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WO1991002061A1 - Peroxydase de la thyroide humaine de recombinaison - Google Patents

Peroxydase de la thyroide humaine de recombinaison Download PDF

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Publication number
WO1991002061A1
WO1991002061A1 PCT/US1990/004289 US9004289W WO9102061A1 WO 1991002061 A1 WO1991002061 A1 WO 1991002061A1 US 9004289 W US9004289 W US 9004289W WO 9102061 A1 WO9102061 A1 WO 9102061A1
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Prior art keywords
thyroid peroxidase
human thyroid
antibody
htpo
tpo
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PCT/US1990/004289
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English (en)
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Basil Rapaport
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Basil Rapaport
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Publication of WO1991002061A1 publication Critical patent/WO1991002061A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to the field of molecular biology and immunology. More particularly, the invention relates to the production of recombinant human thyroid peroxidase in non-thyroidal eukaryotic cells. The invention is further related to methods of using recombinant human thyroid peroxidase, and, in particular, to methods of using recombinant human thyroid peroxidase in diagnosis of immune disorders such as Hashimoto's thyroiditis.
  • Hashimoto's thyroiditis is the most common autoimmune endocrinopathy, affecting, at least subclinically, up to 15% of the adult female population (1,2). Antibodies against a number of thyroid antigens are present in the sera of these patients, including thyroglobulin and the thyroid "microsomal” antigen (3,4). Other antigens of lesser, or uncertain, importance, include the second colloid antigen (3), tubulin (5), DNA (6) and Autoimmune Thyroid Disease-Related Antigen I (ATRA I) (7).
  • ATRA I Autoimmune Thyroid Disease-Related Antigen I
  • Antibodies against the microsomal antigen, which is expressed on the cell surface (8,9), are believed to be of greater importance than those against thyroglobulin in the pathogenesis of Hashimoto's thyroiditis. This is because antimicrosomal antibodies (MSA) are more closely associated with the active phase of the disease (1,10,11) and are complement-fixing (8). These antibodies are, therefore, likely to initiate thyroid cellular damage.
  • MSA antimicrosomal antibodies
  • TPO thyroid peroxidase
  • hTPO Human TPO immunopurified by monoclonal antibodies (mAbs) has been available, but is of limited value because of: (a) inadequate supplies of human thyroid tissue; (b) the difficulties in purification of this membrane-bound antigen; and (c) contamination with other thyroid autoantigens such as thyroglobulin, which is highly abundant. Fragments of hTPO have been generated as recombinant bacterial (0-galactosidase) fusion proteins, and reactivity of a number of Hashimoto patient sera with small fragments of TPO expressed as fusion proteins has been reported (18). Those data, however, are difficult to interpret, because the plaque assays used require extensive pre-adsorption of polyclonal antisera (7) and can yield false positive results.
  • a reported fusion protein originally described as reactive with 19 of 20 Hashimoto patient sera (ref. 18, clone C2) has, upon im unopurification with anti-5- galactosidase mAbs, been found to react with fewer Hashimoto patient sera in an ELISA assay (40).
  • bacterial fusion proteins too, have been of limited value because: (a) no combination of fragments has been found that reacts with all Hashimoto's sera; (b) the conformation of the fusion protein may differ from that of the native protein; and (c) the bacterial products may be toxic when added to immune cells in culture.
  • the present inventor achieved expression of recombinant hTPO in non-thyroidal eukaryotic cells. Like native hTPO, this recombinant hTPO is enzymatically active, is expressed on the cell surface, and is not a fusion protein.
  • the recombinant hTPO of this invention is recognized in a specific manner by sera from patients with Hashimoto's thyroiditis that contain "anti icrosomal" antibodies. All 36 Hashimoto patient sera selected to represent a range of antimicrosomal antibody levels seen in this disease were reactive with the eukaryotic-expressed recombinant hTPO of the invention. It is an object of the present invention, then, to provide for a convenient and economical source of recombinant hTPO, which does not suffer from the disadvantages associated with the immuno-purified native protein or with the recombinant fusion protein previously available.
  • the present invention thus provides a number of important advances in the characterization of the human thyroid microsomal antigen, and opens the way to substantial further developments in this field.
  • Recombinant, enzymatically-active, human thyroid peroxidase has been generated in non-thyroidal eukaryotic cells.
  • the conformation of this protein is not encumbered by the ⁇ - galactosidase fusion partner.
  • the TPO is glycosylated. The demonstration of functional TPO activity indicates unequivocally that the cDNA previously cloned (16, 17, 18, 19), is indeed TPO.
  • the only thyroidal (or, indeed, human) protein produced by, or found in, the CHO-TPO cells of the present invention is hTPO. Even though human sera from both normal subjects and patients with Hashmoto's thyroiditis contain antibodies that react with some antigen(s) of untransfected CHO cells, only the Hashimoto's patient sera react with the recombinant hTPO.
  • the present invention also sheds light on previous observations that the microsomal antigen appeared as a doublet when analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blot (33,41,42). It was not known whether the doublet represented two separate proteins or the partial degradative product of a single protein. Kimura et al . observed two forms of hTPO mRNA and cDNA, and suggested the possibility of alternate splicing of the initial TPO transcripts (19). Nagaya a et al . reported the existence of four different forms of hTPO mRNA transcripts in cultured Graves' thyroid cells after TSH stimulation (43). The present discovery of a doublet as the product of a single, intron-less, hTPO gene argues strongly against the likelihood of alternate splicing.
  • the present inventor observed, under the same conditions, that the major im- munogenic form of recombinant hTPO in transfected CHO cells is about 200 kD in mass which is converted upon reduction to a single band of about 110 kD. This difference may be related to varied expression of hTPO in different cell types (human and CHO). However, it was also reported that a 200 kD protein was produced by subjecting the extracted human thyroid microsomal 107 kD protein major band to PAGE under non- reducing conditions (41).
  • the present finding of a diminished 110 kD signal after reduction of the recombinant hTPO protein is in accordance with other findings using the native microsoma antigen (45,46).
  • human TPO in its native state, exists either as a multimer or in association with another membrane protein of similar size. Epitope recognition by autoantibodies may be conformation-dependent.
  • hTPO The derived amino acid sequence of hTPO suggested to the present inventor the presence in recombinant full-length hTPO and thus, in naturally-occurring hTPO, of a signal peptide, as well as a putative hydrophobic membrane-spanning region (transmembrane domain) at the carboxyl terminus of the protein (amino acid residues 846-870) (16, 17, 19, 54).
  • Naturally- occurring hTPO has been shown to be a thyroidal cell surface protein.
  • Recombinant, enzymatically active hTPO is also cell membrane-associated in stably transfected non-thyroidal eukaryotic cells (48).
  • the present inventor hypothesized that the signal peptide directs the human TPO through the cell membrane, but that the hydrophobic region of hTPO becomes embedded in the cell membrane, thereby preventing secretion from the cell.
  • hTPO secretion of recombinant hTPO for the purposes of the present invention, it is meant that the recombinant hTPO expressed by a host cell is directed through and dissociated from the host cell membrane. There has heretofore been no functional proof that the hTPO hydrophobic region 846-870 cor ⁇ responds to a transmembrane domain.
  • the present invention demonstrates the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation.
  • the insertion, by site-directed mutagenesis, of a stop codon immediately upstream of this putative transmembrane domain converts hTPO into a secreted protein that is enzymatically active and immunologically intact.
  • the stop codon By introducing the stop codon, the hTPO was truncated by 85 residues, removing the carboxyl terminus (933 amino acids).
  • Mutated hTPO cDNA inserted into a eukaryotic expression vector, was stably transfected into CHO cells.
  • CHO cells expressing the truncated hTPO protein secreted im unoprecipitable TPO into the culture medium after 4 hours of chase, with levels accumulating progressively over a 24 hour period.
  • CHO cells expressing wild-type hTPO released no i - munoprecipitable TPO into the culture medium.
  • the secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells.
  • TPO enzymatic activity was present in conditioned medium from CHO cells transfected with the mutated hTPO, but was absent in conditioned medium from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO.
  • the secreted form of hTPO can be used to generate large amounts of soluble TPO protein for use in structural and immunological studies, as well as for diagnostic uses.
  • recombinant, enzymatically active, TPO, or a functional or chemical derivative thereof recombinant, enzymatically active, TPO, or a functional or chemical derivative thereof.
  • hTPO produced by non- thyroidal eukaryotic cells.
  • recombinant hTPO that is enzymatically active, immunologically intact and secretable, or a functional or chemical derivative thereof.
  • Yet another embodiment of the invention comprises a plasmid selected from the group consisting of pECE-HTPO, pHTP0(Ml)-ECE-SV2-DHFR, pHTP0-DHFR-2B, pHTP0-DHFR-4C and pHTP0-DHFR-4C-MTX.
  • non- thyroidal eukaryotic cell transformed with any of these plasmids, as well as methods of producing hTPO comprising culturing the transformed cell under conditions allowing expression of the hTPO and recovering the hTPO.
  • the invention provides for an antibody against the hTPO of the invention.
  • a method of detecting hTPO in a sample comprising contacting the sample with an antibody against full-length recombinant hTPO or an antibody against a secretable hTPO, wherein the antibody is detectably labeled, so as to form a complex between the hTPO in the sample and the detectably labeled antibody, and detecting the complexed or uncomplexed labeled antibody.
  • kit for the detection of hTPO in a sample comprising container means comprising one or more containers, wherein one of the containers comprises detectably labeled antibody against hTPO.
  • a method of detecting antibodies to hTPO in a sample comprising contacting the sample with full-length recombinant hTPO or secretable recombinant hTPO so as to form a complex between an hTPO-specific antibody in the sample and the recombinant hTPO, and detecting the complexed antibody.
  • a kit for the detection of antibodies to hTPO in a sample comprising container means comprising one or more containers, wherein one of said containers comprises recombinant hTPO.
  • FIG. 1 Construction of the expression plasmid pHTPO- ECE.
  • pHTPO-BS (upper right) was digested with Not I, the ends blunted with the Klenow fragment of DNA polymerase I, and the DNA subsequently digested with Xba I.
  • the released Bluescript vector was further digested with Sea I to obtain good separation on agarose gel electrophoresis because of the similar size of this vector (2.95 kb) and the HTPO cDNA fragment (3.1 kb).
  • the mammalian expression vector pECE (25) (upper left) was digested with Eco RI, the ends blunted with the Klenow fragment of DNA polymerase I, and the DNA subsequently digested with Xba I.
  • pHTPO-BS and pECE fragments were then ligated using T4 DNA ligase (26).
  • Figure 2 Fluorescence-activated cell sorter (FACS) analysis of CHO cells transfected with pHTPO-ECE. CH0-HTP012b cells were processed as described herein.
  • Panel A Cells exposed to phycoerythrin (PE)-labeled second antibody alone, without prior exposure to human serum.
  • Panel B Cells incubated in serum (1:100) from a patient with Hashimoto's thyroiditis (ELISA value of 1.779) without subsequent incubation in PE-labeled second antibody.
  • PE phycoerythrin
  • Panel C Cells sequentially incubated in the Hashimoto's serum described in panel B and in PE-labeled second antibody.
  • Panel D As in panel C, except that serum from a normal individual, lacking antimicrosomal antibodies, was used.
  • Panels E and F The same data as in panels C and D plotted to show the forward scatter. These data indicate that the relative sizes of the cell populations reacting with the normal and the Hashimoto's sera are the same.
  • Figure 3 Linear regression analysis of ELISAs using antibodies against human thyroidal microsomes or against recombinant human TPO.
