Classifications

• • 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
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/75—Agonist effect on antigen
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/575—Hormones
  • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]

Definitions

• the invention relates to antibodies, particularly human antibodies and fragments thereof that bind the thyroid stimulating hormone receptor (TSHR), especially in humans, and their uses in diagnostic and therapeutic roles.
• TSHR thyroid stimulating hormone receptor
• the thyroid gland is, as is well known, one site of metabolic control within the body. Cancer of the thyroid gland is not particularly common, but the high rate of disease re-occurrence necessitates long term surveillance. Usually, during treatment for cancer of the thyroid, the majority of the thyroid tumor is removed, but a small amount often remains that must be treated by radiotherapy. Following surgery, it is necessary to treat the patient with thyroid hormone, as the patient will no longer produce this.
• One role of the thyroid gland is to take up iodine from the body. Hence, it should be possible to treat any remaining tumor cells with radioactive iodine. Unfortunately, though, thyroid cancer cells do not take up iodine well.
• the patient in order for the radioactive iodine to work, the patient has to either be treated with recombinant TSH or have thyroid hormone treatment withdrawn in order to elevate natural TSH levels, to stimulate iodine uptake.
• thyroid hormone treatment withdrawn in order to elevate natural TSH levels, to stimulate iodine uptake.
• a significant proportion of the treated patients fail to respond to recombinant TSH after some time.
• recombinant TSH is very expensive and not all patients may be offered this treatment. Withdrawal of thyroid hormone has quite unpleasant side effects for the patient, particularly fatigue, muscle cramps, puffiness and constipation. It would be beneficial if a new cheaper treatment could be found that stimulated iodine uptake, without causing such unpleasant side effects.
• Graves' disease is a common antibody-mediated disorder in which the primary target antigen has been identified as the thyroid follicular cell surface receptor for thyroid stimulating hormone (TSHR).
• TSHR thyroid stimulating hormone
• One group of anti-TSHR antibodies behave as agonists, mimicking the action of the natural ligand, TSH, on the receptor and are known as thyroid stimulating antibodies (TSAbs).
• the TSAbs hyperstimulate the thyroid follicular cells to secrete thyroxine resulting in hyperthyroidism.
• Another group of anti-TSHR antibodies may act as antagonists of TSH binding to the receptor which may occasionally lead to hypothyroidism.
• Neutral class antibodies to the TSHR that have neither agonist nor antagonist activity have also been described, although their role in disease remains to be clarified.
• the TSHR belongs to the family of G protein coupled receptors (GPCRs) with a large extracellular domain, a seven transmembrane (TM) region and a short cytoplasmic tail.
• GPCRs G protein coupled receptors
• the TM region of the GPCRs is responsible for the transmission of the activating signal by regulating small secondary messengers such as cAMP, diacylglycerol and inositolphosphate (D?3). It is likely that the mode of TSHR activation and the consequent intracellular regulatory cascade may ultimately be responsible for the variations observed in different patients with regard to toxic hyperplasia (gland enlargement, goitre), extrathyroidal complications and response to treatment.
• the TSHR is unique among the large family of GPCRs in undergoing complex post-translational modifications such as cleavage into two disulphide-linked subunits, known as the A-subunit and the B-subunit.
• the A-subunit of 53kD, corresponding to the ectodomain of TSHR is of special interest as it preferentially binds TSAbs and it has been proposed that the resulting, cleaved fragment released into the bloodstream may be the primary stimulus for provoking autoimmunity in susceptible individuals.
• TSAbs as monoclonal antibodies (mabs) has been a long sought goal, but has proved to be extraordinarily difficult to achieve.
• the establishment of experimental animal models of hyperthyroid Graves' disease has led to the development of IgG mabs with limited TSAb activity.
• a human IgG mab to TSHR developed from a patient with Graves' disease, was described with powerful thyroid activity in the nanogram range.
• the human mab acted as a full agonist by activating the TSHR to maximal stimulation equivalent to that achieved with sub-saturating concentrations of TSH.
• a murine mab developed from an experimental model, with similar efficacy, which behaves as a full agonist for the TSHR has also been described.
• the mab was pathogenic in antibody transfer experiments in vivo and reportedly led to a lymphocytic infiltrate of the thyroid gland.
• the determinants on the receptor for the full agonist and other stimulatory and blocking murine mabs are dependent on the conformational, three dimensional folding of the ectodomain, residing in a region rich in leucine repeats within the horseshoe structure.
• the inventors have surprisingly developed two antibodies that have an extraordinarily high affinity for TSHR and consequently powerfully stimulate TSHR.
• the two mabs show full agonist activity to the TSHR, but also show subtle differences in their behavior at low concentrations of IgG in terms of antagonistic activity for the TSHR.
• the mabs are pathogenic in vivo when transferred into mice whereby a single injection of microgram quantities of IgG induces rapid hypersecretion of thyroxine leading to sustained hyperthyroidism with considerable morphological changes, but with minimal mononuclear cell infiltrate in the thyroid glands.
• an antibody that binds to the TSHR, particularly to human TSHR with high affinity and specificity.
• an antibody comprising a CDR comprising an amino acid sequence having substantial homology to an amino acid sequence selected from the sequences shown in figures:
• an antibody comprising a first CDR comprising an amino acid sequence having substantial homology to an amino acid sequence selected from the sequences shown in figures:
• a second CDR comprising an amino acid sequence having substantial homology to an amino acid sequence selected from the sequences shown in figures:
