Methods for Purifying Selected CEA Family Member Proteins

Summary of Invention

[0042] This invention provides methods for purifying proteins that are selected CEA family members, including CEA, engineered CEA, CEA cross-reacting antigens, and proteins having at least about 86% amino-acid identity with CEA. Proteins are purified from many starting materials, by cation-exchange chromatography below pH 4.0 and size-exclusion chromatography. The purification methods of this invention do not utilize perchloric acid or antibody affinity steps. After purification, the proteins are of about 90% purity, substantially free of cross-reacting antigens, substantially free of CA19-9, substantially free of endotoxins and substantially free of antibodies. Preferably, after purification, the proteins are of about 90% purity, preferably at least about 95% purity, and more preferably the proteins are of at least about 98% purity, and more preferably the proteins are of about 100% purity. This invention provides the purified proteins of this invention in suitable pharmaceutical carriers, formulated for vaccines, and for use as reference standards.

Brief Description of Drawings

[0043]
FIG. 1 Reverse phase HPLC results showing purification of about 98% purity CEA from pooled cell culture harvests.

[0044]
FIG. 2 Reverse phase HPLC results showing purification of about 99% purity CEA from partially purified ascites fluid.

[0045]
FIG. 3 Reverse Phase HPLC results of TriChem Resources CEA product, 10690B, showing a purity of about 50%.

Detailed Description

[0046] As used herein, "substantially free of endotoxins" refers to less than about 1000 endotoxin units (EU) per mg protein.

[0047] As used herein, "substantially free of antibodies"refers to no detectable antibodies by methods known to the art.

[0048] As used herein, "endotoxin"refers to the lipopolysaccharide complex associated with the outer envelope of Gram-negative bacteria.

[0049] As used herein, "substantially free of cross-reacting antigens" refers to less than about 10% in a purified sample of the protein of interest, of molecules that are known to the art to cross-react immunologically with said protein of interest. Preferably there is less than about 5% and more preferably there is less than about 2%, and more preferably there is about 0%.

[0050] As used herein, "carcinoembryonic antigen"refers to the CEA protein, a 180kD glycosylated protein, encoded by the CEACAM5 gene, Genbank Accession Number M17303.

[0051] As used herein, "engineered" refers to a DNA molecule or the protein encoded by that DNA molecule, that has been cloned, or is recombinant. Some engineered DNA molecules and corresponding proteins are modified by techniques known to the art, such as by recombinant DNA techniques. Engineered molecules may contain mutations such as point mutations, missense mutations, insertions, deletions, and/or rearrangements.

[0052] As used herein, "epitope" refers to the smallest peptide recognized by an antibody or T cell. Epitopes can be synthesized chemically or translated in vivo or in vitro.

[0053] As use herein, "antigen"refers to a molecule that is recognized by an antibody or T cell.

[0054] As used herein, "cross-reacting antigens" refers to two or more antigens that can be recognized by a single antibody or T cell. Cross-reacting antigens therefore share similar epitopes.

[0055] As used herein, "non-specific cross-reacting antigen"refers to the NCA protein, a glycosylated protein, which is encoded by the CEACAM6 gene (Genbank accession numbers M29541and M18728).

[0056] As used herein, "carbohydrate antigen 19-9" refers to the epitope recognized by antibodies that recognize the Sialyl Lewis A protein.

[0057] As used herein, "AG128375"refers to the prostate-specific CEA family member cloned by AlphaGene in 2001 (http://www.alphagene.com/new_cea.htm). AG128375 refers to the gene and the corresponding protein.

[0058] As used herein, "nucleotide"refers to a single unit of deoxyribonucleic acid, DNA, or ribonucleic acid, RNA.

[0059] As used herein, "point mutation"refers to a single changed nucleotide in a sequence of DNA or RNA when compared to a reference.

[0060] As used herein, "missense mutation"refers to a point mutation that causes a change in the encoded amino acid.

[0061] As used herein, "insertion" refers to the addition of nucleotides to the interior or end of a gene causing the addition of amino acids to the encoded protein.

[0062] As used herein, "deletion" refers to the removal of nucleotides from the interior or end of a gene causing the absence of amino acids to the encoded protein.

[0063] As used herein, "immunogenicity"refers to the ability of a molecule to elicit an immune response.

[0064] As used herein, "pharmaceutical composition"refers to a composition formulated, as is known to the art, for pharmaceutical use.

[0065] As used herein, "vaccine"refers to a composition including adjuvants, that, when given to patients, may elicit an immune response.

[0066] As used herein, "ion-exchange chromatography" refers to the separation of molecules based on their charge. The ions in the mobile phase are separated by electrostatic interactions with the stationary phase.

[0067] As used herein, "cation"refers to a positively charged molecule.

[0068] As used herein, "anion"refers to a negatively charged molecule.

[0069] As used herein, "resin"refers to an insoluble solid used in chromatography.

[0070] As used herein, "anion-exchange chromatography"refers to a technique of separating selected anions from a solution using a positively charged resin.

[0071] As used herein, "cation-exchange chromatography" refers to a technique of separating selected cations from a solution using a negatively charged resin, without also using a positively charged resin.

[0072] As used herein, "mixed-bed ion-exchange chromatography" refers to a technique of separating selected anions and cations from a solution using a mixture of positively and negatively charged resins.

[0073] As used herein, "DEAE"refers to one or more diethylaminoethyl groups which when attached to a suitable resin, is a positively charged anion exchanger, useful at pH 2-9.

