Patent Application
20130183313
The present invention relates to antibodies which are reactive with the thyrotropin (TSH) receptor (TSHR), and in particular, though not exclusively, to antibodies which bind to the TSHR and which can block TSHR stimulation by TSH- or TSHR-stimulating antibodies.
Thyrotropin, or thyroid stimulating hormone (TSH), is a pituitary hormone that regulates thyroid function via the TSHR (Szkudlinski M W, Fremont V, Ronin C, Weintraub B D 2002 Thyroid-stimulating hormone and TSHR structure-function relationships. Physiological Reviews 82: 473-502). The TSHR is a G-protein coupled receptor and is composed of three domains:—a leucine rich domain (LRD), a cleavage domain (CD) and a transmembrane domain (TMD) (Nunez Miguel R, Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Blundell T L, Rees Smith B, Furmaniak J 2004 Analysis of the thyrotropin receptor-thyrotropin interaction by comparative modelling. Thyroid 14: 991-1011). Binding of TSH to the TSHR triggers receptor signalling which leads to stimulation of formation and release of thyroid hormones; thyroxine (T4) and tri-iodothyronine (T3). A negative feedback mechanism involving the levels of T4 and T3 in the circulation controls the release of TSH from the pituitary (and thyrotropin releasing hormone secreted by the hypothalamus) that in turn controls thyroid stimulation and the levels of thyroid hormones in serum.
It is well documented in the art that some patients with autoimmune thyroid disease (AITD) have autoantibodies reactive with the TSHR (Rees Smith B, McLachlan S M, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In a majority of cases, these autoantibodies bind to the TSHR and mimic the actions of TSH thereby stimulating the thyroid to produce high levels of T4 and T3. These autoantibodies are described as thyroid stimulating autoantibodies or TSHR autoantibodies (TRAbs) with stimulating activity or TSH agonist activity. The physiological feedback mechanism of thyroid function control mentioned above is not effective in the presence of such thyroid stimulating autoantibodies and patients present with symptoms of thyroid hyperactivity or thyrotoxicosis (excess of thyroid hormones in serum). This condition is known as Graves' disease. In some patients, TRAbs with stimulating activity are thought to be responsible for interaction with TSHRs in retro-orbital tissues and to contribute to the eye signs of Graves' disease. A human monoclonal autoantibody which acts as a powerful thyroid stimulator (hMAb TSHR1) has been described in detail in patent application WO2004/050708A2.
In contrast in some patients with AITD, autoantibodies bind to the TSHR, prevent TSH from binding to the receptor but do not have the ability to stimulate the TSHR. These types of autoantibody are known as TRAbs with blocking activity or TSH antagonist activity, and patients who have blocking TRAbs in their serum may present with symptoms of an under-active thyroid (hypothyroidism) (Rees Smith B, McLachlan S M, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In particular, TRAbs with blocking activity when present in serum of pregnant women cross the placenta and may block foetal thyroid TSHRs leading to neonatal hypothyroidism and serious consequences for development. Furthermore, TRAbs with blocking activity can be found in breast milk of affected mothers and this may contribute further to clinical hypothyroidism in the baby. To date human monoclonal autoantibodies to the TSHR with TSH antagonist activity have not been available. Consequently, detailed studies of how this type of autoantibody interacts with the TSHR, and how their interactions with the TSHR compare with those of stimulating type of autoantibodies (such as M22) and with TSH, have been limited.
Human chorionic gonadotropin is a hormone produced during pregnancy which has mild thyroid stimulating effects.
Characterisation of the properties of TRAbs with stimulating or blocking activities is of critical importance in studies which aim to improve the diagnosis and management of diseases associated with an autoimmune response to the TSHR. The invention described in patent application WO2004/050708A2 provides details about the properties of a human monoclonal autoantibody with powerful stimulating activity and its interaction with the TSHR. Furthermore, patent application WO2006/016121A discloses a mutated TSHR preparation including at least one point mutation which can be used in the differential screening and identification of patient serum stimulating TSHR autoantibodies, patient serum blocking TSHR autoantibodies and TSH in a sample of body fluid from a patient being screened. Patent application WO2004/050708A2 also describes a mouse monoclonal antibody (9D33) with TSHR blocking activity. 9D33 binds to the TSHR with high affinity (2×1010 L/mol) and is an effective antagonist of TSH, hMAb TSHR1 (M22) and patient serum TRAbs with stimulating or blocking activities (patent application WO2004/050708A2 and Sanders J, Allen F, Jeffreys J, Bolton J, Richards T, Depraetere H, Nakatake N, Evans M, Kiddie A, Premawardhana L D, Chirgadze D Y, Miguel R N, Blundell T L, Furmaniak J, Rees Smith B 2005 Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist. Thyroid 15: 672-682). Although the mouse monoclonal antibody 9D33 shows at least some of the characteristics of patient serum TRAbs with blocking activity, it is a mouse antibody generated by immunisation of an experimental animal with the TSHR and as such may not be truly representative of TSHR autoantibodies generated in the process of an autoimmune response to the TSHR in humans. As a mouse monoclonal antibody, 9D33 would need to be humanised for in vivo applications in humans. This may be disadvantageous in view of the expense and complication involved in the humanisation process.
The present invention results from the production and properties of a human monoclonal autoantibody (5C9) to the TSHR that is an effective antagonist of TSH and of stimulating TRAbs in patient sera. 5C9 has been isolated from the peripheral lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies. The lymphocytes were immortalised by infection with Epstein Barr virus (EBV) and positive clones fused with a mouse/human cell line to generate a stable clone. IgG was purified from supernatants of clone cultures and the ability of 5C9 IgG to bind to the TSHR and influence TSHR activity was assessed. In particular, the ability of 5C9 to inhibit TSH binding to the TSHR, and to inhibit cyclic AMP stimulating activity of TSH was studied. Furthermore, the ability of 5C9 to inhibit binding of stimulating or blocking patient serum TRAbs to the TSHR and to inhibit their biological activity was also assessed. In addition, the use of 5C9 in assays for TSHR antibodies, TSH and related compounds was investigated. Variable region (V region) genes of the heavy (HC) and light chains (LC) of 5C9 were sequenced and the complementarity determining regions (CDRs) assigned.
According to a first aspect of the invention there is provided an isolated human antibody for the TSHR which is an antagonist of TSH.
According to second aspect of the invention there is provided an isolated humanised antibody for the TSHR which is an antagonist of TSH.
An antibody according to either the first or second aspect of the invention is “an antibody according to the invention”.
An antibody according to the invention may be an antagonist of thyroid stimulating antibodies.
An antibody according to the invention may have the TSH antagonist characteristics of patient serum TSHR autoantibodies which are TSH antagonists.
An antibody according to the invention may be an antagonist of TSH and an antagonist of thyroid stimulating antibodies.
An antibody according to the invention may have the antagonistic characteristics of patient serum TSHR autoantibodies which are antagonists of thyroid stimulating antibodies.
An antibody according to the invention may be an inhibitor of binding to TSHR or a portion thereof by TSH, by M22, or antibodies with stimulating activity or antibodies with blocking activity to the TSHR. A TSHR portion may include the LRD or a substantial portion thereof. Preferably, an antibody which prevents such binding.
An antibody according to the invention may be a monoclonal or recombinant antibody, or comprise or consist of a fragment thereof which is an antagonist of TSH. An antibody according to the invention may comprises a VH region which comprises one or more CDRs selected from CDR 1, CDR 2, or CDR 3, shown in
An antibody according to the invention may have a binding affinity for human full length TSHR of about 1010 L/mol. Preferably an antibody according to the invention has a binding affinity for human full length TSHR of about 109 L/mol.
