasks, followed 6 hours later by admixture of 0.3 ml of 0.04 mM aminopterin.
- 10 ml of HAT (hypoxanthine/aminopterin/thymidine) medium are admixed to the flasks.
- viable cells are plated out in 96-well tissue culture plates at about 2x10* viable cells per well (768 total wells) in HAT buffer medium as described in Kennett et al. , Curr. Top. Microbiol. Immunol.. 81:77 (1978).
- the cells are fed seven days after fusion with HAT medium and at -approximately 4-5 day intervals thereafter as needed with HT medium. Growth is followed microscopically, and culture supernatants are collected about two weeks later and assayed for the presence of HDL-specific antibody by solid phase radioimmunoassay (RIA) essentially as described in Curtiss and Edgington J. Biol. Chem. f 257:15213-15221 (1982).
- RIA solid phase radioimmunoassay
- the plates are maintained for 30 minutes at 20°C, the wells emptied by shaking, and blotted dry to form a solid-support, i.e., a solid matrix to which peptide-LDL immunogen is operatively affixed.
- a solid-support i.e., a solid matrix to which peptide-LDL immunogen is operatively affixed.
- each well is then admixed 50 ⁇ l of hybridoma tissue culture supernatant to form a solid-liquid phase immunoreaction admixture.
- the admixture is maintained for 2 hours at 37°C to permit formation of solid-phase immunoreaction products.
- 50 ⁇ l of 125 I-labeled goat anti-mouse IgG at 0.25 ⁇ g protein per ml are admixed to each well to form a labeling reaction admixture. That admixture is maintained for 1 hour at 37°C to permit formation of 125 I- labeled solid-phase immunoreaction products.
- Hybridomas are selected from hybridoma cultures that secrete anti-peptide antibodies into their culture media, and further characterized as described herein.
- AI-4 The hybridoma that bears the laboratory designation 611 AV63C2.111 and secretes the paratopic molecule designated AI-4 was obtained from a fusion of splenocytes from Balb/c mice (Scripps Clinic and Research Foundation Vivarium, La Jolla, Calif.) immunized with Apo/VLDL as discussed in Example 2. The standard fusion protocol was used as discussed in Example 6. The hybridomas so prepared were screened and assayed as discussed hereinbefore, with the one exception that Apo AI/HDL was used as the coated substrate instead of peptide-LDL immunogen. The hybridoma was deposited on March 5, 1985 with the American Type
- the hybridoma that bears the laboratory designation H103D8.1D11 and secretes the paratopic molecule designated AI-11 was obtained from a fusion of splenocytes from Balb/c mice (Scripps Clinic and Research Foundation Vivarium, La Jolla, Calif.) immunized with Apo AI/HDL as discussed in Example 2.
- the standard fusion protocol was used as discussed in Example 6.
- the hybridomas so prepared were screened and assayed as discussed hereinbefore, with the one exception that Apo AI/HDL was used as the coated substrate instead of peptide-LDL immunogen.
- the hybridoma was deposited on September 16, 1986 with the American Type Culture Collection, Rockville, Md. under the ATCC accession number HB 9201 in accordance with the Budapest Treaty on International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
- Ascites fluids were obtained from separate sets of 10- week old Balb/c mice, which had been primed with 0.3 ml of mineral oil and injected intraperitoneally with 5xl0 6 hybridoma cells with the designations 611 AV63C2.111, H103D8.1D11, and HB9570. The average time for development of ascites was 9 days. Following clarification by centrifugation at 15,000 x g for 15 minutes at 23°C, ascites fluids produced by hybridomas were pooled and stored frozen at -20 "C.
- Purified AI-4, AI-11, and AI-18 monoclonal antibodies from the hybridomas were prepared by fast protein liquid chromatography (FPLC) using a Pharmacia Mono Q HR5/5 anion exchange column (Pharmacia Fine Chemicals, Piscataway, NJ) using a 0-0.5 molar (M) NaCl gradient in 10 mM Tris, pH 8.0 following directions supplied with the column.
