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WO1999046574A2 - Anticorps permettant d'evaluer l'etat de modification post-translationnelle de proteine et procedes d'elaboration et d'utilisation correspondants - Google Patents

Anticorps permettant d'evaluer l'etat de modification post-translationnelle de proteine et procedes d'elaboration et d'utilisation correspondants Download PDF

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WO1999046574A2
WO1999046574A2 PCT/US1999/004653 US9904653W WO9946574A2 WO 1999046574 A2 WO1999046574 A2 WO 1999046574A2 US 9904653 W US9904653 W US 9904653W WO 9946574 A2 WO9946574 A2 WO 9946574A2
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antibody
polypeptide
post
protein
translationally modified
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PCT/US1999/004653
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WO1999046574A3 (fr
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Laszlo Otvos
Hildegund Ertl
Magdalena Thurin
Ralf Hoffman
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The Wistar Institute
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the field ofthe invention is compositions and methods of making and using antibodies and polypeptides useful in the detection of posttranslationally modified variants of proteins.
  • the tumor suppressor protein p53 is considered a guardian ofthe genome (Lane, 1992, Nature 358:15-16).
  • the p53 gene encodes a nuclear phosphoprotein that is altered by mutation or deletion in about 50% of human tumors (Hollstein et al., 1991, Science 253:49-53).
  • p53 protein can be divided into five distinct regions.
  • the transactivation domain covers the first 42 amino acid stretch. This region mediates the transcriptional activity of p53, which is directly correlated to its ability to suppress cell growth (Fields and Jang, 1990, Science 249:1046-1049).
  • the next domain is responsible for sequence-specific DNA binding (Kern et al., 1991, Science 252:1708-1711). Recently, an additional functional domain was identified on p53 between the transactivation and the sequence-specific DNA binding domains. Walker and Levine (1996, Proc. Natl. Acad. Sci. USA 93:5335-15340) localized a region between amino acids 61 and 94 that is necessary for efficient growth suppression.
  • the tetramerization domain (amino acids 319-360) of p53 interacts with other p53 protein chains to form the biologically active tetramer, as was shown by peptide mapping (Sakamoto et al., 1994, Proc. Natl. Acad. Sci. USA 91 :8974-8978). This is not a true tetramer, but a di er of d ⁇ mers in which each chain contributes with a ⁇ -pleated sheet and an ⁇ -helix (Clore et al., 1995, Science 265:386-391; Clore et al, 1995, Nature Struct. Biol. 2:321-391; Clubb et al, 1995, Protein Science 4:855-862).
  • the fifth domain (basic domain) carries a high positive charge and interacts non-sequence specifically with DNA. This basic region is located at the C-terminus between amino acids 363 and 386 (Buchman et al., 1988, Gene 70:245-252; Farrel et al., 1991, EMBO J. 10:2879-2887). p53 lacking the last 30 amino acids at the
  • PKC Protein kinase C
  • Ser378 Only phosphorylation of Ser378 has been identified so far by PKC (Takenaka et al., 1995, J. Biol Chem. 270:5405- 5411) although, based on the consensus sequences for this kinase, four additional target residues are located in the basic region of p53 (Ser367, Ser371, Ser376 and Thr377)
  • Phosphorylation ofthe C-terminal region appears to function in the opposite manner.
  • phosphorylation may promote different conformational forms of p53 that interact with distinct tissue-specific factors to regulate gene expression (Mukhopadhyay et al., 1995, Springer- Verlag, New York, pp. 79-8).
  • Another report has suggested that variations in the phosphorylation might affect the half-life of wild- type ⁇ 53 (Steegenga et al, 1996, J. Mol. Biol. 263:103-113).
  • Glycosylation a second type of post-translational modification, has also been identified at the C-terminal domain of p53. Shaw et al. (1996, Oncogene 12:921- 930) mapped O-glycosylation at or around the C-terminal region. Among the many varieties of O-glycosylation, serine and threonine residues sometimes carry single ⁇ - linked GlcNAc moieties. Several lines of evidence indicate that ⁇ -linked O-GlcNAc attachment of serines and threonines is a regulatory modification, perhaps analogous to phosphorylation (Hayes and Hart, 1994, Curr. Opin. Biol. 4:692-696).
  • O-GlcNAc modified proteins are also transiently phosphorylated.
  • practically all O-GlcNAc modified proteins form specific and reversible multimeric complexes with other proteins.
  • O-GlcNAc also appears to be highly immunogenic (Hart et al., 1994, Glycosyl Dis. 1:214-215) and a potential modifier of peptide/protein conformation (Hayes and Hart, 1994, Curr. Opin. Biol. 4:692-696).
  • anti-p53 antibodies Although a number of anti-p53 antibodies are commercially available, their applicability is often limited. For example, polyclonal antibody 421 is not very sensitive in ELISA, or Western-blotting. High sensitivity monoclonal antibodies for the different p53 subdomains would be extremely useful. An even bigger problem is that in spite ofthe major interest in the status of phosphorylation of p53 in the different tissues, cell lines and tumor progression stages, currently there are no phosphate or sugar specific anti-p53 monoclonal antibodies available. Synthetic phosphopeptides and glycopeptides can be used as immunogens, and the resulting phosphopeptide or glycopeptide specific monoclonal antibodies are likely to be invaluable reagents for the study of p53 transformation and processing.
  • Antibodies can distinguish peptides versus phosphopeptides and proteins versus phosphoproteins as extensive experience with neuronal cytoskeletal proteins indicate (Lee et al., 1988a, Proc. Natl. Acad. Sci. USA 85:1998-2002; Lee et al, 1988b, Proc. Natl. Acad. Sci. USA 85:7384-7388; Lang et al., 1992, Biochem. Biophys. Res. Cornmun. 187:783-790). Phosphorylation specific mAbs would be especially attractive in the light of a current report as now described.