[0074] As used herein, "QAE"refers to one or more quaternary aminoethyl groups which when attached to a suitable resin, is a positively charged anion exchanger, useful at pH 2-12.

[0075] As used herein, "CM" refers to one or more carboxymethyl groups which when attached to a suitable resin, is a negatively charged cation exchanger, useful at pH 6-11.

[0076] As used herein, "size-exclusion chromatography" refers to a technique of separating molecules in a solution on the basis of molecular size. Gel filtration is a type of size-exclusion chromatography. A solid phase matrix consists of porous beads that are packed into a column with a mobile liquid phase flowing through the column. The mobile phase has access to both the volume inside the pores and the volume external to the beads. Larger molecules remain in the volume external to the beads, resulting in a shorter flow path, and in a more rapid exit from the column. Smaller molecules that can access the liquid within the pores of the beads are retained longer and, therefore pass more slowly through the column. The liquid leaving the column is collected in fractions. Type of resin used, size of column, and number of fractions collected are some of the variables determining which size of molecules are separated and the extent of separation from other molecules.

[0077] As used herein, the "pI"refers to the isoelectric point, the pH of equal electrical potential, of a protein.

[0078] As used herein, "solvent extraction" refers to a process of separating components of a mixture using a chemical that keeps at least the desired component in solution.

[0079] As used herein, "ethanol precipitation"refers to a method for precipitating a molecule(s) of interest by adding ethanol to a sample solution.

[0080] As used herein, "unstable"refers to a protein that is not stably dissolved in a solution and at least partially precipitates.

[0081] As used herein, "size filtration"refers to filtering a sample to exclude molecules of a certain size. Size filtration can be used to remove molecules, such as proteins, of a certain size from a sample solution, or to concentrate a sample solution by retaining a molecule and removing water. Size filtration can be used to isolate molecules of interest from a solution by first filtering out molecules smaller than the molecule of interest, and then filtering out molecules about the size of the molecule of interest, and collecting the eluent.

[0082] As used herein, "RIA"or "radio-immuno-assay" refers to immuno-affinity assay wherein the molecule(s) of interest or the antibodies are radio-labeled, and the quantitated radioactivity is utilized to quantitate the molecule(s) of interest.

[0083] As used herein, a "kit"refers to an assembled set of reagents and/or equipment, preferably packaged together.

[0084] As used herein, "purify"or "isolate" refers to separating a type of molecule from other molecules in a sample, thereby concentrating that type of molecule.

[0085] As used herein, "pure" refers to a type of molecule separated away from other types of molecules. A percentage is used to indicate the extent of purity.

[0086] As used herein, "HPLC"refers to high-pressure or high-performance liquid chromatography.

[0087] As used herein, "substantially free of cross-reacting antigens" refers to a composition of a protein of interest, said composition also comprising less than 10% of any other molecules that can be recognized by a single antibody or T cell that also recognizes the protein of interest. Preferably "substantially free of cross-reacting antigens"refers to less than 5%, more preferably less than 2%, and more preferably about 0%.

[0088] As used herein, "substantially free of NCA" refers to when the slope of the curve of the purified sample is less then two times the slope of the curve of the negative control on an NCA ELISA test, as described below.

[0089] As used herein, "substantially free of CA19-9"refers to less than 2%.

[0090] As used herein, "protein"refers to a glycosylated polypeptide.

[0091] As used herein, "peptide"refers to glycosylated, covalently-linked amino-acids, which may be a portion of a protein.

[0092] As used herein, "native"refers to a protein or DNA molecule that is identical to the corresponding protein or DNA molecule found in nature.

[0093] As used herein, "solution"refers to an aqueous solution.

[0094] As used herein, "suitable pharmaceutical carrier"refers to any formulation known in the art for delivering pharmaceuticals.

[0095] Suitable pharmaceutical carriers include carriers for vaccines. Immunogenic compositions and/or vaccines may be formulated by any of the means known in the art. They can be typically prepared as injectables or as formulations for intranasal administration, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection or other administration may also be prepared. The preparation may also, for example, be emulsified, or the protein(s)/peptide(s) encapsulated in liposomes. Where mucosal immunity is desired, the immunogenic compositions advantageously contain an adjuvant such as the nontoxic cholera toxin B subunit (see, e.g., United States Patent No. 5,462,734). Cholera toxin B subunit is commercially available, for example, from the Sigma Chemical Company, St. Louis, MO. Other suitable adjuvants are commercially available and may be substituted therefore. It is preferred that an adjuvant for an aerosol immunogenic (or vaccine) formulation be able to bind to epithelial cells and stimulate mucosal immunity.

[0096] Among the adjuvants suitable for mucosal administration and for stimulating mucosal immunity are organometallopolymers including linear, branched or cross-linked silicones, which are bonded at the ends or along the length of the polymers to the particle or its core. Such polysiloxanes can vary in molecular weight from about 400 up to about 1,000,000 daltons; the preferred length range is from about 700 to about 60,000 daltons. Suitable functionalized silicones include (trialkoxysilyl) alkyl-terminated polydialkylsiloxanes and trialkoxysilyl-terminated polydialkylsiloxanes, for example, 3-(triethyoxysilyl) propyl-terminated polydimethylsiloxane. See United States Patent No. 5,571,531, incorporated by reference herein. Phosphazene polyelectrolytes can also be incorporated into immunogenic compositions for transmucosal administration (intranasal, vaginal, rectal, respiratory system by aerosol administration) (See e.g., United States Patent No. 5,562,909).

