WO2025186694A1 - Method and kit for detecting anti-tsh receptor autoantibodies - Google Patents

Abstract

The invention relates to a pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein, and a method for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample, which employs said fusion proteins pair. More specifically, the capture and detection fusion proteins of the pair of the invention comprise an extracellular domain of a human TSHR and a monomeric non-human immunoglobulin Fc domain, or fragment thereof, or a bacterial maltose binding protein (MBP). Also disclosed is a kit for carrying out the method of the invention. In a preferred embodiment, the method of the invention is a sandwich immunoassay.

Description

Method and kit for detecting anti-TSH receptor autoantibodies

Technical field

The present invention pertains to a method and kit for detecting autoantibodies against the TSH receptor (TSHR), particularly anti-TSHR autoantibodies present in a biological fluid sample.

Background art

The human thyroid-stimulating hormone receptor (hTSHR) is a member of the glycoprotein hormone receptor sub-family of the leucine-rich repeat-containing family of G protein- coupled receptors.

TSHR, a member of the class A G-protein-coupled receptors, is essential for the function and growth of the thyroid gland and activates different signaling pathways, required for the synthesis and release of thyroid hormones.

The TSHR structure is constituted by interplaying domains located in different sites of the thyrotrophic cells (the extracellular leucin-rich repeat domain (LRRD) and the hinge region, the intramembrane serpentine domain (SD) and the cytoplasmic tail).

After expression on the thyrocyte cell surface the TSHR undergoes cleavage within the hinge region. The loss of a C-peptide leads to an extracellular A subunit (comprising the LRRD and a part of the hinge region), and a B subunit (comprising the remainder of the hinge region, the SD and the cytoplasmic tail): the shed A subunit is the autoantigen initiating and driving the autoimmune response in Graves disease (GD) (Miguel R.N. et al., Analysis of the thyrotropin receptor-thyrotropin interaction by comparative modeling. Thyroid 2004 Dec;14(12):991-1011; De Bernard S et al. Sequential cleavage and excision of a segment of the thyrotropin receptor ectodomain, J Biol Chem 1999 Jan 1;274(1): 101-107; Rapoport P and McLachlan SM, TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit. A Molecular and Clinical Perspective. Endocr Rev. Feb 2016(1): 23-42). Thyroid-stimulating hormone (TSH) receptor (TSHR) autoantibodies (TRAbs) are detected in nearly all untreated patients with Graves’ disease and are responsible for the pathological features of this disease, i.e. stimulation of thyroid growth and function, onset of orbitopathy, and/or dermopathy (Michalek K. et al., TSH receptor autoantibodies. Autoimmun Rev - 2009, 9: 113-6).

TRAbs are autoantibodies and as such, they are not a molecularly defined analyte but a mixture of high-affinity IgGs that bind selected epitopes of the TSHR that varies among individuals and fluctuates within one individual. Small changes in the level, affinity, or fine specificity of the TRAbs can result in major changes in their capacity to activate the TSHR. Three categories of TRAbs have been described so far: stimulating (TSAbs), blocking (TBAbs), and neutral (N-TRAbs). Their relative concentrations define the clinical picture and the progression of Graves’ disease. Both, stimulating- and blocking-type TRAbs bind to the TSHR LRD (Evans M. et al., Monoclonal autoantibodies to the TSH receptor, one with stimulating activity and one with blocking activity, obtained from the same blood sample, Clin Endocrinol (Oxf) 2010 Sep;73(3):404-12).

In the last 60 years, a variety of laboratory methods have been proposed and employed to detect and measure TRAbs, based on two different principles: bioassays and immunoassays. The former measure functional activity of TRAbs, either stimulating or blocking activity, while the latter measure the binding to the receptor (total TRAbs, T-TRAbs), irrespective of functional discrimination.

Measuring TRAbs is challenging and generations of tests using different TSHR preparations and ligands have been developed over the years, while in parallel, labeling and detection methods have also improved for the immunoassays in general.

Until 1958, the available methods for detection of TRAbs were bioassays based on the original principles of Adams and McKenzie (McKenzie JM, Delayed thyroid response to serum from thyrotoxic patients. Endocrinology 1958;62:865-8; Adams DD, The presence of an abnormal thyroid stimulating hormone in the serum of some thyrotoxic patients. J Clin Endocrinol Metab 1958;18:699-712). Recent technological improvements of these bioassays enable measuring TRAb levels that are highly correlated with GD activity, but their use in clinical practice needs yet optimization and harmonization. For this reason, the use of TSHR bioassays is still restricted to a small number of specialized laboratories all over the world.