The invention helps the skilled addressee to understand the immunological mechanisms which drive development and production of stimulating and blocking TSHR autoantibodies. Additionally, the invention helps the skilled addressee to understand molecular differences between TSHR autoantibodies with thyroid stimulating activity and with blocking activity. In addition, the method of medical treatment and pharmaceutical compositions of the invention provide new treatments for thyroid-related conditions.
A preferred antibody in accordance with the invention is 5C9. 5C9, has been found unexpectedly to inhibit thyroid stimulating hormone receptor constitutive activity, that is to say the production of cyclic AMP in a test system in the absence of thyroid stimulating hormone or M22. This may be particularly advantageous in the treatment of thyroid cancer cells remaining in the thyroid, or in metastases, especially in preventing or delaying regrowth as those cells will grow more rapidly as a consequence of thyroid stimulating hormone receptor constitutive activity.
The term “antibody” and cognate terms, such as “antibodies”, used herein embraces according to context immunoglobulin-based binding moieties such as monoclonal and polyclonal antibodies, single chain antibodies, multi-specific antibodies and also binding moieties, which may be substituted by the skilled addressee for such immunoglobulin-based binding moieties, such as domain antibodies, diabodies, IgGΔCH2, F(ab′)2, Fab, scFv, VL, VH, dsFv, Minibody, Triabody, Tetrabody, (scFv)2, scFv-Fc, F(ab′)3 (Holliger P, Prospero T, Winter G 1993 “Diabodies: small bivalent and bispecific antibody fragments” Proc Natl Acad Sci USA 90: 6444-6448.), (Carter P J 2006 “Potent antibody therapeutics by design” Nat Rev Immunol 6: 343-357).
The term “TSHR” refers to full length human thyroid stimulating hormone receptors having the amino acid sequence shown in
According to another aspect of the invention there is provided a nucleotide comprising:
According to another aspect of the invention there is provided a vector comprising a nucleotide according to the above aspect of the invention.
The vector may be a plasmid, virus or fragment thereof. Many different types of vectors are known to the skilled addressee.
According to another aspect of the invention there is provided an isolated cell including an antibody; nucleotides or/vector according to the invention. The isolated cell may express an antibody according to the invention. Preferably, the isolated cell secretes an antibody according to the invention. Preferably an isolated cell according to the invention is from a stable hetero-hybridoma cell line.
According to a further aspect of the invention there is provided a composition comprising a defined concentration of TSHR autoantibodies and including an antibody according to the invention. Such a composition may comprise a defined concentration of TSHR autoantibodies with TSH antagonist activity, and includes an antibody according to the invention.
Alternatively, a composition according to this aspect of the invention may comprise a defined concentration of TSHR autoantibodies which are antagonists of thyroid stimulating antibodies, and includes an antibody according to the invention. A composition may comprise a defined concentration of TSHR autoantibodies with TSH antagonist activity and which are antagonists of thyroid stimulating antibodies, and includes an antibody according to the invention.
According to another aspect of the invention there is provided a pharmaceutical composition for administration to a mammalian subject for the treatment of a thyroid-related condition comprising an antibody according to the invention, together with a pharmaceutically acceptable carrier. The thyroid-related condition may be selected from thyroid overactivity, Graves' eye disease, neonatal hyperthyroidism, human chorionic gonadotrophin-induced hyperthyroidism, pre-tibial myxoedema, thyroid cancer and thyroiditis.
A pharmaceutical composition according to the invention may be suitable for human administration. Preferably a pharmaceutical composition according to the invention has no significant adverse effect on the immune system of the subject.
A pharmaceutical composition according to the invention may include an additional thyroid stimulating hormone receptor antagonist. A suitable additional thyroid stimulating hormone receptor antagonist is 9D33 as disclosed in WO2004/050708.
Various formats are contemplated for pharmaceutical compositions according to the invention. A pharmaceutical composition according to the invention for use in the treatment of a thyroid-related condition may be in an injectable format. A pharmaceutical composition according to the invention for use in the treatment of pre-tibial myxoedema is preferably in a topical format. A pharmaceutical composition according to the invention for use in the treatment of Graves' eye disease is preferably in the form of eye drops.
Pharmaceutical compositions of this invention comprise any antibody in accordance with the invention of the present invention, with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
The pharmaceutical compositions of the invention may be administered orally, parenterally, by inhalation spray, topically, by eyedrops, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration or administration by injection. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavouring and/or colouring agents may be added.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
According to a further aspect of the invention there is provided a method of producing an antibody according to the invention, the method comprising culturing one or more isolated cells according to the invention whereby the antibody is expressed by the cell. Preferably, the antibody is secreted by the cell.
According to a further aspect of the invention there is provided a method of treating a thyroid-related condition in a mammalian subject, or in cells derived from the subject, the method comprising contacting the subject, or the cells, with an antibody according to the invention.
According to another aspect of the invention there is provided a method of inhibiting thyroid stimulating autoantibodies stimulating the TSHR in the thyroid of a mammalian subject, the method comprising contacting the subject with an antibody according to the invention. Preferably binding of thyroid stimulating autoantibodies to the TSHR is prevented.
According to another aspect of the invention there is provided a method of inhibiting thyroid stimulating autoantibodies binding to extra-thyroidal TSHRs in a mammalian subject, the method comprising contacting the subject with an antibody according to the invention. The extra-thyroidal TSHRs may be in retro-orbital tissues and/or pre-tibial tissue of the subject. The antibody of the invention preferably blocks TSHR autoantibodies binding to extra-thyroidal TSHRs when used in the method.
According to another aspect of the invention there is provided a method of treating thyroid cancer in the thyroid, or in metastases, in a subject or in thyroid cells derived from a subject, the method comprising contacting the cancerous cells with an antibody according to the invention, in order to inhibit constitutive thyroid stimulating hormone receptor activity in the cells. Preferably regrowth of thyroid cancer cells is prevented or delayed.
There is also provided a method of treating thyroid overactivity due to constitutive thyroid activity, in a subject or in thyroid cells derived from a subject, the method comprising contacting the subject or thyroid cells with an antibody according to the invention, in order to inhibit thyroid overactivity due to constitutive thyroid activity.
The subject treated in the various methods of the invention described above is preferably human.
According to another aspect of the invention there is provided the use of an antibody according to the invention in the treatment of a thyroid-related condition. Alternatively there is provided the use of an antibody according to the invention in the preparation of a medicament for the treatment of a thyroid-related condition.
There is also provided an antibody according to the invention for use in medical therapy. In particular, there is provided the use of an antibody according to the invention for use in the treatment of a thyroid-related condition. The thyroid-related condition may be selected from thyroid overactivity, Graves' eye disease, neonatal hyperthyroidism, human chorionic gonadotrophin-induced hyperthyroidism, pre-tibial myxoedema, thyroid cancer and thyroiditis.
According to another aspect of the invention there is provided a method of characterising TSHR antibodies comprising determining binding of a TSHR antibody under test to a polypeptide having a TSHR-related amino acid sequence in which the method involves a method step including the use of an antibody according to the invention. Preferably the method comprises determining the effects of an antibody according to the invention on binding of a TSHR antibody to that polypeptide. The polypeptide having a TSHR-related amino acid sequence preferably comprises full length human TSHR.
According to another aspect of the invention there is provided a method for characterising TSH and related molecules, comprising determining binding of TSH, or a related molecule under test, to a polypeptide having a TSHR-related amino acid sequence, in which the method involves a method step including the use of an antibody according to the invention.
Methods for characterising TSHR antibodies, or TSH and related methods described above may be in an ELISA format.
According to another aspect of the invention there is provided a method of determining TSHR amino acids involved in binding TSHR autoantibodies which act as antagonists, the method comprising providing a polypeptide having a first TSHR-related amino acid sequence to which an antibody according to the invention binds, modifying at least one amino acid in the TSHR-related amino acid sequence and determining the effect of such modification on binding of the antibody.
