EP0604453A1 - Disease associated b-cell epitopes of tpo and use thereof - Google Patents
Disease associated b-cell epitopes of tpo and use thereofInfo
- Publication number
- EP0604453A1 EP0604453A1 EP92916802A EP92916802A EP0604453A1 EP 0604453 A1 EP0604453 A1 EP 0604453A1 EP 92916802 A EP92916802 A EP 92916802A EP 92916802 A EP92916802 A EP 92916802A EP 0604453 A1 EP0604453 A1 EP 0604453A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- tpo
- htpo
- cells
- antibody
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to 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 (Volpe, R., In Werner's The Thyroid, 5th Edition (Ingbar, S.H., et al ., Eds.), J.B. Lippincott Co., Philadelphia, pp. 1266-1291 (1986); Gordin, A., et al.. Acta Endocrinol. 90:33-42 (1979)).
- Antibodies against a number of thyroid antigens are present in the sera of these patients, including thyroglobulin and the thyroid "microsomal" antigen (Doniach, D., et al ., Clin. Endocrinol.
- Antibodies against the microsomal antigen, which is expressed on the cell surface 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 (Volpe,. R., In Werner's The Thyroid. 5th Edition (Ingbar, S.H., et al.. Eds.), J.B.
- 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 autoant genes such as thyroglobulin, which is highly abundant.
- a reported fusion protein originally described as reactive with 19 of 20 Hashimoto patient sera (Libert, R., et al.. EMBO J. 6:4193-4176 (1987), clone C2) has, upon immunopurification with anti-.-galactosidase mAbs, been found to react with fewer Hashimoto patient sera in an ELISA assay (Dinsart, C, et al.. 17th Annual Meeting of the European Thyroid Association. Abstract #235 (1988)).
- 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 "antimicrosomal" 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.
- 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. Unlike bacterial fusion proteins previously reported, the conformation of this protein is not encumbered by the ⁇ - galactosidase fusion partner. Furthermore, unlike the bacterially-produced protein, the TPO is glycosylated. The demonstration of functional TPO activity indicates unequivocally that the cDNA previously cloned (Magnusson, R.P., et al .. J. Biol. Chem. £62:13885-13888 (1987); Magnusson, R.P., et al.. Mol . Endocrinol .
- the present invention also provides for the identification of the 0-cell epitope on thyroid peroxidase associated with autoimmune thyroid disease.
- this aspect of the invention provides a method for identifying the molecular interaction responsible for the ?-cell recognition of thyroid peroxidase.
- the present inventor observed, under the same conditions, that the major im- munogenic form of recombinant hTPO in transfected CH0 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 CH0). 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 (Hamada, N., et al.. J. Clin. Endocrinol. Metab. 61:120-128 (1985)).
- 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)
- hTPO Naturally-occurring hTPO has been shown to be a thyroidal cell surface protein. Recom ⁇ binant, enzymatically active hTPO is also cell embrane- associated in stably transfected non-thyroidal eukaryotic cells (Kaufman, K.D., et al.. J. Clin. Invest. 84:394-403 (1989)).
- 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 hydrophobic region 846-870 corresponds 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).
- hTPO cDNA inserted into a eukaryotic expression vector, was stably transfected into CHO cells. Immunoprecipitation and PAGE of cellular 35 S- methionine-labeled proteins with Hashimoto's patient serum revealed a 105-101 kD doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kD doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable 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 im- 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.
- hTPO produced by non- thyroidal eukaryotic cells.
- 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.
- a 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.
- a 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 (Ellis, L., et al..
- FIG. 1 Fluorescence-activated cell sorter (FACS) analysis of CHO cells transfected with pHTPO-ECE. CHO-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.
- Figure 5. Relative TPO activities observed in CHO cells transfected with pECE-HTPO, pHTP0-DHFR-2B and pHTP0-DHFR-4C, shown plotted against methotrexate concentration.
- FIG. 6 Nucleotide sequence of human TPO gene after site-directed mutagenesis. The mutations incorporated two stop codons, as well as an EcoRl site for confirmation, in the region immediately upstream from the transmembrane region of the human TPO gene.
- FIG. 7 cDNA sequence and derived amino acid sequence of human thyroid peroxidase (Magnusson, R.P., et al., Mol . Endocrinol. 1:856-861 (1987)).
- Figure 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, enzymatically- 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.
- Figure 11 Comparison of 51 sera, selected to provide a spectrum of anti-MSA levels, in terms of their reactivity with Graves' thyroid .microsomes .and recombinant, enzymatically- active human TPO generated in non-thyroidal eukaryotic cells.