  • Figure 4 Linear regression analysis of ELISAs using antibodies against human thyroidal microsomes or against recombinant human TPO, 1/1000 dilution.
  • Cardiff refers to the source of the microsomal antigen of both Figures 3 and 4.
  • FIG. 6 Nucleotide sequence of human TPO gene after site-directed mutagenesis. The mutations incorporated two stop codons, as well as an EcoRI site for confirmation, in the region immediately upstream from the transmembrane region of the human TPO gene.
  • FIG. 8 Schematic diagram showing the expression plasmid pHTP0(Ml)-ECE-SV2-DHFR.
  • FIG. 10 Comparison of 51 sera, selected to provide a spectrum of anti-MSA levels, in terms of their reactivity with Graves' thyroid microsomes and recombinant, enzymaticall - active human TPO generated in non-thyroidal eukaryotic cells.
  • the anti-MSA assay data are expressed as an ELISA index, relative to a standard serum.
  • Data for the anti-hTPO antibody assay are expressed as absolute O.D. units, normalized to a blank well value of 0.000.
  • A serum dilution 1/100 (sera from four normal patients are enclosed within the rectangle);
  • B serum dilution 1/1,000;
  • C serum dilution 1/10,000.
  • FIG. 14 (A) Immunoprecipitation of mutated hTPO in different clones of transfected CHO cells. CHO - non- transfected CHO cells; CHO-TPO - CHO cells transfected with wild type hTPO; CH0-TP0-M1-POOLED - pooled colonies of CHO cells transfected with the mutated form of hTPO; CH0-TP0-M1-D through K - individual colonies of CHO cells, transfected with mutated hTPO, that were selected with cloning cylinders and then expanded. Cells were radiolabeled with 3 ⁇ S-methionine and immunoprecipitated with Hashimoto's thyroiditis serum containing high anti-hTPO antibody levels.
  • FIG. 15 Biosynthesis and processing of TPO. Immuno- precipitation studies were performed with CHO cells expressing wild-type hTPO (upper panel), and with CHO cells transfected with the mutated form of hTPO (lower panel). Pulse for 4 h (0 hours of chase) with 3* 5s-methionine was followed by chase with unlabeled methionine for the indicated periods of time. Immunoprecip tations were then performed on both cell lysates and conditioned media, as indicated.
  • FIG. 16 Human TPO enzymatic activity in the medium of CHO cells after transfection with wild-type hTPO (cell line CHO-TPO 12g) (14) and CHO cells transfected with the mutated form of hTPO (CH0-TP0-M1-K1). Media were collected after 3 days of culture. TPO enzymatic activity in the media was measured by the guaiacol assay. The time course shown refers to the accumulation of oxidized guaiacol substrate in the assay, and not to the kinetics of enzyme secretion into the medium.
  • FIG. 17 T cell clones from the thyroid infiltrate in Graves' disease, expanded in the absence of antigen, recognize recombinant TPO. Clone + autologous irradiated PBL - black bars; clone + PBL + control (untransfected) CHO microsomes - striped bars; clone + PBL + CHO microsomes transfected with TPO - grey bars. Results are expressed as mean cpm of [ 3 H]thymidine incorporation from triplicate cultures. Error bars indicate standard errors of the mean (S.E.M). Similar results were obtained in three or more replicate experiments.
  • Figure 18 Determination of the epitope for the anti- microsomal/TPO monoclonal antibody 20.10.
  • the nucleotide sequences of the 5'- and 3'-ends were determined for 14 clones selected from the hTPO cDNA fragment library. These boundaries are annotated by the numbers assigned to the nucleotides in hTPO previously reported (17).
  • the smallest region of overlap between all 14 clones is from 881-927 b.p.
  • the first two nucleotides in this span do not constitute a complete codon, so the epitope area can be defined as between 883-927 b.p., corresponding to the derived amino acid sequence shown.
  • DESCRIPTION OF THE PREFERRED EMBODIMENTS The nucleotide sequences of the 5'- and 3'-ends were determined for 14 clones selected from the hTPO cDNA fragment library. These boundaries are annotated by the numbers assigned to the nucleotides in hTPO previously reported
  • Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al .. Molecular Biology of the Gene. Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al.. Molecular Cell Biology. Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, B.M., Genes II. John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W., et al..
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • cDNA complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire genome of an organism.
  • a cDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et al .. Molecular Cloning: A Laboratory Manual, supra.
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene.
  • Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines.
  • a presently preferred vector for this purpose is the ⁇ -ZAP vector.
  • vector is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replica- tion in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • DNA expression vector is meant any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome.
  • DNA expression vectors include bacterial plasmids and phages.
  • substantially pure is meant any antigen of the present invention, or any gene encoding any such antigen, which is essentially free of other antigens or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule, such as any of the cDNA sequences of the present invention is meant to refer to any nucleotide subset of the molecule.
  • a “variant” of such molecule is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be "substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc.
  • the moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc.
  • Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences. 16th ed., Mack Publishing Co., Easton, Penn. (1980).
  • a “functional derivative" of a gene of the human TPO antigen of the present invention is meant to include “fragments,” “variants,” or “analogues” of the gene, which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity.
  • a DNA sequence encoding the human thyroid peroxidase of the present invention, or its functional derivatives, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, T., et al .. supra, and are well known in the art.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and trans!ational .regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypept-fde.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal, the initiation of protein synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal, the initiation of protein synthesis.
  • Such regions will normally include those £'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the thyroid peroxidase gene sequence, or (3) interfere with the ability of the thyroid peroxidase gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of the human thyroid peroxidase protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells, although eukaryotic (and, particularly, non-thyroidal eukaryotic) expression is preferred.
  • Preferred prokaryotic hosts include bacteria such as ______ coli , Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia. etc.
  • the most preferred prokaryotic host is J _ coli.
  • Other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may also be utilized. Under such conditions, the protein may not be glycosylated.
  • the procaryotic host must be compatible with the rep!icon and control sequences in the expression plasmid.
  • a prokaryotic cell such as, for example, E. coli, B.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter of the 0-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325, etc.
  • inducible prokaryotic promoters include the major right and left promoters of bacteriophage ⁇ (P
  • E. coli E. coli. the ⁇ -amylase (Ulmanen, I., et al ., J. Bacteriol. 162:176-182 (1985)) and the ⁇ -28-specific promoters of J . subtilis (Gilman, M.Z., et al.. Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)), and Streptomvces promoters (Ward, J.M., et al., Mol. Gen. Genet. 203:468-478 (1986)).
  • Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottes an, S. (Ann. Rev. Genet. 18:415-442 (1984)).
  • ribosome binding sites are disclosed, for example, by Gold, L., et al. (Ann. Rev. Microbiol . 35:365- 404 (1981)).
  • Most preferred hosts are eukaryotic hosts including yeast, insects, fungi, and mammalian cells either in vivo, or in tissue culture. Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CH0-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. CHO cells are presently preferred mammalian host cells. COS cells also are convenient eukaryotic hosts for human thyroid peroxidase expression, as well as for study of the regulation of human thyroid peroxidase expression.
  • transcriptional and trans!ational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression.
  • pro ⁇ moters from mammalian expression products, such as actin, collagen, myosin, etc., may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.
  • Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications including glycosylation.
  • Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • yeast ubiquitin hydro!ase system in vivo synthesis of ubiquitin-human TPO fusion proteins may be accomplished.
  • the fusion proteins so produced may be processed in vivo or purified and processed in vitro, allowing synthesis of the human TPO protein with a specified amrtno terminus sequence.
  • problems associated with retention of initiation codon-derived methionine residues in direct yeast (or bacterial) expression may be avoided.
  • Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized.
  • Known glycolytic genes can also provide- very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.
  • human TPO or functional derivatives thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express human TPO by methods known to those of skill.
  • sequences encoding human TPO may be operably linked to the regulatory regions of the viral polyhedrin protein (Jasny, Science 238: 1653 (1987)).
  • Infected with the recombinant baculovirus, cultured insect cells, or the live insects themselves, can produce the human TPO protein in amounts as great as 20 to 50% of total protein production.
  • caterpillars are presently preferred hosts for large scale human TPO production according to the invention.
  • eukaryotic regulatory regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al., J. Mol. APPI. Gen.
  • the human TPO encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the human TPO protein may occur through the transient expression of the introduced sequence. Alter ⁇ natively, permanent expression may occur through the integra- tion of the introduced sequence into the host chromosome.
  • a non-replicating DNA (or RNA) molecule which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the human TPO protein may occur through the transient expression of the introduced sequence. Alter ⁇ natively, permanent expression may occur through the integra- tion of the introduced sequence into the host chromosome.
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotropic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama, H., Mol . Cel. Biol. 3:280 (1983).
  • the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, ⁇ VX.
  • Such plasmids are, for example, disclosed by Maniatis, T., et al . (In: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids .include pC194, pC221, pT127, etc.
  • Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomvces plasmids include pIJlOl (Kendall, K.J., et al., J. Bacteriol.
  • Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, D., et al ., Miami Wntr. Svmp. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomvces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al.. J. Clin. Hematol. Oncol. 10:39- 48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Expression, Academic Press, NY, pp. 563-608 (1980)).
  • the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate- precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al ., Science 240(4858): 1538 (1988)), etc.
  • biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate- precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran
  • mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al ., Science 240(4858): 1538 (1988)), etc.
  • recipient cells After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the human TPO protein, or in the production of a fragment of this protein. This can take place in the transformed cells as such, or following the induction of these cells to differentiate.
  • the expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cel 1 ul ose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • the human TPO or functional derivative thereof may be isolated by the use of anti-human TPO antibodies. Such antibodies may be obtained by well-known methods, some of which as mentioned hereinafter.
  • antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab')2 fragments) which are capable of binding an antigen.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al.. J. Nucl. Med. 24:316-325 (1983)).
  • Antibodies according to the present invention may be prepared by any of a variety of methods. For example, cells expressing the human TPO protein, or a functional derivative thereof, can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding human TPO.
  • antibodies according to the present invention are mAbs.
  • Such mAbs can be prepared using hybridoma technology (Kohler et al ., Nature 256:495 (1975); Kohler et al.. Eur. J. Immunol. 6:511 (1976); Kohler et al.. Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hvbridomas. Elsevier, N.Y., pp. 563-681 (1981)). In general, such procedures involve immunizing an animal with human TPO antigen.
  • the splenocytes of such animals are extracted and fused with a suitable myeloma cell line.
  • a suitable myeloma cell line may be employed in accordance with the present invention.
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands, J.R., et al . (Gastroenterology 80:225- 232 (1981).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the human TPO antigen.
  • Antibodies according to the present invention also may be polyclonal, or, preferably, region specific polyclonal antibodies. Region specific polyclonal antibodies and methods of using them are described in co-pending U.S. application Serial Number 06/731,470, filed 07 May 1985, the specification of which is incorporated herein by reference as though set forth in full.
  • Antibodies against human TPO according to the present invention are well suited for use in standard immunodiagnostic assays known in the art, including such immunometric or "sandwich” assays as the forward sandwich, reverse sandwich, and simultaneous sandwich assays.
  • the antibodies may be used in any number of combinations as may be determined by those of skill without undue experimentation to effect immunoassays of acceptable specificity, sensitivity, and accuracy for the human TPO antigen or equivalents thereof.
  • Standard reference works setting forth general principles of immunology include Roitt, I., Essential Immunology, Sixth Ed., Blackwell Scientific Publications, Publisher, Oxford (1988); Kimball, J.