A method of modifying an antibody according to the invention, the method comprising modifying at least one amino acid of the antibody and determining an effect of such a modification on binding to a TSHR-related sequence. Preferably modified TSHR antibodies are selected which have an enhanced affinity for the TSHR.
According to another aspect of the invention there is provided a method of identifying molecules which inhibit thyroid stimulating antibodies binding to the TSHR, the method comprising providing at least one antibody according to the invention as reference. Preferably molecules under test which prevent thyroid stimulating antibodies binding to the TSHR are selected.
There is also provided a method of identifying molecules which inhibit thyroid blocking antibodies binding to the TSHR, the method comprising providing at least one antibody according to the invention as reference. Preferably molecules which prevent thyroid blocking antibodies binding to TSHR are selected.
Antibodies and methods in accordance with the invention will now be described, by way of example only, with reference to the accompanying drawings,
Lymphocyte Isolation and Cloning of the Human Monoclonal TSHR Autoantibody 5C9 The monoclonal autoantibody 5C9 was isolated generally using the procedure described in WO2004/050708A2. Lymphocytes were first isolated from a blood sample collected from a patient with postpartum hypothyroidism and high levels of TRAbs (Local Ethical Committee approval was obtained). The lymphocytes were infected with Epstein Barr Virus (EBV) (European Collection of Cell Cultures—ECACC; Porton Down, SP4 0JG,UK) and cultured on mouse macrophage feeder layers as described in WO2004/050708A2. Immortalised lymphocytes secreting TSHR autoantibodies were fused with a mouse/human hybrid cell line K6H6/B5 (ECACC) and cloned four times by limiting dilution to obtain a single colony. The presence of TSHR autoantibody in cell culture supernatants at different stages of cloning was detected by inhibition of 125I-labelled TSH binding to the TSHR (WO2004/050708A2). A single clone producing the TSHR autoantibody was expanded and supernatants from the cultures were harvested for autoantibody purification.
5C9 IgG was purified from culture supernatants using protein A affinity chromatography on MabSelect™ (GE Healthcare, UK) as described in Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Kiddie A, Brereton K, Premawardhana L D, Chirgadze D Y, Nunez Miguel R, Blundell T L, Furmaniak J, Rees Smith B 2004 Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function. Thyroid 2004 14: 560-570) and purity assessed by SDS-polyacrylamide gel electrophoresis (PAGE).
The heavy chain isotype of 5C9 was determined using a radial diffusion assay (The Binding Site; Birmingham, B29 6AT, UK), and the light chain isotype was determined by Western blotting with anti-human kappa chain and anti-human lambda chain specific mouse monoclonal antibodies (Sigma-Aldrich Company Ltd, Poole, UK).
5C9 IgG at 10 mg/mL in 20 mmol/L sodium acetate pH 4.5 was incubated with immobilized pepsin prepared according to the manufacturer's instructions (Perbio Science UK Ltd, Cramlington, UK) for 4½ hours at room temperature with shaking. Thereafter, immobilised pepsin was removed by centrifugation (1000×g, 5 minutes at room temperature) and the supernatant dialysed against 300 mmol/L NaCl, 10 mmol/L Tris-HCl pH 7.5 overnight at 4° C. The dialysed mixture containing 5C9 F(ab′)2 and small amounts of intact IgG was separated using a Sephacryl S-300 High Resolution Matrix (GE Healthcare, Chalfont St Giles, UK). The 5C9 F(ab′)2 preparations purified in this way did not contain intact IgG as judged by SDS-PAGE and HPLC gel filtration analyses.
Furthermore, F(ab′)2 was reduced using a final concentration of 100 mmol/L L-cysteine for 1 hour at 37° C. The reaction was stopped with a final concentration of 50 mmol/L iodoacetamide for 30 minutes at room temperature. The F(ab′) was purified using a Sephacryl S-300 column as above. F(ab′) preparations purified in this way did not contain F(ab′)2 as judged by SDS-PAGE and HPLC gel filtration analysis.
In addition, 5C9 IgG was treated with mercuripapain (Sigma, UK) at an enzyme/protein ratio of 1:100 dialysed into 50 mmol/L NaCl, Tris-HCl pH 9.0 and passed through an anion exchange Sepharose (Q-Sepharose Fast flow from GE Healthcare) column to separate intact IgG or Fc from the Fab preparation. Analysis by SDS-PAGE and gel filtration (Sephacryl S-300 column; as above) indicated that intact IgG was undetectable in the Fab preparation.
5C9 IgG was labelled with 125I as described in Sanders J, Oda Y, Roberts S, Kiddie A, Richards T, Bolton J, McGrath V, Walters S, Jaskolski D, Furmaniak J, Rees Smith B 1999. The interaction of TSH receptor autoantibodies with 125I-labelled TSH receptor. Journal of Clinical Endocrinology and Metabolism. 1999 84: 3797-3802) or with biotin hydrazide (Perbio Science, Cramlington, UK).
Binding inhibition assays were carried out using TSHR coated tubes as described in WO2004/050708A2. In the assay, 100 μL of test sample (MAb preparation, patient serum or unlabelled TSH) and 50 μL of start buffer (RSR Ltd) were incubated in TSHR coated tubes for 2 hours at room temperature with gentle shaking. After aspiration, the tubes were washed and 100 μL of 125I-labelled protein (5×104 cpm) added and incubated for 1 hour at room temperature with shaking. The tubes were then aspirated, washed and counted in a gamma counter.
Inhibition of labelled protein binding was calculated as:—
Control material was a pool of healthy blood donor sera or individual healthy blood donor sera or other materials as indicated in the results of various experiments.
Unlabelled 5C9 IgG in 50 μL of assay buffer (50 mmol/L NaCl, 10 mmol/L Tris pH 7.8 and 1% Triton X-100) and 50 μL of 125I-labelled 5C9 IgG (30,000 cpm in assay buffer) were incubated in TSHR coated tubes for 2 hours at room temperature with shaking (maximum binding occurred under these conditions), aspirated, washed twice with 1 mL of assay buffer and counted in a gamma counter. The concentration of IgG bound vs bound/free was plotted (Scatchard G 1949 The attraction of proteins for small molecules and ions. Annals of the New York Academy of Sciences 51: 660-672) to derive the association constant.
The ability of 5C9 IgG and other preparations to stimulate production of cyclic AMP in Chinese hamster ovary (CHO) cells transfected with the human TSHR was tested as described in WO2004/050708A2. CHO cells expressing either approximately 5×104 or approximately 5×105 TSHR per cell were seeded into 96-well plates at 3×104 cells per well, adapted into DMEM (Invitrogen Ltd, Paisley, UK) without foetal calf serum and then test samples (TSH, IgG or patient serum) added (100 μL diluted in cyclic AMP assay buffer i.e. NaCl free Hank's Buffered Salts solution containing 1 g/L glucose, 20 mmol/L HEPES, 222 mmol/L sucrose, 15 g/L bovine serum albumin and 0.5 mmol/L 3 isobutyl-1-methylxanthine pH 7.4) and incubated for 1 hour at 37° C. After removal of test solutions, cells were lysed and cyclic AMP concentration in the lysates assayed by one of two methods: 1) using a Biotrak enzyme immunoassay system from GE Healthcare, Chalfont St Giles, UK; or 2) using Direct Cyclic AMP Correlate—EIA kits from Assay Designs; Cambridge Bioscience, UK. Results are expressed as pmol/mL of cyclic AMP in the cell lysate (200 μl) or as Pmol per cell well.
The ability of 5C9 IgG and other preparations to inhibit the stimulating activity of porcine (p) TSH, native human (h) TSH and recombinant human (rh) TSH, MAb M22 and patient serum TRAbs in CHO cells expressing TSHRs was assessed. This was carried out by comparing the stimulatory effect of TSH, M22 or TRAbs in the absence and in the presence of 5C9 IgG (or other preparations being tested). The assay was carried out as described above except 50 μL of 5C9 (or other preparations being tested) diluted in cyclic AMP assay buffer was added to the cell wells followed by 50 μL of TSH or M22 or patient serum (diluted as appropriate in cyclic AMP assay buffer) and incubated and tested as for the stimulating assay described above.