- Purified Mabs were concentrated using an Amicon stirred ultrafiltration cell (Danvers, MA; PM 30 membrane) to a concentration of 1 mg/ml, dialyzed into PBS (phosphate- buffered saline, pH 7.2) and stored at -70°C.
- Hybridomas secreting anti-peptide antibodies as described in Example 6A are injected into 10-week old
- Radioiodination of HDL, Apo Al and immunochemically purified goat anti-mouse Ig was performed enzymatically utilizing the Iodogen iodination procedure and Iodogen obtained from Pierce Biochemicals. The Iodogen iodination was utilized to characterize the antigens and antibodies for the solid phase radioimmunoassay as discussed below.
- the Apo Al CNBr fragment specificity of MAB AI-4, MAB AI-11 and MAB AI-18 was determined by Western blot analysis according to the method in Curtiss et al. , Proceeding of the Workshop on Lipoprotein Heterogeneity, Ed. by Lippel, NIH Publication No. 87-2646 p. 363-377 (1987). Briefly CNBr fragmentation was performed on isolated Apo Al dissolved in 90% formic acid. CNBr was added in a 13,000 molar excess and the reaction mixture was maintained about 15 hours at about 20 degrees C.
- Apo Al polypeptides AI84-111, AI85-111, AI90-111, AI93-111, AI94-111, AI-94-125, AI96-111, AI99-111, and AI99-114 were tested for immunoreactivity with monoclonal antibody AI-11 in a direct binding ELISA.
- 50 ⁇ g/ml of each polypeptide was dissolved in PBS to form a peptide coating solution, of which 150 ⁇ l was admixed into the wells of a flexible polyvinyl chloride microtiter plate (Immulon) . The wells were then maintained about 16 to 20 at 4°C to permit the peptide to absorb onto (coat) the walls of the wells.
- the plate was then dried for 1 hour at 37°C followed by addition of 100 ⁇ l of PBS containing 0.5 ⁇ g/ml horseradish peroxidase conjugated AI-11 antibody to form a solid-liquid phase immunoreaction admixture.
- the resulting solid-liquid phase immunoreaction admixture was maintained at 20°C for 1 hour to permit formation of a solid-phase polypeptide-containing immunoreaction product.
- the wells were then washed 3 times with rinsing buffer to remove unbound antibody.
- the amount of immunoreaction product present in the solid phase was then determined by admixing two hundred microliters of OPD substrate into each well to form a developing-reaction admixture. The admixture was maintained for 30 minutes at about 20°C. Subsequently, 50 ⁇ l of 4 N H 2 S0 4 were admixed into each well to stop the developing- reaction, and the resulting solution was assayed for absorbance at 450 nanometers using a microtiter plate reader (Dynatech) to detect the amount of formed immunoreaction product.
- Apo Al polypeptides AI85-111, AI94-125, and AI96-111 were found to be specifically immunoreactive with the monoclonal antibody AI-11 in the above direct binding ELISA.
- the Apo Al polypeptides AI90-111, AI93-111 and AI99-114 were also recognized by the antibody but with decreased specificity.
- the Apo Al polypeptide AI99-111 was not bound by the antibody.
- a competition ELISA was performed with AI96-111 as the test synthetic polypeptide in comparison to Apo Al-containing serum and purified Apo AI/HDL.
- Microtiter plates were coated with AI96-111 as described hereinbefore.
- 50 ⁇ l of a fluid sample (i.e., an Apo Al-containing fluid sample) or standard (i.e., an Apo Al polypeptide) to be assayed were admixed into the polypeptide AI96-lll-coated well simultaneously with 50 ⁇ l of HRPO-conjugated AI-11 antibody to form an immunoreaction admixture.
- results of this assay indicate that MAB AI-11 immunoreacts with AI96-111 present in the fluid sample but has a greater affinity for native Apo Al in serum and purified HDL.
- MAB AI-4 The immunoreactivity of MAB AI-4 for native Apo AI/HDL and various synthetic polypeptides was examined by a competitive RIA performed as follows:
- the conserved native epitope defined by MAB AI-4 includes the amino acid residues at positions 99-114 at a minimum, because peptide AI99-114 immunoreacted with MAB AI-4. However, peptide AI99-121 immunoreacted with MAB AI-4 nearly as well as Apo AI/HDL, indicating that the residues 99-121 define a preferred epitope on Apo Al.