- Figure 12 Intra-assay variability of anti-hTPO antibody ELISA at standard (1/100) serum dilution. Mean ⁇ standard deviation of 10 iterations of anti-hTPO antibody ELISA results for three autoimmune sera selected to represent low, medium, and high autoantibody 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 ⁇ S-methionine was followed by chase with unlabeled methionine for the indicated periods of time. Immunoprecipitations 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 CH0-TP0 12g) (Kotani, T., et al.. J. Clin. Endocrinol. Metab. 62:928-933 (1986)) 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 (Magnusson, R.P., et al__, Mol. Endocrinol. 1:856-861 (1987)).
- 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.
- FIG. 19 Determination of the epitope recognized by TPO MAb 47.
- the nucleotide sequences of the 5'- and 3'-prime ends were determined for 18 clones in the TPO cDNA fragment library (see Materials and Methods) recognized by MAb 47.
- the smallest region of overlap between all 18 clones is from 2219- 2247 basepairs in the human TPO cDNA sequence, coding for the indicated amino acids.
- FIG. 20 Western blot analysis of human TPO, using TPO MAb.
- Recombinant human TPO expressed in Chinese hamster ovary cells was used as antigen under denaturing and reducing conditions (see Materials and Methods). After polyacrylamide gel electrophoresis and transfer to the membranes, the membranes were probed with the indicated antibodies.
- TPO MAb 1, 2, 9, 15, 18, 24, 30, 40, 47, 53, 59, 60 and 64 are mouse MAbs generated against native undenatured human TPO (Ruf, J., et al.. Endocrinology 125:1211-8 (1989)).
- the controls are mouse MAbs raised against denatured human TPO (Portmann, L., et al.. J. Clin. Invest. 81:1217-1224 (1988)) (A and B) and control mouse ascitic fluid (C). The sizes of the mol wt markers are shown on the left, and that of recombinant human TPO is indicated by the arrow.
- 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, Men o 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.. Principles of Gene Manipulation: An Introduction to Genetic Engineering.
- cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
- cloning it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
- cDNA is meant 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 fragments, variants, and variants, 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.
- 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.
- 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 translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
- 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 5'-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 E__ col i , Bacillus, Streptomvces, 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 replicon and control sequences in the expression plasmid.
- a prokaryotic cell such as, for example, E. coli, B. subtilis. Pseudomonas, Streptomvces. etc.
- Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
- constitutive promoters examples include the jjrt 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 ⁇ (PL and PR), the tr ⁇ , recA. lacZ. lad, and gal promoters of E. coli. the ⁇ -amylase (Ulmanen, I., et al .. J. Bacteriol.
- J _ subtilis 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. £03:468-478 (1986)).
- Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. .Biochimie 68:505-516 (1986)); and Gottesman, 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 translational regulatory signals 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- oters 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 hydrolase 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 amino 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.
- Production of 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)).
- live insects are to be used, caterpillars are presently preferred hosts for large scale human TPO production according to the invention.
- eukaryotic regulatory regions Such 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. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al ..
- yeast gal4 gene promoter Johnston, S.A., et al.. Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al.. Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)). Of these, presently the most preferred is the SV40 promoter.
- 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
- Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In:
- Suitable Streptomvces plasmids include pIJlOl (Kendall, K.J., et al.. J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as ⁇ CZl (Chater,
- 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. Svmo. 19:265-274 (1982); Broach, J.R., In: J_he 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., CgU £8:203-204 (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-cellulose, phosphocellulose, polyribocyti- dylic 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') 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.
- 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 £56:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al.. Eur. J. Immunol. 6_:292 (1976); Hammerling et al..
- 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.
- Hybridoma A New Dimension in
- 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.
- an anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
- An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. 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 antigenie 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 hemocyanin (KLH) and used to immunize additional BALB/c mice.
- Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for the hPTO epitope.
- 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 gamma counter or a scintillation counter.
- Isotopes which are particularly useful for the purpose of the present invention are 3 Hf 125 I ⁇ 32p, 35 S ⁇ 14 Cj 51 Cr> 36 clj 57 C ⁇ j 58 Co> 59 Fe and
- fluorescent labeling compounds fluoroscein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
- the antibodies of the invention also can be detectably labeled using fluorescent emitting metals such as l 52 Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal che ating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine- tetraacetic acid (EDTA). 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.
- fluorescent emitting metals such as l 52 Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal che ating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediamine- tetraacetic acid (EDTA).
- DTPA diethylenetriaminepentaacetic acid
- EDTA ethylenediamine- t
- chemiluminescent labeling compounds examples include luminal, isoluminol, thero atic 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 to 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
- immunometric assay or sandwich immuno- assays.
- tb 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 radiolabeled 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.