Other MAbs and sera from patients with blocking type TRAbs were tested in this assay in addition to 5C9.
The variable region genes of the 5C9 heavy and light chains were determined as described in WO2004/050708A2, using total RNA prepared from 1×107 hetero-hybridoma cells secreting 5C9 IgG to produce mRNA for RT-PCR (reverse transcriptase PCR) reactions. Specific IgG1 HC and kappa LC sense and antisense strand oligonucleotide primers were designed using the Medical Research Council's V-base (http://vbase.mrc-cpe.cam.ac.uk/) and synthesised by Invitrogen (Paisley, PA4 9RF, UK). The RT reaction was carried out at 50° C. for 15 minutes followed by 40 cycles of PCR at 94° C. for 15 seconds, 50° C. for 30 seconds and 72° C. for 30 seconds. DNA products were cloned into pUC18 and sequenced by the Sanger-Coulson method (Sanger F, Nicklen S, Coulson A R 1977 DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences of USA 74: 5463-5467). V region sequences were compared with available sequences of human Ig genes using Ig blast (http://www.ncbi.nlm.nih.gov/igblast/).
The methods used to introduce specific mutations into the TSHR sequence have been described in patent application WO2006/016121A. Furthermore, the transfection of mutated TSHR constructs into CHO cells using the Flp-In system is also described in WO2006/016121A.
Flp-In-CHO cells expressing either wild type or mutated TSHRs were seeded into 96 well plates and used to test the ability of 5C9 preparations to block the stimulating activity of TSH, M22 or patient serum TRAbs as described above.
5C9 IgG at 2.55 mg/mL was dialysed into 100 mmol/L sodium phosphate buffer pH 8.5 and reacted with EZ-Link NHS-LC-Biotin (Perbio) using a molar ratio of IgG to biotin of 1/10. Test serum samples (75 μL) were incubated in TSHR-coated ELISA plate wells (RSR Ltd) for 2 hours at room temperature with shaking (500 shakes per minute). After removing the test samples and washing, biotin labelled 5C9 IgG (2 ng in 100 μL) was added and incubation continued for 25 min at room temp without shaking. The wells were emptied, washed and 100 μL streptavidin-peroxidase (10 ng in 100 μL; RSR Ltd) was added and incubated for 20 min at room temperature without shaking. The wells were then washed three times, the peroxidase substrate tetramethyl benzidine (TMB; 100 μL; RSR Ltd) was added and incubated for 30 minutes at room temperature in the dark without shaking. 50 μL of 0.5 mol/L H2SO4 was then added to stop the reaction and the absorbance of each plate well was read at 450 nm using an ELISA plate reader. Inhibition of 5C9 IgG-biotin binding was calculated as:—
Lymphocytes (27×106) obtained from 20 mL of patient's blood were infected with EBV and plated out at 1×106 cells per well in a 48 well plate on feeder layers of mouse macrophages.
On day 13 post EBV infection the plate well supernatants were monitored for inhibition of 125I-TSH binding. Cells from the positive wells were expanded and fused with the K6H6/B5 hybridoma cell line and plated out in 96 well plates. One clone stably producing antibody with 125I-TSH binding inhibiting activity was obtained and re-cloned 4 times. The monoclonal antibody, designated as 5C9, purified from hetero-hybridoma culture supernatants was subclass IgG1 with kappa light chains.
The ability of different concentrations of 5C9 IgG to inhibit binding of labelled TSH or labelled M22 or labelled 5C9 itself to the TSHR is shown in Table 1. As shown in Table 1, 12% inhibition of 125I-TSH binding was observed with as little as 0.005 μg/mL of 5C9 IgG and the inhibition increased in a dose dependent manner up to 84% inhibition at 100 μg/mL of 5C9. This can be compared to 125I-TSH binding inhibition by donor serum IgG; 13% inhibition at 0.05 mg/mL increasing in a dose dependent manner to 94% inhibition at 1 mg/mL. In the case of donor plasma, 16% inhibition of 125I-TSH binding was observed at 1:160 dilution in healthy blood donor pool serum and 95% inhibition at 1:10 dilution.
5C9 IgG also had an effect on binding of 125I-M22 IgG to the TSHR coated onto the tubes (Table 1). 9% inhibition of 125I-M22 IgG binding was observed at 0.01 μg/mL of 5C9 IgG and increasing concentrations resulted in a dose dependent increase of inhibition up to 85% at 100 μg/mL. Donor serum IgG was effective at 0.01 mg/mL causing 9% inhibition and the effect increased in a dose dependent manner to 89% inhibition at 1 mg/mL. Donor serum plasma showed 13% inhibition of 125I-M22 IgG binding at 1:320 dilution and 91% inhibition at 1:10 dilution.
Unlabelled 5C9 IgG was able to inhibit 125I-5C9 binding to the TSHR coated tubes in a dose dependent manner (11% inhibition at 0.005 μg/mL up to 88% inhibition at 100 μg/mL) (Table 1). Binding of 125I-5C9 was also inhibited by donor serum IgG (15% inhibition at 0.05 mg/mL and 91% inhibition at 1 mg/mL) as well as dilutions of donor plasma (10% inhibition at 1:320 dilution and 92% at 1:10 dilution).
The ability of 5C9 IgG to block TSH and M22-mediated stimulation of cyclic AMP in CHO cells expressing the TSHR is shown in Tables 2a-c. Porcine TSH (3 ng/mL) strongly stimulated cyclic AMP production (19,020±2154 fmol/cell well; mean±SD; n=3) (Table 2a). In the presence of 0.1 μg/mL of 5C9 IgG the stimulating activity of porcine TSH was reduced to 11874±4214 fmol/cell well (mean±SD; n=3) and the inhibiting effect was dependent on 5C9 concentration with only 2,208±329 fmol/cell well of cyclic AMP produced in the presence of 1 μg/mL of 5C9 (Table 2a). The lymphocyte donor serum also had a strong inhibiting effect on TSH mediated cyclic AMP stimulation in CHO-TSHR cells. As shown in Table 2a, inhibition of cyclic AMP production down to approximately 6000 Enol/cell well occurred in the presence of donor serum at 1:10 dilution (total serum IgG concentration at this dilution of 1.43 mg/mL) compared to 19000 fmol/cell well in the absence of serum. This effect corresponded to the effect of approximately 0.37 μg/mL of purified 5C9 IgG (calculated from the dilution curve of the effect of different concentrations of 5C9 IgG shown in Table 2a). This indicates that purified 5C9 IgG is approximately 3900 times more active than the donor serum IgG in terms of ability to block the ability of TSH to stimulate cyclic AMP production.
Fragments of 5C9, such as 5C9 F(ab′)2 and 5C9 Fab were also effective inhibitors of TSH stimulation. In particular, TSH stimulation inhibiting activity of 5C9 IgG, 5C9 F(ab′)2 and 5C9 Fab at 100 μg/mL were the same (Table 2b). At 10 μg/mL all three preparations: 5C9 IgG, 5C9 F(ab′)2 and 5C9 Fab were also potent inhibitors of TSH stimulating activity, however, 5C9 IgG appeared to be more effective than 5C9 F(ab′)2 or 5C9 Fab (Table 2b).
M22 Fab (3 ng/mL) is a potent stimulator of cyclic AMP (9,432±822 fmol/cell well) (Table 2c) and in the presence of 5C9 the stimulating effect of M22 Fab was inhibited in a dose dependent manner, with cyclic AMP levels reduced to 1,298±134 fmol/cell well in the presence of 0.1 μg/mL of 5C9 IgG. Complete inhibition of M22 stimulation occurred at 100 μg/mL of 5C9 (Table 2c).