[0097] The active immunogenic ingredients are often mixed with excipients or carriers, which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. The concentration of the immunogenic polypeptide in injectable, aerosol or nasal formulations is usually in the range of 0.2 to 5 mg/ml. Similar dosages can be administered to other mucosal surfaces.

[0098] In addition, if desired, the vaccines may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants, which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP); N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE); and RIBI, which contains three components extracted from bacteria: monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies (especially IgG, IgM or IgA) directed against the immunogen resulting from administration of the immunogen in vaccines, which comprise the adjuvant in question. Such additional formulations and modes of administration as are known in the art may also be used.

[0099] All references cited herein are included in their entirety to the extent that they are not inconsistent with the disclosure herein.

[0100] This invention provides methods for purifying a protein, which is a selected CEA family member, including CEA, proteins having at least 86% amino-acid identity with CEA, and CEA cross-reacting antigens. Proteins are purified from many starting materials, by cation exchange chromatography below pH 4.0 and size exclusion chromatography. The purification methods of this invention do not utilize PCA or antibody affinity steps. After purification, the proteins are of at least 90% purity, substantially free of cross-reacting antigens, substantially free of CA19-9, substantially free of endotoxins, and substantially free of antibodies. Preferably after purification, the proteins are of at least 95% purity, more preferably the proteins are of at least 98% purity, and even more preferably the proteins are of at about 100% purity. This invention also provides methods for using such purified CEA family member proteins as reference standards or as pharmaceuticals, such as vaccines. This invention also provides methods for purifying engineered CEA family member proteins, such as CEA mutagenized to contain an agonistic immunogenic epitope.

[0101] It has not previously been recognized that CEA and CEA family members are stable at such low pH, that contaminants, such as protein aggregates, are unstable at such low pH, and that the method of combining the techniques used in this invention is so effective at purifying these proteins.

[0102] The methods of this invention yield CEA that is at least 90% pure and substantially free of NCA, CA19-9, endotoxins, and other cross-reacting antigens. Preferably, after purification, CEA is at least 95% pure, more preferably CEA is at least 98% pure, and more preferably CEA is about 100% pure.

[0103] The methods of this invention yield NCA that is at least 90% pure and substantially free of CEA, CA19-9, endotoxins, and other cross-reacting antigens. Preferably, after purification, NCA is at least 95% pure, more preferably NCA is at least 98% pure, and more preferably NCA is about 100% pure.

[0104] The methods of this invention yield AG128375 that is at least 90% pure and substantially free of CEA, NCA, CA19-9, endotoxins, and other cross-reacting antigens. Preferably, after purification, AG128375 is at least 95% pure, more preferably AG128375 is at least 98% pure, and more preferably AG128375 is about 100% pure.

[0105] The methods of this invention yield CAP1-6D-CEA that is at least 90% pure and substantially free of CEA, NCA, CA19-9, endotoxins, and other cross-reacting antigens. Preferably, after purification, CAP1-6D-CEA is at least 95% pure, more preferably CAP1-6D-CEA is at least 98% pure, and more preferably CAP1-6D-CEA is about 100% pure.

[0106] In one embodiment, the method of purifying a selected CEA family member to be at least 90% pure, preferably at least 95% pure, more preferably at least 98% pure, more preferably about 100% pure, and substantially free of cross-reacting antigens, CA19-9, and endotoxins comprises:·providing a starting material solution comprising a CEA family member protein,·performing cation-exchange chromatography (CEC) at below pH 4·performing size-exclusion chromatography (SEC) The chromatography steps can be performed in either order, but preferably the CEC is performed first on the provided starting material solution, and SEC is performed second using the eluent from the CEC. When the SEC is performed first, there is an equally pure but lower yield of the protein of interest.

[0107] Starting material solutions comprising CEA family member proteins can be obtained from many sources. Starting materials include: cells, tissue, fluids, and solids. Such starting materials are from cell culture or organisms, human or non-human. These cells or organisms contain a native and/or engineered CEA family member protein(s). Fluids are aqueous solutions and include cell culture aspirate and bodily fluids, such as ascites fluid. Solids include non-tissue solids such as feces.

[0108] ATCC cell lines containing CEA include: CCL-218, CCL-221, CCL-222, CCL-225, CCL-227 through CCL-231, CCL-233 through CCL-235, CCL-237, CCL-238, CCL-244, CCL-247, CCL-248, CCL-249, CCL-250, CCL-250.1, CCL-252 through CCL-255, CL-187, CL-188, CRL-1682, CRL-1687, CRL-1803, CRL-1837, CRL-1864, CRL-1918, CRL-2119, CRL-2134, CRL-2158, CRL-2159, CRL-5822, CRL-5971, CRL-5973, and CRL-5974. Two cell lines that are particularly useful in the practice of this invention are ATCC #CCL-253, designated NCI-H508, and ATCC-248, designated T84.