A variety of immunoassay methods have been proposed and employed to detect and measure TRAbs. Shewring et al. in the early 1980s firstly described a competitive radioactive immunoassay (Shewring G and Rees Smith B. An improved radioreceptor assay for TSH receptor antibodies. Clin Endocrinol (Oxf)1982;17:409-17). The progressive improvement of the analytical schemes and of the key biological reagents (receptors of different species and tissues, preparation of antigenic source, types of tracers, etc.) has brought, through three generations of methods, to the improvement of the assay performances (Tozzoli R., The increasing clinical relevance of thyroid-stimulating hormone receptor autoantibodies and the concurrent evolution of assay methods in autoimmune hyperthyroid. J Lab Precis. Med., 2016; 3, 27).

First generation of assays for TRAbs (1980-1999) were based on competitive schemes in liquid phase, and used native (porcine or human) TSH receptor (TSHR) and radiolabeled, or enzyme-coated, bovine TSH as tracer. Since such assays were performed manually, they took days of assay time.

In the late 1990s, two teams were successful in producing monoclonal antibodies to the TSHR that were useful for receptor immobilization onto solid phases of plastic tubes or enzyme-linked immunosorbent assay (ELISA) plates without changing the functional TSHR conformation (Sanders J et al., The interaction of TSH receptor autoantibodies with ¹²⁵I- labelled TSH receptor, J Clin Endocrinol Metab. 1999 Oct;84(10):3797-802 Costagliola S. et al., Second generation assay for thyrotropin receptor antibodies has superior diagnostic sensitivity for Graves' disease. J Clin Endocrinol Metab. 1999 Jan;84(l):90-7). This led to a new generation of TRAb assays (solid phase in-vitro assays). Based on that, a “porcine TRAb assay” as well as a “human TRAb assay” was made commercially available. Additionally, non-isotopic TRAb assays were developed and introduced into the market, e.g. luminescence based human (LIA) and peroxidase based porcine TRAb assays (ELISA). Both liquid and solid phase TRAb assays typically employ bovine TSH as tracer.

In 2002, Sanders et al. reported the development of a thyroid stimulating monoclonal antibody of human origin (TSMAb), named M22 (Sanders J et al, Thyroid-stimulating monoclonal antibodies. Thyroid. 2002 Dec; 12(12): 1043-50). M22 was produced from lymphocytes of a GD patient and was found to be most suitable for a new TRAb assay technique. This TSMAb was employed to substitute bovine TSH used in all previous liquid and solid phase TRAb assays. In detail, TRAbs of patient sera inhibit the binding of M22 to immobilized porcine TSHR. This novel assay technique is currently available as ELISA and as fully automated CLIA platforms, in two different versions: The first variant is a “short version” with directly labeled M22 and consequently a lower number of wash steps. The second variant is characterized by the use of indirectly labeled M22 and includes, therefore, more wash steps. TRAbs are detected by their ability to inhibit M22 binding (Rees Smith B. et al., A new assay for thyrotropin receptor autoantibodies. Thyroid 2004; 14:830). Unfortunately, there is a relatively high inter-method variability of M22 based TRAb assay values despite the strict calibration to NIBSC 90/672.

TRAb tests can also be divided into two main categories depending on the detection method used: competition immunoassays and non-competitive (two-site) immunoassay. Competition immunoassays detect all types of anti-TRAbs by measuring their ability to compete with a labeled ligand (TSH or a monoclonal antibody (M22) to TSH-R) for binding to the TSH receptor.

With the aforementioned improvements in assay sensitivity, ease of performance, and relatively rapid turnaround times, there has been a focus on the use of TRAbs methods for routine clinical use. An additional advance has been the availability of international standard material and the use of anti-TSH-R MAbs instead of TSH as calibration material, resulting in a close correlation among the assays from different manufacturers, with clinical sensitivity and specificity of 97% and 99%, respectively, for the diagnosis of GD versus other causes of hyperthyroidism in both adults and children.

To date, a majority of in vitro diagnostics (IVD) immunoassays for TSHR autoantibodies available on the market are competitive binding assays, which are primarily based on displacement of either a labeled bovine TSH or an anti-TSHR Mab as above described.

