WO1995008336A1 - Anticorps a l'ophtalmopathie de graves, antigene orbitaire de l'ophtalmopathie de graves, et ses procedes d'utilisation - Google Patents

Anticorps a l'ophtalmopathie de graves, antigene orbitaire de l'ophtalmopathie de graves, et ses procedes d'utilisation Download PDF

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WO1995008336A1
WO1995008336A1 PCT/US1994/010756 US9410756W WO9508336A1 WO 1995008336 A1 WO1995008336 A1 WO 1995008336A1 US 9410756 W US9410756 W US 9410756W WO 9508336 A1 WO9508336 A1 WO 9508336A1
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Prior art keywords
graves
ophthalmopathy
antigen
antibody
immunoglobulin
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PCT/US1994/010756
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English (en)
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Basil Rapoport
Sandra M. Mclachlan
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Nichols Institute Diagnostics
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Priority to AU77328/94A priority Critical patent/AU7732894A/en
Publication of WO1995008336A1 publication Critical patent/WO1995008336A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the fields of immunology and recombinant genetics. More particularly, the present invention relates to Graves' ophthalmopathy associated antibody and Graves' ophthalmopathy orbital antigen; immunoglobulin genes which are associated with autoi munity in humans; products and processes involved in the cloning, preparation, and expression of genes for the antibody and the antigen; and to diagnostic and therapeutic uses thereof.
  • Graves' ophthalmopathy is a common, disfiguring autoimmune disorder of unknown etiology.
  • target cell(s) or autoantigen(s) involved Weetman, A.P., Autoimmunity 12:215- 222. (1992) and Kendler, D.L., et al . , Clin . Endocrinol . 35:539- 547. (1991)) .
  • the present invention relates to Graves' ophthalmopathy associated immunoglobulin proteins and orbital tissue antigen(s) and processes involved in the cloning, preparation and expression of genes for these immunoglobulins and antigen(s); antibodies (anti-antibody, anti-idiotypic antibody) with specificity to these immunoglobulins; antibodies with specificity to these antigen(s); and nucleotide probes corresponding to these immunoglobulin proteins and antigen(s) .
  • the present antibodies and probes are useful for detecting the presence of orbital tissue antigen in an orbital tissue sample.
  • the present antigen polypeptide is useful for detecting Graves' ophthalmopathy antibodies in an orbital tissue sample as well as for producing antibodies thereto.
  • the present invention relates to the identification, characterization and sequencing of cDNA's and genomic fragments which encode Graves' ophthalmopathy immunoglobulin proteins and orbital tissue antigen(s), which are present in orbital tissue infiltrating B cells and orbital tissue antigens which are present in other orbital tissue cells, in patients with Graves' ophthalmopathy.
  • genetic sequences encoding the present immunoglobulin proteins can be used to identify the present antigen. Once identified these antigen(s) can be isolated and the genetic sequences of the antigen(s) can be readily determined by one of ordinary skill in the art using known techniques. These antigen peptides and fragments thereof can then be used to detect the present immunoglobulin and to produce antibodies thereto.
  • the invention also provides for expression vectors containing such genetic sequences, hosts transferred with such expression vectors, and methods for producing the genetically engineered or recombinant immunoglobulin proteins and antigen polypeptides.
  • the invention also provides antibodies which specifically recognize present immunoglobulin proteins (anti-antibody, anti-idiotypic antibody) and the present antigen(s) .
  • the present immunoglobulin or antigen cDNA and recombinant protein are useful for making antibodies which specifically recognize the present immunoglobulin protein and antigen, respectively.
  • the present invention also relates to pharmaceutical compositions containing the present anti-idiotypic antibody raised against the present immunoglobulin protein(s) .
  • the present invention further relates to methods of treating Graves' ophthalmopathy, whereby a therapeutically effective amount of a pharmaceutical composition containing the present anti-idiotypic antibody raised against the present Graves' ophthalmopathy associated immunoglobulin protein(s) , is administered to a patient having Graves' ophthalmopathy.
  • the present invention also relates to immunoglobulin fragments of the present ophthalmopathy associated immunoglobulin whose genes are generally associated with autoimmunity, immunoglobulin fragments whose genes are generally associated with autoimmunity, and genes whose sequences are substantially homologous to such autoimmunity associated gene sequences; products and processes involved in the cloning, preparation and expression of such genes, and to diagnostic and therapeutic uses of such gene products.
  • autoimmune associated genes include the ger line genes KL012, DP10, hvl263, S4343 and DP54, and
  • Such autoimmune associated immunoglobulin fragments of the present ophthalmopathy associated immunoglobulin include OF7K.3, OF7K.16, OF7K.11, OF7K.9, OF7K.19, OF7K.17, OF7K.7, OF7H1.2 and OF7H1.19 having the sequences as shown in Figures 3-6.
  • H chain and an immunoglobulin kappa light ( ) chain cDNA library from the orbital tissue of a patient with active Graves' ophthalmopathy were constructed.
  • Analysis of 15 H (IgGl) and 15 L (kappa) chains revealed a restricted spectrum of variable (V) region genes. 14/15 V kappa genes were - 94% homologous to the closest known germline gene, KL012. 13/15 H chain genes were 91% and 90% homologous to the closest germline genes, DP10 and hvl263, respectively.
  • These germline genes also code for other autoantibodies to striated muscle (KL012) and thyroid peroxidase (KL012 and hvl263) .
  • the present inventors have discovered that particular germline genes and genes substantially homologous thereto, are associated with autoimmunity in humans.
  • FIG. 1 illustrates L chain gene PCR products from Graves' thyroid (T) , blood (B) , orbital fat tissues from 7 patients (F1-F7) and orbital muscle from patient 7 (M7) .
  • "-" Control lane lacking template.
  • L-DNA ladder illustrates lambda L chain products obtained using VI + Cl oligonucleotide primers.
  • Fig. 1(B) illustrates kappa L chain products obtained using Vk + Ck primers.
  • Fig. 1(C) illustrates beta-actin products.
  • Fig. 2 illustrates IgGl H chain PCR products from blood (B) , orbital fat (patients 1-7, Fl-7) and orbital muscle from patient 7 (M7) .
  • FIG. 2(A) illustrates IgGl H chain products obtained for cDNA from blood (B) , orbital Fl-6, M7 and F7 using VHla/3a and Cgl oligonucleotide primers.
  • Fig. 2 (B) illustrates IgGl H chain products obtained for cDNA from Fl and F7, M7 and Graves' thyroid (T) using Vc/d + Cgl and VH6 + Cgl primers.
  • FIG. 3 illustrates the nucleotide sequence (Fig. 3(A)) [SEQ ID NOS: 1 - 8] and the derived amino acid sequence (Fig. 3(B)) [SEQ ID NOS: 9 - 16] of the VK genes in clones OF7K.3, 9, 11, 16, 17 and 19.