- IgG j , 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 o 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 i munoadsorbents 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: 9 c, 123 I ⁇ 111 IN , 131 I ⁇ 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 ⁇ g ⁇ , 55 ⁇ 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 antigenie 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.
- TPO-related peptides include at least a portion of a T cell epitope of TPO (such as the NP-7 epitope of Example XII).
- Useful peptides include a sequence of about 5 or more amino acids of TPO, or derivatives of such peptides, which are capable of binding to the TCR of a TPO-specific T cell.
- such a peptide can inhibit antigen presentation to T cells, or other antigen-specific cell-cell (e.g., 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.
- T cells or other antigen-specific cell-cell (e.g., 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.
- Another embodiment of the invention provides for a pharmaceutical preparation comprising the above peptides.
- a method of treating autoimmune disease including but not limited to Hashimoto's thyroiditis, which comprises administering to a patient suffering from such disease a pharmaceutical prepara ⁇ tion comprising a TPO-related peptide.
- 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. 158:52-60 (1988); Hoso. 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 _3£:843-845 (1988); Euroo. 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 mediate 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 disease such as Hashimoto's thyroiditis, is provided 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 Immunolooic 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 .. i. EXD. Med. 169: 27 (1989). If the autoimmune T cells can be eliminated or prevented from reacting with the TPO, the effects of thyroiditis may be greatly alleviated. 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. (Han, J.H., et al.. Biochem. £6:1617-1625 (1987)).
- Double-stranded cDNA was synthesized from 15 ⁇ g mRNA as described by Gubler and Hoffman (Gubler, U., et al.. Gene £:263-269 (1983)).
- 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 (Han, J.H., et al.. Biochem. 16:1617-1625 (1987)).
- 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.
- 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). 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 5-3060 of human thyroid peroxidase cDNA, including the start of translation and the poly-A tail.
- DNA sequence was determined from this double- stranded plasmid using the Sequenase kit and protocol (United States Biochemical, Cleveland, OH). Sequence within the cDNA was confirmed to be identical to human TPO cDNA at the 5' and 3' ends and in the regions adjacent to 10 oligonucleotide primers distributed throughout the cDNA (Magnusson, R.P., et al.. Mol. Endocrinol. 1:856-861 (1987)).
- the mammalian cell expression vector pECE (Ellis, L., et al.. Cell 45:721-732 (1986)) 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. (Maniatis, T., et al.. Molecular Biology: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). EXAMPLE IV Transfection of Chinese Hamster Ovary Cells with pHTPO-ECE
- 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 (Chen, C, et al .. Mol. Cell. Biol. 7:2745-2752 (1987)).
- 20 ⁇ g pHTPO-ECE plus 2 ⁇ g pSV2-neo (28) 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.
- Total cellular RNA was extracted by the method of
- RNA was el ectrophoresed in formaldehyde gels as described by Maniatis et al . (Maniatis, T. , et al .. Molecular Bioloov: A Laboratory Manual . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
- 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/ l 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 (Bradford, M.M., Anal. Biochem. 72:248-254 (1976)) and 50 ⁇ g protein/lane electrophoresed on a 7.5% polyacrylamide SDS gel (Laemmli, U.K., Nature 227:680-689 (1970)). 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.
- 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 (Jansson, R., et al.. Clin. EXD. Immunol. 63:80-86 (1986)). 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
- Membranes were then processed as described above, except that alkaline phosphatase-conjugated goat anti-human IgG, Fc fragment specific (Cappel, Organon Teknika Corp., West Chester, PA) was used as the second antibody with nitroblue tetrazolium (0.3 mg/ml) and 5-bromo-4-chloro-3-indolyl- phosphate (0.15 mg/ml) in lOOmM Tris, pH 9.5, 100 mM NaCl, 5 mM MgCl 2 .
- alkaline phosphatase-conjugated goat anti-human IgG, Fc fragment specific Cappel, Organon Teknika Corp., West Chester, PA
- CH0-HTP012b cells were processed as described by Ellis £t al. (Ellis, L., et al.. Cell 45:721-732 (1986)). 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.
- guaiacol unit is defined as a _A470 of 1.0 per minute which is equivalent to 150 nmols guaiacol oxidized per minute (Chance, B., et al .. In Methods in Enzvmology (Colowick, S.P., et al .). Academic Press, New York 1:764-775 (1955)).
- One unit of iodide peroxidase is defined as a _A353 of 1.0 per minute which corresponds to 43 nmols 13- formed per minute (Magnusson, R.P., et al.. J. Biol. Chem. 259:13783-13790 (1984)).