However, despite the significant improvements achieved in recent years (new solid phases, tracers and assay schemes), TRAb automated immunoassays have still significant limitations. Immunoassays in a competitive format, such as e.g. Brahms™ TRAK human Kriptor and Roche Elecsys® Anti-TSHR Cobas® have low detection sensitivity. Additionally, due to the low stability of the forms of TSH receptor antigens used in the competition schemes, these assays make use of lyophilized reagents with limited stability upon resuspension, thereby requiring pre-analytical resuspension steps that prevent high throughput (e.g. Brahms™ TRAK human Kriptor and Roche Elecsys® Anti-TSHR Cobas®). Only Abbott, very recently, launched a ready-to-use competitive TRAb assay in the automated platform Alinity (Lee DJW et al, Evaluation of the Abbott Alinity i Thyroid- Stimulating Hormone Receptor Antibody (TRAb) Chemiluminescent Microparticle Immunoassay (CMIA). Diagnostics 2023, 13(16), 2707; Choski H et al., Analytical performance of Abbott’s ARCHITECT and Alinity TSH-receptor antibody (TRAb) assays. Clin Chem Lab Med 2023; 61(8): el52-el55).

Key limitations of the competitive assays as above described have been overcome with the development of bridge immunoassays for the detection of TSHR-autoantibodies. Specifically, the “bridge” technology exploits the well-known concept that antibodies of the IgG class carry two antigen-binding domains, through which said antibodies can bind, and thus bridge, two distinct antigen molecules. Consequently, bridge immunoassays allow for TRAbs detection because one arm of the target antibody binds to a capture receptor on a solid phase, and the other arm binds to a detection receptor, resulting in a detectable signal.

WO 2015/193387 discloses a bridge assay for the detection of stimulating autoantibodies against the TSH receptor, which makes use of TSH receptor chimeras as capture and detection reagent. In these chimeras, the TSHR region that binds blocking or neutral autoantibodies has been replaced with the corresponding domain of a rat luteinizing hormone/chorionic gonadotropin receptor (LHCGR). In the bridge assay disclosed in WO 2015/193387, the determination of the TSHR autoantibodies is preferably accomplished by means of an indirect detection system using a TSHR chimera fused to secretory alkaline phosphatase.

Also the commercially available Siemens Immulite® 2000 TSI bridging immunoassay is based on indirect chemiluminescence (CL) detection. In this assay, a detection chimeric protein is used that contains the Alkaline Phosphatase (AP) enzyme fused to the TSH receptor to generate chemiluminescence upon addition of the substrate molecules.

However, indirect detection methods suffer from the limitations that enzyme label reagents are not very stable and may easily be affected by the change of storage conditions. New generation chemiluminescence immunoassays (CLIA), which allow superior immunoanalytical performances, are indeed generally based on reporter molecules consisting of an antibody, or protein antigen, labeled with the chemiluminescent substrate molecule (e.g. isoluminol or acridinium esters derivatives), rather than with an enzyme. In the case of TRAb CLIA assays, however, direct labeling of the TSH receptor with a luminophore molecule can be particularly challenging, because of the dramatic impairment of the binding properties of TSH receptor towards the autoantibodies caused by conventional labeling procedures.

W02020022776 discloses a fusion protein comprising an immunoglobulin Fc region or a carboxyl terminal cap (C-CAP) bound to a thyrotropin receptor (TSHR) fragment, and the use thereof for the treatment or prevention of Graves’ disease.

The studies described in Holthoff HP et al, “Thyroid-stimulating hormone receptor (TSHR) fusion proteins in Graves’ disease”, J Endocrinol. 2020 Aug 1;246(2): 135-147, show the binding of a His-tagged TSHR or a dimeric TSHR-Fc to the anti-TSHR monoclonal antibody M22 and inhibition of monoclonal M22-dependent cAMP formation by both chimeras. The disclosed TSHR-Fc fusion protein comprises two TSHR A domains at relatively fixed steric orientation.

Summary of the invention Thus, there exists a need in the art for a reliable method for detecting TSH receptor autoantibodies (TRAb), particularly circulating TRAbs, that does not suffer from the drawbacks and limitations of the methods of the prior art.

In particular, there is a need for a sensitive method that allows a timely and high throughput detection of TSHR autoantibodies in test samples, thereby enabling safe use of this method in clinical applications, particularly for the diagnosis of Grave’s disease.

Further, there is a need for a method with improved assay consistency and precision, which enables more accurate results in diagnostic assessments.