  • MAb BP53.12 in the control cells, and three additional isoforms were observed to be expressed at significant levels only in apoptotic cells (Maxwell et al., 1996, Electrophoresis 17:1772-1775).
  • MAb BP53.12 recognized the N-terminal domain of p53, where a large number of potential phosphorylation sites can be found. As the phosphate specificity of this antibody is unknown, it is likely that some important phosphate forms ofthe antigens remained undetected in the system.
  • the main antigenic regions of p53 are the extreme termini (Legros et al., 1994, Oncogene 9:2071-2076), where most ofthe proposed phosphorylation sites are found. Lane et al.
  • Another application of synthetic phosphopeptides and glycopeptides is the screening of anti-p53 autoantibodies in sera obtained from cancer patients. Ten percent of breast cancer patients have circulating antibodies directed against the p53 protein, and p53 is overexpressed in the cells of these patients (Davidoff et al., 1992,
  • the terminal domains of p53 wherein most ofthe phosphorylation sites are located are the most immunodominant regions ofthe protein.
  • the highest number of patients having p53 autoantibodies had lung and pancreas carcinomas, two cancer types which are known to have a high frequency of p53 gene mutations, not all patients having p53 antibodies had mutated genes.
  • the mechanism by which p53 is presented to the immune system is still unknown (Lubin et al., 1993, Cancer Res. 53:5872-5876).
  • An analysis ofthe post-translational modifications ofthe antigens recognized by the anti-p53 autoantibodies is long overdue.
  • mAbs monoclonal antibodies
  • mAbs and polyclonal antibodies are the reagents of choice to characterize the active or inactive status of tumor suppressor p53 (Donehower and Bradley, 1993, Biochim. Biophys. Acta 1155:181-205; Lin and Simmons, 1990,Virology 176:302- 305; Medcalf and Milner, 1993, Oncogene 8:2847-2851; Hall and Milner, 1995, Oncogene 10:561-567).
  • pAb 421 a polyclonal antibody termed pAb 421.
  • the invention includes a method of making an antibody which specifically binds with a post-translationally modified p53.
  • the method comprises the steps of a) administering to an animal a polypeptide comprising a portion ofthe p53 having a post-translationally modified amino acid residue and an immunogenicity enhancer; b) eliciting an antibody response thereto; and c) obtaining the antibody from the animal.
  • the antibody is a polyclonal antibody.
  • the method further comprises d) preparing a monoclonal antibody from the animal.
  • the p53 comprises a post-translationally modified amino acid residue having a modification selected from the group consisting of phosphorylation, glycosylation and prenylation.
  • the p53 is abnormally post-translationally modified in a disease.
  • the polypeptide comprises the p53 and the immunogenicity enhancer.
  • the immunogenicity enhancer is a T-helper cell determinant, even more preferably, the T-helper cell determinant is 3 ID.
  • the polypeptide further comprises a peptide spacer.
  • the portion ofthe p53, the immunogenicity enhancer, and the spacer are cosynthesized. Even more preferably, the immunogenicity enhancer is 3 ID.
  • the invention further includes an antibody made by a method comprising the steps of a) administering to an animal a polypeptide comprising a portion ofthe p53 having a post-translationally modified amino acid residue and an immunogenicity enhancer; b) eliciting an antibody response thereto; and c) obtaining the antibody from the animal.
  • the invention includes an antibody made by a method comprising the steps of a) administering to an animal a polypeptide comprising a portion ofthe p53 having a post-translationally modified amino acid residue and an immunogenicity enhancer; b) eliciting an antibody response thereto; and c) obtaining the antibody from the animal, wherein the polypeptide comprises a peptide spacer and an immunogenicity enhancer being 3 ID wherein the polypeptide, the spacer and 3 ID are cosynthesized.
  • polypeptide comprising a portion of a p53 comprising a post-translationally modified amino acid residue having a modification selected from the group consisting of phosphorylation, glycosylation and prenylation and an immunogenicity enhancer.
  • the portion ofthe p53 is at the C-terminus ofthe polypeptide, and is covalently linked to the immunogenicity enhancer, and further wherein the immunogenicity enhancer is at the N-terminus ofthe polypeptide.
  • the polypeptide further comprises a peptide spacer between the portion ofthe p53 and the immunogenicity enhancer.
  • the length ofthe polypeptide is from about 20 to about 45 amino acid residues. More preferably, the length ofthe polypeptide is 41 amino acid residues.
  • the invention further includes a monoclonal antibody which binds specifically to a post-translationally modified variant of p53, wherein the immunogen used to produce the antibody is the post- translationally modified variant of p53 in combination with an immunogenicity enhancer.
  • the post-translationally modified variant of p53 comprises a modification selected from the group consisting of phosphorylation, glycosylation and farnesylation.
  • the post-translationally modified variant of p53 comprises a biphosphorylated C-terminus wherein Ser378 and Ser392 are phosphorylated.
  • the post-translationally modified variant of p53 comprises a monophosphorylated C-terminus. Additionally included is a monoclonal antibody which binds specifically to a peptide comprising a phosphorylated p53, a peptide spacer, and 3 ID.
  • the invention further includes a method of assessing the post-translational modification status of a p53 obtained from a human patient.
  • the method comprises the steps of a) obtaining a biological sample from a patient comprising the p53; b) contacting an antibody made by aforementioned method with the p53; c) forming an antigen-antibody complex between the p53 and the antibody, and d) detecting the antigen-antibody complex, wherein the presence ofthe complex defines the post-translational modification status ofthe p53.
  • the invention includes a method of determining the presence or absence of an autoantibody specific for a post-translationally modified p53 in a biological sample of a human patient.
  • the method comprises the steps of a) obtaining a biological sample from the patient; b) contacting the biological sample with a polypeptide comprising a portion of a p53 comprising a post-translationally modified amino acid residue having a modification selected from the group consisting of phosphorylation, glycosylation and prenylation and an immunogenicity enhancer; c) forming an antigen-antibody complex between the polypeptide and the autoantibody, and d) detecting the antigen-antibody complex; wherein the presence ofthe complex is an indication that the autoantibody is present in the biological sample.