• an antibody comprising a heavy chain comprising one or more CDRs having an amino acid sequence having substantial homology to an amino acid sequence selected from the sequences shown in figures:
• the invention further provides an antibody comprising a light chain comprising one or more CDRs having an amino acid sequence having substantial homology to an amino acid sequence selected from the sequences shown in figures: 12, 13, 14, 32, 33 and 34.
• an antibody comprising an amino acid sequence having substantial homology to an amino acid sequence selected from the sequences shown in figures: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 35, 36, 37, 38, 39, 40, 41, 42 and 43.
• the invention also provides an antibody comprising a heavy chain variable region having substantial homology to an amino acid sequence selected from the sequences shown in figures: 20, 21, 22, 23, 39 and 40.
• the antibody has a light chain variable region having substantial homology to the amino acid sequence shown in figures 24.
• the antibody has a light chain variable region having substantial homology to the amino acid sequence shown in figure 41.
• an antibody comprising a light chain variable region having substantial homology to an amino acid sequence selected from the sequences shown in figures: 24 and 41.
• an antibody comprising a CDR encoded by a nucleotide sequence having substantial homology to a nucleotide sequence selected from the sequences shown in figures:
• an antibody comprising:
• an antibody comprising a heavy chain comprising one or more CDRs encoded by a nucleotide sequence having substantial homology to a nucleotide sequence selected from the sequences shown in figures: 44, 45, 46, 47, 61 and 62.
• an antibody comprising a light chain comprising one or more CDRs encoded by a nucleotide sequence having substantial homology to a nucleotide sequence selected from the sequences shown in figures:
• the invention further provides an antibody encoded by a nucleotide sequence having substantial homology to a nucleotide sequence selected from the sequences shown in figures: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 66, 67, 68, 69, 70, 71, 72 and 73.
• an antibody comprising a heavy chain variable region encoded by a nucleotide sequence having substantial homology to a nucleotide sequence selected from the sequences shown in figures: 56, 57, 58, 59, 71 and 72.
• the antibody comprises a light chain variable region encoded by a nucleotide sequence having substantial homology to the nucleotide sequence shown in figure 60.
• the antibody comprises a light chain variable region encoded by a nucleotide sequence having substantial homology to the nucleotide sequence shown in figure 73.
• an antibody comprising a light chain variable region encoded by a nucleotide sequence having substantial homology to a nucleotide sequence selected from the
• an antibody that binds to the same epitope as an antibody according to other aspects of the invention.
• antibody is well known in the art. Herein it means an immunoglobulin or any functional fragment thereof. It encompasses any polypeptide that has an antigen-binding site. It includes but is not limited to monoclonal, polyclonal, monospecific, polyspecific, nonspecific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies.
• antibody encompasses antibody fragments such as Fab, F (ab 1 ) 2, Fv, scFv, Fd, dAb, and any other antibody fragments that retain antigen-binding function. Typically, such fragments would comprise an antigen- binding domain.
• the term “antibody” means a whole antibody molecule, namely two heavy chains, each with one variable region and three constant regions, and two light chains, each with one variable region and one constant region.
• Intact antibodies are also known as immunoglobulins (Ig). As indicated above, intact antibodies comprise light chains and heavy chains. Light chains are classified into two isotypes, and heavy chains are classified into five isotypes (A, D, E, G, and M). Some heavy chain isotypes are further divided into isotype subclasses, e. g.,lgGl, IgG2, IgG3, and IgG4. It is particularly preferred that the antibodies of the invention are IgG antibodies. In particular, IgG2b and IgG2a antibodies are preferred.
• the domain and three dimensional structures of different antibodies are known in the art.
• the light chain is composed of a constant domain (C) and an N-terminal variable domain (V).
• the heavy chain is composed of three or four constant domains (C H ), a hinge region, and a N- terminal variable domain (V H ).
• the C H adjacent to the VH domain is designated CHI.
• the VH and VL domains contain four regions of conserved sequence called framework (FR) regions (FRl, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence called complementarity determining regions (CDR).
• the CDRs (CDRl, CDR2, and CDR3) contain most of the antibody amino acids that specifically binds antigen.
• Heavy chain CDRs are denoted Hl 5 H2, and H3, while light chain CDRs are denoted Ll, L2, and L3.
• the term CDR is well known in the art. One skilled in the art would be able to recognise CDRs in an antibody or fragment by using Kabat numbering and the amino acids found either side of the CDRs.
• the Fab fragment (Fragment antigen-binding) consists of VH, C H I, VL and -C L domains covalently linked by a disulfide bond between the constant regions.
• the Fv fragment is smaller and consists of V H and VL domains non-covalently linked.
• a single chain Fv fragment (scFv) can be constructed.
• the scFv contains a flexible polypeptide that links the C-terminus of V H to the N-terminus of V L , or the C-terminus of V L to the N-terminus of V H .
• a 15-mer (GIy 4 Ser) 3 peptide may be used as a linker, but other linkers are well known.
• the antibodies of the invention are preferably able to bind to the Thyroid Stimulating Hormone Receptor (TSHR), especially the human TSHR.
• TSHR Thyroid Stimulating Hormone Receptor
• the antibodies also preferably cross react with the mouse TSHR. Further they are preferably able to agonise that receptor, that is to stimulate the production of thyroid hormone. It is possible to screen for these functions using techniques well known in the art.
• a functional fragment is an antibody fragment that is still able to bind TSHR. Further, a functional fragment is preferably able to agonise TSHR.
