# US 9,751,940 B2 · Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto

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- Number: US 9,751,940 B2
- Document ID: US9751940B2
- Status: Granted
- Kind: B2
- Application number: 13/673,878
- Filed: 2012-11-09
- Published: 2013-04-11
- Granted: 2017-09-05
- Earliest claimed: 2001-08-23
- Assignees: RSR Limited
- Applicants: RSR Limited
- Inventors: Bernard Rees Smith; Jadwiga Furmaniak; Jane Fina Sanders
- Web page: https://patentgrep.com/patent/US9751940B2

## CPC

- C07K 16/26: against hormones {; against hormone releasing or inhibiting factors}
- A61P 5/14: of the thyroid hormones, e.g. T3, T4
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/723: {G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor}
- C07K 16/2869: {against hormone receptors (for antibodies against neuromediator receptors C07K16/286)}
- G01N 33/564: for pre-existing immune complex or autoimmune disease {, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N 33/76: Human chorionic gonadotropin {including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/505: {comprising antibodies}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention is concerned with epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto.

## Claims

### Claim 1

A binding partner for a TSH receptor, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
binds to an epitope on the TSH receptor;
has a binding affinity for the TSH receptor of at least about 108 molar−1;
is positive for thyroid stimulating activity at a concentration of 20 μg/ml in a cyclic AMP thyroid cell assay, wherein the binding partner is classified as positive if the thyroid stimulating activity of the binding partner is greater than 180 percent in the cyclic AMP thyroid cell assay, said percent thyroid stimulating activity comprising 100×(ratio of cyclic AMP produced in the thyroid cell assay in the presence of said binding partner to cyclic AMP produced in the thyroid cell assay in the presence of sera pooled from healthy blood donors); and
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube and wherein the TSH receptor is from a species selected from a group consisting of porcine, bovine, feline, canine, mouse, rat and ovine and wherein the monoclonal antibody comprises a VH domain comprising the three CDRs from SEQ ID NO:67 paired with a VL domain comprising the three CDRs from SEQ ID NO:68; or a VH domain comprising the three CDRs from SEQ ID NO:69 paired with a VL domain comprising the three CDRs from SEQ ID NO:70; or a VH domain comprising the three CDRs from SEQ ID NO:71 paired with a VL domain comprising the three CDRs from in SEQ ID NO:72.

### Claim 2 (depends on 1)

The binding partner according to claim 1, wherein the binding partner is positive for thyroid stimulating activity at a concentration of 2 μg/ml in the cyclic AMP thyroid cell assay.

### Claim 3 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody is a recombinant antibody.

### Claim 4 (depends on 1)

The binding partner according to claim 1, wherein the antigen binding fragment comprises a Fab fragment, Fv fragment, F(ab')2 fragment, or scFv.

### Claim 5 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:67 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:68.

### Claim 6 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:69 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:70.

### Claim 7 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:71 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:72.

### Claim 8 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody comprises:
a VH domain wherein CDR1 comprises amino acid residues 31 to 35 of SEQ ID NO: 67, CDR2 comprises amino acid residues 50 to 66 of SEQ ID NO:67, and CDR3 comprises amino acid residues 99 to 109 of SEQ ID NO:67; and
a VL domain wherein CDR1 comprises amino acid residues 24 to 34 of SEQ ID NO:68, CDR2 comprises amino acid residues 50 to 56 of SEQ ID NO:68, and CDR3 comprises amino acid residues 89 to 97 of SEQ ID NO: 68.

### Claim 9 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody comprises:
a VH domain wherein CDR 1 comprises amino acid residues 31 to 35 of SEQ ID NO:69, CDR 2 comprises amino acid residues 50 to 66 of SEQ ID NO:69, and CDR 3 comprises amino acid residues 99 to 109 of SEQ ID NO:69, and
a VL domain wherein CDR1 comprises amino acid residues 24 to 34 of SEQ ID NO:70, CDR2 comprises amino acid residues 50 to 56 of SEQ ID NO:70, and CDR3 comprises amino acid residues 89 to 97 of SEQ ID NO: 70.

### Claim 10 (depends on 1)

The binding partner according to claim 1, wherein the monoclonal antibody comprises:
a VH domain wherein CDR1 comprises amino acid residues 31 to 35 of SEQ ID NO: 71, CDR2 comprises amino acid residues 50 to 66 of SEQ ID NO:71, and CDR 3 comprises amino acid residues 99 to 109 of SEQ ID NO:71, and
a VL domain wherein CDR1 comprises amino acid residues 24 to 34 of SEQ ID NO:72, CDR2 comprises amino acid residues 50 to 56 of SEQ ID NO:72, and CDR3 comprises amino acid residues 89 to 97 of SEQ ID NO: 72.

### Claim 11 (depends on 1)

A pharmaceutical composition comprising a binding partner according to claim 1 and one or more pharmaceutically acceptable carriers, diluents or excipients therefor.

### Claim 12 (depends on 11)

The composition according to claim 11, further comprising recombinant human TSH.