  • Figure 1 is an image of a gel showing the purification of murine p53 protein expressed by Sf9 insect cells infected with recombinant baculovirus using a mAb p53-18 immunoaffinity column. Column fractions were analyzed by SDS-PAGE. The far left column shows the position ofthe molecular mass markers. Lanes 1-8 correspond to fractions 1-8, respectively, eluted with glycine buffer, pH 2.9. The gel was stained with Coomassie Blue.
  • Figure 2 is a graph depicting results from a conformation-sensitive enzyme-linked immunosorbent assay of unphosphorylated and diphosphorylated p53 C-terminal peptides.
  • the unphosphorylated peptide is represented by triangles, and the diphosphorylated peptide is represented by circles.
  • Open symbols correspond to peptides plated from water, and closed symbols correspond to peptides plated from TFE.
  • the dilution ofthe ascites fluid containing the antibody was 1 : 1000.
  • Figure 3 is an illustration of a helical wheel representing the structure of p53 peptide 371-393.
  • the inner circle corresponds to the first 18 amino acids
  • the outer circle corresponds to amino acids 19-23, overlapping amino acids 1-5.
  • Ser378 (in position 8) and Ser392 (at the outer circle in position 4) are marked with asterisks.
  • an element means one element or more than one element.
  • an "immunodominant region" of a protein means a portion of a protein having a stretch of amino acid residues which comprise an epitope that stimulates an immune response in an animal.
  • post-translationally modified amino acid residue means an amino acid residue having any ofthe post-translational modifications described herein.
  • immunogenicity enhancer is meant any composition capable of recruiting T-cells to assist B-cells in an immunological response.
  • immunogenicity enhancers are peptides, which may be synthetic or naturally occurring. The peptides may be unmodified or may be modified containing amino acid residues that are phosphorylated or glycosylated.
  • An example of an immunogenicity enhancer is the peptide 3 ID, which represents an epitope ofthe rabies virus nucleoprotein in mice ofthe H 2k haplotype.
  • binds specifically to means an antibody or antigen (e.g., a polypeptide), which binds in an ELISA or Western Blotting method without binding to a control target, or which binds in an ELISA method to result in an optical density (O.D.) value greater than 0.10, or which binds in an ELISA method to result in an O.D. value of at least twice the background O.D. value.
  • an antibody or antigen e.g., a polypeptide
  • peptide spacer means a tripeptide sequence separating portions of a synthetic polypeptide.
  • T-helper cell determinant means a peptide sequence that stimulates T-helper cells.
  • cosynthesized means prepared in one continuous solid-phase synthesis.
  • amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
  • antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab) 2 , as well as single chain antibodies and humanized antibodies (Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc.
  • synthetic antibody as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.
  • protein typically refers to large polypeptides.
  • peptide typically refers to short polypeptides.
  • polypeptide sequences the left-hand end of a polypeptide sequence is the amino-terminus, or "N-terminus”; the right-hand end of a polypeptide sequence is the carboxyl-terminus, or "C-terminus.”
  • variant means one form or chemical species of a molecule, such as a protein, or a polypeptide, wherein the molecule may exist in one or more structurally related and similar forms.
  • a multi-phosphorylated variant of p53 protein is a form ofthe p53 protein comprising more than one phosphate group on the polypeptide.
  • a monophosphorylated p53 polypeptide is a form of a polypeptide comprising a portion ofthe p53 protein having only one phosphate group in the polypeptide.
  • post-translationally modified means a biochemical modification to a protein, polypeptide or amino acid residue, wherein the modification is made using a chemical synthetic method as described herein, or an enzyme in a cell or other living system after the translation of a nucleic acid transcript encoding the protein or polypeptide or amino acid residue into a polypeptide.
  • Such modifications include, for example, glycosylation, phosphorylation and prenylation.
  • Glycosylation can be effected, for example, by the process of exposing a polypeptide to enzymes which effect glycosylation, e.g. mammalian glycosylating enzymes, whereby a sugar moiety, e.g., N-acetyl glucosamine, is added to a hydroxyl group on an amino acid residue (e.g. serine, threonine, tyrosine) of a polypeptide.
  • Such modifications also include, for example, phosphorylation.
  • Phosphorylation can be effected synthetically as described herein, or by exposing a polypeptide to enzymes which effect phosphorylation, e.g. Phosphokinase C, whereby a phosphate group is added to a hydroxyl group on an amino acid residue (e.g., serine, threonine or tyrosine) of a polypeptide.
  • a "post-translationally modified" p53 protein may be a p53 protein that is phosphorylated at one or more serine residues along the polypeptide by a kinase enzyme after the p53 protein is translated from a nucleic acid transcript to a polypeptide in a cell.
  • a post-translationally modified p53 polypeptide may be a polypeptide comprising a portion ofthe p53 protein that is phosphorylated at one or more serine residues along the polypeptide by a chemical synthetic method as described herein.
  • multi-phosphorylated means a variant (as described herein) of a polypeptide comprising more than one amino acid residue having a phosphate group.
  • a “multi-phosphorylated variant of p53” includes a form ofthe protein p53 having a phosphate group on both ofthe serine amino acid residues Ser378 and Ser392 ofthe polypeptide.
  • biphosphorylated means a variant (as described herein) of a polypeptide comprising two amino acid residues having a phosphate group.
  • a "biphosphorylated p53 C-terminus polypeptide” means a p53 polypeptide comprising the C-terminus ofthe p53 protein, having a phosphate group on two amino acid residues (e.g., Ser378 and Ser392) ofthe p53 C-terminus.
  • “monophosphorylated” means a variant (as described herein) of a polypeptide comprising one amino acid residue having a phosphate group.