- 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), gentamicin (48 ⁇ g/ml) and 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 minutes 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 (Bradford, M.M., Anal. Biochem. 72:248-254 (1976)). Yield of microsomal protein was approximately 100-200 ⁇ g per 100 mm dish of confluent cells.
- 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 minutes at room temperature. After aspiration, the wells were washed twice in Tris buffer, once in Tris-Tween buffer, and once in Tris buffer. Seru samples were diluted 1/100, 1/1000 or 1/10,000 in PBS, 5 g/L BSA.
- the non-coding strand of human TPO cDNA in the phagemid Bluescript (Stratagene, San Diego, CA), was used as a template for oligonucleotide-directed mutagenesis.
- a 52 bp mutagenic primer (5'-AGGCTCCCTCGGGTGAC ⁇ GAA ⁇ CCCATGTAGCTGGCTGCTCTGCTGATCG-3'), synthesized by the Molecular Genetics Core Facility, San Francisco Veterans' Administration Medical Center, was designed to generate two stop codons directly upstream of the putative membrane-spanning region of the protein.
- TGA and TAG codons were created at 2629-2631 bp and 2641-2643 bp in human TPO cDNA (Magnusson, R.P., et al.. Mol. Endocrinol. 1:856-861 (1987)), 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-HTP0, to generate pHTP0(Ml)-ECE-SV2-DHFR.
- This plasmid contains components of the expression vectors pECE (Ellis, L., et al.. Cell 45:721-732 (1986)) and pSV2-dhfr (Lee, F., et al.. Nature 294:228-232 (1981)), provided by Dr. William Rutter (University of California, San Francisco) and Dr. Gordon Ringold (Syntex, Palo Alto), respectively.
- pSV2-DHFR-ECE-HTP0 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. Enzymes. Restriction enzymes, T4 DNA ligase and DNA polymerase I, Klenow fragment were obtained alternatively from Bethesda Research Laboratories (Gaithersburg, MD), New England Biolabs (Beverly, MA) or Boehringer-Mannheim (Indianapolis, IN).
- membrane protein or 30 ⁇ g of deoxycholate (DOC)-extracted protein from pHTPO- ECE-transfected cell lines (CH0-HTP04, CH0-HTP012, 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 (Portmann, L., et al .. J. Clin. Invest. 81:1217-1224 (1988)).
- TPO activity in the CH0-TP012 clones was approximately the same as TPO activity in TSH- stimulated Graves' thyroid cells in monolayer culture (Table I).
- 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.
- the specificity of the 200 kD and 110 kD bands discussed above was demonstrated in two separate experiments utilizing wild-type, non-TPO-transfected, CHO cells.
- recombinant human TPO antigen of the present invention In order to further examine its specificity, recombinant human TPO was compared with Graves' thyroid microsomes as a source of antigen in an ELISA procedure.
- the recombinant hTPO was present in 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 (Kaufman, K.D., et al.. J. Clin. Invest. 84:394-403 (1989)), 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 (Kaufman, K.D., et al.. J. Clin. Invest. 84:394-403 (1989)). 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.
- pDHFR-TP0-4C- MTX The relative TPO activities observed in CHO cells transfected with pECE-HTPO, pHTP0-DHFR-2B and pHTP0-DHFR-4C are shown plotted against methotrexate concentration in Figure 5. Further, one particular subclone, designated pDHFR-TP0-4C- MTX, has been found to express relatively greater amounts of TPO than any other construct so far isolated, and, in this regard, comprises the best mode presently contemplated of expressing human TPO in CHO cells.
- the plasmid pDHFR-TP0-4C- MTX was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 on October 3, 1989, with accession number CRL 10250.
- FIG. 5 shows that, with increasing methotrexate concentrations, a plateau is reached for CHO expression of TPO by the pHTP0-DHFR-2B and pHTP0-DHFR-4C plasmids. While not intending to be bound by any particular theory, one possible explanation for this observation is that the expressed full length 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.
- Immunoreactive TPO was absent from the cell surface of the CH0-TP0-M1 cells, as demonstrated by the lack of immunofluorescence when these cells were pre-incubated with Hashimoto's thyroiditis serum and fluorescently-tagged goat anti-human IgG antibody, unlike CHO cells transfected with wild-type hTPO (Kaufman, K.D., et al.. J. Clin. Invest. 84:394-403 (1989)).
- Amounts of radiolabeled, immunoprecipitable wild-type hTPO protein at 24 hours of chase were similar to baseline (0 hours).
- the observed decrease in signal in CH0-TP0-M1-K1 cell lysates from 4 to 24 hours.is paralleled by an increase in signal in the medium of these cells, supporting the concept of a secreted protein, which, accordingly, comprises another embodiment of the present invention.