• antigen-binding site means the part of an antibody that specifically binds antigen.
• the part of the antigen that is recognised and bound by the antibody is referred to as the "epitope".
• An antigen-binding domain usually comprises variable regions from both the light chain (V L ) and the heavy chain (V H ), but it does not have to comprise both.
• Antigen-binding fragments include Fab fragments (monovalent fragments consisting of the V L , V H , C L and C HI domains); F(ab') 2 fragments (bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments (the two V H and CH I domains); Fv fragments (VL or V H domains, dAb fragments (Ward et al., (1989) Nature 341: 544-546), one or more complementarity determining regions (CDR); and single chain Fvs.
• the various antibody fragments can be obtained using conventional techniques known to those with skill in the art. It is possible to screen for the functionality of the fragments, e.g.
• the antibodies according to the invention are preferably chimeric, humanised (CDR grafted or reshaped).
• chimeric refers to antibodies in which the whole of the variable regions of a mouse or rat antibody are expressed along with human constant regions. This provides the antibody with human effector functions and also reduces immunogenicity (HAMA) caused by the murine Fc region.
• HAMA immunogenicity
• Humanised antibodies also called CDR grafted or reshaped antibodies
• CDR grafted or reshaped antibodies are an alternative to chimeric antibodies in which only the complimentarity determining regions from the rodent antibody V-regions are combined with framework regions from human V-regions. The idea is that these antibodies should be more human-like than chimeric and thus perhaps less immunogenic than chimeric antibodies.
• Transgenic mice have been created which have a repertoire of human immunoglobulin germline gene segments. These mice when immunised thus make human like antibodies. B cells from such immunised mice may be used in the production of monoclonal antibodies.
• the antibodies and nucleic acids of the invention are preferably isolated.
• isolated refers to a molecule that is substantially free of its natural environment.
• an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it was derived.
• the term also refers to preparations where the isolated protein is sufficiently pure for pharmaceutical compositions; or at least 70-80% (w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; or at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.
• substantially homologous means that the relevant amino acid or nucleotide sequence (e. g.
• CDR (s), V H or V L domain) will be identical to or have minor differences to the specifically defined sequences. Minor differences include minor amino acid changes, such as 1 or 2 substitutions in a 5 amino acid sequence of a specified region.
• the second antibody has the same specificity and has at least 50% of the affinity of the same.
• sequences substantially identical or homologous e. g. , at least about 85% sequence identity
• the sequence identity can be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher.
• substantial homology preferably means at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
• homology may be at least 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
• Sequences including constant regions may have less homology, for example, 75%, 80%, 85%, 87%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher.
• Substantially identical or homologous sequences also include nucleic acid sequences that will hybridize under selective hybridization conditions (e. g., highly stringent hybridization conditions), to the complement of the specifically defined strand.
• the percent identity can be determined by standard alignment algorithms, for example, the Basic Local Alignment Tool (BLAST) described by Altshul et al.( (1990) J. MoI. Biol., 215: 403-410); the algorithm of Needleman et al.( (1970) J. MoI. Biol., 48: 444-453); or the algorithm of Meyers et al.((1988) Comput. Appl. Biosci. , 4: 11-17).
• the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ( (1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0). This would be known by those skilled in the art.
• stringent describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3. 1-6.3. 6.
• Antibodies can be made by any method known in the art. A preferred method is using traditional hybridoma techniques (Kohler and Milstein (1975) Nature, 256: 495-499). For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. No limitation is placed on the present invention as to method of production or source of antibody.
• the invention provides antibodies that bind to TSHR. It is further envisaged that one skilled in the art could create more antibodies by altering the V H and/or V L sequence(s) provided. Such antibodies may be derived by a skilled person using techniques known in the art and are also encompassed by the invention. For example, modifications such as amino acid substitutions, deletions, or additions can be introduced into any part of the antibody, providing functionality remains. Changes may be introduced into the framework regions, especially to, for example improve the stability of the antibody. Changes may also be introduced into the CDRs to alter the antibody's affinity for the TSHR. The affinity of an antibody for the TSHR may be tested using standard techniques known in the art.
• V H and V L sequences are envisaged in particular. Such changes will produce molecules having functional and chemical characteristics similar to those of the antibodies from which the modifications are made. Conservative modifications are modifications unlikely to dramatically change the shape or function of the antibody, such as replacing one amino acid with another amino acid that has similar characteristics, e.g. replacing a hydrophobic amino acid with another hydrophobic amino acid.
• natural amino acids may be used, as may non-naturally occurring amino acids that have been created by, for example, chemical synthesis.
• the antibodies according to the invention may be linked to other molecules.
• antibodies may be linked to a protein or to a nonproteinaceous polymer such as polyethylene glycol, polypropylene glycol, and polyoxyalkylenes.