  • detecting it is intended to include determining the presence or absence of a substance or quantifying the amount of a substance.
  • the term thus refers to the use of the materials, compositions, and methods of the present invention for qualitative and quantitative determinations.
  • the isolation of other hybridomas secreting mAbs of the same specificity as those described herein can be accomplished by the technique of anti-idiotypic screening. Potocmjak, et al ., Science 215:1637 (1982).
  • an anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an rantibody.
  • An Id antibody can be prepared by immunizing ah animal of the same species and genetic type (e.g.
  • the immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).
  • an anti-Id antibody which is specific for iditoypic determinants on a given mAb, it is then possible to identify other B cell or hybridoma .clones sharing that idiotype. Idiotypic identity between the antibody product of two clones makes it highly probable that the antibody products of the two clones recognize the same antigenic epitopes.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id.
  • mAbs generated against the hTPO antigen may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice.
  • Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs.
  • the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemoc anin (KLH) and used to immunize additional BALB/c mice.
  • Sera from these mice will contain anti-anti-Id antibodiess that have the binding properties of the original mAb specific for the hPTO epitoape.
  • the anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as hTPO.
  • the hybridoma cells of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production. Briefly, cells from the individual hybridomas are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired mAbs.
  • MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Antibodies according to the present invention are particularly suited for use in immunoassays wherein they may be utilized in liquid phase or bound to a solid phase carrier.
  • the antibodies in these immunoassays can be detectably labeled in various ways.
  • labels and methods of labeling known in the art.
  • Examples of the types of labels which can be used in the present invention include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemilumi- nesCent compounds, bioluminescent compounds and metal chelates.
  • Those of ordinary skill in the art will know of other suitable labels for binding to antibodies, or will be able to ascertain the same by the use of routine experimentation.
  • the binding of these labels to antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art.
  • antibodies according to the present invention can be detectably labeled is by linking the antibody to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric or fluoro- metric means.
  • enzymes which can be used to detectably label antibodies include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotin-avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • detectably labeled antibodies also can be detected by labeling the antibodies with a radioactive isotope which then can be determined by such means as the use of a gam a counter or a scintillation counter.
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H, 125 If 32p, 35 s> 14 C ⁇ 51 Crf 36 cl f 57 C ⁇ ) 58 C ⁇ ) 59 Fe and
  • fluorescent labeling compounds fluoroscein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, al lophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibodies of the invention also can be detectably labeled using fluorescent emitting metals such as l ⁇ Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as d i ethyl enetri ami nepentaaceti c acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA di ethyl enetri ami nepentaaceti c acid
  • EDTA ethylenediaminetetraacetic acid
  • Antibodies also can be detectably labeled by coupling them to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of the chemical reaction.
  • chemiluminescent labeling compounds examples include luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester , and dioxetane.
  • Bioluminescent compound may be used to label the antibodies according to the present invention.
  • Biolumi- nescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a biolumi- nescent antibody is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the assay t ⁇ be used.
  • assays which can be incorporated in kit form are many,- and include, for example, competitive and non- competitive assays.
  • Typical examples of assays which can utilize the antibodies of the invention are radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), and immunometric, or sandwich, immunoassays.
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • ELISA enzyme-linked immunosorbent assays
  • sandwich, immunoassays sandwich, immunoassays.
  • immunometric assay or "sandwich immuno ⁇ assay” it is meant to include simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art.
  • antibodies according to the present invention will be useful in other variations and forms of assays which,are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
  • the incubation medium usually added with the labeled soluble antibody.
  • the “blockers” are added to assure that non-specific proteins, protease, or human antibodies to mouse immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radio!abeled indicator antibody, to yield false positive or false negative results.
  • the selection of "blockers” therefore adds substantially to the specificity of the assays described in the present invention. It has been found that a number of nonrelevant (i.e. non ⁇ specific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g.
  • IgGj, IgG2 a , IgM, etc. can be used as “blockers.”
  • concentration of the "blockers” (normally 1-100 ⁇ g/ ⁇ l) is important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in human serum.
  • the buffer system containing the "blockers” needs to be optimized.
  • Preferred buffers are those based on weak organic acids, such as imidazole, HEPPS, MOPS, TES, ADA, ACES, HEPES, PIPES, TRIS, and the like, at physiological pH ranges.
  • Somewhat less preferred buffers are inorganic buffers such as phosphate, borate or carbonate.
  • known protease inhibitors should be added (normally at 0.01-10 .g/ml) to the buffer which contains the "blockers.”
  • solid phase immunoadsorbents which have been employed and which can be used in the present invention.
  • Well known immunoadsorbents include glass, polystyrene, polypropylene, dextran, nylon and other materials, in the form of tubes, beads, and microtiter plates formed from or coated with such materials, and the like.
  • the immobilized antibodies can be either covalently or physically bound to the solid phase immunoadsorbent, by techniques such as covalent bonding via an amide or ester linkage, or by adsorption.
  • suitable solid phase immunoadsorbents and methods for immobilizing antibodies thereon or will be able to ascertain such, using no more than routine experimentation.
  • labels such as radionuclides may be bound to antibodies according to the present invention either directly or by using an intermediary functional group.
  • An intermediary group which is often used to bind radioisotopes which exist as metallic cations to anti ⁇ bodies is diethylenetriaminepentaacetic acid (DTPA).
  • DTPA diethylenetriaminepentaacetic acid
  • Typical examples of ⁇ metallic cations which are bound in this manner are: 99»Tc, 123 I, n ⁇ IN, »l It 97 Ru> 67 Cu> 67 Ga and 68 Ga .
  • the antibodies of. the invention can also be labeled with non ⁇ radioactive isotopes for purposes of diagnosis.
  • Elements which are particularly useful in this manner are l ⁇ Gd, ⁇ A , 162 Dy, 52 Cr and 56 Fe.
  • the hTPO-encoding DNA sequence of the present invention may be used as a DNA probe to isolate or detect complementary DNA sequences according to well-known hybridization methods.
  • the human antigen genes may then be cloned and expressed in a host to give the human antigen. This human antigen may then be used in diagnostic assays for the corresponding autoantibody.
  • the antigen of the invention may be isolated in substantially pure form employing antibodies according to the present invention.
  • an embodiment of the present invention provides for substantially pure hTPO, characterized in that it is recognized by and binds to the anti-hTPO antibodies of the present invention.
  • the present invention provides a method of isolating or purifying hTPO by forming a complex with one or more antibodies directed against hTPO.
  • the substantially pure hTPO of the present invention may in turn be used to detect or measure antibody to hTPO in a sample, such- as serum or urine.
  • a sample such- as serum or urine.
  • one embodiment of the present invention comprises a method of detecting the presence or amount of antibody to hTPO in a sample, comprising contacting the sample containing the antibody to hTPO with detectably labeled hTPO, and detecting the label.
  • immunoreactive fractions and immunoreactive analogs of hTPO also may be used.
  • immunoreactive fraction is intended any portion of the hTPO antigen which demonstrates an equivalent recognition by, or binding to, an antibody directed against hTPO.
  • immunoreactive analog is intended a protein which differs from hTPO by one or more amino acids, but which demonstrates an equivalent recognition by, or binding to, an anti-hTPO antibody.
  • autoimmune thyroiditis As in Hashimoto's thyroidits, such a self antigen can be any epitope of TPO which is recognized by a receptor on a T cell capable of helping a B cell make an anti-TPO antibody, or a T cell involved in the autoimmune process by any other known mechanism (see below).
  • T cells are readily available from the thyroid, for example in Graves' disease in the form of infiltrates extracted from thyroidectomy specimens. By studying such infiltrates, it is possible to examine the antigenic specificities of T cells selected in vivo for their pathogenic relevance.
  • the infiltrating T cells can act as T helper (Th) cells, responding to TPO epitopes, and helping B cells make specific anti-TPO antibodies.
  • T helper (Th) cells can mediate a cell-mediated immune response and act on thyroid epithelial cells either directly or via the local release of cytokines. This may lead to destruction of thyroid epithelial cells, when cytotoxic T cells specific for TPO are activated, or via an inflammatory response mediated by a different T cell class. Disruption of the activation or action of such T cells would serve to inhibit the production of anti-TPO antibodies, on the one hand, or of thyroid epithelium-damaging T cells on the other.
  • One embodiment therefore provides a peptide which is capable of binding to the T cell receptor (TCR) of a TPO- specific T cell.
  • TCR T cell receptor
  • Such a peptide would include at least a T cell epitope of the TPO (such as the NP-7 epitope of Example XII).
  • Useful peptides would include a sequence of about 5 or more amino acids of from TPO, or derivatives of such peptides, which are capable of binding to the TCR of a TPO-specific T cell.
  • TCR T cell receptor
  • T-T or T-B interactions in the immune system which are needed for generation of either anti-TPO antibodies or TPO-specific cell- mediated immunity.
  • Acha-Orbea H., et al .
  • Kumar, V. et al ..
  • Ann. Rev. Immunol. 7:657-682 (1989); Urban, J.L. et al.. Cell 54:577-592 (1989); Wraith, D.C., et aK (Cell 57:709-715 (1989); Wraith, D.C., et al..
  • Another embodiment of the invention provides for a pharmaceutical preparation comprising the above TPO-related peptide.
  • a method of treating autoimmune thyroiditis such as Hashimoto's disease, is provided which involves administering a pharmaceutical preparation comprising , the peptide which capable of binding to a TPO-specific T cell and preventing its activation or action.
  • An alternate peptide-based therapeutic strategy contemplated within the scope of the present invention is directed to vaccines comprising TPO-specific T cells (Cohen, I.R., Immunol. Rev. 94:5-21 (1986); Prog. Immunol. VI:491-499 (1986); Scientific Amer. 258:52-60 (1988); HOSP. Prac. pp. 57-64 (February 15, 1989); Cohen, I.R., et al .. Immunol . Today 9:332-335 (1988)) and peptides mimicking the TCR of such TPO-specific T cells (Vandenbark, A.A. et al . , Nature 341:541-544 (1989); Howell, M.D. et al..
  • Such preparations are administered to an individual to prevent or suppress an autoimmune response to TPO by inducing a state of "counter-autoimmunity.”
  • Such counter- autoimmunity is thought to be mediated by T cells which are specific to the TCR of the autoimmune (i.e., TPO-specific) T cell (Cohen, supra. Vandenbark et al .. supra, and Sun, D. et al.. Nature 332:843-845 (1988); Europ. J. Immunol. 18:1993- 1999 (1988)).
  • the invention is therefore directed to T cells specific for TPO capable of acting as a "vaccine” and inducing a state of counter-autoimmunity.
  • Another embodiment includes TCR- mimieking peptides of such T cells.
  • Yet another embodiment is directed to the T cells induced by such TPO-specific T cell and TCR peptide vaccines which mediated the counter-autoimmune effects or down-regulate TPO-specific T cells.
  • Another embodiment of the invention provides for a pharmaceutical preparation comprising such a T cell vaccine, TCR peptide, or counter-autoimmune T cell.
  • a method of treating autoimmune thyroiditis such as Hashimoto's disease, which includes the use of a pharmaceutical preparation comprising either a TPO-specific T cell vaccine, a TCR peptide vaccine, or a counter-autoimmune T cell specific for TPO-specific T cells.
  • An additional embodiment of the present invention is directed to a T suppressor (Ts) lymphocyte capable of interacting specifically with an anti-TPO B cell or T cell, leading to suppression of an anti-TPO immune response.