Scatchard analysis indicated that 125I-labelled 5C9 bound to the TSHR with an association constant of 4×1010 L/mol.
The ability of serum TRAbs to inhibit 125I-5C9 IgG binding to TSHR coated tubes is shown in Table 3 and
Binding of 125I-5C9 IgG to the TSHR was not markedly inhibited (inhibition range 3.4-18.9%) by sera from 10 different healthy blood donors (N1-N10, Table 3). Sera from 40 patients with Graves' disease (G1-G40, Table 3) all positive for TSHR autoantibodies in 125I-TSH and 125I-M22 inhibition assays (Table 3) inhibited 125I-5C9 binding to TSHR coated tubes (inhibition range 22.0-85.2%) to a greater extent than sera from healthy blood donors (Table 3). The ability of patient serum TRAbs to inhibit 125I-5C9 IgG, 125I-TSH or 125I-M22 IgG binding to the TSHR was comparable with a Pearson correlation coefficient r=0.95 (125I-5C9 IgG versus 125I-TSH;
These experiments show that 5C9 IgG binding to the TSHR is inhibited effectively by serum TRAbs and that the inhibiting effect of serum TRAb on 5C9 IgG binding is similar to their inhibiting effect on TSH or M22 binding.
Table 4 shows the inhibition of 125I-5C9 IgG binding by different dilutions of patient serum TRAbs with TSH blocking activity (B1-B5) and patient serum TRAbs with powerful thyroid stimulating activity (S1, S2, S4). Binding of 125I-5C9 IgG was inhibited in a dose dependent manner by sera B1-B5 sera as well as by sera S1, S2 and S4. The same blocking and stimulating sera also inhibited 125I-TSH and 125I-M22 IgG binding in a dose dependent manner, furthermore the percentage of inhibition with all three labelled ligands were comparable at the same dilutions of sera (Table 4a & b).
These results indicate that TSHR autoantibodies with both stimulating and blocking activities inhibit 5C9 binding to the TSHR.
Inhibition of 125I-5C9 IgG Binding to the TSHR by Mouse MAbs with TSH Binding Inhibiting Activity
The ability of different mouse TSHR MAbs with 125I-TSH binding inhibiting activity to inhibit 125I-5C9 binding to the TSHR was tested and compared with the effect on 125I-M22 IgG binding (Table 5). As shown in Table 5 all MAbs that had ability to inhibit 125I-TSH and 125I-M22 IgG binding also inhibited 125I-5C9 binding although in the case of some MAbs the inhibiting effect on 125I-5C9 and 125I-M22 binding was weaker than that on 125I-TSH binding.
These experiments suggest that there is a considerable overlap between the binding sites on the TSHR for 5C9 and those for mouse TSHR MAbs which have the ability to inhibit TSH binding.
As shown in Table 2, 5C9 IgG was able to block TSH or M22 stimulation of cyclic AMP levels in CHO cells expressing TSHRs. In a different series of experiments the effect of 5C9 IgG on the stimulating activity of patient serum TRAbs was tested and the results are shown in Table 6a. Sera T1-T9 and T11-T18 stimulated cyclic AMP production in CHO-TSHR cells and incubation with a control MAb IgG (2G4 specific for human thyroid peroxidase) had no effect on their stimulating activities. However, in the presence of 5C9 IgG (50 μL of 200 μg/mL), the stimulating activity of all sera tested was markedly reduced (Table 6a).
Dose response effects of 6 different sera (T1, T6, T3, T19, T20, T21) are shown in Tables 6b-g. In these experiments, concentrations of 5C9 IgG ranging from 0.1 μg/mL to 100 μg/mL caused a dose dependent reduction of serum stimulating activity and the effect of 5C9 IgG was comparable to the effect of 9D33, a mouse monoclonal antibody to the TSHR with blocking activity (described in WO2004/050708A2) in all sera tested except serum T3. In the case of T3 serum (Table 6a & 6d) about 50% inhibition of cyclic AMP production in the presence of 100 μg/mL of 5C9 IgG was observed, whereas 100 μg/mL of 9D33 IgG resulted in almost complete inhibition. This suggested that there might be some minor differences between the epitopes recognised by 5C9 and 9D33.
Effect of 5C9 IgG on Basal (i.e. Non-Stimulated) Cyclic AMP Production in CHO Cells Expressing TSHR
As well as inhibiting the stimulating activity of TSH and TSHR antibodies, 5C9 inhibited the amount of cyclic AMP produced in the absence of these thyroid stimulators. In particular, Table 6b shows 1207±123 fmol/cell well of cyclic AMP produced in the presence of 100 μg/mL control monoclonal IgG (2G4) reduced to 301±38 fmol/cell well in the presence of 100 μg/mL of 5C9 IgG. The effects of 9D33 IgG were less with 721±183 fmol/cell well produced in the presence of 100 μg/mL 9D33 IgG. Similar results were obtained in the separate experiments shown in Tables 6d, 6e, 6f and 6g. This indicates that 5C9 IgG has a marked effect on the basal or constitutive activity of the TSHR.
The effects of single amino acid mutations in the TSHR on 5C9 ability to block cyclic AMP stimulating activity of porcine TSH in CHO-TSHR cells are shown in Table 7. In particular, the effect of 5C9 on stimulation of cyclic AMP production was studied in CHO cells expressing the TSHR with the following residues mutated to alanine: Lys 58, Ile 60, Arg 80, Tyr 82, Thr 104, Arg 109, Lys 129, Phe 134, Asp 151, Lys 183, Gln 235, Arg 255, Trp 258, Ser 281. In addition, the effect of a change of charge mutation was studied in the case of TSHR residues: Arg80Asp, Asp151Arg, Lys183Asp, Arg255Asp, in which in accordance with conventional notation the amino acid residue which is replaced, and its position in the primary sequence polypeptide, is indicated before the replacement amino acid residue. Previous studies have shown that change of charge mutation of TSHR Asp160Lys caused a loss of responsiveness of the TSHR to TSH while the response to M22 was not affected (patent application WO2006/016121A). Consequently, the effect of TSHR Asp160Lys mutation on 5C9 biological activity was studied using M22 as a stimulator of cyclic AMP in CHO-TSHR cells (Table 71).
Out of all the TSHR mutations studied, only three mutations were found to affect the ability of 5C9 to act as an antagonist. Mutation of Lys129 to Ala (Table 7h) resulted in a complete loss of the ability of 5C9 IgG to block TSH stimulation of cyclic AMP production.
Also TSHR mutation Lys183Ala caused a partial reduction of 5C9 IgG blocking activity; 28% inhibition of TSH stimulation was observed at 1 μg/mL when tested with TSHR Lys183Ala mutation compared to 84% inhibition with wild type TSHR (Table 7m). Even at 100 μg/mL of 5C9 IgG, only partial inhibition of TSH stimulation (43%) was detectable in the experiments with TSHR Lys183Ala mutation whereas at this concentration a complete blocking of TSH stimulating activity (93%) was observed in the experiments with wild type receptors (Table 7m). When positively charged Lys 183 was mutated to negatively charged aspartic acid, the effect on 5C9 biological activity was similar to that observed with Lys183 Ala mutation (Table 7m). This suggests that Lys183 is important for 5C9 biological activity. In the case of Asp151Ala mutation, a slight reduction of 5C9 IgG blocking activity: 49% inhibition at 1 μg/mL compared to 88% inhibition with wild type (Table 7j) was observed. However, in the presence of 100 μg/mL of 5C9 IgG the activity was the same as with wild type TSHR. When negatively charged Asp151 was mutated to positively charged arginine, a significant reduction in 5C9 IgG blocking activity was not observed (Table 7k).