  • the closest putative germline gene, KL012 (Pargent, W. , et al., Eur. J. Immunol . , 21:1821- 1827 (1191)) is shown at the top.
  • S43434 is a VK 1 gene used in a myasthenia gravis striational muscle autoantibody of thymic B cell origin (Victor, K.D. , et al., Eur. J. Immunol . 22:2231-2236 (1992)). *Identical to OF7K.5, 10, 12, 15, 18, 20, 23 and 25.
  • FIG. 4 illustrates the nucleotide sequence (Fig. 4(A)) [SEQ ID NOS: 17 - 18] and the derived amino acid sequence (Fig. 4(B)) [SEQ ID NOS: 19 - 20] of the VK gene in clone OF7K.7.
  • the closest putative germline gene, VK005 (Straubinger, B., et al., J. Mol . Biol . , 155:23-24 (1988)) is shown at the top.
  • FIG. 5 illustrates the nucleotide sequence (Fig. 5(A)) [SEQ ID NOS: 21 - 23] and the derived amino acid sequence (Fig. 5(B)) [SEQ ID NOS: 24 -26] of the VH gene in clone OF7H1.2.
  • the closest putative germline gene, DP10 The closest putative germline gene, DP10
  • FIG. 6 illustrates the nucleotide sequence (Fig. 6(A)) [SEQ ID NOS: 30 - 31] and the derived amino acid sequence (Fig. 6(B)) [SEQ ID NOS: 32 - 33] of the VH gene in clone OF7H1.19.
  • the closest putative germline gene, DP54 Tomlinson, I.M., et al . , J. Mol . Biol . , 227:776-798 (1992)
  • Fig. 6(C) [SEQ ID NOS: 34 - 35] illustrates the nucleotide and the derived amino acid sequences of the D region of this group of H chain genes. *Identical to OF7H1.25.
  • Gene By the term “gene” is intended a DNA sequence which encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific peptide. Further, the term includes intervening, non-coding regions, as well as regulatory regions, and can include 5' and 3 ' ends.
  • Gene sequence is intended to refer generally to a DNA molecule. As used herein, the terminology is meant to include both a DNA molecule which contains a non-transcribed or non-translated sequence. The term is further intended to include any combination of gene(s), gene fragment (s), non-transcribed sequence(s), or non-translated sequence(s) which are present on the same DNA molecule.
  • the present sequences may be derived form a variety of sources including DNA, cDNA, synthetic DNA, RNA, or combinations thereof.
  • Such gene sequences may comprise genomic DNA which may or may not include naturally occurring introns.
  • genomic DNA may be obtained in association with promoter regions or poly A sequences.
  • the gene sequences, genomic DNA or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified form suitable. cells (i.e., orbital tissue infiltrating B cells, orbital tissue cells) by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
  • 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 DAN sequence complementary to an RNA molecule of interest, carried in a cloning vector. This term includes genes from which the intervening sequences have been removed.
  • Recombinant DNA By the terminology “recombinant DNA” is meant a molecule that has been recombined by in vitro splicing cDNA or a genomic DNA sequence.
  • Cloning is meant the use of in vi tro 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 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 , Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 2d. ed. (1989) .
  • 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 LAMBDA- ZAP vector.
  • Cloning Vehicle A plasmid or phage DNA or other DNA sequence which is able to replicate in a host cell.
  • the cloning vehicle is characterized by one or more endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the DNA, which may contain a marker suitable for use in the identification of transformed cells. Markers include for example, tetracycline resistance or ampicillin resistance.
  • the word vector can be used to connote a cloning vehicle.
  • expression control sequence is a sequence of nucleotides that controls or regulates expression of structural genes when operably linked to those genes. They include the lac systems, the trp system major operator and promoter regions of the phage lambda, the control region of fd coat protein and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells.
  • Expression vehicle is a vehicle or vector similar to a cloning vehicle but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
  • the cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.
  • Expression control sequences will by depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • promoter is intended to refer to a DNA sequence which can be recognized by an RNA polymerase. The presence of such a sequence permits the RNA polymerase to bind and initiate transcription of operably linked gene sequences.
  • promoter region is intended to broadly include both the promoter sequences as well as all gene sequences which may be necessary for the initiation of transcription. The presence of a promoter region is, therefrom sufficient to cause the expression of an operably linked gene sequence.
  • operably linked means that the promoter controls the initiation of expression of the gene.
  • a promoter is operably linked to a sequence of proximal DNA if upon introduction into a host cell the promoter determines the transcription of the proximal DNA sequence or sequences into one or more species of RNA.
  • a promoter is operably linked to a DNA sequence if the promoter is capable of initiating transcription of that DNA sequence.
  • prokaryote is meant to include all organisms without a true nucleus, including bacteria.
  • Eukaryote is meant to include organisms and cells which have a true nucleus, including mammalian cells.
  • host is meant to include not only prokaryotes, but also such eukaryotes as yeast and filamentous fungi, as well as plant and animal cells.
  • the term includes an organism or cell that is the recipient of a replicable expression vehicle.
  • substantially homologous refers to the ability of a first DNA sequence encoding Graves' ophthalmopathy associated antibodies or Graves ophthalmopathy orbital antigen, to hybridize to a second DNA sequence encoding the foregoing, respectively, under stringent conditions, for example, at about 0.Ix sodium citrate sodium chloride buffer (SSC) at a temperature of about 65 * C.
  • SSC 0.Ix sodium citrate sodium chloride buffer
  • a “functional derivative” of the present antigen or antibody is a protein which possesses a biological activity (either functional or structural) or immunological characteristics that are substantially similar to a biological activity or immunological characteristics of non- recombinant antigen or antibody. Such a functional derivative may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional derivative” is intended to include the "fragments,” “variants,” “analogues,” “homologues, " or “chemical derivatives” of a molecule.
  • Fragment of a molecule such a the present antibody or antigen, is meant to refer to any variant of the molecule.
  • variants of a molecule are meant to refer to a molecule substantially similar in structure and biological activity or immunological characteristics to either the entire molecule, or to a fragment thereof. Thus, provided that two molecules possess a similar activity, they are considered variants as that therm is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the sequence of amino acid residues is not identical.
  • Analog of a molecule is meant to refer to a molecule substantially similar in function to either the entire molecule or to a fragment thereof.
  • 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' 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 described in Remington's Pharmaceutical Sciences (1980) . Procedures for coupling such moieties to a molecule are well known in the art.
  • Anti-Antibody is intended an antibody directed against antigenic determinants on other antibody (immunoglobulin) molecules.
  • Anti-idiotypic Antibody By the terminology “anti- idiotypic antibody” is intended an antibody directed against an idiotypic determinant of another antibody.