  • a "monophosphorylated p53 C-terminus” means a p53 polypeptide comprising the C-terminus ofthe p53 protein, having a phosphate group on one amino acid residue (e.g., Ser378) ofthe p53 C-terminus.
  • biological sample means a cell, a tissue or a biological fluid which is obtained from a living organism, or from a culture of cells or viruses.
  • the invention includes a method of making antibodies which bind with high specificity to a post-translationally modified variant of a physiologically important protein.
  • the method includes the preparation of a polypeptide comprising an immunogenicity enhancer, such as a T-helper cell determinant, and a portion of a protein having a post-translationally modified amino acid residue.
  • the antibodies can be polyclonal or monoclonal.
  • the antibodies can also be synthetic.
  • the protein can be any protein, but is preferably a protein that displays post-translationally modified variants which may be prevalent in a disease state, such as cancer.
  • a preferred protein is the tumor-suppressor protein p53.
  • the post-translationally modified variant ofthe protein can be a protein having a phosphorylated amino acid residue, or more than one phosphorylated amino acid residue, for example, a serine residue having a phosphate group attached thereto.
  • the post-translationally modified variant can also be a protein having a glycosylated amino acid residue, or more than one glycosylated amino acid residue, for example, a serine residue having a O-linked N-acetyl glucosamine attached thereto.
  • the post-translationally modified variant can also be a farnesylated amino acid residue.
  • the post-translationally modified variant may comprise multiple or mixed post-translationally modified amino acid residues, such as a protein which has several phosphorylated serine residues and several glycosylated serine residues.
  • the post-translationally modified variant may comprise a modified amino acid residue at any amino acid residue along the protein that is susceptible to enzymatic or chemical modification.
  • the modification can be naturally occurring, such as by enzymatic phosphorylation by a protein kinase or a glycosylating enzyme. Also, the modification can be chemically created, such as by purifying a protein and chemically adding a phosphate group to a serine amino acid residue which is hydroxylated. Modified amino acid residues are commercially available.
  • the post-translationally modified variant can also be a portion ofthe protein, such as an immunodominant region of a protein.
  • the post- translationally modified variant can also be a portion of a protein that does not comprise an immunodominant region.
  • the post-translationally modified variant can be a portion of a protein that is hidden or unexposed to the surface ofthe protein under normal physiological conditions.
  • the protein p53 has a region called the central core which is unexposed to the surface under normal cellular conditions, and thus, is inaccessible to immune response components. This buried or inaccessible region is an example of a portion of a protein that is not immunodominant.
  • the method ofthe invention for making antibodies which specifically bind with the post-translationally modified variant of a protein comprises administering to an animal a polypeptide comprising a portion ofthe protein having a post-translationally modified amino acid residue and an immunogenicity enhancer.
  • the polypeptide may be administered by injection to an animal for developing an immune response.
  • the animal is preferably a mouse.
  • An example of such mice are female C3H/Hc mice.
  • the animal may be any animal capable of generating antibodies to a polypeptide.
  • the polypeptide may be found as a naturally occurring peptide, or may be synthesized by a peptide synthesis method.
  • the polypeptide may be synthesized by any method of synthesis known to the skilled artisan.
  • Polypeptides are preferably synthesized on solid-phase using automated synthesizers employing standard Fmoc-methodology (Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161-214). Phosphorylated serine and threonine residues are incorporated as Fmoc-Ser(PO 3 HBzl)-OH or Fmoc-Thr(PO 3 HBzl). This improved "synthon" strategy for phosphopeptide synthesis is superior to "global" phosphorylation.
  • peracetylated Fmoc-Ser/Thr- ⁇ (GlcNAc)-OH is incorporated into the peptide as any other amino acid, except that no excess ofthe acetylating agent is used.
  • the serine derivative is marketed by Bachem California.
  • the threonine derivative and the serine derivative as well, if necessary, is synthesized using the method of Filira et al. (1990, Int. J. Pept. Protein Res. 36:86-96).
  • ⁇ -GlcNAc can be added to side-chain unprotected Ser (and presumably Thr) still on the resin through glucose-oxazoline (formed in situ) after the peptide chain assembly is completed (Hollosi et al, 1991 , Tetrahedron Lett.
  • a polypeptide spacer may be placed between the portion ofthe protein having the post-translationally modified amino acid residue and the immunogenicity enhancer.
  • the purpose ofthe peptide spacer is to prevent unnatural conformational induction in the post-translationally modified protein portion that can happen when it is linked covalently to the highly helical 3 ID sequence.
  • the 3 ID immunogenicity enhancer is placed at the N-terminus of the polypeptide, and the portion ofthe post-translationally modified protein is at the C-terminus ofthe polypeptide, and between the two is placed a peptide spacer.
  • An example ofthe peptide spacer is the tripeptide glycine-alanine-glycine (Gly-Ala-Gly).
  • the polypeptide may be any length such that it is long enough to be sufficiently immunogenic, and so that antipeptide antibodies will primarily recognize the presence or absence ofthe phosphate or carbohydrate on the post-translationally modified protein portion. If the polypeptide is too long, this will not be the case.
  • the entire length ofthe polypeptide, including the immunogenicity enhancer is from about 20 to about 45 amino acid residues long. In a preferred embodiment where the polypeptide includes a portion of post-translationally modified p53 protein, the length ofthe polypeptide is 41 amino acids long.
  • Another important concern in the design ofthe polypeptide used in the method ofthe invention is the stability ofthe polypeptide in the serum ofthe animal into which it will be injected to raise antibodies.
  • tests ofthe polypeptide in mouse serum for stability against protease digestion should be carried out prior to injection ofthe polypeptide.
  • serum stability tests can be carried out as follows.
  • the synthesized polypeptide comprising post-translationally modified amino acid residues can be incubated with mouse serum at about 25% mouse serum at 37° C for several hours. Serum proteins can then be precipitated with a precipitating agent such as 15% trichloroacetic acid.