  • Ts T suppressor
  • Such suppression could be of TPO-specific antibody production or of TPO-specific T cell-mediated thyroid damage such as that mediated by cytotoxic T cells or in a TPO-specific delayed hypersensitivity response.
  • the invention is directed to an epitope of TPO capable of inducing antigen-specific Ts cells and its use in generating Ts cells and in treating autoimmune thyroiditis.
  • Another embodiment is a TPO-specific Ts in a pharmaceutical preparation.
  • Yet another embodiment is directed to a method of treating autoimmune thyroiditis, such as Hashimoto's disease, comprising administering a pharmaceutical preparation comprising a TPO epitope capable of inducing Ts cells.
  • An additional embodiment is a method of treating autoimmune thyroiditis by administering a pharmaceutical preparation comprising TPO-specific Ts cells capable of suppressing an anti-TPO response.
  • suppressor cells see, for example, Green, D., et al .. Ann. Rev. Immunol. 1: 439 (1983) and Benacerraf, B., In: The Biology of Immunologic Disease. HP Publishing Co., Inc., NY, pp. 49-62 (1983).
  • the present invention allows the determination of the T cell epitope or epitopes of TPO (see Example XII, below) using standard techniques commonly known to those of ordinary skill in the art. Further, the present invention makes possible the characterization of the autoimmune TCR specific to the TPO using methods described in, for example, Burns, F., et al .. ______
  • T cells that will accomplish this objective may be generated which are specific for the autoimmune TCR for TPO using methods described in, for example, Acha-Orbea, H., et al . , Ann. Rev. Immunol . 7: 371 (1989).
  • a thyroid cDNA library was constructed to maximize the inclusion of full-length cDNA in the coding orientation.
  • Hyperplastic thyroid tissue was obtained from a patient undergoing thyroidectomy for Graves' disease.
  • mRNA was isolated according to the method of Han et al. (23).
  • Double- stranded cDNA was synthesized from 15 ⁇ g mRNA as described by Gubler and Hoffman (24). Not I and Xba I linker- primers/adaptors were incorporated into the cDNA to create those restriction sites at the 5' and 3' ends, respectively, of the cDNA (23).
  • the cDNA was size-selected ( > 1 kb) by agarose gel (Seaplaque, FMC, Rockland, ME) electrophoresis, digested with Not I and Xba I, ligated into Not I- and Xba I- cut bacteriophage lambda-Zap using T4 DNA ligase, and packaged (Giga-Pak Gold, Stratagene, San Diego, CA).
  • the resulting phage library contained a total of 2 x 10 4 recombinant clones before amplification.
  • EXAHPLE II EXAHPLE II
  • the amplified cDNA library was plated at a density of 4 x 10 4 pfu per 150 mm diameter dish and probed using the insert from a partial human TPO cDNA clone (clone 19, ref. 17). Two positive bacteriophage clones were isolated. A Bluescript phagemid containing the human TPO cDNA insert was generated from one of these clones using the helper phage R408, according to the Stratagene protocol. The resulting recombinant Bluescript plasmid (pHTPO-BS) contained bases
  • the mammalian cell expression vector pECE (25) was obtained from Dr. William Rutter (U.C.S.F.). Human TPO cDNA was cloned into the multiple cloning site of this vector as described in* Figure 1. Enzyme reactions and DNA manipulations were performed as described in Maniatis et al . (26).
  • Chinese hamster ovary cell line CH0-K1 was maintained in Hams' F-12 medium supplemented with 10% fetal bovine serum, penicillin (125 units/ml), streptomycin (100 ⁇ g/ml) and amphotericin-B (2.5 ⁇ g/ml).
  • Transfection and selection with G-418 was carried out by the method of Chen and Okayama (27).
  • 20 ⁇ g pHTPO-ECE plus 2 ⁇ g pSV2-neo (28) (from Dr. John Baxter, U.C.S.F.) were used for the transfection. Control transfections with 20 ⁇ g pECE plus 2 ⁇ g pSV2-neo, and 20 ⁇ g pSV2-neo alone, were performed concurrently.
  • Transfected CHO cells were extracted to obtain soluble protein.
  • Five 100 mm diameter dishes were washed 3 times with calcium-magnesium free phosphate-buffered saline (PBS). After aspiration, 5 ml of 0.5% Triton X-100 in the same buffer, supplemented with 10 ⁇ g/ml leupeptin, 0.5 mg/ml bacitracin and 2 mM phenylmethylsulfonyl fluoride (all from Sigma, St. Louis, M0), were added to the first dish. This initial cell solution was scraped and transferred successively to the other 4 dishes of cells. The cell solution was then tumbled for 1 hour at 4 C.
  • Protein content was determined (30) and 50 ⁇ g protein/lane electrophoresed on a 7.5% polyacrylamide SDS gel (31). Proteins were electrotransferred (30 V x 5 hours, or 250 mA overnight) to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) in an electroblotting apparatus (Hoeffer, San Francisco, CA) containing 25 mM Tris, 192 mM glycine, 20% methanol. In later experiments, transfer was accomplished using a PoTyblot semi-dry electrotransfer system (American Bionetic, Hayward, CA), according to the directions of the manufacturer.
  • Membranes were rinsed once in TBS (0.1 M Tris, pH 8.0, 0.15 M NaCl), then for 30-60 minutes at room temperature in TBS containing 0.5 % Tween 20 (Sigma, St. Louis, M0). After 3 further rinses with TBS-Tween, the blots were probed as described by Young and Davis (32) using a 1:250 dilution of a.mouse mAb against the thyroid microsomal antigen (33), followed by a 1:250 dilution of horseradish peroxidase- conjugated goat anti-mouse IgG antibody (Sigma, St. Louis, M0).
  • CH0-HTP012b cell extracts were probed using a panel of polyclonal Hashimoto's thyroiditis sera, provided by Dr. S. M. McLachlan, University of Wales, Cambridge. Antimicrosomal antibody titers had previously been determined by enzyme-linked immunosorbant assay (ELISA) in the presence of excess thyroglobulin (11). Multiple Hashimoto's thyroiditis sera were applied to a single filter overnight at 4 ⁇ C using a Miniblotter 45 manifold (Immunetics, Cambridge, Mass.).
  • ELISA enzyme-linked immunosorbant assay
  • CH0-HTP012b cells were processed as described by Ellis et al. (34). In brief, cells from a 100 mm diameter dish were detached by mild trypsinization, and the cells rinsed and pelleted (5 minutes at 100 x g, 4°C) in Ham's F12 medium, 10% fetal calf serum (see above). The cells were resuspended in 0.2 ml of phosphate-buffered saline (PBS), lOmM Hepes, pH 7.4, 0.05% Na azide (buffer A).
  • PBS phosphate-buffered saline
  • lOmM Hepes pH 7.4, 0.05% Na azide
  • Serum to be tested (2ul) was added for 30 minutes at 4°C, followed by two rinses in buffer A with 2% fetal calf serum and resuspension in 0.2 ml of the same solution.
  • 25 ul of goat anti-human IgG, Fc specific, affinity- purified, R-Phycoerythrin-labeled (Caltag, South San Francisco, CA) were added for another 30 minutes at 4 ⁇ C.
  • the cells were analyzed on a fluorescence-activated cell sorter.
  • Human TPO activity was assayed following extraction from cell microsomes with trypsin and deoxycholate as previously described (35). In later experiments, a more rapid method was used. Cells were suspended with a rubber scraper in 1.5 ml calcium-magnesium free Dulbecco's phosphate-buffered saline and protein determined on a 5 ul aliquot. The cells were then pelleted in a icrocentrifuge for 2 minutes. Cold 0.1% deoxycholate (0.2 ml/mg cellular protein) was added for 10 minutes. The extract was microcentrifuged for 5 minutes and the supernatant removed for assay.
  • One guaiacol unit is defined as a _A470 of 1.0 per minute which is equivalent to 150 nmols guaiacol oxidized per minute. (36).
  • One unit of iodide peroxidase is defined as a _A353 of 1.0 per minute which corresponds to 43 nmols 13- formed per minute (37).
  • CHO-HTPO 12b Chinese hamster ovary cells (clone CH0-HTPO 12b) expressing enzymatically-active human TPO has been described above. These cells, had been transfected with the recombinant plasmid pHTPO-ECE, constructed by the insertion of a full-length human TPO cDNA into the expression vector pECE.
  • CHO-HTPO 12b and CH0-K1 (control, non- transfected) cells were grown in Ham's F-12 medium supplemented with 100 g/L fetal bovine serum (FBS), penicillin (125 units/ml), genta icin (48 ⁇ g/ml) and amphotericin-B (2.5 ⁇ g/ml).
  • FBS fetal bovine serum
  • penicillin 125 units/ml
  • genta icin 48 ⁇ g/ml
  • amphotericin-B 2.5 ⁇ g/ml
  • Cells were grown to confluence in 100 mm dishes, the cells were rinsed three times with Dulbecco's calcium- magnesium free, phosphate-buffered saline (PBS), and then scraped into a solution containing 10 mM Tris, pH 7.4, 0.25 M sucrose, 2 mM phenylmethyl sulfonyl fluoride, 10 ⁇ g/ml leupeptin, 0.5 mg/ml bacitracin (Buffer A). Cells were homogenized for 20 seconds with a Polytron, centrifuged for 15 min at 10,000 x g, 4 ⁇ C, and the supernatant then centrifuged for 1 hour at 100,000 x g, 4 ⁇ C.
  • PBS Dulbecco's calcium- magnesium free, phosphate-buffered saline
  • microsomal pellet was resuspended in 0.5 ml of Buffer A, homogenized in a Dounce homogenizer, and then frozen at -80 ⁇ C until use. Protein content was determined by the method of Bradford (30). Yield of microsomal protein was approximately 100-200 ⁇ g per 100 mm dish of confluent cells.
  • Multiwell micro-ELISA plates (Dynatech Labs, Chantilly, VA) were coated (overnight at 4 ⁇ C) with 4 ⁇ g CHO-HTPO 12b or CH0-K1 microsomal protein per well in coating buffer (0.05 M sodium bicarbonate, pH 9.3, 0.02% sodium azide).
  • the wells were then rinsed twice in 0.2 M Tris, pH 7.4, 0.15 M NaCl (Tris buffer), once in 0.2 M Tris, pH 7.4, 0.15 M NaCl, 0.05% Tween 20 (Tris-Tween buffer), and once in Tris buffer.
  • 100 ul of PBS, 50 g/L bovine serum albumin (BSA) (Sigma, St. Louis, M0) were added to each well and incubated for 20 min at room temperature. After aspiration, the wells were washed twice in Tris buffer, once in Tris-Tween buffer, and once in Tris buffer.
  • Serum samples were diluted 1/100, 1/1000 or 1/10,000 in PBS, 5 g/L BSA. 100 ul of the diluted serum sample were added per well in duplicate and incubated for 1 hour at 37°C. The wells were then washed three times with PBS. 100 ul of peroxidase-conjugated, affinity-purified, goat anti-human IgG, Fc fragment specific antibody (Cappel, Organon Teknika Corp., West Chester, PA.), diluted 1/500 in PBS, 250 g/L FBS, were added to each well and incubated for 1 hour at 37 ⁇ C. The wells were then washed four times with Tris-Tween buffer.
  • TGA and TAG codons were created at 2629-2631 bp and 2641-2643 bp in human TPO cDNA (17), respectively.
  • an Eco RI restriction site (GAATTC, at 2630-2635 bp) was created together with the first (TGA) stop codon.