  • idiotope is intended an idiotypic determinant, i.e., an antigenic determinant on a variable domain of an immunoglobulin molecule.
  • Idiotype By the term “idiotype” is intended a set of one or more idiotopes that distinguish a clone of immunoglobulin producing cells from other clones. Idiotypes occur in the variable domains of immunoglobulin molecules and may be within, near to, or outside of the antigen binding site; antibodies to idiotypes located within or near the antigen biding site will prevent the immunoglobulin from combining with the antigen.
  • Idiotype-anti-idiotype Network By the terminology 0 "idiotype-anti-idiotype network" is intended a B-cell regulatory mechanism. Activation of a B cell results in a clone of plasma cells producing immunoglobulin of a single idiotype, which because it was previously present in very small quantities, can be recognized as "nonself" and results s in the production of anti-idiotypic antibodies directed against its idiotypic determinants. There can also be anti- anti-idiotypic antibodies directed against the second antibodies, antibodies directed against them, and so forth. These antibodies react with antigen receptors on B cells and T helper and suppressor cells, as well as with circulating antibodies, to enhance or suppress production of the initial antibody by various mechanisms.
  • Suitable orbital tissue samples include orbital fat, connective or muscle tissue samples.
  • This invention comprises amino acid sequences of Graves' ophthalmopathy associated immunoglobulin proteins, orbital antigen, genetic sequences coding for such mRNA, expression vehicles containing the genetic sequences, hosts transformed therewith and recombinant immunoglobulin proteins or antigen(s) and antisense RNA produced by such transformed host expression.
  • the invention further comprises antibodies directed against such immunoglobulin proteins or antigen(s) .
  • genetic sequences is intended to refer to a nucleic acid molecule (preferably DNA) .
  • Genetic sequences which are capable of encoding the immunoglobulin proteins or antigen(s) are derived from a variety of sources. These sources include genomic DNA, cDNA, synthetic DNA, and combinations thereof.
  • the preferred .source of the genomic DNA or mRNA is orbital tissue infiltrating B cells or orbital tissue cells. The mRNA may then be used to obtain cDNA by techniques known to those skilled in the art.
  • Probes may be synthesized based on the amino acid sequence of the immunoglobulin proteins or antigen(s) by methods known in the art.
  • the Graves' ophthalmopathy associated immunoglobulin protein, orbital antigen, or immunoglobulin fragment genomic DNA of the invention may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with the 5' promoter region of the immunoglobulin proteins or antigen(s) gene sequences and/or with the 3' transcriptional termination region. Further, such genomic DNA may be obtained in association with the genetic sequences which encode the 5' non-translated region of the immunoglobulin protein or antigen mRNA and/or with the genetic sequences which encode the 3' non-translated region.
  • Graves' ophthalmopathy immunoglobulin proteins or antigen(s) genomic DNA can be extracted and purified from the orbital tissue including orbital tissue infiltrating B cells of humans having Graves' ophthalmopathy, or from any cell that expresses the immunoglobulin protein or antigen(s) by means well known in the art (for example, see Guide to Molecular Cloning Techniques. S.L.
  • immunoglobulin protein or antigen mRNA can be isolated from any cell which produces or expresses the immunoglobulin protein or antigen, and used to produce cDNA by means well known in the art (for example, see Guide to Molecular Cloning Techniques, S.L. Berger, et al . , eds., Academic Press (1987)) .
  • the mRNA preparation used will be enriched in mRNA coding for such immunoglobulin protein or antigen(s), either naturally, by isolation from cells which are producing large amounts of the protein, or in vitro, by techniques commonly used to enrich mRNA preparations of specific sequences, including for example sucrose gradient centrifugation, or PCR.
  • cDNA can then be prepared for example, by reverse transcription. The cDNA can then be amplified by PCR using suitable primers (Chazenbalk, G.D., et al., J. Clin . Invest . , 92 : 62-14 (1993) .
  • DNA preparations either human genomic DNA or cDNA
  • suitable DNA preparations are randomly sheared or enzymatically cleaved, respectively, and ligated into appropriate vectors to form a recombinant gene (either genomic or cDNA) library.
  • a DNA sequence encoding the immunoglobulin protein or antigen(s) or its functional derivatives may be inserted into a DNA vector in accordance with conventional techniques, including blunt-ending or staggered-ending 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 Sambrook, et al . , ( In : Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 2d. ed. (1989)), and are well known in the art.
  • an immunoglobulin and/or antigen(s) clone can be identified by any means which specifically selects for immunoglobulin protein or antigen(s) DNA such as, for example, (a) by hybridization with an appropriate nucleic acid probe(s) containing a sequence specific for the DNA of this protein, or (b) by hybridization-selected translational analysis in which native mRNA which hybridizes to the clone in question is translated in vi tro and the translation products are further characterized, or, (c) if the cloned genetic sequences are themselves capable of expressing mRNA, by immunoprecipitation of a translated immunoglobulin or antigen product produced by the host containing the clone.
  • Oligonucleotide probes specific for the immunoglobulin proteins or antigen(s) which can be used to identify clones to this protein can be designed from knowledge of the amino acid sequence of the immunoglobulin proteins or antigen(s) .
  • the sequence of amino acid residues in a peptide is designated herein either through the use of their commonly employed three-letter designations or by their single-letter designations. A listing of these three-letter and one-letter designations may be found in textbooks such as Biochemistry, Lehninger, A., Worth Publishers, New York, NY (1970) .
  • the amino acid sequence is listed horizontally, the amino terminus is intended to be on the left end whereas the carboxy terminus is intended to be at the right end.
  • the residues of amino acids in a peptide may be separated by hyphens. Such hyphens are intended solely to facilitate the presentation of a sequence.
  • the peptide fragments are analyzed to identify sequences of amino acids which may be encoded by oligonucleotides having the lowest degree of degeneracy. This is preferably accomplished by identifying sequences that contain amino acids which are encoded by only a single codon.
  • amino acid sequence may be encoded by only a single oligonucleotide sequence
  • amino acid sequence may be encoded by any of a set of similar oligonucleotides.
  • all of the members of this set contain oligonucleotide sequences which are capable of encoding the same peptide fragment and, thus, potentially contain the same oligonucleotide sequence as the gene which encodes the peptide fragment
  • only one member of the set contains the nucleotide sequence that is identical to the exon coding sequence of the gene.
  • this member is present within the set, and is capable of hybridizing to DNA even in the presence of the other members of the set, it is possible to employ the unfractionated set of oligonucleotides in the same manner in which one would employ a single oligonucleotide to clone the gene that encodes the peptide.
  • one or more different oligonucleotides can be identified from the amino acid sequence, each of which would be capable of encoding the present immunoglobulin proteins or antigen(s) .