These and other needs are addressed by the present invention, which in a first aspect provides an in vitro method for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample, as defined in appended claim 14.

The method of the invention is based on the use of a pair of recombinant fusion proteins able to recognize and bind to autoantibodies produced in response to an immune reaction to a TSH-receptor.

Accordingly, another aspect of the invention is a pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein, wherein said capture fusion protein and said detection fusion protein both comprise an extracellular domain of a human TSHR and a fusion partner selected from:

(i) a monomeric Fc domain of an immunoglobulin and

(ii) a bacterial maltose binding protein (MBP), wherein the TSHR extracellular domain consists of the amino acid sequence of SEQ ID NO. 1 or a variant thereof comprising up to five amino acid substitutions relative to the amino acid sequence of SEQ ID NO. 1, wherein the monomeric Fc domain is a non-human Fc domain or a fragment thereof, the monomeric Fc domain or the fragment thereof comprising up to fifteen amino acid substitutions relative to the wild type Fc domain, wherein said amino acid substitutions result in the Fc domain or fragment thereof not being capable of Fc domain dimerization, wherein the bacterial MBP consists of the amino acid sequence of SEQ ID NO. 2 or a variant thereof comprising up to fifteen amino acid substitutions relative to the amino acid sequence of SEQ ID NO. 2, wherein the human TSHR extracellular domain is linked to the fusion partner, optionally through a peptide linker.

Further aspects of the invention are an isolated nucleotide sequence encoding a capture fusion protein or a detection fusion protein as above defined, an expression vector comprising said nucleotide sequence, and/or a host cell comprising said expression vector.

Still another aspect of the invention is a kit for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample, the kit comprising a pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein as above defined.

Other features and advantages of the present invention are defined in the appended claims, which form an integral part of the description.

Detailed description

As disclosed in more detail below, the present invention provides a pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein, each fusion protein comprising a ligand-binding domain of a human thyroid stimulating hormone receptor (TSHR). The fusion proteins of the pair of the invention are suitable to be employed as capture and detection reagents in an assay for the high-throughput detection of TSHR autoantibodies in biological samples.

The term “pair” as used herein refers to a set of two anti-TSHR autoantibody-binding fusion proteins as above defined, which are intended to be concomitantly used as a capture reagent and a detection reagent in an antibody immunoassay method.

As used herein, the term “capture” refers to an immunoassay reagent capable of binding selectively to a target analyte, such as an anti-TSHR autoantibody, thus enabling said analyte to be captured and isolated from a biological sample.

As used herein, the term “detection” refers to an immunoassay reagent capable of binding selectively to a captured target analyte, such as an anti-TSHR autoantibody, thereby allowing detecting the presence and/or measuring a target analyte in a biological sample.

As used herein, the term “immunoassay” refers to an in vitro method employing immune system’s components to detect, quantify and/or measure the presence of a target analyte.

As used herein, the expression “antibody immunoassay” refers to an immunological assay wherein the target analyte is an antibody.

As used herein, the expression “sandwich immunoassay” refers to an immunoassay wherein the target analyte is an antibody capable of forming a bridge between the capture reagent and detection reagent, resulting in the detection and/or measurement of the analyte. More specifically, the assay takes full advantage of the two antigen binding sites on each antibody that allows them to form a bridge between a capture reagent, optionally immobilized on a solid support, with a detection reagent, optionally added in a detection step.

According to the invention, both the capture fusion protein and detection fusion protein comprise an extracellular domain of a human TSHR (hereinafter referred to also as a TSHR ectodomain) that consists of the amino acid sequence as set forth in SEQ ID NO. 1. More specifically, the amino acid sequence of SEQ ID NO. 1 corresponds to residues 21-261 of the amino acid sequence of the human thyroid stimulating hormone receptor accessible from the NCBI database under accession number XP 054232650.1 (SEQ ID NO. 29). As known in the art, the N-terminal leucine-rich region of TSHR, encompassing amino acids 21-261, is the major target of TRAbs, primarily through antibody binding to discontinuous and conformational epitopes of the receptor (Chazenbalk GD et al., Engineering the human thyrotropin receptor ectodomain from a non-secreted form to a secreted, highly immunoreactive glycoprotein that neutralizes autoantibodies in Graves’ patients’ sera. The Journal of Biological Chemistry. Vol. 272, No. 30, Issue of July 25, pp. 18959-18965, 1997; Frank CU et al., Bridge technology with TSH receptor chimera for sensitive direct detection of TSH receptor antibodies causing Graves’ disease: analytical and Clinical Evaluation; Horm Metab Res 2015; 47: 880-888; Balucan FS et al., Thyroid autoantibodies in pregnancy: their role, regulation and clinical relevance, J Thyroid Res.2013). Accordingly, the TSHR ectodomain in the capture and detection fusion proteins of the pair the invention is able to recognize and bind autoimmune thyroid-stimulating antibodies.