[0109] Starting materials can be centrifuged, such as at about 4900g for about 30 minutes; the supernatant can comprise the starting material solution. Without wishing to be bound by any theory, the Applicants believe the centrifugation prevents the aggregation of mucins. Starting material cells can be optionally solubilized before centrifugation. Tissues and fluids are optionally homogenized and/or solubilized before centrifugation. Solubilization and homogenization are performed by methods known to the art including but not limited to using detergents, sonicators, and physical homogenizers. Starting material solutions also include samples of CEA family member proteins of less than 90% purity or not substantially free of cross-reacting antigens, CA19-9, or endotoxins. Such samples may have been partially purified by methods using PCA or antibody affinity steps. When the protein of interest is CEA, such a starting material solution may be 10695B from TriChem Resources (West Chester, Pennsylvania), a partially purified CEA product. Starting materials are chosen such that the above methods are successful at purifying the protein of interest to at least 90%, preferably 95%, more preferably 98%, and more preferably about 100%, and substantially free of cross-reacting antigens, CA19-9, and endotoxins.

[0110] Cation-exchange chromatography used in the methods of this invention excludes the use of anion-exchange chromatography resins such as in mixed-bed ion-exchange chromatography. Cation-exchange chromatography comprises:·providing the starting material solution·quantitating total protein in the starting material if not already known and if necessary, concentrating the starting material·if necessary, adjusting the starting material solution to comprise CEC start buffer·applying the starting material solution comprising CEC start buffer to a chromatography column packed with cation-exchange resin, preferably at least about 1ml resin per 23mg total protein in the adjusted starting material solution, preferably at a rate less than about 20cm/hr·washing the column with CEC start buffer until the unbound contaminating materials are washed out and the protein of interest remains bound, preferably at a rate less than about 40cm/hr·washing the column, preferably with CEC elution buffer, until the protein of interest is released, and collecting the protein of interest.

[0111] The starting material solution may be concentrated and quantitated by any method known to the art. The preferred CEC start buffer contains from 0M NaCl to about 200mM NaCl and about 10mM H3PO4, at below pH 4.0. Use of a pH below 4.0 decreases aggregation of some proteins. The CEC start buffer preferably contains about 0.2M NaCl when the protein of interest is CEA. The pH of the CEC start buffer varies from about pH 3.0 to below pH 4.0, and is preferably about pH 3.0 when the protein of interest is CEA. The starting material solution may be adjusted by concentrating it, dialyzing it, and adding necessary chemicals. Any cation-exchange resin known to the art to be effective between pH 2 and pH 4 may be used, such as Fractogel EM SO3 (EMD SO3-650 M, product No. 16882-1, CIX from EM Science, Gibbstown, New Jersey). Before applying the adjusted starting material solution, the column is preferably washed with a buffer similar to the CEC start buffer, such as with 10mM H3PO4 and 0.2M NaCl at about the pH of the CEC start buffer. The adjusted starting material solution may be applied to the chromatography column at any rate effective to provide the required purity, preferably a rate of about 20cm/hr. The column is then washed with enough start buffer to remove substantially all the unbound proteins. The column is typically washed out with about 4 volumes of start buffer at no more than 40cm/hr. The composition of the elution buffer is chosen such that the protein of interest is eluted with sufficiently few contaminants so that if followed by SEC, the protein of interest is at least 90% pure, preferably 95% pure and more preferably 98% pure. The CEC elution buffer preferably comprises 10mM H3PO4 and from about 0.3M NaCl to about 1M NaCl and is at pH below 4.0. Preferably the NaCl concentration is about 1M, when the protein of interest is CEA. Preferably the pH is about 3.0. The column is typically washed with 1-2 volumes CEC elution buffer. The cation-exchange chromatography step achieves over 100-fold purification when using very low purity starting material solutions. The buffers are chosen as appropriate to obtain the required purity.

[0112] Size-exclusion chromatography comprises:·concentrating the eluent from the CEC step as necessary·if necessary, adjusting the concentrated eluent to also comprise SEC buffer·applying the adjusted eluent to a prepared chromatography column packed with an appropriate size-exclusion resin ·washing the column with SEC buffer until the protein of interest is eluted·collecting the fraction(s) containing the protein of interest substantially free of contaminantsThe eluent from the CEC step may be concentrated by any method known to the art to between about 10mg/ml and about 25 mg/ml. SEC buffer is any suitable buffer known to the art. The SEC buffer typically contains about 0.1M NaCl and about 10mM Na2HPO4 at about pH 8.0. When the protein of interest is CEA, the column is preferably about 2.6cm (diameter) by about 60cm (length) and the resin is preferably Pharmacia Superdex 200. The column and any other glassware are typically first washed with 0.2N NaOH, a depyrogenation step that is standard to the art for preventing endotoxin contamination. The column is then typically washed with about 5 column volumes of SEC buffer at about 30 cm/hr. The size of the column, the resin used, and the number of fractions collected modified as necessary to obtain the required purity. When a possible contaminant is of a similar size to the protein of interest, a larger column and a better-distinguishing resin are used, and more and smaller fractions are collected, as is standard in the art. The known size (kd) of the protein of interest is used to determine which fraction(s) to collect. When the protein of interest is CEA, the fractions starting at 0.25 column volumes are collected. This peak is at least 90% pure, preferably 95% pure, more preferably 98% pure, more preferably about 100% pure, and substantially free of cross-reacting antigens and endotoxins. The fraction may be concentrated by any method known to the art and is preferably lyophilized. The pellet thus produced may be resuspended in an appropriate buffer to comprise the purified sample.

[0113] These methods are effective when the starting material contains CEA family member proteins that can be obtained at the required purity by cation-exchange chromatography below pH 4.0 and size-exclusion chromatography. For the size-chromatography step, the resin, the size of the column, and the number and size of the fractions collected are chosen to obtain the required purity of the protein of interest from the selected starting material. Alternatively, a different starting material is chosen. Using larger columns, separating the eluent into more fractions, and other methods known to the art are methods that can be used to better separate similarly-sized proteins. For example, when NCA is purified by the methods of this invention, the equivalent of Sephadex G200, G150 or G100 resin is used, and the fraction containing molecules the size of NCA is collected during size-exclusion chromatography.