- a particularly preferred polypeptide for use in diagnostic competition ELISA or RIA is peptide AI94-125.
- Peptide AI99-121 was prepared having an E in residue position 104 in place of the usual residue F and was shown to immunoreact with MAB AI-4.
- Apo Al peptides defined by MAB AI-4 that have either an E or an F residue at position 104 will immunoreact with MAB AI-4 and are useful in diagnostic methods when used in conjunction with MAB AI-4.
- Apo Al polypeptides immunoreactive with MAB AI-11 according to the above competition RIA are summarized in Figure 6.
- the conserved native epitope defined by MAB Al- 11 includes the amino acid residues at positions 96-111, because peptide AI96-111 immunoreacted with MAB AI-11.
- Proteoliposomes containing lecithin, 14-C- cholesterol and Apo Al were prepared in the following manner. 7.7 mg of phosphatidylcholine (egg yolk lecithin) were dried under nitrogen gas in a 13 x 100 glass test tube. 116 ⁇ g of cholesterol from a 1 mg/ml solution in ethanol and 78.3 ⁇ g of 14-C-cholesterol from a 0.29 mg/ml solution in benzene with a specific activity of 0.04 mCi/ml were dried in the same tube without coming in contact with the dried lecithin. To this tube was added 0.3 ml of a 725 mM solution of sodium cholate in Tris HC1 buffer (10 mM
- Tris 140 mM NaCl, 1 mM EDTA-tetrasodium salt, pH 7.2), 2.5 ml Tris HC1 buffer and 0.8 ml of a 1 mg/ml solution of purified Apo Al in Tris HC1 buffer.
- the admixture was vortexed for 60 seconds and then mixed on a rotating wheel at room temperature for 1 hour.
- the admixture was dialyzed overnight against 500 ml of Tris HC1 buffer which was changed 5 times.
- the proteoliposomes were adjusted to a volume of 4 ml after dialysis and stored in the refrigerator.
- the 4 mis of proteoliposomes contained 9.78 x 10 "6 mole lecithin, 3.0 x 10 "7 mole cholesterol, 2.01 x 10 " 7 mole 14-C-cholesterol and 3.14 x 10 "8 mole Apo Al. Molar ratios of lecithin:cholesterol:Apo Al were 250:12.5:0.8.
- the LCAT-mediated cholesterol esterification assay was performed in duplicate.
- To glass screw-cap tubes was added 100 ⁇ l of the prepared proteoliposome solution, 125 ⁇ l of a 2% solution of human serum albumin in Tris HC1 buffer, monoclonal antibodies in amounts from 15.6 ⁇ g to 500 ⁇ g/tube and Tris HC1 buffer to a final volume of 455 ⁇ l.
- the tubes were capped, vortexed and maintained for 30 minutes in a 37"C water bath.
- the admixture was vortexed for 20 seconds, and the upper layer was removed to a 13 x 100 glass tube.
- To the original reaction mixture was added 3 ml more of the hexane/cholesterol/cholesteryl linoleate solution.
- This admixture was vortexed, and the upper layer was removed and added to the first extraction.
- the contents of both extractions were dried under nitrogen gas or evaporated overnight.
- the dried contents of the tubes containing the extracted material was dissolved in 50 ⁇ l of chloroform and spotted onto Empore thin layer chromatography sheets (TLC) . The tube was rinsed with an additional 50 ⁇ l and added to the first spot.
- TLC Empore thin layer chromatography sheets
- the TLC sheets were developed in a solvent of hexane:ethyl ether in a 60:40 ratio. The sheets were air dried and exposed to iodine to visualize the origins. The sheets were then placed on Kodak X-OMAT autoradiography for an overnight exposure. Areas on the TLC sheets corresponding to radioactive signals on the film were cut and placed in scintillation vials with 3 ml of scintillant (PPD-PDPDP in toluene) . The 14-C radioactive label was detected in a beta counter. Fractional esterification rates (FER) were determined from the scintillation counts where FER is expressed as cholesteryl ester cpm over cholesterol plus cholesteryl ester cpm.