- conditioned media were tested for TPO enzymatic activity.
- 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).
- Fig. 16 TPO activity in conditioned media from CHO cells expressing wild-type hTPO (Fig. 16) was clearly present in lysates of these cells, as previously described (Kaufman, K.D., et al .. J. Clin. Invest. 84:394-403 (1989)).
- 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.
- 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 62 ⁇ 8 cpm; T cells.+ TEC: 6108 ⁇ 1040 cpm.
- 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.
- TPO cDNA 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.. Euroo. 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.
- 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.
- TPO polypeptides 200-500 b.p. in length, coding for TPO polypeptides of 66-166 amino acids.
- the entire hTPO protein comprises 933 amino acids.
- TPO polypeptide fragments are expressed as bacterial fusion proteins, so called because the protein is a hybrid of a 10 kD fragment of J-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 NotI (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 mM MnCl 2 and bovine serum albumin, 100 ug/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
- BBL DNAase I (0.1 ng DNase/ ⁇ g cDNA)
- bovine serum albumin 100 ug/ml.
- TPO cDNA fragments 200-500 b.p. in length were recovered by electroelution.
- the PCR-generated DNA was labeled with 3 2 p - ⁇ CTP to a specific radioactivity of 0.8 x 10 9 cp / ⁇ g DNA using the random primer method (Multiprime; Amersham, Arlington Heights, IL).
- the screening procedure employed standard techniques (Maniatis, T., et al ., Molecular Biology: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)), 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 (Seto, P., et al.. _ s . Clin. Invest. 80:1205-1208 (1987)). 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
- TP0-CH0 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.
- PBS phosphate-buffered saline
- PMSF ImM phenylmethyl sulfonylfluoride
- 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 (Schardt, C.W., et al.. J. Immunol. Methods 55:155-168 (1982)).
- Nucleotide sequence analysis of selected clones PIaque- 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 (Maniatis, T., et al.. Molecular Bioloov: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)), 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 (Sanger, F., et al.. Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)). Nucleotide sequence analysis was performed using the software provided by Bionet.
- the new TPO cDNA fragment sublibrary constructed contained 3.8 x 10 6 recombinant clones, with an effective (correct orientation and reading frame) size one-sixth of this number. The insert sizes were confirmed to be in the 200-500 b.p. range.
- the monoclonal antibody spanned the same region (746-1,150 b.p.) of the hTPO gene (Magnusson, R.P., et al.. Mol. Endocrinol. 1:856-861 (1987)) ( Figure 18).
- the epitope recognized by the monoclonal antibody lies within this 15 amino acid span.
- 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.
- 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.
- the 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 (Kaufman, K.D., et al.. J. Clin. Invest. 84:394-403 (1989)).
- 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 0-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 (Kaufman, K.D., et al.. J. Clin. Invest. 84:394-403 (1989)). 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 J-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 (Male, D.K., et al .. Immunol. 54:419-426 (1985); Fukuma, N., et al.. Immunol . 67:129-131 (1989)) or TPO (Libert, F., et al.. EMB0_i.6:4193-4196 (1987); Ludgate, M., et al.. J. Clin. Endocrinol. Metab.
- 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.
- Example XIII a human TPO cDNA sublibrary was constructed expressing random fragments of the protein (each 66-166 amino acids in length) (Mehra, V., et al.. Proc. Natl. Acad. Sci. USA 83:7013-7017 (1986)).
- serum from patients with Hashimoto's disease with high titers of anti-TPO antibodies failed to recognize any of these TPO protein fragments.
- TPO fragments in this library were recognized by a mouse monoclonal antibody (MAb) against denatured human TPO.
- MAb mouse monoclonal antibody
- This example presents the determination of the disease- associated B-cell epitopes on TPO, using a panel of 13 MAb generated against nondenatured human TPO (Ruf, J., et al.. Endocrinology - 125:1211-8 (1989)).
- the binding of some of these MAb to native TPO is inhibited by anti-TPO antibodies in the serum of patients with autoimmune thyroid disease (Ruf, J., et al.. Endocrinology 125:1211-8 (1989)), indicating that these particular MAb epitopes correspond to or are in the vicinity of the disease-associated epitopes. Determination of the epitopes for some of the TPO MAb in the panel could, therefore, delineate molecular domains of the autoimmune thyroid disease-associated B-cell epitopes.