The effect of TSHR mutations on 5C9 activity can be compared to the effects of TSHR mutations on 9D33 activity. As described in patent application WO2006/016121A, TSHR mutations Lys 58, Arg 80, Tyr 82, Arg 109, Lys 129 and Phe 134 had an effect on 9D33 activity. None of these mutations except Lys129, however, also had an effect on 5C9 activity. Furthermore, none of the mutations except Lys 129 that affected M22 activity (Arg 80, Tyr 82, Glu 107, Arg 109, Lys 129, Phe 130, Lys 183, Tyr 185, Arg 255 and Trp 258) as described in the patent application WO 2006/016121A affected 5C9 activity. In addition, Lys 183 mutation had a partial effect on 5C9 activity and M22 activity but had no effect on 9D33 activity.
These results indicate that there are differences in terms of the TSHR residues important for interaction with the thyroid stimulating human autoantibody M22, with mouse blocking antibody 9D33 and with human blocking autoantibody 5C9. Consequently, a combination of 5C9 with other TSHR antibodies with antagonist activities (such as 9D33) may be a particularly effective means of inhibiting the stimulating activity of patient serum TRAbs, other stimulators and/or TSHR constitutive activity.
Sequence analysis of the genes coding for 5C9 indicated that the HC V region genes were from the VH3-53 family, the D genes from the D2-2 family and the J genes from the JH4 family. In the case of the LC, V region genes were from the 012 family and J region genes from the JK2 germline. The HC nucleotide and amino acid sequences are shown in
There are somatic mutations in the HC gene sequence compared to the germline sequences; in particular 1 silent mutation in FWR1, 2 replacement mutations in CDR2, 1 silent and 1 replacement mutation in CDR3 and 1 silent mutation in FWR4. However, the HC V region sequence is characterised by two insertions; one 6 base pairs long between the V and D genes and one 15 base pairs long between the D and J genes. Consequently, the HC CDR1 is 5 amino acids long, CDR2 is 16 amino acids long and the CDR3 is 18 amino acids long (
In the LC sequence there are: 1 silent mutation in FWR1, 1 replacement mutation in CDR1, 1 replacement mutation in CDR3 and a 6 base pairs long insertion between the V and J genes. The LC CDR1 is made up of 11 amino acids, CDR2 of 7 amino acids and CDR3 of 10 amino acids (
An example of an ELISA based on 5C9 IgG-biotin binding to TSHR coated plate wells for detection of TSHR autoantibodies is shown in Table 8. In this assay all samples positive for inhibition of TSH-biotin binding were also positive for 5C9 IgG-biotin binding. Furthermore, the absorbance signal, the percent inhibition and the derived units/L values were comparable in the TSH-biotin and 5C9-biotin assays (Table 8).
As shown in Table 9, 5C9 IgG was able to block the stimulation of cyclic AMP production by all five of the TSMAbs (1, 2, 4, 5 and 7) tested. For example TSMAb1 (Table 9) stimulated cyclic AMP levels to 18.94±7.4 pmol/mL while in the presence of 100 μg/mL 5C9 IgG only 1.24±0.07 pmol/mL of cyclic AMP was produced. This can be compared to 16.5±1.1 pmol/mL cyclic AMP levels in the presence of 100 μg/mL of the control MAb 2G4 (Table 9).
The cyclic AMP levels shown in Tables 9, as well as in Tables 10-15 below, are expressed in pmol/mL i.e. the levels of cyclic AMP per cell well are: pmol/mL÷5 (representing 200 μL of sample from each well assayed).
The ability of 5C9 IgG to block the cyclic AMP stimulation of porcine TSH is shown in Table 2 and Table 10. In addition 5C9 IgG showed the ability to inhibit cyclic AMP stimulation of both native human TSH (NIBSC reference preparation 81/565 from National Institute for Biological Standards and Control, South Mimms, Potters Bar EN6 3QG UK) and recombinant TSH (NIBSC reference preparation 94/674) (Table 10). In particular stimulation of cyclic AMP production by 100 ng/mL of either recombinant or native human TSH requires 0.1-1.0 μg/mL of 5C9 IgG to obtain complete inhibition of cyclic AMP production. At the time of blood collection for 5C9 isolation the levels of circulating TSH in the donor serum were 160 mU/L (approximately 32 ng/mL), the results obtained (Table 2a and Table 10) indicate that this level of circulating TSH would be completely blocked in the presence of 32-320 ng/mL of 5C9 IgG in the serum. The levels of TSHR autoantibodies in the donor serum were estimated, using inhibition of 125I-M22 binding to the TSHR (as described in Nakatake N, Sanders J, Richards T, Burne P, Barrett C, Dal Pra C, Presotto F, Betterle C, Furmaniak J, Rees Smith B 2006 Estimation of serum TSH receptor autoantibody concentration and affinity. Thyroid 16: 1077-1084), to be 1700 ng/mL (120 ng/mg) i.e. several fold higher than the concentration of 5C9 required for blocking of thyroid stimulation by TSH.
Effect of 5C9 IgG on Basal (i.e. Non-Stimulated) Cyclic AMP Activity in CHO Cells Expressing TSHR with Activating Mutations S281I, 1568T and A623I
5C9 IgG was able to reduce the amount of cyclic AMP produced in CHO cells expressing TSHR with activating mutations when thyroid stimulators (i.e. TSH or TSHR antibodies) were absent. As shown in Table 11a the basal cyclic AMP concentration in CHO cells expressing the TSHR with activating mutation S281I was 9.90±1.51 pmol/mL in the absence of 5C9 and this was decreased to 4.17±0.60 pmol/mL in the presence of 0.01 μg/mL 5C9 IgG and to 3.44±0.63 pmol/mL in the presence of 1 μg/mL 5C9 IgG. The blocking mouse TSHR MAb 9D33 had little effect as did the control MAb 2G4 (Table 11a).
Similar results were obtained with the TSHR activating mutation 1568T (Table 11b), which showed a basal cyclic AMP concentration of 21.39±5.31 pmol/mL. This decreased to 5.29±0.75 pmol/mL on addition of 1 μg/mL of 5C9 IgG compared to 20.52±0.95 pmol/mL and 21.65±1.99 pmol/mL in the case of addition of 2G4 IgG and 5C9 IgG, respectively. In the case of a third TSHR activating mutation studied i.e. A623I with basal cyclic AMP concentration of 36.89 pmol/mL addition of 1 μg/mL of 5C9 IgG reduced the cyclic AMP levels to 16.43±1.27 pmol/mL compared to little effects with 1 μg/ml of control IgG 2G4 (28.96±2.29 pmol/mL) or 1 μg/mL of 9D33 IgG (40.09±7.73 pmol/mL) (Table 11c).
These results indicate that 5C9 unlike the mouse blocking MAb 9D33 has a marked effect on cyclic AMP production associated with the TSHR activating mutations even when the mutations are in different parts of the TSHR (i.e. S281I in the extracellular domain, 1568T in the second extracellular loop of the transmembrane domain and A623I in the third intracellular loop of the transmembrane domain).
The human TSHR blocking MAb 5C9 and the mouse TSHR blocking MAb 9D33 at concentrations as low as 1 μg/mL have the ability to block TSHR cyclic AMP stimulating activity of TSH in CHO-TSHR cells as shown in previous experiments and in Table 12. The effects of the 9D33 IgG and 5C9 IgG on TSH mediated stimulation of cyclic AMP were additive as shown in Table 12; Experiments 1-5). The same additive effect was observed when two different concentrations of TSH (3 ng/mL and 0.3 ng/mL) were used for stimulation (Table 12; Experiments 1-3 and Experiments 4 and 5, respectively).