• Linking antibodies to such molecules is well known in the art and may be carried out by standard methods. Linking antibodies to such molecules can have an effect on certain characteristics of the antibodies, for example half life in blood.
• antibodies include detectable or functional tags or labels, such as enzymatic labels, e.g. horseradish peroxidase or alkaline phosphatase, radiolabels and chemical moieties e.g. biotin.
• detectable or functional tags or labels such as enzymatic labels, e.g. horseradish peroxidase or alkaline phosphatase, radiolabels and chemical moieties e.g. biotin.
• the antibodies may also be linked to toxic agents such as toxins, cytostatic or cytotoxic molecules and radioisotopes. Alternatively, the antibodies may be linked to other antibodies.
• the antibodies are linked to radioactive iodine.
• Linking such molecules to antibodies is well known in the art and may be achieved by standard techniques, for example by covalent attachment.
• the invention also provides methods of making antibodies, including a method of generating an antibody or functional fragment thereof comprising: a) providing a repertoire of nucleic acids encoding a variable domain that either includes a CDRl, CDR2 or CDR3 encoding region to be replaced or lacks a CDRl, CDR2 or CDR3 encoding region; b) combining the repertoire with a donor nucleic acid having a nucleotide sequence substantially homologous to a sequence selected from the sequences in figures:
• the term "repertoire” refers to a genetically diverse collection of nucleotide sequences derived wholly or partially from sequences encoding immunoglobulins.
• the sequences may be generated by the method given above, or by rearrangement in vivo of the V, D, and J segments of heavy chains, and the V and J segments of light chains.
• the sequences can be generated from a cell in response to which rearrangement occurs, e. g., in vitro stimulation.
• part or all of the sequences may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, e. g. , U. S. Patent 5,565, 332.
• the method may additionally comprise selecting an antibody that binds TSHR from the expressed antibodies and isolating it. It may include the step of selecting an antibody that agonises TSHR from the expressed antibodies and isolating it.
• the invention also provides isolated nucleic acids encoding antibodies according to the invention including nucleotides encoding the CDRs, variable domains and other functional fragments of such antibodies, and substantially homologous sequences.
• the nucleic acids may comprise DNA or RNA, and they may be synthetic (completely or partially) or recombinant (completely or partially).
• nucleotide sequences provided and references thereto encompass DNA molecules with the specified sequence, and encompass RNA molecules with the specified sequence in which U is substituted for T.
• a nucleic acid may encode any part of the antibody for example, a CDR, a variable region, a light chain, a heavy chain, an scFv, a Fab, the entire antibody or any other functional fragment thereof.
• nucleic acid having substantial homology to a sequence selected from: the sequences shown in figures:
• nucleic acids of the invention are substantially homologous to the sequences provided.
• sequences are preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homologous to the sequences provided.
• the invention also provides constructs such as plasmids, vectors, transcription or expression cassettes, which comprise at least one nucleic acid according to the invention.
• an antibody comprising culturing host cells under appropriate conditions so they express the antibody from the nucleic acid.
• any desired fragment or antibody may be isolated and/or purified using any suitable technique, then used as appropriate.
• Suitable host cells include mammalian cells, insect cells, plant cells, yeast cells, or prokaryotic cells, e.g., E coli.
• Mammalian cells available in the art for heterologous polypeptide expression include lymphocytic cell lines (e. g. , NSO), HEK293 cells, Chinese, hamster ovary (CHO) cells, COS cells, HeLa cells, baby hamster kidney cells, oocyte cells.
• the antibodies of the invention are monoclonal antibodies.
• Monoclonal antibodies may be produced by standard methods, as first described by Kohler and Milstein.
• the antibodies may be produced using a hybridoma.
• a hybridoma There is provided a first hybridoma having ECACC accession number 06032901. Also provided is a second hybridoma having ECACC accession number 06032902.
• an antibody produced by a hybridoma according to the invention or a functional fragment thereof.
• an antibody comprising culturing a hybridoma according to the invention under conditions that allow expression of the antibody and isolating the antibody from the culture.
• a hybridoma is a cell created artificially by fusion of a tumour cell with a B-lymphocyte. Such cells are produced in the standard method of producing monoclonal antibodies, as first described by Kohler and Milstein.
• the antibodies of the invention have multiple uses. Firstly, they may be used to further the studies into TSHR and Graves' disease.
• the antibodies also have therapeutic and diagnostic uses.
• the antibodies may be used to target cancer cells, especially thyroid tumor cells and metastases of thyroid tumors.
• the antibodies may be used to deliver radioactive compounds such as radioactive iodine to the tumor cells ("magic bullets") or to stimulate the tumor cells to take up radioactive iodine for both diagnostic and therapeutic purposes.
• radioactive iodine such as radioactive iodine
• they are killed. They can also be identified by scanning for radioactivity using either the magic bullet approach or the radioactive iodine uptake procedure.
• composition comprising an antibody according to the invention.
• composition is suitable for administration to patients.
• it may comprise one or more appropriate pharmaceutical excipient(s) such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.
• suitable pharmaceutical excipient(s) such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.
• suitable pharmaceutical excipients such as solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents.
• Other active compounds may also be included.
• the pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. It may be possible to create compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes. For example, the administration may be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