  • the mutagenesis procedure was performed according to the protocol of the manufacturer (Muta-gene phagemid in vitro mutagenesis kit, Biorad, Richmond, CA) to generate the plasmid pHTPO(Ml)-BS.
  • the cDNA was excised by digestion of pHTPO(Ml)-BS with Not I, the ends blunted with the Klenow fragment of DNA Polymerase I, and the cDNA liberated by digestion with Xba I.
  • the mutated cDNA (3.05 Kb) was substituted for wild-type human TPO cDNA in the plasmid pSV2- DHFR-ECE-HTPO, to generate pHTP0(Ml)-ECE-SV2-DHFR.
  • This plasmid contains components of the expression vectors pECE (25) and pSV2-dhfr (53), provided by Dr. William Rutter (University of California, San Francisco) and Dr. Gordon Ringold (Syntex, Palo Alto), respectively.
  • pSV2- DHFR-ECE-HTPO was digested with Sal I, the ends blunted with the Klenow fragment of DNA polymerase I, and the hTPO cDNA released by digestion with Xba I.
  • the remaining vector (pSV2- DHFR-ECE) was treated with bacterial alkaline phosphatase, gel purified, and recovered in SeaPlaque agarose (FMC BioProducts, Rockland ME). Mutated hTPO cDNA, also recovered in SeaPlaque agarose, was ligated into this vector.
  • membrane protein or 30 ⁇ g of deoxycholate (DOC)-extracted protein from pHTPO-ECE-transfected cell lines (CH0-HTP04, CHO- HTPO ⁇ , CH0-HTP014, CH0-HTP017), from a control cell line co- transfected with pECE and pSV2-neo, and from another control cell line transfected with pSV2-neo alone, were subjected to SDS polyacrylamide gel electrophoresis under reducing conditions.
  • the proteins were electrotransferred to nitrocellulose membranes and then probed, as described herein, with a mouse mAb against the thyroid microsomal antigen (33).
  • Comparison of the recombinant TPO signals on western blots performed under reducing and non-reducing conditions revealed the following with reduction: (a) loss of the 200 kD broad band; (b) alteration of the 110 kD signal so that it no longer clearly represents a doublet; and ⁇ (c) lessening of the specific signals so that some of the weaker sera become negative.
  • a non-immune serum described above that reacted with a band of approximately 110 kD represents a wild-type CHO protein, and not TPO.
  • recombinant human TPO was compared with Graves' thyroid microsomes as a source of antigen in an ELISA procedure.
  • the recombinant hTPO was present 1n microsomes prepared from a non-thyroidal, non- human eukaryotic cell line which cannot, therefore, contain thyroid-specific antigens other than hTPO. Nevertheless, because sera from patients with autoimmune thyroiditis contain antibodies against numerous antigens, some of which may be present in Chinese hamster ovary (CHO) cells (48), each serum sample was also assayed against microsomes prepared from control, non-.transfected CHO cells.
  • CHO Chinese hamster ovary
  • the anti-hTPO antibody ELISA data were also expressed as the difference between values obtained using the CHO-HTPO microsomes and the CH0-K1 microsomes as antigen, to correct for possible interference by anti-CHO cell antibodies (48). No significant change was found in the correlation between the thyroid microsomal and the recombinant hTPO assays using these revised data at each of the three serum dilutions.
  • the precision of the anti-hTPO antibody ELISA was assessed using three sera chosen to represent a spectrum of autoantibody potency.
  • the intra-assay coefficients of variation (CV) for these sera were 5.12%, 7.39%, and 10.2%, respectively.
  • the inter-assay CV's (7 iterations for each serum) were 5.36%, 7>.63%, and 7.29%, respectively.
  • CHO cell expression of human TPO can be significantly increased by employing a different plasmid.
  • a dihydrofolate reductase (DHFR)-TPO construct has been made in which both genes (DHFR and TPO) are driven by the SV40 promoter ( Figure 4). Screening of CHO cells transfected with these constructs has produced two plasmids, designated pHTP0-DHFR-2B' and pHTP0-DHFR-4C, which presently express three-fold more antigen than that achieved using the pECE-HTPO plasmid.
  • TPO gene is toxic to the host CHO cells, resulting in selection for DHFR, but against TPO, at higher methotrexate concentrations.
  • the result of such selection might be that DHFR is amplified while TPO is deleted.
  • the full length TPO gene is membrane-associated, the present inventor hypothesized that it may be possible to increase TPO production in CHO cells if the expressed protein could somehow be dissociated from the membrane. Accordingly, experiments have been undertaken to generate a secretable form of human TPO, by identifying and eliminating the wild-type transmembrane sequence from the gene.
  • TPO activity (1.0 guaiacol U/10 ml medium) was clearly present in the culture medium from the CHO cells expressing the mutated form of hTPO (Fig. 16).
  • TPO activity there was no detectable enzymatic activity in conditioned media from CHO cells expressing wild-type hTPO (Fig. 16), despite strong TPO activity present in lysates of these cells, as previously described (48).
  • TPO Thyroid Peroxidase
  • Infiltrating mononuclear cells were extracted from the thyroidectomy specimen of a 26 year old female (CX81:HLA-A1, 2; B8, 37; DR3; DRw52; DQw2) with persistently relapsing Graves' disease and a high titer of antithyroid microsomal antibodies (1:640) by enzyme digestion followed by overnight incubation and separation of the non-adherent cells as previously described (Londei, M. et al ., Science 228:85-89 (1985)).
  • the activated cells were selectively expanded by growth in recombinant IL-2 (Ajinomoto - 20 ng/ml) and 10% human serum in RPM1-1640 (Gibco) for one week.
  • Cells were further expanded, nonspecifically with the addition of irradiated autologous peripheral blood lymphocytes as feeder cells, 0KT3 monoclonal antibody (30 ng/ml) and IL-2 for two weeks prior to cloning at limited dilution (0.5 cells/well) with 0KT3/IL-2 and DR-matched antigen presenting cells (APC). Further expansion and maintenance of all clones was by 1-2 weekly restimulations with 0KT3/IL-2 and HLA unmatched irradiated feeder cells. Cells were assayed at the end of the feeding cycle and a minimum of 5 days after their last exposure to IL-2.
  • Proliferation assays were performed over 3 days in triplicate microtiter wells. Irradiated autologous PBL (2 - 5 x 10 4 ) were added to 10 4 clone T cells in 200 ⁇ l of 10% human serum. 1 ⁇ l of neat microsome (protein concentration 5 mg/ml) was added per well. 1 ⁇ Ci of [ 3 H]thymidine was added for the final 6 hours of the assay prior to harvesting onto glass fiber filters and scintillation counting. Peripheral blood mononuclear cells purified by sucrose gradient centrifugation (Lymphoprep - Nycomed) were incubated at 10 5 cells per well in microtiter wells containing 200 ⁇ l 10% human serum. Control or TPO microsomes in 1-2 ⁇ l were added per well as above. Cultures were incubated for 5-6 days and pulsed with [ 3 H]thymidine in the last 6-18 hours prior to harvesting and scintillation counting.
  • T cells In vivo ⁇ activated thyroid infiltrating T cells were selected by growth in recombinant IL-2. The resultant population was then further expanded non-specifically by stimulation with anti-ClB3 antibodies (0KT3) in combination with IL-2. Lines so derived consistently showed a marked response to autologous thyroid epithelial cells in the absence of added antigen-presenting cells (APC). For example, the following levels of T cell stimulation, measured as incorporation of radiolabeled thymidine, were observed: T cells: 51 ⁇ 3 cpm; Thyroid epithelial cells (TEC): 62 ⁇ 8 cpm; T cells + TEC: 6108 + 1040 cpm.
  • TEC thyroid epithelial cells
  • T cell clones were obtained by plating the lines at limiting dilution (0.5 cells/well) followed by further expansion with IL-2 and 0KT3. In this way, antigen-specific selection was avoided prior to screening of the clones.
  • the complete sequence of human TPO cDNA was cloned into the mammalian expression vector pECE and transfected into Chinese Hamster Ovary (CHO) cells as described above. These transfected cells express high levels of immunoreactive and enzymatically active TPO. Microsomes prepared from transfected CHO cells were found to induce significant proliferation of 5 of 24 clones derived from the intrathyroidal population ( Figure 17A). These cells showed no response to untransfected CHO microsomes ( Figure 17A).
  • peripheral blood T cells from the same individual, from other Graves' patients, or from normal controls, responded to both transfected and untransfected preparations (Figure 17B).
  • PBL reactivity to CHO cell derived proteins is not unexpected as similar reactivity has been described with other xenogeneic cell extracts (Van Vliet, E. et al .. Europ. J. Immunol. 19:213-216 ,(1989)).
  • it demonstrates the difference in antigenic repertoire between thyroid infiltrating and peripheral blood T cells, as at no time was any response to untransfected CHO microsomes seen with thyroid-derived T cells ( Figure 17A and Table IV).
  • NP-7 (10 ⁇ g/ml) or control or TPO microsomes (0.5 to 1 ⁇ l) were added per well as indicated.
  • Autologous irradiated PBL or EBV-transformed B cells were used as antigen presenting cells (APC) at an APC:T cell ratio of between 2 and 5.
  • Results are the mean cpm ( ⁇ S.E.M.) of triplicate wells. Positive results were confirmed in 2 to 7 different experiments.
  • Clones were further screened using a panel of 10 synthetic peptides based on the TPO sequence, selected using two T cell motif algorithms (Rothbard, J.B., Ann. Inst.
  • T cell epitopes 535-551 These results also define the site of a T cell epitope within TPO (residues 535-551) and provide evidence for the presence of at least two distinct epitopes on a single target molecule in the same individual. Such information is very important . for the design of appropriate peptide-based immunotherapy, as discussed above.
  • the nucleotide sequences of seven randomly selected clones recognized by mAb-47 were determined. All the clones spanned the same region of the TPO cDNA, overlapping in the region of 2180-2171 bp. This region encodes 30 amino acids (at position 698-728) in the TPO protein.
  • Anti-TPO mAb-47 is unique among 13 mAbs tested in that it recognizes a continuous epitope on TPO. The other mAbs presumably recognize discontinuous epitopes. The competitive binding to TPO of mAb-47 and naturally occurring anti-TPO autoantibodies suggests that mAb-47 defines a natural, disease-associated TPO epitope.
  • TPO is an extremely large antigen (approximately 107 kD), and these techniques have not allowed definition of the precise epitopes involved.
  • the present inventor therefore undertook to screen, with sera from patients with Hashimoto's thyroiditis, a bacteriophage (lambda-Zap) human thyroid cDNA expression library containing large numbers of hTPO cDNA fragments. Each of these fragments is 200-500 b.p. in length, coding for TPO polypeptides of 66-166 amino acids.
  • the entire hTPO protein comprises 933 amino acids.
  • These TPO polypeptide fragments are expressed as bacterial fusion proteins, so called because the protein is a hybrid of a 10 kD fragment of 0-galactosidase linked to the thyroid protein component.
  • TPO cDNA fragment library construction A full-length (3.05 kb) cDNA clone as described above for human thyroid peroxidase was released from its Bluescript vector (Stratagene, San Diego, CA.) by digestion with EcoRI (BRL Laboratories, Gaithersburg, MD) and Notl (Boehringer, Mannheim, West Germany). Because both vector and insert are of similar length, the Bluescript was further digested with Seal (New England Biolabs, Beverly, MA.). The TPO cDNA was purified by agarose gel electrophoresis and electroelution.