[0114] The percent purity of the purified sample is determined by a quantitative assay method known to the art. Typically this involves quantitating the protein of interest in the sample and quantitating the total protein in the sample.

[0115] (amount of protein of interest / amount of total protein) * 100 = percent purity of protein of interestPercent purity is typically determined by Reverse Phase HPLC. Reverse Phase HPLC is performed on equipment such as the Waters 2690 Alliance (Milford, MA) using a column such as the Zorbax® Reliance Column 300SB-C18 (Hewlett Packard, Palo Alto, CA) a 3.5 micron, 4.6mm x 15cm column. To a sample to be tested, trifluoroacetic acid (TFA) is added to the sample until the sample contains 0.1% TFA. The TFA-added sample is injected onto the column. Sample Buffer, SB, containing deionized water and 0.1% TFA, is injected and the column is washed for 5 minutes. A linear gradient of SB to Elution Buffer, EB, containing 100% Acetonitrile and 0.1% TFA, is run over 60 minutes. 100% EB is held for another 5 minutes. The flow rate is 0.5ml/min. The components of the sample are detected at a 211nm wavelength, using a Waters PDA detector (Milford, MA). The output is recorded in graphical format, the curve is integrated, and the percent purity of the protein of interest is calculated.

[0116] (area under the peak for the protein of interest / area under the entire spectrum) * 100 = percent purity of protein of interestThe possible contaminants, including cross-reacting antigens, are assayed by any means known to the art. Cross-reacting antigen levels are less than 10% total and preferably less than about 2% each. Preferably cross-reacting antigen levels are less than about 5% total, and more preferably they are less than about 2% total. Endotoxin levels are less than 1000EU but preferably less than about 100EU.

[0117] The percent purity of the protein of interest is usually determined immediately following purification.

[0118] Both CEA and NCA are collected together from the cation-exchange chromatography column. All members of the CEA gene family when matured, including glycosylated, have similar charge properties and behave similarly during cation-exchange chromatography.

[0119] The amount of CA19-9 in a sample can be quantitated with the CA19-9 Centocor® CA 19-9TM RIA system which is a solid phase radioimmunoassay. Polystyrene beads coated with 1116-NS-19-9 antibody are incubated with the specimen, standards, and controls. During this incubation, reactive determinants present in the specimen or controls are bound to the antibody on the solid phase. Unbound materials present in the specimen are removed by aspiration of the fluid and washing of the beads. The tracer, composed of 125I labeled 1116-NS-19-9 antibody, quantifies the number of reactive determinants. Unbound labeled antibody is removed by aspiration of the fluid and washing of the beads. The bound radioactivity is proportional to the concentration of the 1116-NS-19-9 reactive determinants in the specimen within the working range of the assay. A standard curve is obtained by plotting the CA 19-9 assay results of the standards vs. bound radioactivity. The CA 19-9 assay results of unknowns and controls, run concurrently with the standards, can be determined from the standard curve.

[0120] The amount of NCA in a sample can be quantitated using an ELISA format comparing the unknown amount of NCA to the known amount of CEA. A 50μl of a final concentration of 2μg CEA/ml of the CEA test sample was loaded onto a 96-well ELISA plate and allowed to bind at 37oC overnight. A monoclonal antibody against NCA, preferably B6.2 from NIH, was serially diluted in a range from 0.16 μg/ml to 10 μg/ml and a monoclonal antibody against CEA, preferably COL-1 from NIH, is serially diluted in a range from 0.16 μg/ml to 10 μg/ml. 50 μl of prepared B6.2 dilutions were added to half of the plate that was bound with CEA antigen, logging which dilution is added to each well. 50 μl of COL-1 dilutions were added to the second half of the plate, again logging which dilution is added to each well. The mixture was allowed to incubate at 37oC for one hour. The plate was washed with a solution of 1% bovine serum albumin phosphate buffered saline. An appropriate dilution of Horseradish Peroxidase-labeled anti-mouse secondary antibody was prepared. 50 μl was added to the plate and allowed to incubate for one hour at 37oC. The plates were once again washed, and 100 μl of the colorimetric detection reagents A and B were added to each well, and the plate was allowed to incubate for 5-15 minutes at room temperature. A 1N H2SO4 stop reagent was added to each well, and the absorbance was read at 450nm. The NCA result is determined by calculating the slope of the curve of the plotted absorbances at 450nm. A sample is substantially free of NCA when the slope of the line formed by plotting the antibody concentration versus the absorbance, is less than double the slope of the negative control line.

[0121] The amount of endotoxins in a sample can be quantitated with the Quantitative Chromogenic Limulus Amebocyte Lysate (LAL), QCL-1000®, test kit, catalog number 50-648U, license number 709, from BioWhittaker, a CAMBREX company. This assay utilizes a modified LAL and a synthetic color producing substrate to detect endotoxin chromogenically. The Chromogenic LAL test is a quantitative test for gram-negative bacterial endotoxin. A sample is mixed with the LAL supplied in the test kit and incubated at 37oC for six minutes. The reaction is stopped with stop reagent. If endotoxin is present in the sample a yellow color will develop. The absorbance of the sample is determined spectrophotometrically at 405-410 nm. Since this absorbance is in direct proportion to the amount of endotoxin present, the concentration of endotoxin is calculated from a standard curve. If the concentration of endotoxin in the test sample is greater than 1 EU/ml, the sample is diluted 5-fold and retested.