• Solutions or suspensions used for intradermal or subcutaneous application typically include at least one of the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite ; chelating agents such asethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose.
• the pH can be adjusted with acids or bases.
• Such preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials.
• Solutions or suspensions used for intravenous administration include a carrier such as physiological saline, bacteriostatic water, CremophorELT"" (BASF, Parsippany, NJ), ethanol, or polyol.
• a carrier such as physiological saline, bacteriostatic water, CremophorELT"" (BASF, Parsippany, NJ), ethanol, or polyol.
• the composition must be sterile and fluid for easy syringability. Proper fluidity can often be obtained using lecithin or surfactants.
• the composition must also be stable under the conditions of manufacture and storage. Prevention of microorganisms can be achieved with antibacterial and antifungal agents, e. g. , parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.
• isotonic agents sucrose
• polyalcohols mannitol and sorbitol
• sodium chloride may be included in the composition.
• Prolonged absorption of the composition can be accomplished by adding an agent which delays absorption, e. g. , aluminium monostearate and gelatin.
• Oral compositions include an inert diluent or edible carrier.
• the composition can be enclosed in gelatin or compressed into tablets.
• the antibodies can be incorporated with excipients and prepared as tablets or capsules, for example.
• the oral composition may also contain, for example, a binder, an excipient, a lubricant and flavourings.
• compositions may also be administered by a transmucosal or transdermal route.
• antibodies that comprise a Fc portion may be capable of crossing mucous membranes in the intestine, mouth, or lungs (via Fc receptors).
• Transmucosal administration can be accomplished through the use of lozenges, nasal sprays, inhalers, or suppositories.
• Transdermal administration can also be accomplished through the use of composition containing ointments, salves, gels, or creams known in the art.
• penetrants appropriate to the barrier to be permeated are used.
• antibodies are delivered in an aerosol spray from a pressured container or dispenser, which contains a propellant (e. g., liquid or gas) or a nebulizer.
• a propellant e. g., liquid or gas
• a nebulizer e.g., nebulizer
• antibodies of this invention are prepared with carriers to protect the antibodies against rapid elimination from the body.
• Biodegradable polymers e. g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid
• Methods for the preparation of such pharmaceutical compositions are known by those skilled in the art.
• Antibodies or compositions according to the invention may be administered in therapeutically effective amounts, as determined, based on, for example, the patient's weight, gender, age and medical condition.
• the antibodies or compositions may be administered in a single dose, as a bolus or as continuous therapy.
• the term "effective amount” refers to a dosage or amount that is sufficient to stimulate TSHR activity to produce thyroid hormone or to stimulate the uptake of iodine.
• subject and “patient” are intended to include human and non- human animals.
• Subjects may include a human patient having a thyroid cancer or a metastasis of thyroid cancer.
• non-human animals of the invention includes all vertebrates, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.
• an antibody or functional fragment thereof according to the invention for use in therapy.
• the invention provides the use of an antibody or functional fragment thereof according to the invention, in the preparation of a medicament for the diagnosis or treatment of cancer.
• cancer refers especially to cancers of the thyroid gland and to metastases of such cancers.
• Also provided is a method of locating tumor cells comprising:
• a method of locating tumor cells comprising:
• the radioactive compound is readioactive iodine.
• a method of treating tumor cells comprising:
• Antibodies according to the invention may also be used in assays, such as competition assays for the presence of anti-TSHR antibodies.
• assays such as competition assays for the presence of anti-TSHR antibodies.
• Also provided is a method of assaying for the presence of anti-TSHR antibodies comprising: a) providing a support to which TSHRs are bound; b) applying labeled antibodies according to the invention to the support; c) applying a test sample to the support; and d) assaying the displacement of the antibodies.
• the labeled antibodies bind to the TSHRs and the amount of antibodies bound may be measured. If there are anti-TSHR antibodies in the test sample, they will compete with labeled antibodies to bind to the TSHRs. The amount of anti-TSHR antibodies in the sample can be assayed by measuring the difference in amount of labeled antibodies bound before and after application of the sample. Competition assays of this nature are well known and appropriate techniques and support apparatus could be used by those skilled in the art.
• a labeled antibody is an antibody to which a detectable label has been attached. Suitable labels are well known in the art and examples are discussed above.
• test sample is a sample of serum taken from the patient.
• TS Abs thyroid stimulating antibody activities
• KSAbI IgG and KSAb2 IgG and Fab fragments assessed by bio-assay in CHO cells stably transfected with human TSHR.
• Thyroid stimulating blocking activity of KSAbI (D) and KSAb2 ( ⁇ ) IgG measured in JP09 cells using a sub-saturating concentration of 40 ⁇ U bTSH.
• the TSBAb activity expressed as % inhibition of TSH induced cAMP, was calculated as described in Materials and Methods. Different concentrations of KSAbI and KSAb2 IgG were examined to ensure that the blocking activity was not dependent upon the antibody concentration. The results shown are representative of at least two independent experiments performed in triplicate. Figure 3
• TSH binding inhibiting immunoglobulins TSH binding inhibiting immunoglobulins (TBII) activity of KSAbI IgG ( ⁇ — ⁇ ) and Fab fragments ( ⁇ - • -- • ⁇ ) and KSAb2 IgG ( ⁇ - ⁇ ) and Fab fragments ( ⁇ * ⁇ -.-.-. + ⁇ ) using TRAK (DYNOtest human) kits.
• TKII TSH binding inhibiting immunoglobulins