- FER Fractional esterification rates
- Purified MABs AI-4 and AI-11 were prepared as described in Example 6C.
- Purified MABs AI-9, AI-16 and AI-18 were generated from fusions of splenocytes from mice immunized with Apo AI/HDL similarly to the methods described in Example 6C.
- the MABs AI-9 and AI-16 were used as control MABs in the LCAT-mediated cholesterol esterification assay and are known to immunoreact with CNBr4 and the Apo Al amino acid residues sequence AI1-15, respectively.
- the MAB AI-18 immunoreacts with the Apo Al amino acid residue sequence AI95-105.
- Proteoliposomes that contained 48 ⁇ g/ml of Apo A-I and approximately 300,000 cpm/tube were incubated with a 4- fold molar excess of each antibody and 1% HSA for 30 minutes at 37°C. Mercaptoethanol and LPDP were added for a further 1 hour incubation at 37°C. The extent of antibody binding was assessed following precipitation by an additional incubation with 2 ml of Tachisorb M (Calbiochem, La Jolla, CA) for 60 min. at room temperature on a platform shaker. The tubes were then centrifuged at 1500 x g for 20 min. Suprenatants were aspirated and the pellets were washed with 1 ml of cold PBS and recentrifuged.
- Tachisorb M Calbiochem, La Jolla, CA
Abstract
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CA002084677A CA2084677C (en) | 1990-06-07 | 1991-06-07 | Apo ai polypeptides, antibodies, and immunoassays |
AU81064/91A AU662885B2 (en) | 1990-06-07 | 1991-06-07 | APO AI polypeptides, antibodies, and immunoassays |
JP3511924A JPH06508344A (en) | 1990-04-07 | 1991-06-07 | Apo AI polypeptides, antibodies and immunoassays |
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WO2003045993A2 (en) * | 2001-11-23 | 2003-06-05 | Syn.X Pharma, Inc. | Protein biopolymer markers predictive of type ii diabetes |
US8138313B2 (en) | 2007-06-15 | 2012-03-20 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Treatment of tumors using specific anti-L1 antibody |
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CN110494561A (en) * | 2017-03-30 | 2019-11-22 | 希森美康株式会社 | Anti- ApoA1 antibody |
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US5055396A (en) * | 1987-11-03 | 1991-10-08 | Scripps Clinic And Research Foundation | Diagnostic methods and systems for quantifying apo ai |
AU3413989A (en) * | 1988-03-29 | 1989-10-16 | Scripps Clinic And Research Foundation | Diagnostic methods and systems for quantifying apo ai |
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1991
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Non-Patent Citations (3)
Title |
---|
Journal of Biological Chemistry, Volume 263, No. 27, issued 25 September 1988, CURTISS et al., "Localization of two epitopes of apolipoprotein A-I that are exposed on human high density lipoproteins using monoclonal antibodies and synthetic peptides", pages 13779-13785, see entire document. * |
Journal of Lipid Research, Volume 31, issued 1990, MARCOVINA et al., "Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression", pages 375-384, see entire document. * |
See also references of EP0500826A4 * |
Cited By (5)
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WO2003045993A2 (en) * | 2001-11-23 | 2003-06-05 | Syn.X Pharma, Inc. | Protein biopolymer markers predictive of type ii diabetes |
WO2003045993A3 (en) * | 2001-11-23 | 2003-11-20 | Syn X Pharma Inc | Protein biopolymer markers predictive of type ii diabetes |
US7125678B2 (en) | 2001-11-23 | 2006-10-24 | Nanogen, Inc. | Protein biopolymer markers predictive of type II diabetes |
US8138313B2 (en) | 2007-06-15 | 2012-03-20 | Deutsches Krebsforschungszentrum Stiftung Des Offentlichen Rechts | Treatment of tumors using specific anti-L1 antibody |
US9260521B2 (en) | 2007-06-15 | 2016-02-16 | Medigene Ag | Treatment of tumors using specific anti-L1 antibody |
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PT97914B (en) | 1998-11-30 |
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