  • the probability that a particular oligonucleotide will, in fact, constitute the actual immunoglobulin or antigen coding sequence can be estimated by considering abnormal base pairing relationships and the frequency with which a particular codon is actually used (to encode a particular amino acid) in eukaryotic cells.
  • Such "codon usage rules" are disclosed by Lathe, et al . , J.
  • the suitable oligonucleotide, or set of oligonucleotides, which is capable of encoding a fragment of the immunoglobulin protein or antigen(s) gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) may be synthesized by means well known in the art (see, for example, Synthesis and Application of DNA and RNA, S.A. Narang, ed. , 1987, Academic Press, San Diego, CA) and employed as a probe to identify and isolate the cloned immunoglobulin proteins or antigen(s) gene by techniques known in the art. Techniques of nucleic acid hybridization and clone identification are disclosed by Maniatis, et al .
  • the above-described DNA probe is labeled with a detectable group.
  • detectable group can be any material having a detectable physical or chemical property. Such materials have been well-developed in the field of nucleic acid hybridization and in general most any label useful in such methods can be applied to the present invention.
  • radioactive labels such as P, H,
  • the oligonucleotide may be radioactively labeled, for example, by "nick-translation" by well-known means, as described in, for example, Rigby, et al . , J. Mol . Biol . 113 :231 (1977) and by T4 DNA polymerase replacement synthesis as described in, for example, Deen, et al . , Anal . Biochem . 135:456 (1983) .
  • polynucleotides are also useful as nucleic acid hybridization probes when labeled with a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • a non-radioactive marker such as biotin, an enzyme or a fluorescent group.
  • the actual identification of immunoglobulin protein or antigen sequences permits the identification of a theoretical "most probable" DNA sequence, or a set of such sequences, capable of encoding such a peptide.
  • a DNA molecule or set of DNA molecules, capable of functioning as a probe(s) for the identification and isolation of clones containing the immunoglobulin protein or antigen gene.
  • a library is prepared using an expression vector, by cloning DNA or, more preferably cDNA prepared from a cell capable of expressing the immunoglobulin protein or antigen, into an expression vector.
  • the library is then screened for members which express the immunoglobulin protein or antigen, for example, by screening the library with antibodies to the immunoglobulin protein or antigen protein.
  • the above discussed methods are, therefore, capable of identifying genetic sequences which are capable of encoding immunoglobulin proteins or antigen(s) or fragments thereof.
  • Such expression identifies those clones which express proteins possessing characteristics of the immunoglobulin proteins or antigen(s) . Such characteristics may include the ability to specifically bind antibody to the immunoglobulin proteins or antigen(s) and the ability to elicit the production of antibody which are capable of binding to the immunoglobulin proteins or antigen(s) .
  • the cloned immunoglobulin or antigen encoding sequences obtained through the methods described above, and preferably in a double-stranded form, may be operably linked to sequences controlling transcriptional expression in an expression vector, and introduced into a host cell, either prokaryote or eukaryote, to produce recombinant immunoglobulin or antigen or a functional derivative thereof.
  • a host cell either prokaryote or eukaryote
  • the present invention encompasses the expression of the immunoglobulin or antigen, or a functional derivatives thereof, in prokaryotic or eukaryotic cells, and particularly, eukaryotic expression is preferred.
  • Preferred prokaryotic hosts include bacteria such as E. coli , Bacillus, Streptomyces , P ⁇ eudomona ⁇ , Salmonella, Serratia, etc.
  • the most preferred prokaryotic host is E. coli .
  • Other enterobacteria such as Salmonella typhimurium or Serratia marcescen ⁇ , 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.
  • Graves' ophthalmopathy immunoglobulin or antigen protein or a functional derivative thereof
  • a prokaryotic cell such as, for example, E. coli , B . subtilis, Pseudomonas, Streptomyces, etc.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible) .
  • constitutive promoters examples include the int promoter of bacteriophage lambda, the J a promoter of the Beta-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 lambda
  • E. coli E. coli , the alpha-amylase (Ulmanen, I., et al., J. Bacteriol . 152:176-182 (1985)) and the sigma-28-specific promoters of B. subtilis (Gilman, M.Z., et al . , Gene 32:11- 20 (1984) ) , the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In : The Molecular Biology of the Bacilli , Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward, J.M., et al . , Mol . Gen . Genet .
  • Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol . 1:277-282 (1987)); Cenatiempo, Y. (Biochimie dS ⁇ OS- ⁇ ie (1986)); and Gottesman, S. (Ann . Rev. Genet . 15:415-442 (1984)) .
  • ribosome binding sites are disclosed, for example, by Gold, L., et al . (Ann . Rev. Microbiol . 35:365-404 (1981)) .
  • Especially preferred eukaryotic hosts are mammalian cells either in vivo, in animals or in tissue culture.
  • Mammalian cells provide post-translational modifications to recombinant immunoglobulin or antigen which include folding and/or glycosylation at sites similar or identical to that found for the native immunoglobulin or antigen.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains expression control sequences which contain transcriptional regulatory information and such sequences are “operably linked” to the nucleotide sequence which encodes the polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • Two DNA sequences are said to be operable linked if induction of promoter function results in the transcription of the immunoglobulin or antigen encoding sequence mRNA and 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 expression regulatory sequences to direct the expression of the immunoglobulin or antigen mRNA, antisense RNA, or protein, or (3) interfere with the ability of the immunoglobulin or antigen template 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.
  • regulatory regions needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing control sequences will include a region which contains a promoter for transcriptional control of the operably linked gene.
  • Expression of the immunoglobulin or antigen in eukaryotic hosts requires the use of regulatory regions functional in such hosts, and preferably eukaryotic regulatory systems.
  • a wide variety of transcriptional and translational regulatory sequences can be employed, depending upon the nature of the eukaryotic host.
  • the transcriptional and translational regulatory signals can also be derived from the genomic sequences of viruses which infect eukaryotic cells, such as adenovirus, bovine papilloma virus, Simian virus, herpes virus, or the like.
  • these regulatory signals are associated with a particular gene which is capable of a high level of expression in the host cell.
  • control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such amethionine.
  • Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell. Promoters from heterologous mammalian genes which encode mRNA product capable of translation are preferred, and especially, strong promoters such as the promoter for actin, collagen, myosin, etc., can be employed provided they also function as promoters in the host cell.
  • Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, et al . , J.
  • TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, et al . Nature (London) 250:304-310 (1981)); in yeast, the yeast gal4 gene promoter (Johnston, et al . Proc . Natl . Acad. Sci . USA 75:6971-6975 (1982); Silver, et al . , Proc . Natl . Acad. Sci . USA 51:5951-5955 (1984) ) or a glycolytic gene promoter may be used.
  • a fusion product of the immunoglobulin or antigen may be constructed.
  • the sequence coding for the immunoglobulin or antigen may be liked to a signal sequence which will allow secretion of the protein from or the compartmentalization of the protein in, a particular host.
  • signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable to subsequent removal.
  • the native signal sequence for this protein may be used.
  • Transcriptional initiation regulatory signals can be selected which allow for repression or activation, so that expression of the operably linked 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.
  • Translational signals are not necessary when it is desired to express immunoglobulin or antigen antisense RNA sequences.
  • the non-transcribed and/or non-translated regions 3 ' to the sequence coding for the immunoglobulin or antigen can be obtained by the above-described cloning methods.
  • the 3'-non-transcribed region may be retained for its transcriptional termination regulatory sequence elements; the 3-non-translated region may be retained for its translation termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells. Where the native expression control sequence signals do not function satisfactorily in the host cell, then sequences functional in the host cell may be substituted.
  • the vectors of the invention may further comprise other operably linked regulatory elements such as enhancer sequences, or DNA elements which confer tissue or cell-type specific expression on an operably linked gene.
  • the immunoglobulin or antigen DNA encoding sequence and an operably linked promoter is introduced into a recipient eukaryotic cell as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or a closed covalent circular molecule which is incapable of autonomous replication, the expression of the immunoglobulin or antigen may occur through the transient expression of the introduced sequence.
  • a non-replicating DNA (or RNA) molecule which may either be a linear molecule or a closed covalent circular molecule which is incapable of autonomous replication
  • Genetically stable transformants may be constructed with vector systems, or transformation systems, whereby immunoglobulin or antigen DNA is integrated into the host chromosome. Such integration may occur de novo within the cell or, in a most preferred embodiment, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, with retroviral vectors, transposons or other DNA elements which promote integration of DNA sequences in chromosomes.
  • a vector is employed which is capable of integrating the desired gene sequences into a mammalian host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector in the chromosome, for example, the marker may provide biocide resistance, e.g., resistance to 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.
  • the introduced sequence is 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, as outlined below.
  • 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 eukaryotic plasmids include those derived from the bovine papilloma virus, vaccinia virus, SV40, and, in yeast, plasmids containing the 2-micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, et al . , Miami Wntr. Symp. 15:265-274 (1982); Broach, J.R., In : The molecular Biology of the Yeast Saccharomyces : Life Cycle and Inheri tance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-470 (1981); Broach, J.R. , Cell 25:203-204 (1982); Bollon, et al .
  • the DNA construct(s) is introduced into an appropriate host cell by any of a variety of suitable means, including transfeetion.
  • 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 immunoglobulin or antigen, or in the production of a fragment of this protein.
  • This expression can take place in a continuous manner in the transformed cells, or in a controlled manner, for example, expression which follows induction of differentiation of the transformed cells (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like) .
  • the expressed protein is isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • the immunoglobulin or antigen DNA encoding sequences obtained through the methods above, will provide sequences which, by definition, encode the immunoglobulin or antigen, or functional derivatives thereof and which may then be used to obtain immunoglobulin or antigen antisense RNA genetic sequences as the antisense RNA sequence will be that sequence found on the opposite strand of the strand transcribing the peptide core's mRNA.
  • the antisense DNA strand may also be operably linked to a promoter in an expression vector such that transformation with this vector results in a host capable of expression of an immunoglobulin or antigen antisense RNA in the transformed cell.
  • Antisense RNA and its expression may be used to interact with an endogenous immunoglobulin or antigen DNA or RNA in a manner which inhibits or represses transcription or translation of the immunoglobulin or antigen genes in a highly specific manner.
  • Use of antisense RNA probes to block gene expression is discussed in Lichtenstein, C, Nature 333:801- 802 (1988) .
  • An antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of a hapten which can be recognized and bound by an antibody.
  • An antigen may have one, or more than one epitope.
  • An "antigen” is capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • the specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • 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) 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 : 16-325 (1983)) .
  • the antibodies of the present invention have specificity to one or more epitopes present on the antigen peptide, or an idiotype on the present immunoglobulin.
  • the antibodies of the invention can be polyclonal or monoclonal, provided that they are made with the present immunoglobulin or antigen polypeptide or fragment thereof an immunogen. Both of these types of antibodies as well as the present Graves' ophthalmopathy associated immunoglobulin itself, can be utilized in the multiple applications described herein below.
  • the present antibodies can be used to detect the presence of the present immunoglobulin or antigen in a orbital tissue sample.
  • the present immunoglobulin can be detected by contacting the sample with an imaging-effective amount of the present detectably labeled appropriate antibody and detecting the label, thereby establishing the presence of the present immunoglobulin or antigen in the sample. Detection can be carried out by imaging in vivo .
  • the present immunoglobulin or antigen can also be detected by known immunoassay techniques, including, for example, RIA, ELISA, etc., using the present appropriate antibodies.
  • the antibodies of the present invention are prepared by any of a variety of methods.
  • cells expressing the present immunoglobulin or antigen, or a fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding the present immunoglobulin or antigen.
  • an immunoglobulin or antigen fragment is chemically synthesized and purified by HPLC to render it substantially free of contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of high specific activity.
  • Polyclonal antibodies can be generated in any suitable animal including, for example, mice, rabbits or goats.
  • the present immunoglobulin or antigen peptide can be injected by itself or linked to appropriate immunoactivating carriers, such as Keyhole's limpet hemocyanin (KLH) .
  • KLH Keyhole's limpet hemocyanin
  • Monoclonal antibodies can be prepared in various ways using techniques well understood by those having ordinary skill in the art. For example, monoclonal antibodies can be prepared using hybridoma technology (Kohler, et al . , Nature 255:495 (1975); Kohler, et al . , Eur. J. Immunol . 6:511 (1976); Kohler, et al . , Eur. J. Immunol . 6: 292 (1976); Hammerling, et al . , In : Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp.
  • hybridomas which produce monoclonal antibodies which enable the detection of the present immunoglobulin or antigen can be easily produced and isolated with minimal screening.
  • Hybridomas producing monoclonal antibodies specific for epitopes which are found on the present immunoglobulin or orbital antigen are most effectively produced by first immunizing an animal from which hybridomas can be produced such as, for example, a Balb/c mouse, with initial subcutaneous injections of Freund's adjuvant, followed by booster injections within a few days. The fusion can be carried out using any of the techniques commonly known to those of ordinary skill in the art.
  • the screening of the hybridomas to determine which ones are producing monoclonal antibodies specific for the present peptide is straightforward and can be done either in a standard ELISA or RIA format.
  • a standard ELISA or RIA format For example, in an RIA screening format the culture supernatant, or ascites fluid from a hybridoma producing monoclonal antibody is reacted
  • 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 raised against the present antigen or with the present ophthalmopathy associated immunoglobulin 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 idiotypic determinants on a given mAb, it is then possible to identify other B cell or hybridoma clones sharing that idiotype. Idiotypic identity between the antibody product of two clones makes it highly probable that the antibody products of the two clones recognize the same antigenic epitopes.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id.