- TPO Fragment Library The construction of the TPO random fragment cDNA library (3.8 x 10 6 plaque-forming units) has been described previously. Immunoscreening of the library was performed by standard techniques, as previously described, using 13 mouse MAb generated against native human TPO (Ruf, J., et al.. Endocrinology 125:1211-8 (1989)). Positive clones were plaque-purified and used to generate Bluescript plasmids for nucleotide sequencing of the cDNA inserts (Sanger, F., et al.. Proc. Natl. Acad. Sci. USA Z4:5463-5467 (1977)).
- MAb binding was detected with horseradish peroxidase-1inked sheep antimouse immunoglobulin G F(ab')2 (Amersham International, Aylesbury, Buckinghamshire, United Kingdom) diluted 1:1,000 using 0.5 mg/mL 4-chloro-l-naphthol, 0.57 mg/mL imidazole, 17% methanol, and 0.42% hydrogen peroxide as substrate.
- TPO MAb 47 and TPO MAb 20.10 (Portmann, L., et al., J. Clin. Invest. 81:1217-1224 (1988)) all were strongly positive.
- TPO MAb 20.10 (Portmann, L., et al.. J. Clin. Invest. 81:1217-1224 (1988)), generated against the denatured protein and previously shown to recognize a linear epitope between TPO amino acids 266-281 (Finke, R., et al.. J. Clin.
- the present data demonstrate that only one (no. 47) of a panel of 13 MAb generated against native human TPO reacts with random 66-166 amino acid fragments of the 933-amino acid TPO molecule. Consistent with this observation, only MAb 47 recognizes intact TPO after denaturation and reduction, although all 13 MAb in this panel recognize native nondenatured human TPO (Ruf, J., et al .. Endocrinology 115:1211-8 (1989)). In agreement with our findings, MAb 47 is unique in this panel of TPO Mab, in that it was the only MAb whose binding to TPO could not be abolished by dithiothreitol treatment of the protein.
- TPO MAb 47 (amino acids 713-721) is different from that for TPO MAb 20.10 (amino acids 266-281). Furthermore, TPO MAb 20.10 reacts only with denatured TPO (Portmann, L., et al .. J. Clin. Invest. 81:1217- 1224 (1988)).
- 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-HTPO.
- 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/ml).
- 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 CH0-HTP0-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. (J. Immunol. Methods 55:155-168 (1982)), 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'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 mM 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 ⁇ g.
- 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) were 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 CHO-TPO 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.
- 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 (Ruf, J., et al.. Acta Endocrinol . Suppl. 281:49-56 (1987)) and the microsomal antigen (Kajita, Y., et al.. FEBS Lett. 187:334-338 (1985)) are bound to the lectin concanavalin A.
- CHO cells stably expressing human hTPO (CHO-TPO 12g) (Kaufman, K.D., gt a!... J. Clin. Invest. 84:394-403 (1989)) 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/ml 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. 35 S-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 mM 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
- 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 were 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 Laem li 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 mM 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 mM EDTA and 1% ⁇ - mercaptoethanol). As a control, to monitor degradation of the hTPO, 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 35 S-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 Europaeus (UEA-F) agarose-bound lectins (all purchased from Vector Laboratories, Burlingame, CA).
- Con A Concanavalin A
- PNA peanut agglutinin
- WGA wheat germ agglutinin
- RCA1 Ricinus communis agglutin 1
- Ulex Europaeus Ulex Europaeus
- samples (0.3 ml) were diluted to 10 ml in Buffer A (20 mM Tris HCl, 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 C Cl2- 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 HCl, 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- acetylglucosamine; PNA and RCA1, D-galactose; Con 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 (Magnusson, R.P., et al .. Mol. Endocrinol. 1:856-861 (1987); Kimura, S., et al .. Proc. Natl. Acad. Sci. USA 84:5555-5559 (1987); Libert, F., et al.. Nucl . Acids Res. 15:6735 (1987)) 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 (0-1inked).
- 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 5 S-methionine, followed by immunoprecipitation with anti-hTPO antibodies present in the serum of patients with Hashimoto's thyroiditis.
- 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.
- N-linked glycans by cleaving the glycosidic linkage between the two N-acetyl glucosamine (GlcNac) residues in the chitobiose core, increased the electrophoretic mobility of the hTPO doublet to approximately 110 kD and 105 kD.
- 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.
- hTPO 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-linked oligosaccharides with terminal galactose residues), peanut agglutinin (terminal Gal-j3-l,3-GalNac) or Ulex europaeus (terminal ot-L-fucose).
- RCA1 Ricinus communis agglutinin 1
- RCA1 highest affinity for bi- and tri-antennary N-linked oligosaccharides with terminal galactose residues
- peanut agglutinin terminal Gal-j3-l,3-GalNac
- Ulex europaeus terminal ot-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.
- 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.