[0122] The amount of CEA in a sample can be quantitated by assaying with an analyzer such as the ACS 180 Analyzer from Bayer (ACS:180 SE, Tarrytown, New York) or the IMX or analyzer from Abbott (AXSYM, Abbott Park, Illinois) that quantitates the amount of CEA in a sample. The Bayer analyzer works in a cuvette by binding the CEA in the sample with rabbit polyclonal anti-CEA antibodies that are labeled with a chemiluminescent reaction component and a mouse monoclonal antibody coupled to magnetic particles. The cuvette is washed while the bound CEA is magnetically retained. Additional reagents are added to initiate the chemiluminescent reaction, which is measured. The amount of chemiluminescence is proportional to the amount of CEA in the sample. The Abbott IMX analyzer assay is based on Microparticle Enzyme Immunoassay technology. The sample is mixed with microparticles coated with mouse monoclonal anti-CEA, and an aliquot is placed on a glass fiber matrix. The microparticles bind to the glass fiber matrix, along with the bound CEA. The matrix is washed. Mouse monoclonal anti-CEA that is bound to alkaline phosphatase is added, it binds to the CEA, and the excess is washed off. The substrate, 4-methylumbelliferyl phosphate, is added and the product of the reaction is quantitated by an optical assembly. The measurement is proportional to the amount of CEA in the original sample. .

[0123] The methods of this invention are useful for purifying CEA proteins, including, native and engineered CEA family members. An example of an engineered CEA is the CEA minigene containing only the N-A3 domains (You, Y.H. (1998) Anticancer Research 18:3193-3202). The methods of this invention are useful for purifying the N-A3 CEA minigene.

[0124] The compositions of this invention are useful for research and for the diagnosis, prognosis, and treatment of diseases. The compositions of this invention are useful as individual reagents or as components in kits, such as those supplied with 180 analyzers. The compositions of this invention are useful as antigens for making polyclonal and monoclonal antibodies. The compositions of this invention are useful as standards or references such as for testing physical properties of other compositions (WO 95/32286, published November 30, 1995) or as a marker in assays (WO 8402983, published August 2, 1984; US Patent No. 4,299,815, issued November 10, 1981). In diagnosis and prognosis, compositions of this invention can be used as reference standards, against which a patient"s level of the corresponding protein(s) is compared. The compositions of this invention can be used as control vaccines, reference standards against which potentially more effective vaccines are compared (Kass, E. et al. (1999) Cancer Research 59:676-683). Using a composition of this invention as a vaccine reference standard to test a sample vaccine comprises: providing a first sample vaccine, providing a purified composition of this invention formulated as a second vaccine, vaccinating a first subject with said first sample vaccine, vaccinating a second subject with said second reference standard vaccine, and comparing the level of immune response provided by said first sample vaccine and to the level of immune response provided by said second vaccine. A composition of this invention is also useful as a vaccine reference standard when the sample vaccine comprises a composition of this invention.

[0125] Currently the N.I.H. is conducting a vaccine trial of the CEA-derived agonist peptide CAP1-6D. As a control against which to compare its effectiveness, a purified CEA composition of this invention, purified by the methods of this invention, is also being used to vaccinate patients. This invention also provides a vaccine that is even more effective than the CAP1-6D peptide vaccine comprising the CAP1-6D epitope in an engineered CEA protein, made by methods standard to the art comprising: engineering a DNA sequence encoding the CAP1-6D peptide (at amino acid 576, N→D) into a full-length CEACAM5 gene, expressing this engineered CAP1-6D-CEACAM5 gene in cells (that don"t contain native CEA protein) that provide appropriate maturation including glycosylation, and purifying corresponding CAP1-6D-CEA by the methods of this invention. A vaccine comprising CAP1-6D-CEA may comprise all appropriate adjuvants, as described above, and maybe given in an appropriate prime/boost cycle, as is known to the art. This invention provides a CAP1-6D-CEA vaccine, comprising a composition of this invention, purified by the methods of this invention.

[0126] This invention also uses the CAP1-6D-CEA composition of this invention as a control, or reference standard, for comparing the effectiveness of other vaccines. This invention also provides examples of other vaccines comprising, for example: CEA containing other agonistically engineered epitopes. Such CEA compositions may contain: other agonistic modifications of CAP1; agonistic modifications of other epitopes, such as CAP2 - CAP6 recognized by HLA-A2, CAP7 recognized by HLA-A3, LCD-2 and CEA-B recognized by HLA-A24, epitopes recognized by HLA-A*0301, HLA-A*0201, and other epitopes recognized by the immune system; agonistic modifications at more than one of these epitopes in each CEA molecule; one or more of the above-mentioned agonistic epitopes inserted into or fused onto the CEA molecule, possibly within the context of an additional domain; or proteins comprising deletions in CEA, such as deletions of particular domains, with one of the remaining domains containing one or more of the above-mentioned agonistic epitopes; of which all vaccine comprise compositions of this invention, purified by the methods of this invention.

[0127] This invention also provides other CEA family members that are engineered to contain agonistic epitopes, the compositions of this invention comprising such engineered CEA family members, the purification of such compositions by the methods of this invention, and the use of such compositions as vaccines. This invention also provides the use of such compositions comprising native CEA family members as controls or reference standards against which to compare the effectiveness of such vaccines.