  • mAbs generated against the present 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 antigen epitope.
  • the anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated.
  • 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 which are produced in humans, or are "humanized” (i.e., non-immunogenic in a human) by recombinant or other technology such that they will not be antigenic in humans, or will be maintained in the circulating serum of a recipient for a longer period of time.
  • Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e., chimeric antibodies) (Robinson, et al . , International Patent Publication PCT/US86/02269; Akira, et al . , European Patent Application 184,187; Taniguchi, M. , European Patent Application 171,496; Morrison, et al . , European Patent Application 173,494; Neuberger, et al . , PCT Application WO 86/01533, Cabilly, et al . , European Patent Application 125,023; Better, et al .
  • Suitable "humanized" antibodies can be alternatively produced as described by Jones, et al . , Nature 321:552-525 (1986); Verhoeyan, et al . , Science 234:1534 (1988), and Beidler, et al . , J. Immunol . 141:4053-4060 (1988) .
  • the present Graves' ophthalmopathy associated immunoglobulin, fragments thereof or autoimmune associated fragments thereof, or autoimmune associated immunoglobulin fragments, orbital antigen, or antibodies thereto can be utilized in immunoassays for the detection of the orbital antigen or ophthalmopathy associated immunoglobulin, respectively, in an orbital tissue sample.
  • the present immunoglobulin or fragment thereof can be used to detect the present orbital antigen; the present orbital antigen, or fragment thereof, can be used to detect antibodies thereto in an orbital tissue sample; or antibodies against the present immunoglobulin can be used to detect the present immunoglobulin in an orbital tissue sample, etc.
  • Autoimmune associated fragments of the present invention can be used to generally detect an antigen associated with an autoimmune condition such as thyroid peroxidase in Graves' disease.
  • the immunoassays can be competitive or sandwich, or as is otherwise well known and they all depend on the formation of antibody-antigen immune complex. These assays are well known to those of skill in the art.
  • the antibodies or antigen can be immobilized or labeled. There are many carriers to which the antibodies/antigen can be bound for immobilization and which can be used in the present invention.
  • Well-known carriers include but are not limited to glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for purposes of the invention.
  • Those skilled in the art will know many other suitable carriers for binding the antibodies or antigen peptide(s), or will be able to ascertain such, using routine experimentation.
  • one or more of the antibodies or antigen(s) peptide(s) will be coupled with a detectable label such as an enzyme, radioactive isotope, fluorescent compound, chemiluminescent compound, or bioluminescent compound.
  • a detectable label such as an enzyme, radioactive isotope, fluorescent compound, chemiluminescent compound, or bioluminescent compound.
  • the antibodies or antigen peptide(s) can be bound to an enzyme.
  • This enzyme when later exposed to its substrate will react to the substrate in such a manner as to produce a chemical moiety which can be detected, as, for example, spectrophotometric or fluorometric means.
  • enzymes that can be used to detectably label are amalate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeastalcoholdehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.
  • an antibody or antigen can also be detected by labeling the antibody or antigen peptide with a radioactive isotope.
  • the presence of the radioactive isotope could then be determined by such means as the use of a gamma counter or a scintillation counter.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.
  • Another way in which the antibody or antigen peptide can be detectably labeled is by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody or antigen peptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, aromatic-acridiniu ester, imidaxole, acridinium salt, and oxalate ester.
  • Other suitable chemiluminescent labels are described in the following U.S. co-pending applications: U.S. Serial No.
  • a bioluminescent compound may also be used to label the antibody or antigen peptide.
  • Bioluminescence is a special type of chemiluminescence which is found in biological systems and in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent binding partner would be determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase, and aequorin.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of said container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a first antibody bound to an insoluble or partly soluble carrier.
  • a second container may comprise soluble, detectalby-labeled second antibody, in lyophilized form or in solution.
  • the carrier means may also contain a third container means comprising a detectalby-labeled third antibody in lyophilized form or in solution.
  • a kit can be used for sandwich assays. See, e.g., David, et al . , USP 4,376,110 herein incorporated by reference.
  • the carrier means may also contain a plurality of containers each of which comprises different, predetermined amounts of the present antigen peptide. These latter containers can then be used to prepare a standard curve into which can be interpolated the results obtained from the sample containing the unknown amount of the present orbital antigen.
  • Imaging can be carried out in vi tro or in vivo .
  • In vitro imaging can be done with the labels mentioned previously.
  • In vivo imaging is done with diagnostically effective labeled antibodies or antigen(s) .
  • diagnostically effective means that the amount of detectably labeled antibody or antigen administered is sufficient to enable detection of the site of orbital antigen or ophthalmopathy associateed antibody presence when compared to a background signal.
  • the dosage of detectably labeled antibody or antigen(s) for diagnosis will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, counterindications, if any, and other variables, to be adjusted by the individual physician. Dosage can very from 0.01 mg/kg to 2,000 mg/kg, preferably 0.1 mg/kg to 1,000 mg/kg.
  • diagnostically labeled means that the immunoglobulin or antigen has attached to it a diagnostically detectable label.
  • imaging labels and methods of labeling There are many different imaging labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include radioactive isotopes and paramagnetic isotopes.
  • the type of detection instrument available is a major factor in selecting a given radionuclide.
  • the radionucleotide chosen must have a type of decay which is detectable for a given type of instrument.
  • any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention.
  • a radionuclide used for in vivo diagnosis is that the half-life of a radionucleotide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host is minimized.
  • a radionuclide used for in vivo imaging will lack a particulate emission, but produce a large number of photons in a 140-200 ke V range, which may be readily detected by conventional gamma cameras.
  • radionucleotides may be bound to antibody or antigen either directly or indirectly by using an intermediary functional group.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody or antigen are diethylenetriaminepentaacetic acid (DTPA) and ethlenediaminetetracetic acid (EDTA) .
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethlenediaminetetracetic acid
  • Typical examples of metallic ions which can be bound to immunoglobulins are
  • the antibodies or antigen(s) used in the method of the invention can also be labeled with paramagnetic isotopes for purposes of in vivo diagnosis.
  • Elements which are particularly useful as in magnetic resonance imaging (MRI)
  • Preparations of the imaging antibodies or antigen(s) for administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non- aqueous solvents are propyleneglycol,. polyethyleneglycol, vegetable oil such as olive oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media, parenteral vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. See, generally, Remington ' ⁇ Pharmaceutical Science, 16th ed. Mac Eds. 1980.
  • compositions containing a therapeutically effective amount of the present anti- idiotypic antibody raised against the present Graves' ophthalmopathy associated antibody are useful for treating patients with Graves' ophthalmopathy.