  • the pellets from the precipitation can be centrifuged and the supernatant can then be loaded onto a reversed-phase-HPLC method for analysis of degradation ofthe polypeptides by serum proteases.
  • the method ofthe invention for making antibodies which specifically bind with post-translationally modified variants of protein also comprises the step of obtaining antibodies from the immunized animal.
  • Antibodies can be obtained from the animal using methods well known in the art and are described, for example, in Harlow et al, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York.
  • the generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom.
  • Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al.,1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY and in (Tuszynski et al, 1988, Blood 72:109-115). Quantities ofthe desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. However, proteins expressed by molecular biology techniques are often not appropriately phosphorylated or glycosylated. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
  • the invention also includes the polypeptide described above which is useful in generating antibodies which are specific for a post-translationally modified variant of a protein.
  • this polypeptide comprises a portion of a protein having a post-translationally modified amino acid residue, wherein the modification is a modification selected from the group consisting of phosphorylation, glycosylation and farnesylation.
  • the portion ofthe protein may comprise multiple post-translational modifications in more than one ofthe above categories.
  • the portion ofthe protein may comprise more than one post-translationally modified amino acid residue that is phosphorylated, and also more than one amino acid residue that is glycosylated.
  • the polypeptide also comprises an immunogenicity enhancer as described above.
  • the immunogenicity enhancer may be a peptide or a glycosylated peptide, and may be any composition capable of eliciting T-helper cell response in an immunological reaction.
  • the portion of a post-translationally modified protein and the immunogenicity enhancer parts ofthe polypeptide may be cosynthesized, or may synthesized separately and then covalently linked.
  • the methods of synthesis may be as described above.
  • the polypeptide may comprise a portion ofthe p53 protein comprising a post-translationally modified amino acid residue, or several post-translationally modified amino acid residues that are phosphorylated and covalently linked to an immunogenicity enhancer.
  • the immunogenicity enhancer may be the peptide 3 ID.
  • the peptide 3 ID is located at the N-terminus ofthe polypeptide, and is separated by a peptide spacer from the post-translationally modified portion ofthe p53 protein, which is at the C-terminus ofthe polypeptide.
  • the overall polypeptide length is from about 20 to about 45 amino acid residues long.
  • the invention also includes highly sensitive and highly specific monoclonal antibodies which are specific for post-translationally modified variants of biological proteins such as the p53 protein.
  • the invention includes a highly sensitive monoclonal antibody which is highly specific for a double phosphorylated form of p53 protein, which double phosphorylated form is prevalent in cancer patients or normal patients.
  • the invention includes a monoclonal antibody which binds specifically to a post-translationally modified variant ofthe tumor suppressor protein p53.
  • the post-translationally modified variant ofthe tumor suppressor protein p53 may be any variant ofthe tumor suppressor protein p53 having one or more amino acid residues which are post-translationally modified. Such post-translationally modified amino acid residues may be phosphorylated, glycosylated, or farnesylated, or any combination thereof.
  • mAb p53-18 is a monoclonal antibody highly specific and highly sensitive to the post-translationally modified variant of p53 which is biphosphorylated at the C-terminus at the amino acid residues Ser378 and Ser392.
  • the invention also includes a method for assessing the post-translational modification status of a protein, such as p53, obtained from a patient.
  • a protein such as p53
  • the patient may be any animal, however, the preferred patient is a human.
  • the protein may be obtained from a human patient in a serum sample, a tissue biopsy sample, or a cell sample (i.e., a biological sample).
  • the method may be used to assess the presence or absence of a certain post-translationally modified protein variant which is correlated with a disease state or stage of tumor progression.
  • the method comprises obtaining the protein sample from the human patient and contacting antibodies which are highly specific and highly sensitive for the desired post-translationally modified protein variant as described in the invention with the protein sample obtained from the patient.
  • an antigen antibody complex is then formed between the monoclonal antibodies described above and the protein sample described above, by incubating both together according to standard methods.
  • the antigen antibody complex is then detected using an immunological technique such as an ELISA method, immunoprecipitation, or Western
  • the highly specific and highly sensitive monoclonal antibodies are used to detect the presence of, or to assess the quantity of, a post-translationally modified variant ofthe protein to which the monoclonal antibody is specific.
  • the monoclonal antibody specific for a variant of p53 protein which is biphosphorylated at the C-terminal region is used to screen a serum sample from a human cancer patient for the presence of biphosphorylated p53 protein.
  • the invention also includes a method for determining the presence or absence of an autoantibody specific for a post-translationally modified protein variant, such as phosphorylated p53, in a biological sample, e.g., serum, obtained from a patient.
  • a serum sample is obtained from a patient, preferably a human patient, and is assessed for the presence of an autoantibody as follows.
  • Polypeptides ofthe invention comprising an amino acid residue having a post-translational modification are prepared as described herein.
  • the polypeptide prepared is one known to specifically bind the autoantibody sought to be detected in the patient's serum as determined by methods described herein.
  • An antigen antibody complex is then formed between the polypeptide and the serum sample by incubating both together according to standard methods.
  • the antigen antibody complex is then detected using an immunological technique such as an ELISA method, immunoprecipitation, or Western Blotting methods.
  • the presence ofthe autoantibody may be correlated with a disease state or stage of tumor progression in a human.
  • Example 1 Design, preparation and use of synthetic polypeptides
  • the length ofthe polypeptide prepared should be long enough to be sufficiently immunogenic, but not so long that the anti-peptide antibodies will primarily recognize the presence or absence ofthe phosphate or carbohydrate.
  • the peptide families described herein for studying phosphorylated epitopes of p53 were successful in generating p53 specific antibodies. Polypeptides were synthesized having
  • the peptide families described in Table 2 were synthesized (modified amino acids are marked with asterisk; phosphopeptides are printed in italics, and glycopeptides are underlined).
  • the modified amino acids were placed at least five amino acids away from either terminus (except the penultimate residue), so that phosphorylation or glycosylation efficiently modified the recognitional characteristics.