  • the cDNA was then digested (6 minutes at room temperature) into small random-sized fragments with DNAase I (0.1 ng DNase/ ⁇ g cDNA) (BRL) in 20 mM Tris-HCl, pH 7.5, 1.5 M MnCl 2 and bovine serum albumin, 100 ⁇ g/ml . After electrophoresis in 2% SeaPlaque agarose (FMC Bio Products, Rockland, ME), TPO cDNA fragments 200-500 b.p. in length were recovered by electroelution.
  • DNAase I 0.1 ng DNase/ ⁇ g cDNA
  • PCR (30 cycles) was for 1 minute at 94°C, 2 minutes at 55 ⁇ C and 1 minute at 72 ⁇ C.
  • the PCR-generated DNA was labeled with 3 2 ⁇ - ⁇ CTP to a specific radioactivity of 0.8 x 10 9 cpm/ ⁇ g DNA using the random primer method (Multiprime; Amersham, Arlington Heights, IL).
  • the screening procedure employed standard techniques (26), with final washes of 30 minutes (x 2) at 55'C in 0.1 x SSC, 1% SDS buffer (1 x SSC in 150 NaCl, 15 mM Na citrate, pH 7.5).
  • Autoradiography of the nitrocellulose filters was performed with Kodak XAR-5 film.
  • TPO sub-librarv The lambda- Zap library containing TPO cDNA fragments, plated in E. coli Y1090 at about 3 x 10 4 pfu per 150 mm diameter Petri dish, was screened as previously described (20). In brief, after 3.5 hours at 42'C, nitrocellulose filters soaked in lOmM isopropyl-thio-beta-D-galactopyranoside (IPTG) were overlayed for 3.5 hours at 37"C.
  • IPTG isopropyl-thio-beta-D-galactopyranoside
  • Antisera from 13 Hashimoto's thyroiditis patients with high titer antimicrosomal antibodies were used under a variety of different conditions at a dilution of »1:200.
  • screening of the lambda-ZAP libraries provided very little background -with such sera, and, in general, pre-adsorption was not required to reduce this non-specific background.
  • Y1090 proteins were immobilized on nitrocellulose filters.
  • affinity-purified anti-TPO antibodies, prepared using recombinant hTPO expressed on the surface of Chinese hamster ovary (CHO) cells also were used as immobilized antigen (48).
  • TPO-CHO cells (approximately 10 8 ) were resuspended by light trypsinization, diluted in PBS containing 10% calf serum, pelleted (5 minutes at 1,000 x g), and resuspended in the diluted antibody for 1 hour at 4 ⁇ C. Unbound antibody was removed by pelleting the cells, followed by a rinse in ice-cold PBS.
  • the cells were resuspended and incubated for 15 minutes at 4°C in 150 mM acetic acid in PBS containing 0.05% Na azide and 1 mM PMSF. NaOH and 1 M Tris, pH 7.5, were added to neutralize the acetic acid, and the cells and particulate material were removed by centrifugation (5 minutes at 1,000 x g, and then for 30 minutes at 100,000 x g, 4°C), leaving the affinity-purified antibody in the supernatant. The efficiency of the affinity purification was approximately 50%, as measured by ELISA (49).
  • the detection systems for antibody bound to fusion proteins were as previously described (20), using the following antisera: For the mouse antimicrosomal monoclonal antibody, peroxidase-conjugated, affinity-purified goat anti- mouse IgG (heavy and light chain specific) (Cappel , Organon, West Chester, PA.) at a dilution of 1:300; For the polyclonal human antisera, anti-human IgG (Fc fragment, gamma chain specific) (Cappel) at a dilution of 1:300. Color was developed with 2.8 mM 4-chloro-l-naphthol (Sigma, St. Louis, M0).
  • the quality of the immunological reagents used in the polyclonal antibody screening procedure was confirmed by their ability to generate a strong signal with eukaryotic recombinant hTPO on western blot analysis (48). Positive clones were plaque-purified to homogeneity. Control screening of potentially positive plaques was performed by omitting the first (anti-TPO) antibody in the screening procedure.
  • Nucleotide sequence analysis of selected clones Plaque- purified lambda-Zap phage were used to generate Bluescript plasmids containing the fragment of TPO cDNA whose respective fusion proteins had been detected by the antisera. This procedure used the helper phage R408 according to the protocol of the manufacturer (Stratagene). Rescued phagemids were used to infect XL!-blue bacteria (Stratagene). Plasmids were prepared from individual colonies (26), and the sizes of the cDNA inserts were assessed by digestion with EcoRI. Nucleotide sequencing of selected plasmid cDNA inserts was performed by the dideoxynucleotide termination method (52). Nucleotide sequence analysis was performed using the software provided by Bionet.
  • Epitope(s) for the antimicrosomal/TPO antibodies in autoimmune thyroid disease Approximately forty screenings of the same TPO cDNA fragment sub-library described above with sera from patients with Hashimoto's thyroiditis did not yield any positive clones. The modifications that were tried included: 1) the use of different host bacteria (BB4, XL1 blue and Y1090) in which to express the TPO fusion proteins; 2) variation in the antibody binding detection system, including the use of anti-human IgG antibody or protein A from different vendors, as well as different incubation times and temperatures; and 3) the use of thirteen different patients' sera with potent anti-TPO activity.
  • BB4, XL1 blue and Y1090 variation in the antibody binding detection system, including the use of anti-human IgG antibody or protein A from different vendors, as well as different incubation times and temperatures.
  • the sera were tested in multiple ways: without bacterial pre-adsorption; following adsorption with bacterial lysate; or after affinity-purification with recombinant hTPO.
  • the monoclonal antibody always yielded the expected number of positive clones.
  • oligo-dT-primed library contains numerous full-length copies of TPO cDNA (3.1 kb), as was demonstrated by the ability to express enzymatically active, antigenically intact TPO, when such cDNA was subcloned from the phage vector into a eukaryotic expression plasmid, and stably-transfected into eukaryotic Chinese hamster ovary cells (48).
  • a potential difficulty with protein expression in a full-length cDNA phage library is that stop codons in the 5'- untranslated region of the cDNA insert may interrupt the translation of the foreign protein, which is inserted downstream of the j8-galactosidase portion of the fusion protein.
  • two additional approaches were attempted. The first was screening of a random-primed human thyroid cDNA lambda-ZAP library, constructed in the same manner as the oligo-dT primed library, with the exception that random primers, rather than oligo-dT, were used for first strand cDNA synthesis. This library contains cDNA clones with a bias against full-length cDNA copies.
  • the second approach was to delete the 5'- untranslated region from the full-length hTPO cDNA clone in the Bluescript plasmid generated from the lambda-Zap clone (48). This deletion was accomplished by digestion of this plasmid with Xhol . thereby releasing 154 b.p. of the 5'-end of hTPO cDNA, leaving the entire TPO protein (minus the signal peptide) remaining in reading frame with the ⁇ - galactosidase component of the Bluescript plasmid.
  • This new plasmid construct was transfected into XLl-Blue host bacteria for fusion protein generation (Stratagene, San Diego CA) and western blot analysis. Neither the random-primed library nor the Xhol deletion mutant generated a hTPO protein that could be recognized by Hashimoto's antisera, or with anti-TPO antibody affinity-purified from these sera using recombinant hTPO.
  • the present data provide the first definition, at a precise molecular level, of an epitope recognized by an antibody against a thyroid autoantigen.
  • Previous studies using polyclonal or monoclonal antibodies against human thyroglobulin (64,65) or TPO (55,56-61) have suggested that these antibodies recognize different regions of the antigen, but no study has been able to localize an epitope to a region of the molecule as small as 15 amino acid residues in size.
  • the minimum size of a B-cell (antibody-recognized) epitope is under discussion, but is believed to be on the order of 5-10 amino acid residues (66). Therefore, the 15 residue span of the present invention is very close to the size of the epitope itself.
  • a remarkable finding in this example is the striking contrast between the positive results with the anti- microsomal/TPO monoclonal antibody, and the inability of naturally-occurring, disease-associated anti-TPO antibodies to recognize the 66-166 amino acid TPO fragments expressed in the library employed.
  • naturally occurring B -cell epitopes may be more conformational , and subject to influence by the secondary or even tertiary structure of the molecule. Disulfide bonds and contiguity of loops' of the folded protein that may be far distant in its linear structure, may contribute to the formation of a B-cell epitope.
  • the present data suggest that the epitope(s) for the disease-associated anti-TPO antibodies are highly conformational .
  • hTPO recombinant human TPO
  • hTPO gene amplification was accomplished with a vector containing the mouse dihydrofolate reductase (dhfr) gene.
  • dhfr mouse dihydrofolate reductase
  • Stably transfected Chinese hamster ovary (CHO) cells were grown in the presence of progressively increasing concentrations of methotrexate (MTX). TPO expression was measured immunologically in an enzyme-linked immunosorbant assay (ELISA) using anti-TPO antibodies.
  • ELISA enzyme-linked immunosorbant assay
  • the blunt-ended, linearized vector and cDNA were ligated together to form the recombinant plasmid pSV2-DHFR-ECE-HTP0.
  • CHO dhfr-cells (CH0-DG44; kindly provided by Dr. Robert Schimke, Stanford University, Palo Alto, CA) were maintained in Ham's F-12 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), gentamicin (40 ug/ml) and amphotericin B (2.5 ug/ral).
  • Transfected cells were selected for in thymidine-, guanidine-, and hyposanthine-free Ham's F-12 medium supplemented with 10% dialyzed fetal calf serum and antibiotics as above. Individual clones were selected with cloning cylinders and 2 clones with high levels of TPO expression (clones CH0-HTP0-2B and CHO-HTP -C4C) were subsequently used for amplification. Methotrexate (MTX) was added to this selective cell culture medium as an initial concentration of 3.3 nM and surviving cells were harvested and expanded. The methotrexate concentration was sequentially increased by 3.33-fold increments until a final concentration of 10,000 nm (100 ⁇ M) was reached. •
  • MTX methotrexate
  • ELISA of CHO-hTPO and CHO-hTPO-Ml cells ELISA of human sera (kindly provided by Dr. Sandra McLachlan, Cambridge, Wales, UK) of control and MTX-treated CHO-hTPO cells was modified from the method of Schardt et al . (49), as described above, using cellular microsomes. Because the hTPO-Ml protein is secreted into the medium of CHO-hTPO-Ml cells, three-day conditioned media were collected from these cells. Proteins from these media were precipitated and treated, as described above.
  • Antigen for ELISA of human sera was applied as 100 ul of the dialyzed protein precipitate per well, approximately 300 ug protein diluted 1:1 in 2 x coating buffer (0.1 M sodium bicarbonate, pH 9.3 + 0.04% sodium azide). Because more than one ELISA was used for all MTX concentrations, values are reported as an ELISA index referenced to 1000 nM MTX values used across assays of each cell type. The same sera were used in ELISAs of each cell type.
  • Genomic DNA extraction of CHO-hTPO-Ml cells Cells from confluent 100 mm diameter dishes of CHO-hTPO-Ml cells surviving at each MTX concentration were frozen and kept at -80 ⁇ C until replated (100 mm dish), grown to confluence, and used for extraction of genomic DNA. Cells were rinsed three times in 5 ml ice-cold Dulbecco's phosphate-buffered saline, calcium- and magnesium-free (PBS-CMF). The cells were then scraped from the dish, recovered by centrifugation for 10 minutes at 2000 rpm, 4 D C.