- 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. Thus, unlike the polypeptide glycosylation pattern in yeast and bacteria, glycosylation in eukaryotic Chinese hamster ovary cells would be very similar, if not identical, to that in human thyroid cells.
- recombinant hTPO in Chinese hamster ovary cells is functionally active, at the same level present in human thyroid cells in monolayer culture (Kaufman, K.D., et al.. _L_ Clin. Invest. 84:394-403 (1989)).
- 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 (Kaufman, K.D., et al.. J. Clin. Invest. ⁇ 4:394-403 (1989)) or by ELISA.
- 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 .-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 (Livingstone, A.M., et al .. Ann. Rev. Immunol. 5:477- 501 (1987)). 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
- Examples XIII and XIV provide for a method of identifying the interaction responsible for the .-cell recognition of thyroid peroxidase.
- a protein which binds to a specific DNA sequence is purified using affinity chromatography.
- affinity chromatography Specifically, the nine amino acid sequence corresponding to residues 713-721 of thyroid peroxidase is immobilized on an appropriate matrix, such as Sepharose, and used as an affinity matrix to purify the proteins which bind to the particular sequence (Arcangioli
- the DNA binding protein is extracted from human ⁇ cells.
- the protein extract, obtained from the ⁇ cell is applied to a column which contains the immobilized DNA sequence of interest. Proteins which are not capable of binding to the DNA sequence are washed off the column.
- Proteins which bind to the DNA sequence are removed from the column using a salt gradient.
- the proteins eluted from such a column are enriched for the proteins which bind to the specific DNA sequences immobilized on the matrix.
- the DNA binding protein is further purified using procedures well known in the art, such as ion exchange chromatography, high performance liquid chromatography, size exclusion chromatography, and the like.
- the protein is assayed, for example, using the well known gel retardation assay (Garner, M.M. et al., Nucl . Acid Res. 9:3047 (1981); Fried, M. et al .. Nucl. Acid Res. 9:6506 (1981)), or other well known methods.
- DNA binding protein is purified, a partial amino acid sequence is obtained from the N-terminal of the protein.
- the protein is tryptically mapped and the amino acid sequence at one of the fragments is determined by methods known in the art.
- the deduced amino acid sequence is used to generate an oligonucleotide probe.
- the encoding sequence can be based on codons which are known to be more frequently used by the organism.
- the probe can consist of a mixture of all the possible codon combination which could encode the polypeptide.
- a probe complementary to the amino acid sequence is used to screen either a cDNA or genomic library for the genomic sequences which encode the DNA binding protein. Once the gene encoding the DNA binding protein has been obtained, the sequence of the DNA is determined according to well known methods. The gene can be used to obtain large amounts of the protein from a recombinant host, or the sequence can be used in mutational analysis to further define the functional regions within the protein which interacts with the DNA.
- proteins which bind to .-cell epitope are isolated by identifying a clone expressing the protein using well known techniques such as Southwestern blotting (Sharp, Z.D. et al .. Biochim Bioohvs Acta. 1048:306-309 (1990); Gunther, C. V. et al.. Genes Dev. 4:667-679 (1990); and Walker, M.D. et al.. Nucleic Acids Res. 18:1159-1166 (1990)).
- a labeled DNA sequence is used to screen a cDNA expression library whose expressed proteins have been immobilized on a filter via colony or plaque transfer.
- the labeled DNA sequences bind to colonies or plaques which express a protein capable of binding to the particular DNA sequence.
- Clones expressing a protein which bind to the labeled DNA sequence are purified and the cDNA insert which encodes the DNA binding protein is isolated and sequenced.
- the isolated DNA can be used to express large amounts of the protein for further purification and study, used in isolating the genomic sequences corresponding to the cDNA, or used to generate functional derivative of the binding protein.
- the present invention is thus directed to DNA binding proteins which can bind to the .-cell epitope and to functional derivatives thereof.
Abstract
L'invention concerne des éptitopes des cellules B associés à la maladie de la peroxydase thyroïdienne humaine, en particulier une séquence de neuf acides aminés correspondant aux acides aminés 713-721 de la peroxydase tyroïdienne humaine de recombinaison.The invention relates to B cell eptitopes associated with human thyroid peroxidase disease, in particular a nine amino acid sequence corresponding to amino acids 713-721 of recombinant human thyroid peroxidase.