• Figure 4 Measurement for antibody affinity (Kd) of (A) KSAbI and (B) KSAB2 IgG by saturating binding experiments using human TSHR coated tubes from TRAK (DYNOtest human) kits as a source of immobilized receptor and 125 I-labeled mab (approximately 2.5 ⁇ Ci/ml) with or without increasing concentrations of unlabeled IgG. IC50 and Kd values were calculated using Excel. The antibody affinity was expressed as reciprocal Kd values (L/mol). Non specific binding using an irrelevant IgG mab (GADl) was ⁇ 5%. All determinations were performed in duplicate samples.
• results show the broad spectrum of inhibitory activity of all sera from Graves' disease patients and TSBAb positive hypothyroid patients with 125 I-labeled KSAbI or KSAb2 IgG confirming that the monoclonal antibodies bound similar autoreactive epitopes on TSHR to those recognized by patients' sera.
• the control normal serum gave negligible inhibition ( ⁇ 15%).
• the TT4 values ( ⁇ g/ml) in individual mice from each group (n 3) are shown at time 0 (prior to injection of mab), 7, 26 and 70 hrs.
• the result of administration of KSAb2 IgG by intraperitoneal injection is shown in (C). Control mice treated with irrelevant IgG
• FIG. 1 Histology of thyroid glands from mice following passive transfer of KSAbI or KSAb2 IgG to induce hyperthyroid disease.
• Panel A thyroid from control GADl treated mouse; panels B and C, KSAbI IgG at doses of 10 and lOO ⁇ g respectively;
• Panel D detailed view of collapsed follicles from panel B;
• Panels E and F KSAb2 IgG at doses of 10 and lOO ⁇ g respectively.
• Magnification x60 Apoptotic epithelial cells within colloid ( — ⁇ ) and follicles without colloid showed epithelial cell lining of columnar and cuboidal cells with multilay ering (--- ⁇ ).
• Figures 8 to 73 show sequences of antibodies that are examples of the invention.
• the figures show various fragments of two antibodies. The fragment to which each figure relates is defined in table 2.
• FIG. 74 SDS Polyacrylamide gel electrophoresis of purified rFab preparations under reducing conditions.
• the H-chain fragment of the rFab is labelled with an arrow.
• M standard molecular weight markers.
• FIG. 75 TSH binding inhibition activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and IgGl 7 respectively) and purified rFab fragments (labelled as Fab 9 and Fab 17 respectively) assessed in TRAK assay.
• FIG 76 Thyroid stimulating antibody activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and IgGl 7 respectively) and purified rFab fragments (labelled as Fab 9 and Fab 17 respectively) assessed in isotonic HBSS buffer containing sucrose.
• Figure 77 Thyroid stimulating antibody activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and IgGl 7 respectively) and purified rFab fragments (labelled as Fab 9 and Fab 17 respectively) assessed in isotonic HBSS buffer containing sucrose.
• Figure 77 Thyroid stimulating antibody activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and IgGl 7 respectively) and purified rFab fragments (labelled as Fab 9 and Fab 17 respectively) assessed in isotonic HBSS buffer containing sucrose.
• Figure 77 Thyroid stimulating antibody activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and Ig
• TSH binding inhibition activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and IgGl 7 respectively), purified rFab fragments (labelled as Fab 9 and Fab 17 respectively) and L- chain swaps of the rFab fragment (labelled as Fab 9.1-17L and Fab 17.4-9L respectively) assessed in TRAK assay.
• Thyroid stimulating antibody activities of KSAbI and KSAb2 IgGs (labelled as IgG 9 and IgG 17 respectively), purified rFab fragments (labelled as Fab 9 and Fab 17 respectively) and L-chain swaps of the rFab fragments (labelled as Fab 9.1-17L and Fab 17.4-9L respectively) assessed in isotonic HBSS buffer containing sucrose.
• TSHR antibodies and thyroid function tests were measured using two different types of TSH binding inhibition assays (porcine TRAK RIA and TRAK II [DYNOtest human] kits) (BRAHMS GmbH, Germany) requiring 50 ⁇ l and lOO ⁇ l neat serum respectively, essentially according to the manufacturer's instructions (1). The results were expressed as percentage inhibition of 125 I-TSH binding.
• TSAb and TSBAb activities were measured by bioassay using CHO cells stably transfected with human TSHR, , 24h after seeding 30,000 cells/well in flat bottomed 96-well plates (1, 2).
• HBSS Hank's buffered solution
• IBMX isobutyl-1-methylxanthine
• Assays were also conducted in physiological sodium chloride containing isotonic HBSS, where sucrose was replaced with 13OmM NaCl.
• Bovine TSH bTSH, Sigma- Aldrich
• test serum 3 ⁇ l
• purified IgG diluted in the appropriate HBSS medium was added to each well in triplicates and incubated for 4h at 37C.
• cAMP released into the medium was measured by RIA (R&D systems) and the results expressed as pmols/ml or stimulation over basal value obtained with medium, as described (2).
• TSB Abs were detected similarly by adding a sub-saturating dose of bTSH (40 ⁇ U/ml) with test sample or control serum and measuring the reduction in TSH mediated stimulation of cAMP, as previously described (2).
• the inter- and intra-assay coefficients of variation for TSAbs have been measured as ⁇ 16% and ⁇ 14% and for TSB Abs were ⁇ 24% and 11% respectively (2).
• Total thyroid hormone (TT4) was determined by RIA with 25 ⁇ l of serum (D S Labs, UK), using serum from four normal BALB/c animals for determination of basal values.
• TSHR-Ad human TSHR holoreceptor
• the linear DNA was co-transformed by electroporation into electrocompetent E coli BJ5183 together with supercoiled plasmid containing viral DNA, pAdEasy-1. Recombinants were selected in kanamycin, extracted and digested with Pad to expose the ITRs and finally transfected into HEK293A cells to generate viral particles.
• Adenovirus containing human TSHR-A subunit (amino acids 1 to 289) (referred to as A-subunit-Ad) used.
• Adenovirus expressing ⁇ -galactosidase was used as control and prepared using the AdEasy system.
• AU virus constructs were propagated in HEK293 cells and purified twice over CsCl gradient centrifugation (3, 4), dialysed against PBS and viral concentration determined by optical absorbance at 280nm. Purified adenoviruses were aliquoted and stored at -80C.
• AU mice were obtained from Harlan UK Ltd.
• TSHR-Ad Recombinant adenovirus expressing the human TSHR holoreceptor