  • the pellet was resuspended in 2 volumes (100-200 ul) 320 mM sucrose, 10 mM Tris-Cl, pH 7.5, 5 mM MgCl2 > 1% Triton X-100, and kept on ice for 30 minutes. The suspension was centrifuged for 15 minutes at 2500 rpm (4 ⁇ C), and the pellet resuspended in 4.5 ml 10 mM NaCl, 10 M Tris-Cl, pH 7.5, 10 mM EDTA.
  • RNAse digestion (addition of 4.5 ⁇ l 10 mg/ml DNase-free RNase for 60 min at room temperature) was followed by proteinase K digestion overnight at 37 ⁇ C (addition of 0.5 ml 10% SDS + 0.1 ml 10 mg/ml proteinase K).
  • the DNA was then extracted two or three times (until the aqueous phase was clear) with 5 ml 0.1 M Tris-buffered phenol, pH 7.4:CHCl3, 4% isoamyl alcohol (1:1), followed by an equal volume extraction with CHCI3, 4% isoamyl alcohol.
  • Genomic DNA yield from a 100 mm dish of confluent cells was 40-160 ug.
  • Genomic DNA (15 ug) from CHO-hTPO-Ml cells was digested with EcoRI, ethanol- precipitated, resuspended in TE buffer, and requantified by 0D at 260 nm. Aliquots of this DNA (1.0, 0.5, or 0.25 ug) ere diluted in 0.5 ml 0.4 N NaOH, 10 mM EDTA, boiled for 10 minutes and placed on ice.
  • Nylon membrane filters (Hybond-N RPN, 3050N, Amersham Corporation, Arlington Heights, IL), rinsed in 0.4 N NaOH, were applied to a slot-blot apparatus (Minifold II, Schleicher & Schuell , Keene, NH) and the wells were rinsed with 0.5 ml 0.4 N NaOH and vacuum dried. Individual 0.5 ml genomic DNA samples were added per well, vacuum was applied briefly, and the wells were rinsed with 0.5 ml 0.4 N NaOH and vacuum dried. The filters were removed, washed briefly in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) and air dried.
  • 2x SSC 0.3 M NaCl, 0.03 M sodium citrate, pH 7.0
  • Genomic DNA was cross-linked to the filters by UV irradiation (UV Stratalinker 2400, Stratagene, La Jolla, CA), and the filters probed with a labeled, PCR-derived, 0.3 kb fragment of the mouse dhfr cDNA, washed, and autoradiograms performed. Following confirmed removal of first label after boiling in O.lx SSC (0.015 M NaCl, 0.0015 M sodium citrate), 0.1% SDS for 1 hour, the filters were reprobed with a labeled 0.56 kb fragment of human TPO cDNA, washed, and photographed.
  • UV irradiation UV irradiation
  • Recombinant plasmids pSV2-dhfr-ece-hTP0 and pSV2-dhfr- ece-hTPO-Ml were transfected into CHO dhfr- cells to produce CH0-TP0 and CH0-TP0-M1 cell lines, respectively. These cell lines were grown in progressively increasing (3.33 fold) MTX concentrations up to .1000 (membrane-associated hTPO) or 10,000 (secreted hTPO), each cycle taking a minimum of three weeks. Cells at each stage of amplification were cryo ⁇ preserved and were replated after the final amplification step for comparison of the levels of immunoreactive hTPO expression.
  • TPO expression increased markedly over baseline beginning at 333 nM MTX, with progressive increments up to the highest concentration of used (10 uM).
  • Slot-blot analysis of genomic DNA from CH0-HTP0-M1 cells using either a dhfr or hTPO DNA probe revealed similar amplification patterns parallel to that of the pattern of TPO protein expression.
  • TPO TPO available from the membrane-associated (CH0-HTP0-2B cells) and secreted protein (CH0-HTP0-M1 cells) for immunological detection in an ELISA.
  • EXAHPLE XV EXAHPLE XV
  • Carbohydrate moieties on hTPO may contribute to the epitopes recognized by anti-hTPO antibodies in Hashimoto's thyroiditis. This is because bacterial fusion proteins, unlike proteins expressed in eukaryotic cells, are not glycosylated. Very little is known about the carbohydrate moieties in hTPO. Human TPO (67) and the microsomal antigen (68) are bound to the lectin concanavalin A. The latter is also partially bound to lentil lectin (68). It is unknown whether the hTPO carbohydrate structures are N-linked, 0- linked, or both. In the present example, the nature of the carbohydrate components of hTPO was examined, and whether or not hTPO carbohydrate plays a role in the structure of naturally occurring epitopes in Hashimoto's thyroiditis.
  • CHO cells stably expressing human hTPO (CHO-TPO 12g) (48) were cultured in 100 mm diameter dishes in F12 medium containing 10% fetal calf serum, 100 U/ml penicillin, 40 ⁇ g/ml gentamicin and 2.5 ⁇ g/ l amphotericin B.
  • subconfluent cells were rinsed twice in phosphate-buffered saline without calcium and magnesium (PBS-CMF), and were then incubated for 15-20 minutes in methionine-free F12 medium (3 ml/dish) containing 10% dialyzed fetal calf serum. 3 5s-methionine (>1100 Ci/mmol; Amersham, Arlington Heights, IL) was then added to the medium (0.2 mCi/ml), and the incubation was continued for 2-4 hours at 37 ⁇ C.
  • PBS-CMF phosphate-buffered saline without calcium and magnesium
  • the medium was removed and the cells were rinsed twice in PBS-CMF, scraped into ice-cold PBS-CMF, pelleted for 10 minutes at 1000 x g (4 ⁇ C), washed once in 10 ml of the same buffer, and the cell pellet resuspended (0.3 ml/dish of cells) in homogenization buffer (50 mM Hepes, pH 7.5, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 2 mg/ml bacitracin, 0.25 M TLCK (N-p-tosyl-1-lysine chloro-methyl ketone) and 0.1 mM leupeptin (Sigma Chemical Co., St.
  • homogenization buffer 50 mM Hepes, pH 7.5, 1% Triton X-100, 0.1 mM phenylmethylsulfonyl fluoride, 2 mg/ml bacitracin, 0.25 M
  • the 1 ml of solubilized cellular proteins was pre- adsorbed twice for 10 minutes at room temperature with 80 ul of 10% IgG-Sorb (Staphylococcus A) (The Enzyme Center, Maiden, MA), followed by removal of the IgG-Sorb by centrifugation for 3 minutes at 10,000 x g in a microfuge.
  • IgG-Sorb Staphylococcus A
  • Hashimoto's thyroiditis sera with high titers (ELISA readings >1.5 O.D. units) of anti-hTPO antibodies were added to a final dilution of 1:200. Similar results were obtained with three separate sera.
  • IgG-Sorb After incubation overnight at 4 ⁇ C, 150 ⁇ l of IgG-Sorb was added, and the tubes rotated end over end for 2-4 hours at room temperature. The IgG-Sorb was recovered by centrifugation for 5 minutes at 10,000 x g, washed 5 times with 1 ml of immunoprecipitation buffer, and then once with 10 mM Tris, pH 7.5, 2 mM EDTA and 0.5% Na dodecylsulfate. Finally, the pellet was resuspended in Laemmli sample buffer (31), with 50 mM dithiothreitol (DTT), boiled for 3 minutes, and applied to 6% polyacrylamide gels. Molecular weight markers (Sigma; St.
  • Enzymatic deglvcosylation of immunoprecipitated human TPO Recombinant, radiolabeled hTPO, immunoprecipitated and bound to IgG-Sorb, as described above, was recovered in enzymatic digestion buffers rather than in Laemmli sample buffer.
  • Enzymatic digestions (18 hours at 37'C) were as follows: endoglycanase F (Boehringer-Mannheim, West Germany, 30 U/ml; in 100 M Na phosphate buffer, pH 6.0, 50 mM EDTA, 0.1% SDS, 1% beta-mercaptoethanol and 1% NP40); endoglycanase H (Boehringer, 0.2 U/ml; in the same buffer as for endo F, except that EDTA was omitted); 0-glycanase (Boehringer, 2.5 U/ml; same buffer as for endo H); and neuraminidase (Sigma, 1 U/ml; in 100 mM Na acetate, pH 5.2, 5 M EDTA and 1% ⁇ - mercaptoethanol).
  • each experiment included a sample incubated in parallel without added enzyme.
  • Lectin affinity chromatography Detergent extracts of CH0-TP0 cells (5-7 100 mm diameter dishes) were radiolabeled with 5s-methionine (see above) and applied to 2 ml bed volume columns of Concanavalin A (Con A), peanut agglutinin (PNA), wheat germ agglutinin (WGA), Ricinus communis agglutin 1 (RCA1) and Ulex E ⁇ ropaeus (UEA-F) agarose-bound lectins (all purchased from Vector Laboratories, Burlinga e, CA).
  • Con A Concanavalin A
  • PNA peanut agglutinin
  • WGA wheat germ agglutinin
  • Ricinus communis agglutin 1 RCA1
  • Ulex E ⁇ ropaeus Ulex E ⁇ ropaeus
  • samples were diluted to 10 ml in Buffer A (20 mM Tris HC1 , pH 7.4, 150 mM NaCl, 0.1% Triton X-100), supplemented with the following for each individual lectin: WGA and RCA1- 1 mM EDTA; Con A - ImM CaCl 2 , 1 mM MnCl 2 ; PNA - ImM CaCl 2 , 1 mM MgCl 2 ; UEA-F - ImM CaCl2- After application to the columns, the unbound proteins were removed by washing with 50 ml of the foregoing Buffer A's.
  • Buffer A (20 mM Tris HC1 , pH 7.4, 150 mM NaCl, 0.1% Triton X-100)
  • Specifically adsorbed proteins were eluted with 25 ml of the following (all at 300 mM):- WGA, N- acetylglucosa ine; PNA and RCA1, D-galactose; Con A, a- methyl-D-mannoside; and UEA-F, ⁇ -fucose. Fractions of 0.5 ml were collected and counted for radioactivity in a liquid scintillation counter.
  • the two fractions containing the peak of the eluted radioactivity were pooled,(1 ml) and subjected to immunoprecipitation with anti-hTPO antibodies in the sera of patients with Hashimoto's thyroiditis, followed by polyacrylamide electrophoresis and autoradiography (see above) .
  • the derived amino acid sequence of human TPO (17, 19, 54) suggests that there are 5 potential glycosylation sites in the extracellular domain of the enzyme. This is based on the tri-peptide algorithm for glycosylation sites of Asn-X- Ser/Thr (X refers to any amino acid; the third position can be either Ser or Thr). Carbohydrate chains can be linked to the Asn residue (N-linked), or to the Ser or Thr residues (0- linked).
  • hTPO was digested with a number of deglycosylating enzymes of varying specificity.
  • proteins in Chinese hamster ovary (CHO) cells expressing recombinant hTPO were radiolabeled with 3 ⁇ S-methionine, followed by immunoprecipitation with anti-hTPO antibodies present in the serum of patients with Hashimoto's thyroiditis.
  • hTPO As observed previously on western blot analysis (48), recombinant hTPO was present as a doublet of approximately 115 kD and 110 kD, with the relative dominance of the 115 kD and the 110 kb bands varying from experiment to experiment.
  • Endo H which acts similarly to endo F on polymannose but differently from endo F on complex glycans, also converted the mobility of hTPO to a 110 kD and 105 kD doublet.
  • O-glycanase and neura inidase which remove 0-1inked glycans and terminal neuraminic acid, respectively, did not alter the mobility of radiolabeled hTPO.
  • Lectin affinity chromatography (69) provided further support for the polymannose nature of the hTPO carbohydrate moieties.