  • Such compositions contain a therapeutically effective amount of the foregoing anti-idiotopic antibody, and may optionally contain one or more suitable pharmaceutically acceptable carriers and/or excipients, known to those of ordinary skill in the art.
  • Administration, dosage and frequency, and length of treatment can be readily optimized for a particular patient by one of ordinary skill in the art.
  • the present pharmaceutical composition can be formulated as a sterile aqueous or non-aqueous suspensions or emulsions, as described above in Section IV, for example for solutions for intravenous administration.
  • Example 1 Example 1
  • Orbital fat/connective tissue was obtained from 7 patients (Fl-7) and orbital muscle from 1 patient (M7) undergoing orbital decompression for Graves' exphthalmos.
  • mRNA was obtained from orbital tissues (-O.lg) using the QuickPrep mRNA Purification kit (Pharmacia, Piscataway, NJ) and cDNA was prepared by reverse transcription with oligo-dT (First Strand Synthesis kit: Stratacyte, La Jolla, CA) .
  • oligo-dT First Strand Synthesis kit: Stratacyte, La Jolla, CA
  • Thyroid cDNA from a Graves' patient (JA) (Chazenbalk, G.D. , et al., J. Clin . Invest . , 52:62-74
  • Lambda L chain DNA was amplified from 6/7 orbital fat samples (Fl, F3-F7) but not from F2 or from blood (Fig. 1 (A) ) .
  • Kappa L chain DNA was only amplified using cDNA from orbital fat from two patients (F6 and F7), as well as muscle from one patient (M7) (Fig. 1(B)) .
  • Both L chains were amplified from the orbital muscle of one patient (M7) .
  • Beta-actin was amplified from all cDNA templates (Fig. 1(C)), indicating the integrity of the mRNA even in the samples not providing a lambda L chain signal (F2 and blood) .
  • the preferential amplification of lambda DNA rather than kappa DNA in the same cDNA preparation from some orbital tissue cDNA is of interest.
  • Individual plasma cells secrete either kappa or lambda L chains but not both.
  • Preferential lambda amplification relates to more efficient priming for V lambda than for V kappa genes.
  • the bias is genuine, it resembles the lambda L chain bias demonstrated immunohistochemically in Graves' thyroid tissue (Jasani, B. , et al . , J. Endocrinol . Invest . 9 (Suppl . 3) :101- 101 (1986)).
  • oligonucleotide primer Vla/3a (Chazenbalk, G.D., et al . , J. Clin . Invest . 52:62-74 (1993)
  • homologous with the 5' regions of the largest VH gene families VHl and VH3 and a downstream primer Cgl (Chazenbalk, G.D. , et al . , J. Clin . Invest . 52:62-74 (1993)) for the constant region of IgGl, only DNA products with orbital muscle cDNA (M7) and orbital fat cDNA (F7) from patient 7 (Fig. 2(A)), were obtained.
  • H and L chain DNA of the size observed can only be amplified form mRNA-derived cDNA. This experiment evidences that L and H chain DNA can be amplified by PCR from orbital tissue of some patients with Graves' ophthalmopathy.
  • DNA amplified in example 1 in a bacteriophage vector and the nucleotide sequences of the V regions of 15 H and 15 L chains randomly selected from these libraries, were determined.
  • DNA amplified in the PCR reaction from cDNA generated by reverse transcription of mRNA isolated from orbital fat/ connective tissue of patient no. 7 (example 1) was used. This patient was a 62 year old male undergoing orbital decompression for malignant Graves' exophthalmos.
  • the oligonucleotide primers used in the PCR are described in example 1. To cover all 6 VH gene families, 5 sets of forward primers were used, including those of Persson, M.A. , et al . , Proc . Natl . Acad. Sci . USA, 55:2432-2436 (1991) , Marks, J.D., et al . , Eur. J. Immunol .
  • the reverse primer (Cgl) hybridizes with the CHI domain-hinge junction of IgGl H chains.
  • the kappa L chain forward primer cross primes with the four VK gene families (Kabat, E.A., et al . , Sequences of protein ⁇ of immunological interest, U.S. Department of Health and Human Services, Ed. 5 (1991) and the reverse primer was to the carboxyl terminus of the C region. All primers contain restriction sites for cloning into the Imminozap bacteriophage vectors (Stratacyte) .
  • Plasmids were prepared from XLl-blue bacteria (Stratagene) infected with the phagemid. Nucleotide sequencing of plasmid cDNA inserts was performed by the dideoxynucleotide termination method described in Sanger, F., et al., Proc Natl . Acad. Sci . USA 74:5463-5467 (1977) .
  • Immunoglobulin H and L chain genes are formed by the combination of a number of smaller genes separated by introns in the germline DNA (reviewed in Brodeur, P.H., Genes encoding the immunoglobulin variable regions . In : Molecular Genetics of Immunoglobulin, edited by Calabi, F. and Newberger, M.S.: Elsevier Science Publishers B.V. , P. 81-84 (1987)) .
  • the antigen binding, or variable (V) region of the human H chain is formed by the combination of one of -100 VH genes, 1 of -20 diversity (D) genes and 1 of 6 joining (JH) genes. This VDJ unit is then joined to the constant region of the H chain.
  • variable region of the human kappa L chain is formed form 1 of -100 VK genes and 1 of 6 JK genes.
  • the combination of multiple units itself generates diversity in the antigen-binding region. Further, diversity is introduced by antigen-driven somatic mutation in these germline genes. Therefore, as described below for orbital tissue immunoglobulin genes, each antibody can be characterized in terms of the germline (or putative germline) genes encoding the VH, D and JH regions of the H chain and the VJ and JK regions of the kappa L chain.
  • Analysis of random H and L kappa V gene sequence from the Graves' orbital tissue revealed them to be quite restricted. Two groups of L chains were identified among the V regions of the 15 kappa genes (Table I, Fig. 3) .
  • VK genes 5 dominant cluster of 14 VK genes contained sequences that were identical or highly related, differing by no more than 4 base pairs from one another. These genes were -94% homologous to the closest known germline gene, L4.KL012, a VK1 (Pargent, W. , et al., Eur. J. Immunol . 21:1821-1827 io (1991) .
  • One of the orbital tissue KL012-like VK genes (OF7K.17) was >96% homologous to VK gene S43434 (Fig. 3) used by a striational muscle autoantibody derived from thymic B cells of a patient with myastheria gravis (Victor, K.D., et al . , Eur. J. Immunol . 22:2231-2236 (1992) .
  • the second VK gene group (Fig. 4) had only one representative (OF7K.7), which was 91% homologous to a VK2 germline gene, VK005 (Straubinger, B., J. Mol . Biol . 155:23-34 (1988) .
  • the two groups of VK genes were combined with different JK segments; JK2 for the KL 012 L chains and
  • the H chain V regions could also be classified into two groups. One group, with closest homology (91%) to VH1 germline gene DP10 (Tomlinson, I.M., J. Mol . Biol .