  • the p53 362-383 peptide family multiple glycosylated or mixed glycosylated and phosphorylated peptides in which the sugar is proximal to another modified amino acid were not prepared because the coupling of back-to-back bulky amino acids was expected to proceed with a very low yield.
  • Peptide 3 ID an immunogenicity enhancer which provides T-helper bystander help in the immunizations, is an immunodominant T-helper cell determinant and is a portion (amino acid residues 404-418) ofthe rabies virus strain ERA nucleoprotein (Ertl et al, 1989, J. Virol. 63:2885-2892).
  • the amino acid sequence of Peptide 3 ID is as follows:
  • mice were inoculated in the hind legs with 20 mg ofthe tandem construct, and boosted two weeks later. After screening the test bleeds, a third immunization was given five days before the fusion of splenocytes with myeloma cells.
  • the anti-peptide mAbs were assessed by ELISA and Western blotting for specific binding to p53 expressed in various cell lines and tissues.
  • the C-terminal p53 fragment described herein was a good immunogen due to the favorable physico-chemical properties of this protein domain.
  • This region of p53 is hydrophilic and assumes various reverse-turn structures, generally considered advantageous features for inducing anti-protein and anti-peptide antibodies (Hopp and
  • Polypeptides were synthesized on solid-phase using a Milligen 9050 (continuous flow) or Rainin PS3 (batch mixing) automated synthesizers employing standard Fmoc-methodology (Fields and Noble, 1990, Int. J. Pept. Protein Res. 35:161- 214). Phosphorylated serine and threonine residues were incorporated as Fmoc- Ser(PO 3 HBzl)-OH or Fmoc-Thr(PO 3 HBzl). The monoalkyl protected Fmoc-Ser and
  • Fmoc-Thr derivatives are currently marketed by Novabiochem, Ltd. (San Diego, CA).
  • peracetylated Fmoc- Ser/Thr- ⁇ (GlcNAc)-OH was incorporated into the polypeptide as if it was any other amino acid, except that no excess ofthe acetylating agent was used.
  • the serine derivative is marketed by Bachem California (Torrance, CA).
  • polypeptide was cleaved from the support using trifluoroacetic acid (TFA):thioanisole (95:5; v/v).
  • TFA trifluoroacetic acid
  • RP-HPLC reversed-phase high performance liquid chromatography
  • Removal ofthe acetyl protecting group was accomplished using diluted NaOH or NaOMe (Bulet et al, 1996, Eur. J. Biochem. 238:64-69; Otvos et al, 1997, In: Solid-Phase Synthesis and Combinatorial Chemical Libraries, Andrews et al, eds, Mayflower Scientific, Kingswinford, in press.).
  • the integrity ofthe synthetic peptides was analyzed by mass spectrometry and phosphate analysis (Zardeneta et al, 1990, Anal. Biochem. 190:340-347).
  • the modified ELISA protocol was used for conformation-sensitive ELISA studies. In these studies, the polypeptides were plated from trifluoroethanol/water mixtures. First, the active dilution range of antibody preparation was determined. The selectivity of a given antibody dilution toward various peptide antigens was assessed. Generally, from
  • Protein concentrations ofthe purified p53 preparations were determined by bicinchoninic acid (Smith et al, 1985, Anal. Biochem. 150:76-85). For tissue samples, the phosphate or sugar specificity ofthe p53 variants was more important that the actual protein load.
  • Circulating anti-p53 autoantibodies obtained from sera from cancer patients and normal controls were analyzed for the phosphorylation and glycosylation status ofthe p53 protein variants by assessing binding to synthesized polypeptides.
  • Anti-p53 antibodies e.g. 421, 1620, D01, BP53.12, 240, 1801, 246,
  • CM- 10 are commercially available from various sources, including Oncogene Science (Cambridge, MA) and Santa Cruz (Santa Cruz, CA).
  • Some of these antibodies are known to cross-react with p53 regions described herein (e.g. antibody BP53.12 recognizes p53 around amino acids 15 and 20).
  • the synthetic polypeptides, phosphopeptides and glycopeptides prepared herein were used to map the identity and the post-translational modification status ofthe epitopes of these and other antibodies.
  • mice Inbred female C3H/He mice at 6-8 weeks of age were used for the immunizations. Mice were inoculated with 10 microgram doses of polypeptide antigens in each hind leg as described in Example 3 herein.
  • Example 2 Synthesis of phosphopolypeptides corresponding to the p53 C-terminus Seven peptides were prepared as described herein corresponding to the C-terminal region of p53. The total number of amino acid residues in the p53 protein is 393, thus, the numbers used herein refer to amino acid residues 1-393. Two peptides were non-phosphorylated, two had a phosphate group on Ser378, one had the phosphate group on Ser376, and two were phosphorylated on both Ser378 and Ser392.
  • the peptides were either 23 or 33 amino acids long.
  • the peptides were purified on RP- HPLC to homogeneity and then characterized by mass spectroscopy (Otvos et al, 1998, Biochim. Biophys. Act. 1404:457-474).
  • Table 3 indicates the peptide retention times obtained using trifluoroacetic acid as ion-pairing reagent in RP-HPLC.
  • the elution behavior ofthe short peptides was scrutinized during various reversed-phase high performance chromatographic conditions (Hoffmann et al, 1997, Anal. Chim. Acta 352:327-333).
  • Antibody 421 has been reported to recognize p53 at its basic domain between amino acids 372-381 (Wade-Evans and Jenkins, 1985, EMBO J. 4:699-706).
  • a double phosphorylated polypeptide was coupled to an immunodominant rabies T-helper cell epitope and was injected into mice to generate monoclonal antibodies according to the method of (Dietzschold et al, 1990, J. Virol. 64:3804-3809).