As shown before, 5C9 and 9D33 also are able to inhibit M22 Fab mediated stimulation of cyclic AMP in CHO-TSHR cells. The effects of the 9D33 IgG and 5C9 IgG on M22 mediated stimulation of cyclic AMP were additive (Table 13 Experiments 1-4). The same additive effect was observed when two different concentrations of M22 Fab (3 ng/mL and 0.3 ng/mL) were used for stimulation (Table 13; Experiments 1 and 2 and Experiments 3 and 4, respectively).
The additive effects of 5C9 IgG and 9D33 IgG were similar for both TSH and M22 mediated stimulation of cyclic AMP production (Tables 12 and 13).
Effect of 5C9 on Basal (i.e. Non-Stimulated) Cyclic AMP Activity in CHO Cells Expressing a High Number of Wild Type TSHRs Per Cell
A CHO cell line expressing approximately 5×105 receptors per cell showed higher levels of basal (i.e. non-stimulated) cyclic AMP compared to a standard CHO cell line (expressing approximately 5×104 TSHR per cell) used in previous experiments (for example Tables 9-13) i.e. 47.1±11.7 pmol/mL compared to approximately 1.0 pmol/mL, respectively. The effect of 5C9 IgG and 9D33 IgG on wild type TSHR basal activity was assessed using the cell line expressing a high number of receptors per cell. Incubation with 9D33 IgG and a negative control antibody to GAD (5B3) resulted in 0-5.3% inhibition of basal cyclic AMP activity (Table 14; Experiment 1) indicating that the blocking mouse MAb 9D33 or control MAb have no effect on basal cyclic AMP production in CHO cells expressing the wild type TSHR. However, in the case of 5C9 IgG a clear inhibition of basal cyclic AMP activity was observed (Table 14; Experiment 2) with 0.1 μg/mL and 10 μg/mL causing 45.7% and 74.6% inhibition respectively. In addition, 5C9 Fab and 5C9 F(ab′) were also effective inhibitors of basal cyclic AMP activity in CHO cells expressing a high number of TSHRs per cell (Table 14 experiment 3). For example, 1 μg/mL and 100 μg/mL of 5C9 Fab showed 39% and 61% inhibition of basal cyclic AMP production, respectively compared to 48% inhibition by 5C9 F(ab′) at 100 μg/mL (Table 14 experiment 3).
Effect of Patient Serum TSHR Autoantibodies with Antagonist (i.e. Blocking) Activity on Basal (i.e. Non-Stimulated) Cyclic AMP Activity in CHO Cells Expressing TSHR with Activating Mutation 1568T
The basal cyclic AMP production by TSHR 1568T cells in the presence of cyclic AMP assay buffer of 20.5±8.7 pmol/mL was essentially unaffected by addition of normal pool sera from healthy blood donors (NPS) or 3 different individual healthy blood donor sera (N1-N3) tested at 1/10 and 1/50 dilution. The basal cyclic AMP production in the presence of NPS and N1-N3 sera showed 0-14% inhibition compared to basal cyclic AMP production in the presence of cyclic AMP assay buffer (Table 15). However, in the presence of 4 different sera with high levels of blocking type TRAbs (B2-B5) 23-89% inhibition of basal cyclic AMP production was observed (Table 15). In the presence of 5C9 IgG (1 μg/mL), 83% inhibition of TSHR 1568T basal cyclic AMP activity was observed. The dose response effect of 2 blocking sera (B3 and B4) on basal cyclic AMP production in CHO cells expressing TSHR with 1568T mutation is also shown in Table 15.
These results indicate that 5C9 has the TSHR blocking activity characteristic of patient blocking TSHR autoantibodies in particular with respect to inhibition of basal cyclic AMP production in the TSHR activating mutant 1568T.
Effect of Patient Serum TSHR Autoantibodies with Antagonist Activity on Basal (i.e. Non-Stimulated) Cyclic AMP Activity in CHO Cells Expressing TSHR with Activating Mutation S281I
The basal cyclic AMP production by TSHR S281I cells in the presence of cyclic AMP assay buffer was 11.2±2.0 pmol/mL and incubation with healthy blood donor pool sera or individual healthy blood donor sera (diluted 1/10 or 1/50) had no effect (Table 16). In contrast, in the presence of 4 different sera with high levels of blocking type of TRAbs (B2-B5) 31-56% inhibition of basal cyclic AMP production was observed (Table 16). 5C9 IgG at 1 μg/mL caused 71% inhibition of basal cyclic AMP activity in the experiments with TSHR S281I.
Effect of Patient Serum TSHR Autoantibodies with Antagonist Activity on Basal (i.e. Non-Stimulated) Cyclic AMP Activity in CHO Cells Expressing TSHR with Activating Mutation A623I
The basal cyclic AMP production in the case of TSHR A623I cells was 43.5±11.2 pmol/mL in the presence of cyclic AMP assay buffer and was essentially unaffected by incubation with healthy blood donor pool or individual sera (Table 17). Incubation with four different sera with high levels of blocking type of TRAbs (B2-B5) caused—1% to 56% inhibition of cyclic AMP in these experiments (Table 17). This can be compared with 49% inhibition by 5C9 IgG at 1 μg/mL in the same experiment.
Effect of Patient Serum TSHR Autoantibodies with Antagonist Activity on Basal (i.e. Non-Stimulated) Cyclic AMP Activity in CHO Cells Expressing Approximately 5×105 Wild Type TSHRs Per Cell
The basal cyclic AMP production in CHO cells expressing higher number of wild type TSHRs per cell was 28.1±0.7 pmol/mL in this series of experiments. When the cells were incubated with healthy blood donor pool or individual sera (N1-N3) at 1/10 dilutions basal cyclic AMP levels ranged between 99% and 146% of cyclic AMP levels in the presence of cyclic AMP assay buffer while at 1/50 dilutions the range was from 93% to 137%. Out of 4 sera with blocking type TSHR autoantibodies tested, one serum (B2) had no effect on basal cyclic AMP production (Table 18). In the case of two sera (B3 and B5) the levels of cyclic AMP increased relative to the levels observed in the presence of cyclic AMP assay buffer (Table 18). It may well be that sera B3 and B5 contain a mixture of TSHR autoantibodies with stimulating and blocking activities. In contrast, serum B4 had a clear inhibiting effect on basal cyclic AMP production at 1/10 and 1/50 dilution i.e. 31% and 61% respectively of basal cyclic AMP levels relative to the levels in the presence of cyclic AMP assay buffer (Table 18). This can be compared to the levels in the presence of 5C9 IgG at 1 μg/mL of 33% relative to the levels in the presence of cyclic AMP assay buffer (Table 18).
Overall 5C9 IgG shows similar effects on basal cyclic AMP production in CHO cells transfected with wild type TSHR or with TSHR with activating mutations to the effects observed with sera from patients positive for blocking type TSHR autoantibodies. However, the effect of individual patient sera varies in the case of different mutations (Table 19). In the case of wild type TSHR some sera show a stimulating effect presumably due to the presence of TSHR stimulating autoantibodies as well as blocking autoantibodies (Table 19).
The effect of 5C9 on stimulation of cyclic AMP production in CHO cells expressing TSHRs with amino acid mutations was extended to include the following mutations to alanine: Asp43, Glu61, His105, Glu107, Phe130, Glu178, Tyr185, Asp203, Tyr206, Lys209, Asp232, Lys250, Glu251, Thr257, Arg274, Asp276 (Table 20 a-p and summarised in Table 21).
Mutation of TSHR amino acids Asp43, Glu61, His105, Glu107, Tyr185, Asp232 and Thr275 to alanine had no effect on 5C9 IgG's ability to inhibit TSH stimulated cyclic AMP production. The ability of 5C9 to inhibit TSH stimulated cyclic AMP production was reduced by mutation of TSHR Phe130, Glu178, Asp203, Tyr206, Lys250, Glu251 and Asp276 to alanine. In the case of 2 mutations Lys209Ala and Asp274Ala, the ability of 5C9 IgG to inhibit TSH mediated cyclic AMP production was enhanced.