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US73804091A | 1991-07-30 | 1991-07-30 | |
US738040 | 1991-07-30 | ||
PCT/US1992/006283 WO1993003146A1 (en) | 1991-07-30 | 1992-07-30 | Disease associated b-cell epitopes of tpo and use thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0604453A1 true EP0604453A1 (en) | 1994-07-06 |
EP0604453A4 EP0604453A4 (en) | 1995-01-11 |
Family
ID=24966322
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP92916802A Withdrawn EP0604453A4 (en) | 1991-07-30 | 1992-07-30 | Disease associated b-cell epitopes of tpo and use thereof. |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0604453A4 (en) |
JP (1) | JPH07503604A (en) |
AU (1) | AU2403292A (en) |
CA (1) | CA2113280A1 (en) |
FI (1) | FI940391A (en) |
NO (1) | NO940298L (en) |
WO (1) | WO1993003146A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0668918A1 (en) * | 1991-08-28 | 1995-08-30 | Institute Diagnostics Nichols | Disease associated human autoantibodies specific for human thyroid peroxidase |
CN105087512A (en) * | 2014-05-13 | 2015-11-25 | 苏州偲聚生物材料有限公司 | Protein for detecting thyroid diseases and partial peptide of protein |
-
1992
- 1992-07-30 AU AU24032/92A patent/AU2403292A/en not_active Abandoned
- 1992-07-30 WO PCT/US1992/006283 patent/WO1993003146A1/en not_active Application Discontinuation
- 1992-07-30 CA CA002113280A patent/CA2113280A1/en not_active Abandoned
- 1992-07-30 EP EP92916802A patent/EP0604453A4/en not_active Withdrawn
- 1992-07-30 JP JP5503701A patent/JPH07503604A/en active Pending
-
1994
- 1994-01-26 FI FI940391A patent/FI940391A/en not_active Application Discontinuation
- 1994-01-27 NO NO940298A patent/NO940298L/en unknown
Non-Patent Citations (7)
Also Published As
Publication number | Publication date |
---|---|
CA2113280A1 (en) | 1993-02-18 |
FI940391A0 (en) | 1994-01-26 |
NO940298D0 (en) | 1994-01-27 |
EP0604453A4 (en) | 1995-01-11 |
WO1993003146A1 (en) | 1993-02-18 |
JPH07503604A (en) | 1995-04-20 |
FI940391A (en) | 1994-03-28 |
NO940298L (en) | 1994-01-27 |
AU2403292A (en) | 1993-03-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP3747063B2 (en) | FK-506 cytosol binding protein | |
KR100454396B1 (en) | Redirection of cellular immunity by protein-tyrosine kinase chimeras | |
US6511665B1 (en) | Antibodies to interleukin-1 receptors | |
Kaufman et al. | Generation of recombinant, enzymatically active human thyroid peroxidase and its recognition by antibodies in the sera of patients with Hashimoto's thyroiditis. | |
JP2005041859A (en) | Anti-cd4 antibody homolog useful for prevention and treatment of aids, arc and hiv infection | |
IE904641A1 (en) | Recombinant thyrotropin receptor | |
JP2002136292A (en) | Novel receptor-type phosphotyrosine phosphatase | |
US5498597A (en) | FKBP-13, an FK506-binding immunophilin | |
EP0654083B1 (en) | Ptp-d subfamily of protein tyrosine phosphatases | |
EP0483281A1 (en) | Recombinant human thyroid peroxidase | |
JPH08508877A (en) | Protein tyrosine phosphatase PTP-S31 | |
JP3588115B2 (en) | Restoration of cellular immunity by receptor chimera | |
US5998172A (en) | Anti-CD6 ligand antibodies | |
US7196181B1 (en) | Sequences encoding novel human thyroid peroxidase proteins and polypeptides | |
US5968768A (en) | CD6 ligand encoding sequence | |
EP0604453A1 (en) | Disease associated b-cell epitopes of tpo and use thereof | |
US5538886A (en) | Receptor-type phosphotyrosine phosphatase-alpha | |
JPH05501952A (en) | TSH receptor | |
Levy et al. | Characterization of murine monoclonal anti-endothelial cell antibodies (AECA) produced by idiotypic manipulation with human AECA. | |
US6747139B1 (en) | Nucleic acid encoding human thyrotropin receptor | |
US5888794A (en) | Receptor-type phosphotyrosine phosphatase-alpha | |
US6627735B2 (en) | Islet cell antigen 1851 | |
US5807714A (en) | Method of production of antigen-specific glycosylation inhibiting factor | |
US20040101528A1 (en) | Type I IL-1 receptors | |
US6682905B1 (en) | Receptor-type phosphotyrosine phosphatase-alpha |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 19940228 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: NICHOLS INSTITUTE DIAGNOSTICS |
|
RHK1 | Main classification (correction) |
Ipc: C12N 15/53 |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 19941123 |
|
AK | Designated contracting states |
Kind code of ref document: A4 Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU MC NL SE |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 19960201 |