  • the diphosphorylated polypeptide was co-synthesized with a Gly-Ala- Gly spacer and peptide 3 ID. After fusion, eight hybridomas were selected. All clones specifically bound to the immunizing peptide antigen with an affinity which was stronger than or equally as strong as the only other available polyclonal antibody 421 (antibody 421) known to specifically bind to the C-terminal region of p53. These data are shown in Table 4. Because monoclonal antibody p53-18 was an IgM, it also cross- reacted with the unphosphorylated polypeptide.
  • mAb p53-l 8 was significantly more sensitive than pAb 421.
  • mAb p53-18 bound the antigen at 100 times greater sensitivity than the pAb 421 polyclonal antibody. This high sensitivity is extremely important in light ofthe very low level of p53 expression in tissues.
  • MAb p53-18 may enable detection of p53 in tissues and cell lines where p53 was previously undetected.
  • mAb monoclonal antibody
  • MAb p53-18 was highly specific for phosphorylated Ser378 and Ser392 at both the protein and the corresponding peptide levels in conventional aqueous environments, however, when the peptide conformation was changed during the assay procedure to that of an ⁇ -helix, mAb p53-18 also detected the unphosphorylated p53 C-terminal fragment.
  • the irnmunodominance of the phosphorylated p53 C-terminus was indicated by the fact that cancer patients' sera preferentially labeled the same sequence as mAb p53-18, i.e., the double phosphorylated peptide antigen.
  • Peptides and phosphopeptides were detached from the solid support using TFA and were purified by reversed-phase high performance liquid chromatography (RP-HPLC) using an aqueous acetonitrile gradient elution system containing 0.1% trifluoroacetic acid as an ion pairing reagent.
  • RP-HPLC reversed-phase high performance liquid chromatography
  • a solution of 82.5%) TFA, 5% water, 5% thioanisole, 5% m-cresol, and 2.5% ethane-diol was used to detach peptides from the solid support. The integrity ofthe peptides and phosphopeptides was verified by mass spectroscopy.
  • Table 6 comprises a list ofthe sequences ofthe synthetic peptides and Table 3 contains the methods used for their characterization.
  • Two sets of polypeptides were synthesized. One set comprised peptides of 23 amino acids in length, and the second set comprised peptides of 33 amino acids in length (the polypeptides were extended to their N-termini).
  • Polypeptides were prepared in the following phosphorylation states: non-phosphorylated, phosphorylated with a single phosphate group on either Ser378 or Ser392, and phosphorylated with phosphate groups on both
  • the 23-mer double phosphorylated polypeptide was co-synthesized such that the immunodominant T-helper cell determinant 3 ID ofthe rabies virus nucleoprotein was at the N-terminus, the B-cell epitope was at the C-terminus, and a peptide spacer was between the two regions.
  • Groups of six week-old female C3H/He mice were inoculated with 10 micrograms of the tandem peptide mixed with 50% complete Freund's adjuvant in each hind leg. Fourteen days after inoculation, the mice received a booster immunization of 2 x 10 micrograms in 50% incomplete Freund's adjuvant.
  • mice Ten days after the booster immunization, the mouse sera were screened and the mice were given a third immunization. At 5 days after the third immunization, mouse splenocytes were fused with myeloma cells using previously described methods (Dietzschold et al, 1990, J. Virol. 64:3804-3809). Hybridomas were grown in selective medium, screened and subcloned according to standard procedures (Goding, 1986, Monoclonal Antibodies: Principles and Practice: Academic Press, Orlando) .
  • mAbs monoclonal antibodies
  • Sepharose 4B Fast Flow (Pharmacia, Uppsala, Sweden). Unreacted functional groups on the matrix were blocked by incubation with 0.1 molar Tris-HCl pH 8.0. The column was washed several times alternating between Tris-HCl buffer having a pH of 8.0, and acetate buffer having a pH of 3.6.
  • Murine p53 protein was obtained from a cellular extract of Sf9 insect cells infected with a recombinant baculovirus expressing murine wild-type p53. Sf9 cells were collected, lysed in PBS containing 0.1% NP-40, and centrifuged at 12,000 * g for 15 minutes.
  • the column was equilibrated with PBS having a pH of 7.6 and comprising 0.1 molar NaCl.
  • the supernatant containing soluble p53 was loaded onto the p53-l 8 immunoaffinity column and the column was washed with phosphate buffer and PBS to elute non-specifically bound proteins.
  • Murine p53 protein was eluted from the immunoaffinity column using a solution comprising 0.1 molar glycine having a pH of 2.9 into tubes containing a tenth of a fraction volume of 1 molar Tris-HCl having a pH of 8.0.
  • the protein composition of each fraction was evaluated using 12% SDS-PAGE in the presence of protein molecular weight markers and staining using Coomassie Blue.
  • Goat anti-mouse IgG coupled with horseradish peroxidase was used as a secondary antibody in a PBS solution containing 0.01 ) Tween 20.
  • Membranes were washed extensively with washing buffer and reacted with chemiluminescence reagent (NEN Life Science Products, Boston, MA) followed by autoradiography using X-OmatTM X-ray films (Kodak, Rochester, NY).
  • the active dilution range of antibody preparation was determined, and selectivity of antibody dilutions for selected peptide antigens was assessed. From about 40 nanograms to 2.5 micrograms of peptide antigen and from about 0.5 nanograms to 1.2 micrograms of protein samples were loaded in each well. Actual peptide concentrations were determined by reversed-phase HPLC (RP-HPLC)
  • ELISA conditions were modified as follows. The peptides were applied to the plate and dried overnight. The plates were washed with a PBS solution at pH 6.8 containing 0.04% Triton XI 00. This solution was used in all subsequent steps. To assess the conformational effects ofthe polypeptide upon specific binding to the antibody, the assay was repeated under identical conditions, except that the peptides were dissolved in trifluoroethanol (TFE) instead of water prior to applying them to the ELISA plate for drying overnight (Lang et al, 1994, J. Immunol. Meth. 170:103-115).