In summary (Tables 7, 20 and 21), 10 TSHR residues Lys129, Phe130, Asp151, Glu178, Lys183, Asp203, Tyr206, Lys250, Glu251 and Asp276 all reduced the ability of 5C9 to inhibit cyclic AMP stimulation by TSH compared to the wild type TSHR. Mutation of TSHR Lys129 and Asp203 showed the greatest effect and caused complete inhibition of 5C9 activity.
The blocking effect at 1 μg/mL 5C9 on the wild type TSHR (92% inhibition of TSH induced cyclic AMP stimulation) was reduced to 4% in the case of TSHR Asp203Ala mutation (Table 22).
Blocking serum B4 activity was unaffected by TSHR Asp203Ala mutation while a slight reduction in percent inhibition of TSH induced cyclic AMP stimulation was seen with blocking sera B2 and B3 in the case of TSHR Asp203Ala compared to the wild type TSHR.
In the case of one serum B5, a marked reduction in percent inhibition of TSH induced cyclic AMP stimulation was observed; i.e. 69% inhibition compared to 30% inhibition in wild type and mutated TSHR respectively.
The effect of TSHR Asp203Ala mutation on 5C9 activity was greater than the effect on the activity of blocking sera, however the blocking activity of 3/4 sera tested was affected to varying degrees. This may indicate that the binding sites for blocking TSHR autoantibodies and 5C9 overlap but there are some differences in the actual TSHR amino acids in contact with different sera.
The experiments described above provide evidence that an antibody in accordance with the invention such as 5C9 is able to block stimulating activity of different thyroid stimulators, including human and mouse TSHR stimulating antibodies, native human and animal TSH and recombinant human TSH. Furthermore, evidence is provided that two different blocking type antibodies, i.e. a human MAb 5C9 and a mouse MAb 9D33 that, when tested individually, have the ability to block TSH or M22 meditated stimulation of cyclic AMP in CHO cells expressing the TSHR, show additive blocking effect on TSH or M22 stimulation when mixed together.
Antibodies in accordance with the invention such as 5C9 have a novel effect on the TSHR basal (i.e. non-stimulated) cyclic AMP activity. These effects have been studied by experiments using TSHR transfected CHO cells having higher levels of basal cyclic AMP i.e. the blocking effect of antibodies in accordance with the invention, such as 5C9, on basal cyclic AMP activity has been confirmed. Furthermore, it has been shown that some sera with TSHR autoantibodies with blocking (antagonist) activity have the ability to block basal cyclic AMP activity in these TSHR transfected cells. The experiments also provide evidence of the blocking effect of antibodies in accordance with the invention, such as 5C9, and serum TSHR blocking autoantibodies on basal cyclic AMP activity associated with activating TSHR mutations.
These results emphasise that 5C9 is a human MAb showing the characteristics of blocking type TSHR autoantibodies i.e. that it is representative of patient serum TSHR autoantibodies associated with autoimmune thyroid disease.
The experiments described also allowed identification of some of the TSHR amino acids important for the blocking activity of antibodies in accordance with the invention.
Overall, the results indicate that antibodies in accordance with the invention, such as 5C9, show similar TSHR binding activity and similar biological effects on TSHR function as TSHR blocking autoantibodies found in different sera from patients with autoimmune thyroid disease. Consequently, having characteristics and biological activity of serum blocking TSHR autoantibodies, antibodies in accordance with the invention, such as 5C9, have applications for inactivation of the TSHR in various clinical conditions. These conditions include TSHR activation mediated by TSH, TSHR activation mediated by thyroid stimulating TSHR autoantibodies, basal (non-stimulated, constitutive) TSHR activity and TSHR activation associated with activating TSHR mutations. Consequently, antibodies in accordance with the invention, such as 5C9 have applications for management and control of the conditions associated with TSHR activation mentioned above; for example Graves' disease, Graves' ophthalmopathy, hyperthyroidism due to TSHR activating mutations, hyperthyroidism due to abnormal levels of TSH (pathological or pharmacological), thyroid cancer and thyroid cancer metastases.
125I-TSH
125I-M22 IgG
125I-5C9
1single determination
aDonor serum was diluted in cyclic AMP assay buffer as indicated
bIgG concentration of undiluted serum determined by nephelometry was 14.3 mg/mL
1mean of duplicate samples
6381 ± 190
1mean of duplicate samples
125I-M22 IgG binding to TSHR coated tubes and
125I-5C9
125I-TSH
125I-M22
where A = binding in the presence of test serum; B = binding in the presence of a pool of healthy blood donor serum (HBD pool).
125I-5C9 in presence of HBD pool gave 20% binding, 125I-TSH in presence of HBD pool gave 12% binding and 125I-M22 in presence of HBD pool gave 17% binding.
125I-M22 IgG, binding to TSHR coated tubes by patient
125I-5C9
125I-TSH
125I-M22
125I-5C9
125I-TSH
125I-M22
where A = test sample; B = HBD pool
In the presence of the HBD pool approximately 20%, 17% and 12% of the 125I-labelled M22 IgG, 5C9 IgG and TSH respectively bound to the TSHR coated tubes.
125I-TSH
125I-M22 IgG
125I-5C9 IgG
where A = % binding in the presence of test sample; B = % binding in the presence of HBD pool.
1mouse TSHR MAb with thyroid stimulating activity
2mouse TSHR MAb which blocks both TSH and TRAb mediated stimulation of cyclic AMP production (see Table 2)
3human MAb to thyroid peroxidase MAb (negative control)
4mouse TSHR MAb with TSH blocking activity (recognises an epitope formed by TSHR amino acids 381-385)
5mouse TSHR MAb with TSH blocking activity (recognises an epitope formed by TSHR amino acids 36-42)
6mouse TSHR MAb with TSH blocking activity (recognises an epitope formed by TSHR amino acids 246-260)
7mouse Tg MAb (negative control)
In the presence of HBD pool, approximately 13%, 24% and 15% of 125I-labelled 5C9 IgG, M22 IgG and TSH respectively bound to the TSHR coated tubes.
1= duplicate determination
1single determination
1mean of duplicate sample
2single determination
1mean of duplicate sample
1mean of duplicate determinations
Assay calibrators 40 U/L, 8 U/L, 2 U/L and 1 U/L are dilutions of M22 IgG in a pool of healthy blood donor sera (HBD pool) with activities in U/L of NIBSC 90/672 assessed by inhibition of labelled TSH binding to TSHR coated tubes. Assay negative control is an HBD pool.
adilution in cyclic AMP assay buffer
b2G4 is a control human monoclonal autoantibody to thyroid peroxidase.
a2G4 is a control human monoclonal antibody to thyroid peroxidase.
aduplicate determination
a5B3 is a human monoclonal antibody to glutamic acid decarboxylase
a5B3 is a human monoclonal antibody to glutamic acid decarboxylase
In the presence of 5C9 IgG at 1 μg/mL, basal cyclic AMP levels decreased to 33% relative to levels in the presence of cyclic AMP assay buffer.
1mean of duplicate determinations
2single determination
a5B3 is a human monoclonal antibody to glutamic acid decarboxylase (GAD) (negative control for 5C9).
aStimulation of cyclic AMP for this experiment was tested using M22 due to lack of response to TSH (see text for details)
aDilution in cyclic AMP assay buffer.
b% Inhibition of TSH induced cyclic AMP stimulation
1mean of duplicate samples
cpTSH used at a final concentration of 3 ng/mL
| Number | Date | Country | Kind |
|---|---|---|---|
| 0702990.3 | Feb 2007 | GB | national |
| 0714036.1 | Jul 2007 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB08/00518 | 2/14/2008 | WO | 00 | 6/3/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60901332 | Feb 2007 | US |