As used herein, the expression “TSHR fusion protein” refers to a capture fusion protein and/or a detection fusion protein according to the invention, which comprises a TSHR ectodomain as above defined.

In certain embodiments, the capture and/or detection TSHR fusion proteins of the pair of the invention comprise a variant of the TSHR domain comprising up to five amino acid substitutions relative to the amino acid sequence of SEQ ID NO. 1, for example one, two, three, four or five amino acid substitutions.

As used herein, amino acid substitutions within a polypeptide are indicated by the wild type amino acid residue, the amino acid position, and the mutant amino acid residue. For example, S70R shall mean a mutation from serine to arginine at position 70 in the polypeptide.

As used herein, a “variant” of a polypeptide comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to the wild type (native) polypeptide sequence. Within the context of the present description, the terms "variant" and "mutant" are used interchangeably.

Preferably, in the capture and/or detection TSHR fusion proteins according to the invention, the TSHR ectodomain variant comprises an amino acid substitution at one or more positions selected from the group consisting of a cysteine at position 4 (C4), a cysteine at position 9 (C9), a tyrosine at position 96 (Y96), and any combination thereof, relative to the amino acid sequence of SEQ ID NO. 1. Mutations in the THSR ectodomain have been described as resulting in increased protein stability and reduced protein aggregation (Chen CR et al., A full biological response to autoantibodies in Graves’ disease requires a disulfide-bonded loop in the thyrotropin receptor N terminus homologous to a laminin epidermal growth factorlike domain. The Journal of Biological Chemistry. Vol. 276, No. 18, Issue of May 4, pp. 14767-14772, 2001; Latif R. et al., Subunit interactions influence TSHR multimerization, Mol Endocrinol, 2010, 24(10):2009-2018).

Preferred amino acid substitutions in the TSHR ectodomain variant of the capture and/or detection TSHR fusion proteins of the pair of the invention, are selected from the group consisting of a cysteine substitution with a serine at position 4 (C4S), a cysteine substitution with a serine at position 9 (C9S), a tyrosine substitution with a serine at position 96 (Y96S), and any combination thereof, relative to the amino acid sequence of SEQ ID NO. 1.

In a particularly preferred embodiment of the invention, the TSHR extracellular domain variant comprises the amino acid substitutions C4S, C9S, Y96S. An exemplary amino acid sequence of a TSHR extracellular domain variant according to this embodiment is set forth as SEQ ID NO. 3.

The capture and detection TSHR fusion proteins according to the invention further comprise a fusion partner selected from (i) a monomeric Fc domain of an immunoglobulin, and (ii) a bacterial maltose binding protein (MBP).

The term “monomeric Fc domain” as used herein refers to a monomeric polypeptide that comprises a sequence of amino acids corresponding to the Fc region of an immunoglobulin heavy chain comprising or consisting of both a CH2 domain and a CH3 domain, and that does not undergo dimerization.

The term "fragment" as used herein in relation to a monomeric Fc domain refers to an amino acid sequence that corresponds to part but not all of the amino acid sequence of said domain.

In one embodiment, the non-human Fc fragment consists of 50 to 400 amino acids, preferably of 80 to 350 amino acids, more preferably of 100 to 300 amino acids. Exemplary lengths of the non-human Fc fragment are of 50, 80, 100, 150, 160, 200, 250, 300, 350, and 400 amino acid residues. For example the non-human Fc fragment consists of 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,

142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,

160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,

196, 197, 198, 199 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,

214, 215, 216, 217, 218, 219 or 220 amino acids.

According to the invention, the monomeric Fc domain is a non-human Fc domain or a fragment thereof. As used herein, the term "Fc domain" is intended to refer broadly to any immunoglobulin Fc region containing a CH2 and CH3 domain such as an IgG, IgM, IgA, IgD or IgE Fc.

Preferably, the Fc domain is a monomeric Fc domain of a non-human IgG.