  • TFE trifluoroethanol
  • Sera obtained from cancer patients and healthy control patients were screened for circulating anti-p53 autoantibodies using the same protocol, except that the washing buffer contained 1 milligram per milliliter of bovine serum albumin and the secondary antibody used was a sheep anti-human IgG.
  • the antigen specificity of mAb p53-18 was characterized by assessing binding of the mAb to 23-mer peptides. One microgram of each of the four peptides, non-phosphorylated, phosphorylated on Ser378, phosphorylated on Ser392, or phosphorylated on both Ser378 and Ser392, were assessed for specific binding to mAb p53-18 using serial dilutions of the ascites fluid that ranged from 1:100 to
  • the other p53 protein variant was expressed in insect cells infected with mouse p53 baculovirus recombinant as described herein, and was expected to be phosphorylated on Ser378 and Ser392 and on many other potential phosphorylation sites.
  • protein E7 of human papilloma virus (HPV)- 16 was expressed using the baculovirus system under conditions substantially identical to those described herein.
  • the mAb p53-18 specifically bound only the phosphorylated p53 protein, and failed to bind to the non-phosphorylated variant.
  • the mAb p53-18 binding to the phosphorylated protein exhibited some "pro-zone” binding behavior, indicating optimal antigen-antibody interactions under these experimental conditions. Positive binding was detected with as little as 0.08 micrograms of p53 protein. Some non-specific binding of mAb p53-18 to the E7 protein was observed, but was well below the level of binding to p53 and did not indicate "pro-zone” binding characteristics.
  • Antibody p53-18 did not cross-react with the non-phosphorylated protein variant, even when as much as 0.3 micrograms of protein was used.
  • the commercially available antibody 421 was used as a positive control in the experiment.
  • Antibody 421 binds to a non-modified (i.e. non-phosphorylated and non-glycosylated) epitope of p53 at an area that includes Ser378 (Shaw et al, 1996, Oncogene 12:921-930).
  • Antibody 421 binds p53 protein with low sensitivity in ELISA, and thus a high concentration of antibody must be used to detect low levels of p53 protein.
  • p53 protein expressed in E. coli (0.25 microgram and higher amounts) was recognized by a 1:50 dilution of polyclonal antibody (pAb) 421.
  • mAb p53-18 The binding of mAb p53-18 to p53 in a Western blotting method was assessed.
  • Antibody 240 recognized an amino acid stretch in p53 around residue 215, and labels p53 from many species, e.g. human, mouse, rat, etc.(Legros et al, 1994, Oncogene 9:2071-2076).
  • Antibody p53-18 also reacted with different forms of p53, including p53 MD (MI234, EG168) and p53 VD (AV135), mutants of mouse p53 comprising amino acid exchanges within the domain having mutations known in cancer
  • mAb p53-18 specifically binds to phosphorylated p53 regardless of the origin of the protein. In brief, the specific binding exhibited by mAb p53-18 was virtually identical to that of mAb 240 to all positive and negative control protein variants tested.
  • mAb p53-18 unlike some other anti-p53 antibodies, can specifically detect p53 protein in Western-blots, and immunoaffinity columns containing mAb p53-18 can isolate C-terminally phosphorylated p53 from various sources.
  • a polyclonal antibody, specific to phosphorylated Ser392 is marketed by New England Biolabs (Beverly, MA, USA). According to the manufacturer, this antibody was obtained by inoculation of rabbits with a C-terminal phosphopeptide conjugated to a carrier protein. This pAb is claimed to specifically bind p53 only after phosphorylation with CKII at Ser392. The phosphate specificity of this pAb was compared with mAb p53-18 against p53 expressed in E. coli, Sf9 insect cells and the four 33-mer peptides with different phosphorylated forms. The polyclonal antibody did not specifically bind to the phosphorylated protein when a protein load of less than 1 microgram was used.
  • p53 was expressed in two systems. To increase the production of p53 compared to mammalian tissues and cell lines, p53 is generally expressed in bacterial (Hupp et al, 1992, Cell 71 :875-886), baculovirus (O'Reilly and Millner, 1988, J. Virol.
  • p53 expressed in E. coli only binds DNA properly after interaction with cellular proteins, including various kinases (Hupp et al, 1992, Cell 71 :875-886).
  • human p53 expressed in baculovirus-infected Sf9 cells displays a two-dimensional electrophoretic mobility pattern (and consequently phosphorylation pattern) identical to wild-type p53 from human cells (Patterson et al,
  • Insect cells appear to contain all protein kinases necessary for phosphorylation of a mammalian protein (Fuchs et al, 1995, ⁇ ur. J. Biochem. 228:625-639), with at least 9 potential p53 sites phosphorylated in this system (Patterson et al, 1996, Arch. Biochem. Biophys. 330:71-79). Accordingly, the p53 originated from the Sf9 cells was expected to have phosphate groups on both Ser378 and Ser392.
  • a novel phosphate-specific mAb to p53 was prepared.
  • This antibody bears the promise to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the status of phosphorylation of the C-terminus of p53 protein in various cell types, solution environments and stages of tumor progression.

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Abstract

L'invention concerne un procédé permettant d'élaborer des anticorps spécifiques pour les variants à modification post-translationnelle de la protéine p53. L'invention concerne également des anticorps polyclonaux et monoclonaux. L'invention concerne en outre un polypeptide utile dans l'élaboration de ces anticorps, qui comprend une partie de protéine à modification post-translationnelle, un espaceur peptidique et un activateur d'immunogénicité.
PCT/US1999/004653 1998-03-11 1999-03-11 Anticorps permettant d'evaluer l'etat de modification post-translationnelle de proteine et procedes d'elaboration et d'utilisation correspondants WO1999046574A2 (fr)

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US7255999B2 (en) * 2001-05-21 2007-08-14 Monogram Biosciences, Inc. Methods and compositions for analyzing proteins
US9939447B2 (en) 2001-05-21 2018-04-10 Monogram Biosciences, Inc. Methods and compositions for analyzing proteins
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