• TSHR-Ad was constructed using the AdEasy Adenoviral Vector system (Quantum Biotechnologies). Briefly, TSHR cDNA (1) was excised from pBluescript IISK- by digestion with Kpnl and Notl and ligated into the adenovirus transfer vector (pShuttleCMV) (Quantum Biotechnologies).
• A-subunit-Ad injections a low dose immunization protocol of 10 9 particles was used (4).
• Female BALB/c mice (16 animals, age 7-8 weeks) were immunized as described and bled one week and three weeks after the second injection and tested individually for TSAb activity.
• the animals with consistently elevated TSAb activity received a third injection of A-subunit-Ad.
• Spleen cell suspensions were fused with X63-Ag8653 myeloma cells at a ratio of 5:1 using PEG fusion medium for hybridoma production (50% solution, Immune Systems Ltd, UK) in RPMI medium containing 20% FCS, 2mM sodium pyruvate, 2mM L-glutamine and 0.01% PSF (all from Invitrogen, UK) and plated into 96 well plates.
• Hybridomas were selected under hypoxanthine, aminopterine and thymidine (HAT medium, Invitrogen) and HT medium (Immune Systems Ltd).
• the supernatants (lOO ⁇ l) from wells showing growth were tested for TBII activity using TRAK II (DYNOtest human) kits.
• Fab fragments were prepared from IgG by digestion with papain (Sigma- Aldrich), using 2.5mg IgG, IM cysteine (25 ⁇ l), 2OmM EDTA (25 ⁇ l) and lmg/ml papain in acetate buffer (5 ⁇ l) and incubated overnight at 37C. (6). Following addition of 10OmM iodoacetamide (1 lO ⁇ l) to terminate the reaction, the digest was mixed with protein-A Sepharose for Ih at 4C. After a brief microfuge centrifugation step, the supernatant was collected and dialysed overnight against PBS. The purity of the Fab fragments was examined by SDS polyacrylamide gel electrophoresis and TSHR reactivity confirmed by assessing TBII activity.
• Iodination of IgG and Fab fragments and displacement studies 0.25nM of IgG or Fab fragments of KSAbI or KSAb2 in lO ⁇ l PBS were labeled with 5 ⁇ l 125 I-Na using iodogen coated tubes by incubating for lOmin at room temperature. Free isotope was removed by gel filtration in Sepharose G25 columns and specific activity calculated (7). Antibody affinity was measured by saturation binding analysis.
• Injection of different doses of purified IgG of KSAbI or KSAb2 was performed by the intravenous and the intraperitoneal route.
• Female BALB/c mice (18 animals, age 7-8 weeks) were treated with a single intravenous injection in the tail vein of KSAbI or KSAb2 IgG in sterile PBS (50 ⁇ l) containing lO ⁇ g or lOO ⁇ g antibody (3 mice per group).
• Another group of mice were treated with a single intraperitoneal injection of KSAb2 IgG in sterile PBS (lOO ⁇ g).
• mice were injected intravenously with isotype matched, lOO ⁇ g IgG mab specific for the islet cell antigen, glutamic acid decarboxylase (mab GAD-I). AU animals were bled at time 7, 28 and 7Oh post injection and serum TT4 levels were determined. Mice were sacrificed at 7Oh and thyroid glands excised for histological analysis.
• Thyroid histology Thyroid glands were fixed in formalin and processed in formalin. Sections were stained with hematoxylin and eosin for morphological analysis. Immunohistochemistry was performed for the detection of B and T cells on the fixed thyroid sections with anti-mouse CD20 followed by detection with the ImmunoCruz anti-goat kit (Santa Cruz Biotechnology), rat anti-mouse CD4 and CD8 mabs followed by detection with biotinylated anti-rat antibody (Vector laboratories, UK) and a strepavidin-biotin peroxidase conjugate (Dako, Denmark). Antigen retrieval was performed prior to staining by pressure cooking at pH 6.0 for CD20 and CD8 antibodies, and at pH 9.0 for CD4 antibody.
• mice were immunized with recombinant adenovirus expressing the TSHR holoreceptor (TSHR-Ad) and the TSHR-A subunit (A-subunit-Ad) to induce Graves' hyperthyroid disease.
• TSHR-Ad TSHR holoreceptor
• A-subunit-Ad TSHR-A subunit
• Monoclonal antibodies Culture supernatants (lOO ⁇ l) were collected from 70-80% confluent wells and tested neat for TBII activity using TRAK II [DYNOtest human) kits. A total of 250 wells were screened, resulting in 3 positive wells (well 9, 98%; well 17, 96% and well 233, 80% inhibition). Upon expansion, the TBII activity of well 233 primary cell line declined rapidly. The remaining two lines were cloned twice at 0.3 cells per well and renamed KSAbI and KSAb2, which have been in continuous culture for >7months. The H- and L-chain subtypes for KSAbI and KS Ab2 were shown to be IgG2b/k and IgG2a/k respectively.
• KSAbI and KSAb2 IgG stimulated cAMP production in CHO cells stably transfected with human TSHR.
• Initial dose response studies were conducted in NaCl-free sucrose containing medium routinely used for its increased sensitivity for detecting TSAbs (8).
• Figure IA in dose response studies both KSAbI and KSAb2 IgG showed typical sigmoid curves by stimulating TSHR to reach > 98% of the response achieved with a sub- saturating dose of bTSH.
• both the mabs show full agonist activity by achieving near maximal cAMP stimulatory responses, they showed differences in their cAMP stimulatory responses at lower doses of IgG.
• KSAbI and KSAb2 showed maximal stimulation of 199 and 183 fold over basal value, with 3 fold stimulation obtained at 1.2ng/ml and 2.2ng/ml IgG respectively ( Figure IA).
• the EC50 values for KSAbI and KSAb2 IgG were determined to be 9.4ng/ml and 93ng/ml respectively ( Figure IA).
• Fab fragments of KSAbI and KSAb2 also gave similar TSAb responses to the intact parental IgG ( Figure IA) and therefore also behaved as full agonists for the TSHR.
• KSAbI and KSAb2 continued to be active at concentrations of ⁇ lng/ml and 3ng/ml respectively.
• the IgGs showed EC50 values in the nM range of 16.5ng/ml and lOOng/ml respectively ( Figure IB).
• Fab fragments of KSAbI and KSAb2 also showed similar efficacies of cAMP stimulation ( Figure IB).
• TSH mediated stimulation (TSBAb) activity
• KSAbI and KSAb2 IgG Blocking of TSH mediated stimulation (TSBAb) activity
• TSH mediated stimulation blocking assay Different concentrations of IgG were examined in the assay to ensure that the blocking activity was not dependent upon the antibody concentration.
• KSAbI showed negligible TSBAb activity
• KSAb2 IgG showed a reproducible > 20% TSBAb activity in all antibody concentrations tested ( ⁇ 30ng/well) equivalent to 240ng/ml, which were below the sub-saturating concentration of agonist activity for KSAb2. Consequently, KSAb2 acted with weak antagonism to TSH mediated stimulation of cAMP ( Figure 2).