Fc domains of IgG antibodies are used in the art as components for fusion proteins to exploit the salient characteristics of antibodies associated with the activity of the fused proteins. In many cases, the Fc region improves the biophysical properties of its fusion partner, i.e. solubility, stability of the protein and, generally, it boosts the expression of the target protein. In addition, the presence of an Fc fragment allows the use of affinity chromatographic methods for purification, avoiding the insertion of other affinity tags to this purpose. Additional advantages are that the Fc fragment can be engineered to have chemical modifications and, in immunoassays, it can be also exploited as binding site for the capture or detection antibody.

In the present invention the use of a Fc domain in monomeric form has the advantage that it avoids undesirable steric hindrance effects that may arise from homo-dimer formation of the TSHR fusion proteins, thereby minimizing possible interference in the binding of autoantibodies to the TSHR domain and improving immunoassay performance.

In the capture and/or detection TSHR fusion proteins according to the invention, the non- human monomeric Fc domain or the fragment thereof comprises up to fifteen amino acid substitutions relative to the wild type Fc domain, for example one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions. According to the present invention, the amino acid substitutions introduced in the Fc domain result in said domain or fragment thereof not being capable of Fc domain dimer formation.

The variations are designated by the amino acid at that position in the wild-type Fc domain followed by the amino acid substituted into that position.

In the present invention, the Fc domain or fragment thereof can be derived from any suitable non-human antibody, for example but without limitation, from an ovine antibody, or an avian antibody, or an amphibian antibody.

In a preferred embodiment, the monomeric Fc domain according to the invention or the fragment thereof are of an ovine IgG, preferably of sheep origin. Advantageously, the use of a non-human monomeric Fc domain such as e.g. a sheep monomeric Fc domain enables to reduce the interference due to the potential presence of anti-IgG autoantibodies in patients’ biological samples.

In this current embodiment, the monomeric ovine Fc domain or the fragment thereof preferably comprises an amino acid substitution at one or more amino acid positions selected from the group consisting of L121, T136, L138, T167, F177, Y179, R181 and any combination thereof, relative to a wild type ovine Fc domain of SEQ ID NO. 4.

In a preferred embodiment, the amino acid substitutions in the monomeric ovine Fc domain or fragment thereof are selected from the group consisting of L121Y, L121S, L121K, T136Y, T136R, T136S, L138A, L138H, T167R, T167K, T167V, F177R, F177E, Y179M, Y179K, Y179A, R181A, R181Y, and any combination thereof, relative to the wild type ovine Fc domain of SEQ ID NO. 4. More preferably, the monomeric ovine Fc domain or fragment thereof comprises the amino acid substitutions L121Y, T136Y, L138A, T167R, F177R, Y179M, and R181A. In an even more preferred embodiment, the monomeric ovine Fc domain or fragment thereof according to the invention comprises or consists of an amino acid sequence selected from SEQ ID NOs. 5-7.

As indicated above, another embodiment of the present invention contemplates a bacterial maltose binding protein (MBP) as fusion partner in the capture and/or detection TSHR fusion proteins of the pair of the invention. MBP is known in the art as suitable fusion partner for protein expression in mammalian cells, particularly in the case of secreted proteins. In fact, despite the considerable mass (40 kDa), MBP lacks sites that may be spuriously N- glycosylated, as well as cysteine residues that may interfere with the correct formation of disulfide bonds in the protein of interest. Bokhove M. et al. (Easy mammalian expression and crystallography of maltose-binding protein-fused human proteins. J Struct Biol. 2016. 194(1): 1-7) describe several MBP mutations that enhance protein solubility, maltose binding affinity and crystallizability.

Preferably, either one or both the capture and detection TSHR fusion proteins of the pair of the invention comprise an Escherichia coli maltose binding protein.

According to the invention, the bacterial MBP in the capture and/or detection THSR fusion proteins consists of the amino acid sequence of SEQ ID NO. 2 or a variant thereof comprising up to fifteen amino acid substitutions, relative to the amino acid sequence of SEQ ID NO. 2. As described in Bokhove M. et al. 2016, MBP mutations may enhance protein solubility and crystallizability, as well as maltose binding affinity.

Preferably, the amino acid substitutions within the bacterial MBP variant are at one or more amino acid positions selected from the group consisting of 12, D82, K83, E172, N173, K239, A312, 1317, E359, K362, D363, R367, and any combination thereof, relative to the amino acid sequence of SEQ ID NO. 2.