[0128] This invention provides vaccines, comprising one or more compositions of this invention. A vaccine comprising two engineered CEA compositions of this invention, each modified at a different immunogenic epitope, such as CAP1-6D-CEA and a CEA agonistically-engineered at an HLA-A24 epitope, both purified by the methods of this inventions, is an example of such a vaccine. This invention also contemplates more than one vaccine, each containing at least one composition of this invention, utilized sequentially in varying prime and boost strategies. This invention also provides purifying CEA family members engineered to contain antagonistic epitopes.

[0129] This invention provides compositions comprising purified CEA family members, native and engineered, from other species, such as those found in primates, guinea pigs, rats, and mice. The methods of this invention are useful for purifying such CEA family member proteins.

[0130] The methods of this invention are useful for purifying proteins of the CEA family that are present in the starting material solutions only containing other CEA family member proteins that are separable by size-exclusion chromatography. For example, the methods of this invention are not useful for purifying CAP1-6D-CEA from a cell line that expresses native CEA, for these products are essentially identical in size and not practically separable by size-exclusion chromatography. The methods of this invention are useful if it is desirable to purify both proteins together, such that the composition comprising both proteins is at least about 90% pure, preferably at least about 95% pure and more preferably at least about 98% pure. The methods of this invention are useful for purifying CAP1-6D-CEA from a cell line that does not express native CEA, including but not limited to such starting materials as a CAP1-6D-CEA transformed mouse cell line. Similarly, the methods of this invention are useful for purifying AG128375 and engineered AF128375 containing agonistic immunogenic epitopes.

[0131] Purified compositions of at least about 90% purity, preferably of at least about 95% purity and more preferably at least about 98% purity, of proteins having at least about 86% amino acid identity with CEA include, but are not limited to, NCA. Both CEA and NCA are cleaved of an N-terminal signal peptide of about 34-amino-acids and a C-terminal peptide, that possibly directs membrane attachment, of about 26 amino acids. Before these peptides are cleaved, CEA and NCA are about 84% identical. Methods for determining amino acid sequence identity are known to the art. CAP1-6D-CEA is 99.8% identical to CEA. The N-A3 minigene is 100% identical.

[0132] All references cited in the present application are incorporated in their entirety herein by reference to the extent not inconsistent herewith.

[0133] It will be appreciated by those of ordinary skill in the art that starting materials, CEA family member proteins, and purification methods other than those specifically disclosed herein are available in the art and can be employed in the practice of this invention. All art-known functional equivalents of starting materials, CEA family member proteins, and purification methods are intended to be encompassed in this invention.

[0134] The following examples are provided for illustrative purposes only, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles which occur to the skilled artisan are intended to fall within the scope of the present invention.

[0135] Example 1 CEA Purification from Human Liver Tissue 130.6g of lyophilized human liver tissue, including tumor tissue, were extracted for CEA at a neutral pH and the sample was brought to a concentration of 0.1M sodium chloride and adjusted to pH 3.0. The CEA containing sample was then applied to Fractogel EM SO3 cation exchange chromatography media equilibrated to pH 3.0 with 10mM glycine and 0.1M sodium chloride, pH 3.0. The total protein of the sample was 29820mg and the column volume was 1250ml giving a load of 23.9mg total protein per milliliter of packing material. The column was washed with the equilibration buffer to wash out all of the non-absorbed protein. The CEA was then eluted with a linear gradient to buffer B: 10mM glycine, 0.5M sodium chloride, pH 3.0. The CEA eluted at about 0.25M sodium chloride, and the CEA content was determined using Bayer"s ACS-180 analyzer.

[0136] The CEA-containing fraction was concentrated to 10-15mg/ml total protein in a YM-30 stirred cell concentrator. The 8.0ml sample was injected onto a Pharmacia Superdex 200 size-exclusion chromatography column with a diameter of 2.6cm and a length of 60cm. The CEA was eluted in 20mM tris, 0.75M sodium chloride, pH 7.5. A single band appeared on SDS-PAGE.

[0137] Example 2CEA Purification from Pooled Cell Culture Harvests 1Two CEA-containing human tumor cell lines, ATCC #CCL-253 and ATCC #CCL-248, were grown for four weeks. Spent media was then collected daily.

[0138] Cell culture spent media containing CEA from both cell lines was harvested, pooled, centrifuged, concentrated in a 30 kDa filter and dialyzed into a low conductivity neutral pH buffer. The sample was then injected onto the cation-exchange chromatography column and eluted as described above at a protein load of 20.8mg/ml. The CEA was eluted at about 0.25M sodium chloride to a purity of approximately 2.5%.

[0139] The CEA-containing fractions were pooled and the sample was concentrated to 30mg/ml total protein. As described above, the sample was injected onto a size-exclusion chromatography column and eluted at a purity of about 98% percent, as determined by reverse phase HPLC, as shown on Figure 1. A single band appeared on SDS-PAGE.