• TSH binding inhibiting immunoglobulin (TBII) TSH binding inhibiting immunoglobulin
• both 125 I-labeled KSAbI and KSAb2 IgG bound the receptor with high affinity, with Kds of 4.5 x 10 10 L/mol and 6.25 x 10 10 L/mol respectively ( Figure 4).
• the labeled IgG and Fab fragments of the mabs were then used as tracers in competition studies to study the epitopes on the receptor.
• KSAbI and KSAb2 IgG were also performed with another panel of anti-TSHR IgG mabs which are specific for linear determinants on the receptor and which show negligible thyroid stimulatory activity (9).
• KSAbI or KSAb2 IgG showed any competition with the mabs AlO, A9 and A7 which are specific for residues located in the amino, middle and the carboxy-terminal regions of the receptor respectively (9) (not shown).
• the stimulatory epitopes on the TSHR are different from the linear epitopes recognized by this panel of anti-TSHR mabs.
• the follicles containing the pale colloid were lined with flattened and attenuated epithelial cells, whilst the follicles without colloid showed epithelial cell lining of columnar and cuboidal cells with multilayering (broken arrows in Figure 7.
• individual necrotic cells were found to be present within the luminal colloid and also within the follicular lining epithelium with picnotic nuclei (shown in Figure 7, panel F).
• histological analysis of the H&E sections revealed no mononuclear cell infiltrate into the glands of the KSAbI and KSAb2 IgG treated animals, irrespective of the dose or the route of administration ( Figure 7, panel B- F). This was further substantiated by immunohistochemical staining of the thyroid glands, whereby staining with antibodies to mouse CD4, CD8 and CD20 failed to identify any B or T cell infiltrate (not shown).
• Heavy and light chain variable region fragments were expressed as rFab fragments in E.coli, purified and tested for binding to TSHR by a TSH binding inhibition assay and by measuring cAMP accumulation following TSHR stimulation. Both fragments bound to TSHR. Differences in binding between the fragments and the antibodies may be due to steric hindrance in the fragments, resulting from the expression system used. Results of the assays are shown in Figures 75 and 76.
• Antibodies are clonally related
• KSAbI and KSAb2 fall into this category of disease associated antibodies as they display (i) full agonist activity for the human TSHR (ii) potency in nanogram quantities of IgG (iii) high affinity for human TSHR in 10 10 L/mol range and (iv) in vivo pathogenicity with the induction of hyperthyroidism.
• KSAbI and KSAb2 have comparable affinities and exhibit differences in cAMP generation, particularly at very low doses of TSH.
• Antibodies with potent TSAb activity are pathogenic and directly responsible for the hyperthyroid status. Examination of serum thyroxine levels in individual mice showed that a number of animals where the hormone levels failed to correlate with serum TSAb activity. This was not surprising since a lack of concordance is also recognized both in human patients and in various models of induced Graves' disease (1, 3, 14, 15).
• histological analysis of the thyroid glands at 7Oh post injection showed morphological changes suggestive of both stimulatory and cytotoxic effects.
• the stimulatory effects were characterized by proliferative epithelial changes as evident by foci of hyperplastic follicles with loss of colloid formation associated with multilayering and luminal collapse.
• the cytotoxic effects were present as individual cell necrosis within the lining epithelium and dying thyrocytes present within the colloid material. These cytotoxic effects are likely to be mediated as a result of the increased thyroid hormone production induced in the gland by the TS Abs, leading to a massive accumulation of hydrogen peroxide within the apical surface of the thyroid follicles (16, 17).
• potent thyroid stimulating mabs such as KSAbI and KSAb2 opens the way to a molecular dissection of the TSHR and the epitopes associated with autoimmune disease.
• Mapping of the conformational epitopes on the TSHR recognized by KSAbI and KSAb2 may be performed by phage random peptide library screening in conjunction with mutagenesis (20). This will help in the characterization of the epitopes associated with thyroid stimulation on the TSHR and their modes of intracellular signaling.
• An understanding of these signaling events may also be relevant to the complications of Graves' disease such as TAO and pretibial myxoedema.
• Table 1 Serum thyroid stimulating antibody activities and thyroxine levels in BALB/c mice immunized with Adenovirus TSHR A subunit, measured one week following second immunization. Eleven animals (68%) were hyperthyroid (shown in bold).
• KSAb2 heavy chain
• KSAb2 light chain
• Thyroid-stimulating antibody is related to Graves' ophthalmopathy, but thyroglobulin-binding inhibitor immunoglobulin is related to hyperthyroidism in patients with Graves'disease. Thyroid 10: 809-813.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Immunology (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• Biochemistry (AREA)
• Hematology (AREA)
• Urology & Nephrology (AREA)
• Cell Biology (AREA)
• Biophysics (AREA)
• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Oncology (AREA)
• Neurology (AREA)
• Biotechnology (AREA)
• Hospice & Palliative Care (AREA)
• Microbiology (AREA)
• Endocrinology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Veterinary Medicine (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Public Health (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Priority Applications (4)

Applications Claiming Priority (4)

Publications (1)

Family

ID=38093407

Family Applications (1)

Country Status (5)

Cited By (1)

Families Citing this family (34)

Citations (3)

Family Cites Families (1)

• 2007
  • 2007-03-29 JP JP2009502214A patent/JP5165672B2/ja active Active
  • 2007-03-29 WO PCT/GB2007/001139 patent/WO2007110648A1/en not_active Ceased
  • 2007-03-29 US US12/294,760 patent/US8603466B2/en active Active
  • 2007-03-29 ES ES07732193.3T patent/ES2482145T3/es active Active
  • 2007-03-29 EP EP07732193.3A patent/EP1999153B1/en active Active

Patent Citations (3)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events