Preferred amino acid substitutions within the bacterial MBP variant are selected from the group consisting of I2T, D82A, K83A, E172A, N173A, K239A, A312V, 1317V, E359A, K362A, D363A, R367N, and any combination thereof.

In a particularly preferred embodiment, the bacterial MBP variant according to the invention comprises the amino acid substitutions I2T, D82A, K83A, E172A, N173A, K239A, A312V, 1317V, E359A, K362A, D363 A, R367N, relative to the amino acid sequence of SEQ ID NO.

2. An exemplary amino acid sequence of a MBP variant according to this embodiment is set forth as SEQ ID NO. 8.

The capture TSHR fusion protein and/or the detection TSHR fusion proteins according to the invention may have a configuration wherein either the N-terminus or the C-terminus of the human TSHR extracellular domain is directly linked to the N-terminus or C-terminus of a fusion partner as above defined. For example, in the TSHR fusion proteins according to the invention the C-terminus of the TSHR ectodomain may be directly linked to the N terminus of the non-human monomeric Fc domain or fragment thereof. In another exemplary configuration of the TSHR fusion proteins of the pair of the invention, the N-terminus of the TSHR ectodomain is directly linked to the C-terminus of the bacterial MBP or variant thereof.

Optionally, the extracellular domain of human TSHR and a fusion partner as above defined are linked through a peptide linker. Specifically, the linker may be a peptide comprising 3 to 20 amino acid residues or consisting of 3 to 20 amino acid residues.

In a specific embodiment of the invention, the TSHR extracellular domain of the capture and/or detection TSHR fusion proteins consists of the amino acid sequence of SEQ ID NO.

3, the monomeric non-human Fc domain consists of an amino acid sequence selected from SEQ ID NOs. 5-7, and/or the bacterial MBP consists of the amino acid sequence of SEQ ID NO. 8.

In a preferred embodiment, the capture and/or detection TSHR fusion proteins of the pair of the invention comprise or consist of the amino acid sequence of SEQ ID NO. 9 (corresponding to SEQ ID NO. 3 directly linked to SEQ ID NO. 5), the amino acid sequence of SEQ ID NO. 10 (corresponding to SEQ ID NO. 1 directly linked to SEQ ID NO. 5), the amino acid sequence of SEQ ID NO. 11 (corresponding to SEQ ID NO. 8 directly linked to SEQ ID NO. 3), or the amino acid sequence of SEQ ID NO. 12 (corresponding to SEQ ID NO. 2 directly linked to SEQ ID NO. 1). In certain embodiments, the capture and/or detection TSHR fusion proteins according to the invention may further comprise additional components such as an affinity tag for purification, a detectable tag, an enzymatic recognition tag, a signal peptide or a peptide that is substrate for chemical or enzymatic site-specific conjugation. Among the most common affinity tags, polyhistidine tags (“His-tag”) attached at the C-terminal or N-terminal of the protein of interest are routinely employed in protein sciences and their use within the context of the present invention is therefore well within the knowledge of the person skilled in the art.

However, other affinity tags such as, for example, Arg5, Strep-tag II, FLAG, fluorescein (FITC), Poly(A), Poly(dT) and biotin may be employed. Techniques for the production of epitope-tagged recombinant proteins are generally known in the art.

Optionally, the capture and/or detection TSHR fusion proteins according to the invention comprise a signal peptide for directing protein secretion, for example a human serum albumin signal peptide.

In other embodiments, the disclosed TSHR fusion proteins are covalently linked (conjugated) to a functional moiety, such as e.g. a capture moiety or a signal generating moiety.

The term “conjugated protein” as used herein refers to a protein to which another chemical group or molecule has been attached by covalent bonding.

The term "capture moiety" is used herein to indicate a chemical molecule that may serve to immobilize the capture TSHR fusion protein on a solid support, preferably by binding to a respective binding partner on the solid support.

Suitable capture moieties for use in the present invention include, for example, biotin and hapten moieties. Biotin-binding partners encompass any compound that is capable of tightly but non-covalently binding to biotin or any biotin compound such as e.g. streptavidin and avidin, as well as derivatives and analogs thereof. Hapten moieties may be recognized by selective binding partners including, for example, hapten-binding antibodies.

Exemplary signal generating moieties for use according to the invention include, but are not limited to, fluorescent compounds, chemiluminescent compounds, radioactive compounds, enzymes, enzyme substrates, molecules suitable for colorimetric detection.