  • the second VH group was 91% homologous to VH3 germline gene DP54 (To linson, I.M., et al . , J. Mol . Biol . 227: 116-198 (1992) (Table 1, Figs. 6(A) and (B) ) .
  • the DPIO-related and DP54-related VH genes are combined with JH5 and JH4 genes, respectively (Table 1) and with different D region genes.
  • DPIO-related H chains used the D region germline gene DHQ52 (Schroeder, H.W., Jr., et al . , Science 235:791-793 (1987)) (Fig. 5(C)), which is preferentially used in development of the human immune system.
  • the D region used by the DP54-related H chains bore no resemblance to known D germline genes (Fig. 6) . Partial nucleotide sequencing of the constant regions confirmed that both groups of H chains were of the IgGl subclass.
  • the antibodies encoded by these genes resemble the IgG class antibodies characteristic of autoimmune thyroid disease (McLachlan, S.M., et al . , Endocr. Rev. 13:192-206 (1992)), which reflect chronic stimulation of the immune system.
  • McLachlan, S.M., et al . , Endocr. Rev. 13:192-206 (1992) which reflect chronic stimulation of the immune system.
  • B cells and T cells may both recognize the same antigen, although their epitopes may be different. Therefore, information on orbital tissue autoantibodies and their genes enable the identification of the respective autoantigen(s) .
  • Immunoglobulins and T cell receptors have similar molecular structures. This investigation of immunoglobulin variable region genes in Graves' orbital tissue is analogous to the analysis of T cell receptor variable gene usage in the thyroid of patients with autoimmune thyroid disease. The possibility of restricted T cell receptor gene usage within the thyroid has generated much interest (Martin, A. , et al . , Thyroid 2:247-261 (1992)). The orbital tissue B cell immunoglobulin genes in the present report enables the identification of the antigens recognized by these T cell receptors.
  • This study provides the first information regarding the immunoglobulin genes expressed by plasma cells in the orbital tissue of Graves' ophthalmopathy. These data enable the identification of the orbital antigen(s) which may be the target of a humoral or a cell-mediated immune response.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

L'invention se rapporte à l'immunoglobuline associée à l'ophtalmopathie de Graves et à l'antigène orbitaire de l'ophtalmopathie de Graves; à des gènes d'immunoglobuline qui sont associés à l'autoimmunité de l'homme; à des produits et des procédés utilisés dans le clonage, la préparation et l'expression des gènes pour l'immunoglobuline et l'antigène; à des anticorps dirigés contre l'immunoglobuline (anti-anticorps et anticorps anti-idiotypique) et l'antigène associés à l'ophtalmopathie; à des procédés de détection de la présence de l'immunoglobuline et/ou de l'antigène (des antigènes) associés à l'ophtalmopathie; et à des compositions pharmaceutiques contenant des anticorps anti-idiotypiques. La présente invention se rapporte également à l'ADN génomique isolé, à l'ADNc, à l'ARN antisens et à l'ARN contenant la séquence de l'immunoglobuline ou de l'antigène (des antigènes).
PCT/US1994/010756 1993-09-22 1994-09-22 Anticorps a l'ophtalmopathie de graves, antigene orbitaire de l'ophtalmopathie de graves, et ses procedes d'utilisation WO1995008336A1 (fr)

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US7364735B1 (en) * 1998-05-08 2008-04-29 Stichting Sanquin Bloedvoorziening Method for diagnosis and treatment of haemophilia A patients with an inhibitor

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WO1986001533A1 (fr) * 1984-09-03 1986-03-13 Celltech Limited Production d'anticorps chimeriques
EP0184187A2 (fr) * 1984-12-04 1986-06-11 Teijin Limited Chaîne lourde d'immunoglobuline chimère souris-humaine et chimère de l'ADN codant celle-ci
EP0125023B1 (fr) * 1983-04-08 1991-06-05 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
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US4376110A (en) * 1980-08-04 1983-03-08 Hybritech, Incorporated Immunometric assays using monoclonal antibodies
EP0125023B1 (fr) * 1983-04-08 1991-06-05 Genentech, Inc. Préparations d'immunoglobuline recombinante, méthodes pour leur préparation, séquences d'ADN, vecteurs d'expression et cellules d'hôtes recombinantes
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WO1986001533A1 (fr) * 1984-09-03 1986-03-13 Celltech Limited Production d'anticorps chimeriques
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Title
ACTA ENDOCRINOLOGICA, Volume 121, Number Suppl. 2, issued 1989, CAMPBELL, "Immunology of Graves' Ophthalmopathy: Retrobulbar Histology and Histochemistry", pages 9-16. *
ARTHRITIS AND RHEUMATISM, Volume 32, Number 1, issued January 1989, CHEN et al., "Characterization of Two Immunoglobulin VH Genes That Are Homologous to Human Rheumatoid Factors", pages 72-76. *
AUTOIMMUNITY, Volume 12, issued 1992, WEETMAN, "Thyroid-Associated Ophthalmopathy", pages 215-222. *
BIOCHEM. BIOPHYS. RES. COMM., Volume 179, Number 1, issued 30 August 1991, PORTOLANO et al., "A Human Fab Fragment Specific For Thyroid Peroxidase Generated by Cloning Thyroid Lymphocyte-Derived Immunoglobulin Genes in A Bacteriophage Lambda Library", pages 372-377. *
CLINICAL ENDOCRINOLOGY, Volume 35, issued 1991, KENDLER et al., "A 64 kDA Membrane Antigen is a Recurrent Epitope for Natural Autoantibodies in Patients with Graves' Thyroid and Ophthalmic Diseases", pages 539-547. *
ENDOCRINE REVIEWS, Volume 12, Number 2, issued 1992, McLACHLAN et al., "The Molecular Biology of Thyroid Peroxidase: Cloning, Expression and Role as Autoantigen in Autoimmune Thyroid Disease", pages 192-206. *
EUR. J. IMMUNOL., Volume 21, issued 1991, PARGENT et al., "The Human Immunoglobulin X Locus, Characterization of the Duplicated O Regions", pages 1821-1827. *
J. CLINICAL INVESTIGATION, Volume 90, issued September 1992, PORTOLANO et al., "Recognition by Recombinant Autoimmune Thyroid Disease-Derived Fab Fragments of a Dominant Conformational Epitope on Human Thyroid Peroxidase", pages 720-726. *
TIBTECH, Volume 11, issued February 1993, HARRIS et al., "Therapeutic Antibodies-The Coming of Age", pages 42-44. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7364735B1 (en) * 1998-05-08 2008-04-29 Stichting Sanquin Bloedvoorziening Method for diagnosis and treatment of haemophilia A patients with an inhibitor

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