[0140] Example 3CEA Purification from TriChem Resources Partially Purified CEAPartially purified CEA, product # 10695B (lot # 01H0801FP) was received from TriChem Resources. The sample was centrifuged, concentrated using a 30kDa hollow fiber cartridge, and injected onto the Superdex 200 SEC column. The fractions coming off the column were pooled, labeled A-G and assayed. The purity of the CEA did not meet requirements, therefore the B fraction was concentrated a second time and reprocessed by injecting it onto the SEC column again, to achieve a higher purity CEA. The sample still did not meet percent purity standards. All of the CEA that was eluted from the two SEC runs was pooled and injected onto the a CIX (Fractogel EM SO3 EMD SO3-650 M, product No. 16882-1, CIX from EM Science, Gibbstown, New Jersey) containing CEC column. Fractions 8 and 9 were pooled to obtain 100% of the CEA injected onto the column. The purity was determined to be 100% by reverse phase HPLC. Contaminants were measured to be : CA19-9 = less then 0.31% and less than 0.2 mg/mg total protein using Centocor® CA 19-9TM RIA, NCA = negative using enzyme immunoassay, and endotoxin = 468 EU/mg total protein using BioWhittaker LAL QCL-1000 (Cambrex, East Rutherford, New Jersey). The sample was finally processed by concentrating the sample in a 30kDa filter and diafiltering into PBS. A final pre-bottle assay was performed and the contaminants measured. An SDS-PAGE gel showed the CEA band present near the top of the gel without any contaminating bands.

[0141] Example 4Purification of CEA from Liver Tissue 2Approximately 5.0 g of lyophilized human liver tissue, including tumor tissue, was extracted with a PBS buffer at pH 7.5 to obtain an 80 ml starting material solution containing 5292.8 mg total protein with only 0.2 mg of CEA. 25 ml of the sample was injected onto a column containing 90ml Fractogel EM sulfate cation exchange chromatography media equilibrated in start buffer: 10mM H3PO4, 200mM NaCl at pH 3.0. The total protein load on the column was 18.3 mg/ml with a CEA load of 0.063mg total. The unbound proteins were washed with start buffer for 4 column volumes and the CEA was eluted in a gradient to elution buffer: 1.0M NaCl, 10mM H3PO4 PH 3.0 over 3-column-volumes. The small amount of CEA was then concentrated and injected onto a column containing Pharmacia Superdex 200. A purified peak of CEA was eluted, as observed by HPLC.

[0142] Example 5Purification of CEA from Cell Culture MediaAn ATCC #CCL-253, NCI H508, cell culture was harvested and the one-day harvest centrifuged, concentrated, and diafiltered into a low conductivity neutral pH buffer. The sample was adjusted to pH 3.0 and injected onto a column containing 90ml Fractogel EM sulfate cation exchange chromatography media equilibrated in start buffer: 10mM H3PO4, 200mM NaCl at pH 3.0. The total protein load was 1.7mg/ml and the CEA load was 1.35mg total. The non-absorbed proteins were washed with 4 column volumes of start buffer and CEA was eluted in a 3-column-volume gradient to elution buffer. The CEA eluted at approximately 25% of the gradient at a purity of 66%. The CEA fraction was concentrated to 2.2ml and 0.816mg total protein, and injected onto a 1.6cm/60cm column containing 120ml of Superdex 200 resin. The CEA was eluted at 30cm/hr in eluent: 10mM sodium phosphate and 0.1M sodium chloride pH 8.0. The 1.1mg of CEA was determined to be greater than 98% pure by reverse phase HPLC and SDS PAGE.

[0143] Example 6Purification of CEA from Partially Purified Ascites FluidPartially purified CEA was obtained from Tri-Chem Resources (West Chester, PA, product code 10695B, lot #05J2601FP). The fluid was adjusted to pH 3.0 and injected onto the column containing 90ml Fractogel EM sulfate cation exchange chromatography media equilibrated in start buffer: 10mM H3PO4, 200mM NaCl at pH 3.0. The non-absorbed protein was washed off the column with 4 volumes of start buffer, and the CEA was eluted with a 3-column-volume gradient to elution buffer. The CEA was eluted at 37% purity. The CEA containing fractions were concentrated to 11.6 mg/ml total protein and injected onto a 2.6cm/100cm column containing Superdex 200 resin equilibrated in eluent. The CEA-containing fractions were concentrated and injected onto the SEC column a second time as a polishing step and to determine purity. As shown on Figure 2, the sample was eluted and determined to be of about 99% purity by SDS-PAGE and reverse phase HPLC.

[0144] Example 7Purification of CEA from Pooled Cell Culture Harvests 2Two cell culture harvests were pooled, centrifuged, concentrated, and diafiltered in low conductivity, neutral pH buffer. The sample contained 9571 mg total protein and 17 mg CEA. The sample was brought to 0.2M NaCl, adjusted to pH 3.0, and injected onto a column containing 600ml Fractogel EM sulfate resin. The total protein load was 15.95 mg/ml. The non-absorbed proteins were washed with 4 volumes of start buffer, and the CEA was eluted in a 3-column-volume gradient to 100% elution buffer. The CEA eluted at a purity of approximately 10%. The CEA-containing fractions were concentrated to 16 ml, and injected onto a Superdex 200 column and eluted with eluent. The sample was collected, pooled, and injected onto the SEC column as a polishing step to determine purity. The sample was determined to be greater than 98% pure by SDS-PAGE and reverse phase HPLC.

[0145] Example 8Reverse Phase HPLC of TriChem Resources CEA product, 10690BA sample of TriChem Resources (West Chester, PA) CEA product, 10690B, was tested for purity by reverse phase HPLC. TriChem Resources' literature suggests their product is 95% pure, however our testing by reverse phase HPLC demonstrated this product to only be of about 50% purity, as shown in Figure 3. About ten peaks were detected.

Methods for Purifying Selected CEA Family Member Proteins

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