### Claim 13

A kit for screening for autoantibodies to a TSH receptor in a sample of body fluid obtained from a subject suspected of suffering from, susceptible to, having or recovering from autoimmune disease associated with an immune reaction to a TSH receptor, said kit comprising:
(a) a soluble or solubilized TSH receptor comprising the amino acid sequence of amino acid residues 47 to 61 of SEQ ID NO:17 or SEQ ID NO:22; and
(b) one or more binding partners, wherein the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
binds to an epitope on the TSH receptor wherein the TSH receptor is from a species selected from a group consisting of porcine, bovine, feline, canine, mouse, rat and ovine;
has a binding affinity for the TSH receptor of at least about 108 molar−1;
is positive for thyroid stimulating activity at a concentration of 20 μg/ml in a cyclic AMP thyroid cell assay and wherein the binding partner is classified as positive if the thyroid stimulating activity of the binding partner is greater than 180 percent in the cyclic AMP thyroid cell assay, said percent thyroid stimulating activity comprising 100×(ratio of cyclic AMP produced in the thyroid cell assay in the presence of said binding partner to cyclic AMP produced in the thyroid cell assay in the presence of sera pooled from healthy blood donors); and
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube and wherein the monoclonal antibody comprises the VH domain as shown in SEQ ID NO:67 paired with the VL domain as shown in SEQ ID NO:68; or the VH domain as shown in SEQ ID NO:69 paired with the VL domain as shown in SEQ ID NO:70; or the VH domain as shown in SEQ ID NO:71 paired with the VL domain as shown in SEQ ID NO:72.

### Claim 14 (depends on 13)

The kit according to claim 13, wherein the one or more binding partners comprises a detectable label.

### Claim 15 (depends on 13)

The kit according to claim 13, wherein the kit further comprises a tube for contacting the sample of body fluid from the subject and the TSH receptor so as to permit the TSH receptor to interact with either autoantibodies to a TSH receptor present in the sample, or the one or more binding partners.

### Claim 16 (depends on 13)

The kit according to claim 13, wherein the kit further a labelled binding partner for TSH receptor autoantibodies, a competitor capable of competing with autoantibodies to a TSH receptor in the interaction thereof with a TSH receptor, or a detectable label.

### Claim 17

A binding partner for a TSH receptor, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
has a binding affinity for the TSH receptor of at least about 108 molar−1;
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube; and
has heavy chain variable region CDRs contained within a heavy chain variable domain having an amino acid sequence selected from the group consisting SEQ ID NOs: 67, 69, and 71.

### Claim 18 (depends on 17)

A binding partner for a TSH receptor of claim 17, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof further comprising light chain variable region CDRs contained within a light chain variable domain having an amino acid selected from the group consisting of SEQ ID NOs 68, 70, and 72.

### Claim 19

A binding partner for a TSH receptor, the binding partner comprising an isolated monoclonal antibody or antigen binding fragment thereof that:
binds to an epitope on the TSH receptor;
has a binding affinity for the TSH receptor of at least about 108 molar−1;
is positive for thyroid stimulating activity at a concentration of 20 μg/ml in a cyclic AMP thyroid cell assay, wherein the binding partner is classified as positive if the thyroid stimulating activity of the binding partner is greater than 180 percent in the cyclic AMP thyroid cell assay, said percent thyroid stimulating activity comprising 100×(ratio of cyclic AMP produced in the thyroid cell assay in the presence of said binding partner to cyclic AMP produced in the thyroid cell assay in the presence of sera pooled from healthy blood donors); and
inhibits TSH binding, the binding partner inhibiting binding of TSH to the TSH receptor in a coated tube binding assay by 61% to 83.3% at a concentration of 2 μg/ml to 20 μg/ml wherein the TSH receptor is immobilized on a surface of the tube and wherein the monoclonal antibody comprises a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:67 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:68; a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:69 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:70 or a VH domain comprising amino acid residues 9 to 120 of SEQ ID NO:71 and a VL domain comprising amino acid residues 9 to 107 of SEQ ID NO:72.

### Claim 20

A binding partner for a TSH receptor, the binding partner comprising a monoclonal antibody or fragment thereof comprising a Fab domain for a TSH receptor, wherein the antibody is capable of binding to a TSH receptor so as to stimulate the TSH receptor, which comprises:
an antibody heavy chain (HC) selected from the group consisting of:
a HC comprising (SEQ ID NO:67), (SEQ ID NO:69) or (SEQ ID NO:71); and/or
an antibody light chain (LC) selected from the group consisting of:
a LC comprising (SEQ ID NO:68), (SEQ ID NO:70) or (SEQ ID NO:72).

### Claim 21 (depends on 20)

The binding partner comprising a monoclonal antibody or fragment thereof of claim 20, wherein the binding partner comprising a monoclonal antibody or fragment thereof comprises at least one HC comprising (SEQ ID NO:67), (SEQ ID NO:69) or (SEQ ID NO:71) and at least one LC comprising (SEQ ID NO:68), (SEQ ID NO:70) or (SEQ ID NO:72).

### Claim 22 (depends on 20)

An antibody or fragment according to claim 20, wherein the HC and LC are paired, and wherein the HC in (SEQ ID NO:67) is paired with an antibody LC in (SEQ ID NO:68) to provide an antibody binding site, comprising both these heavy and light chains for a TSH receptor, or wherein an antibody HC as shown in (SEQ ID NO:69) is paired with an antibody LC in (SEQ ID NO:70) to provide an antibody binding site comprising both these heavy and light chains for a TSH receptor, or wherein an antibody HC in (SEQ ID NO:71)is paired with an antibody LC in (SEQ ID NO:72) to provide an antibody binding site comprising both heavy and light chains for a TSH receptor.