  • radiolabeled, recombinant hTPO was retained only on concanavalin A-Sepharose, which binds with high affinity to N-linked oligosaccharides in which at least two outer mannose residues are either unsubstituted, or are substituted only at position C-2 by another sugar.
  • Bound hTPO could be eluted with 300 mM ⁇ -methyl-D-mannoside.
  • TPO did not bind to wheat germ agglutinin (specificity for terminal and internal GlcNac and terminal neuraminic acid), Ricinus communis agglutinin 1 (RCA1) (highest affinity for bi- and tri-antennary N-l inked oligosaccharides with terminal galactose residues), peanut agglutinin (terminal Gal-J-1,3- GalNac) or Ulex europaeus (terminal ⁇ -L-fucose).
  • RCA1 Ricinus communis agglutinin 1
  • RCA1 highest affinity for bi- and tri-antennary N-l inked oligosaccharides with terminal galactose residues
  • peanut agglutinin terminal Gal-J-1,3- GalNac
  • Ulex europaeus terminal ⁇ -L-fucose
  • Radiolabeled recombinant hTPO was first partially purified by concanavalin A-Sepharose affinity chromatography, next digested with three different glycanases, and finally subjected to immunoprecipitation with anti-hTPO antibody in Hashimoto s thyroiditis serum. Complete removal of the N- 1inked carbohydrate chains distal to the chitobiose core with endo F and endo H did not prevent antibody binding. In view of the complexity of these experiments, it is important to note the completeness of N-glycanase treatment. Thus, after deglycosylation, all of the hTPO immunoprecipitated was as the smaller (110 kD and 105 kD) doublet. As a further control, digestion with O-glycanase led to the immunoprecipitation of an unaltered hTPO form (115 kD and 110 kD).
  • thyroid microsomal antigen (68) and immunopurified, non-recombinant human TPO (67) are bound by concanavalin A.
  • the present inventor is not aware of other data on the nature of the oligosaccharide (glycan) moieties in human TPO.
  • the present data provide new information on this subject.
  • the present data indicate that the oligosaccharides present in hTPO do not significantly influence the epitopes recognized by anti-hTPO antibodies in the sera of patients with autoimmune thyroid disease, primarily Hashimoto's thyroiditis.
  • An assumption inherent in the present example is that the glycan components of recombinant hTPO are structurally the same as those in TPO present in human thyroid cells in vivo. While it cannot be excluded that Chinese hamster ovary cells may glycosylate the hTPO polypeptide chain in a manner different from human thyroid cells, it is likely that any such differences would be minor.
  • glycosylation in eukaryotic Chinese hamster ovary cells would be very similar, if not identical, to that in human thyroid cells. Further support for this assumption is that recombinant hTPO in Chinese hamster ovary cells is functionally active, at the same level present in human thyroid cells in monolayer culture (48). In addition, virtually all sera from patients with Hashimoto's thyroiditis that contain anti-microsomal antibodies can recognize this form of recombinant human TPO on western blot analysis (48) or by ELISA.- Thus, by definition, the recombinant human TPO of the present invention contains the relevant, disease- associated epitopes on hTPO.
  • oligosaccharide components In hTPO are not part of the "natural" epitopes recognized by anti-hTPO antibodies in the sera of patients with autoimmune thyroiditis.
  • the glycosylated portion of the molecule could influence the interaction of the antibody with its epitope(s), such as by altering the affinity of this interaction.
  • This three-dimensional configuration may be lacking in peptide fragments, or may be altered by the 0-galactosidase component of the bacterial fusion protein.
  • the present data relate to the recognition of epitopes on TPO by antibodies (B-cell epitopes). These B-cell epitopes are now recognized to be distinct from epitopes presented to T-cells in a major histocompatibility antigen (MHC) restricted manner (71). B-cell epitopes are likely to be important in mediating damage by the immune system to the thyroid gland, while T-cell epitopes are likely to be relatively more important in the initiation of the autoimmune process.
  • MHC major histocompatibility antigen
  • Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis. J. Clin. Invest. 81:657-660.
  • Endo Y Nakano J, Horinouchi K, Ohtaki S, Izumi M, Ishikawa E. An enzyme immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum. Clin Chim Acta. 1980;103:67-77.

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Abstract

L'invention concerne la peroxydase de la thyroïde haumaine de recombinaison (HTPO). Elle concerne également des séquences d'ADN codant une protéine hTPO tronquée, sécrétable et ayant une action enzymatique, ainsi que des vecteurs, y compris des plasmides, comprenant ces séquences, et des hôtes transformés par lesdits vecteurs. L'expression eukaryotique non thyroïdale a été obtenue. hTPO selon l'invention, et des anticorps dirigés contre hTPO sont décrits et sont utiles pour la détection, le diagnostic et la thérapie de maladies immunes, en particulier la thyroïdite de Hashimoto.
PCT/US1990/004289 1989-07-31 1990-07-31 Peroxydase de la thyroide humaine de recombinaison WO1991002061A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0421139A2 (fr) * 1989-09-05 1991-04-10 Nippon Suisan Kaisha, Ltd. Procédé pour la production de la péroxydase thyroide humaine
WO1993023073A1 (fr) * 1992-05-19 1993-11-25 The Regents Of The University Of Michigan Regions d'epitopes de thyroide-peroxydase
EP0668918A4 (fr) * 1991-08-28 1994-11-28 Institute Diagnostics Nichols Auto-anticorps humains associes a une pathologie specifique a la peroxidase thyroidique humaine.
WO1995002824A1 (fr) * 1993-07-13 1995-01-26 B.R.A.H.M.S Diagnostica Gmbh Procede et materiel de determination quantitative de la peroxydase de la thyroide humaine
US5705400A (en) * 1992-03-07 1998-01-06 Rsr Limited Assay for adrenal autoantigen
EP2518166A2 (fr) 2005-05-20 2012-10-31 Veridex, LLC Dosage moléculaire par aspiration de la thyroïde au moyen d'une aiguille fine
CN112501141A (zh) * 2020-10-22 2021-03-16 重庆中元汇吉生物技术有限公司 一种增加人甲状腺过氧化物酶产量的试剂和表达方法

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Publication number Priority date Publication date Assignee Title
JP6277684B2 (ja) * 2013-11-18 2018-02-14 大日本印刷株式会社 細胞積層体の製造方法
JP2018078906A (ja) * 2018-01-17 2018-05-24 大日本印刷株式会社 細胞積層体の製造方法

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EP0139417B1 (fr) * 1983-08-30 1989-07-26 Genentech, Inc. Vaccins à base de protéines liées à des membranes et procédé pour leur préparation

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CA2024562C (fr) * 1989-09-05 2000-09-26 Shinya Yamashita Cellule cho transformee avec un plasmide recombinant exprimant de la perroxydase thiroidienne humaine et methode de preparation

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EP0139417B1 (fr) * 1983-08-30 1989-07-26 Genentech, Inc. Vaccins à base de protéines liées à des membranes et procédé pour leur préparation

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Cell, Vol. 45, issued 6 June 1986, ELLIS et al., "Replacement of Insulin Receptor Tyrosine Residues 1162 and 1163 Compromises Insulin-Stimulated Kinase Activity and Uptake of 2-Deoxyglucose", pages 721-732, see e.g., Figure 1. *
F.T. MAGGIO, "Enzyme-Immunoassay", published 1980 by CRC Press (Florida), see pages 167-170 and 190-194, especially Figures 1 and 2. *
Febs Letters, Vol. 190, No. 1, issued October 1985, CZARNOCKA et al., "Purification of the Human Thyroid Peroxidase and its Identification as the Microsomal Antigen Involved in Autoimmune Thyroid Diseases", pages 147-152, see entire document. *
Journal Of Biological Chemistry, Vol. 262, No. 29, issued 15 October 1987, MAGNUSSON et al., "Molecular Cloning of the Structural Gene for Porcine Thyroid Peroxidase", pages 13885-13888, see especially page 13887. *
Journal Of Clinical Endocrinology And Metabolism, Vol. 62, No. 1, issued 1986, KOTANI el al., "Detection of Autoantibodies to Thyroid Peroxidase in Autoimmune Thyroid Diseases by Micro-Elisa and Immunoblotting", pages 928-933, see e.g., Abstract. *
Journal Of Clinical Endocrinology And Metabolism, Vol. 63, No. 2, issued 1986, OHTAKI et al., "Characterization of Human Thyroid Peroxidase Purified by Monoclonal Antibody-Associated Chromatography", pages 570-576, see e.g., Abstract. *
Journal Of Clinical Investigations, Vol. 80, issued October 1987, SETO et al., "Isolation of a Complementary DNA Clone for Thyroid Microsomal Antigen", pages 1205-1208, see e.g., page 1207. *
Nature, Vol. 294, issued 19 November 1981, LEE et al., "Glucocorticoids Regulate Expression of Dihydrofolate Reductase cDNA in Mouse Mammary Tumor Virus Chimeric Plasmids", pages 228-232, see e.g., Figure 2. *
Nucleic Acid Research, Vol. 15, No. 16, issued 1987, LIBERT et al., "Complete Nucleotide Sequence of the Human Thyroperoxidase-Microsomal Antigen cDNA", page 6375. *
See also references of EP0483281A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0655502A1 (fr) * 1989-09-05 1995-05-31 Nippon Suisan Kaisha, Ltd. Cellules de type CHO transformées par un plasmide recombinant exprimant la péroxydase thyroid humaine de type sécretée et leur préparation
EP0421139A3 (en) * 1989-09-05 1992-09-02 Nippon Suisan Kaisha, Ltd. Method of measuring antimicrosormal antibody and process for the production of human thyroid peroxidase used for the measurement
EP0421139A2 (fr) * 1989-09-05 1991-04-10 Nippon Suisan Kaisha, Ltd. Procédé pour la production de la péroxydase thyroide humaine
US7196181B1 (en) 1990-01-30 2007-03-27 Quest Diagnostics Investments, Inc. Sequences encoding novel human thyroid peroxidase proteins and polypeptides
EP0668918A1 (fr) * 1991-08-28 1995-08-30 Institute Diagnostics Nichols Auto-anticorps humains associes a une pathologie specifique a la peroxidase thyroidique humaine
EP0668918A4 (fr) * 1991-08-28 1994-11-28 Institute Diagnostics Nichols Auto-anticorps humains associes a une pathologie specifique a la peroxidase thyroidique humaine.
US5705400A (en) * 1992-03-07 1998-01-06 Rsr Limited Assay for adrenal autoantigen
US5998153A (en) * 1992-05-19 1999-12-07 The Regents Of The University Of Michigan Thyroid peroxidase epitopic regions
US6528059B1 (en) * 1992-05-19 2003-03-04 Regents Of The University Of Michigan Thyroid peroxidase epitopic regions
WO1993023073A1 (fr) * 1992-05-19 1993-11-25 The Regents Of The University Of Michigan Regions d'epitopes de thyroide-peroxydase
WO1995002824A1 (fr) * 1993-07-13 1995-01-26 B.R.A.H.M.S Diagnostica Gmbh Procede et materiel de determination quantitative de la peroxydase de la thyroide humaine
EP2518166A2 (fr) 2005-05-20 2012-10-31 Veridex, LLC Dosage moléculaire par aspiration de la thyroïde au moyen d'une aiguille fine
CN112501141A (zh) * 2020-10-22 2021-03-16 重庆中元汇吉生物技术有限公司 一种增加人甲状腺过氧化物酶产量的试剂和表达方法

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