When a functional moiety is conjugated to the detection TSHR fusion protein according to the invention, the covalent link preferably occurs at a site located outside the TSHR ectodomain, more preferably at amino acid residues remote from said domain. For example, the covalent link may occur at a site within the fusion partner of the detection TSHR fusion protein, or at the N-terminus or C-terminus of said fusion partner.

In a preferred embodiment of the invention, only one single molecule of a functional moiety is attached per molecule of the detection TSHR fusion protein via site-specific conjugation, preferably at a protein site laying outside the TSHR ectodomain. Conventional methods based on random protein conjugation have little control over the location or orientation of the modification, leading to highly heterogeneous products with varying activity. Challenges associated with molecule conjugation to random sites of a protein include partial or full blockade of protein active site resulting in significant loss of protein bioactivity along with protein unfolding and/or loss of conformational epitope (Braun AC et al., Bioorthogonal strategies for site-directed decoration of biomaterials with therapeutic purpose. Journal of Controlled Release. 2018, 273, 68-85).

In the present invention, the conjugation of a single functional molecule to the detection TSHR fusion protein at a specific site located outside the TSHR extracellular domain, and preferably distant from this domain, allows the detection TSHR fusion protein according to the invention to preserve its folding and physico-chemical properties, without affecting fusion protein ability to specifically bind TSHR autoantibodies.

Several strategies for site-directed conjugation of proteins are available in the art and can be in principle used for the purpose of the present invention (Gong Y et al., 2015 Recent advances in bioorthogonal reactions for site-specific protein labeling and engineering. Tetrahedron Letters 56, 2123-2132; Braun AC et al., 2018. Bioorthogonal strategies for site- directed decoration of biomaterials with therapeutic purpose. Journal of Controlled Release. 273, 68-85; Massa S, 2019, Bioconjugation Methods and Protocols. Methods in Molecular Biology Vol 2033).

Both chemical and enzymatic strategies, aimed at site-directed functionalization or decoration of biologies, can be envisioned through rational engineering of proteins. Chemical conjugation methods rely on the presence of a distinguishing functional group within the protein. This distinguishing functional group can be a unique natural or unnatural amino acid (De Graaf AJ et al., Nonnatural amino acids for site-specific protein conjugation, Bioconjug. Chem. 20 (2009) 1281-1295). The introduction of a Cys residue - through site- directed mutagenesis - offers an elegant method for site-specific conjugation but is generally restricted to proteins with no naturally occurring free cysteine. Alternatively, artificial or unnatural amino acids can be incorporated into proteins and used for chemoselective conjugation. Chemical conjugation strategies can be based on: (i) metal-catalyzed reactions, (ii) photocatalytic reactions and (iii) bioorthogonal reactions proceeding without a need for catalysts. The most relevant and versatile metal-catalyzed bioorthogonal reaction is represented by the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), more commonly known as click chemistry, which links an azide with an alkyne to form a 1,2,3- triazole (Kolb HC et al., Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem. Int. Ed. 2001,40, 2004 ± 2021). Copper-free click chemistry reactions can be also performed, using structurally constrained alkyne groups, like a cyclooctyne group (Jewett JC et al., Rapid Cu-free click chemistry with readily synthesized biarylazacyclooctynones, J. Am. Chem. Soc. 132 (2010) 3688-3690). A click chemistry based site-specific conjugation can be therefore carried out by introducing a non-coded amino acid bearing an alkyne or azido group (Nguyen DP et al., Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA (CUA) pair and click chemistry. J. Am. Chem. Soc. (2009) 131(25):8720- 1; Lang K et al., Cellular incorporation of unnatural amino acids and biorthogonal labeling of proteins, Chem. Rev. 114 (2014) 4764-4806) into the target protein sequence for subsequent post-translation conjugation. A variety of other different catalyst-free reactions, based on the introduction of unnatural amino acids, are described in literature, like the oxime, the hydrazone or the Staudinger ligations. Like metal catalysts, light can be used to activate functional groups for bioconjugations, like the light-induced 1,3-dipolar cyclodaddition, in which the diaryl-tetrazole incorporated into the protein undergoes a reaction upon irradiation with UV light to form in situ a nitrile imine that reacts with an alkene to form a stable pyrazoline cycloadduct (Song W et al., A photoinducible 1,3-dipolar cycloaddition reaction for rapid, selective modification of tetrazole-containing proteins, Angew. Chem. 120 (2008) 2874-2877).

Enzyme-catalyzed ligation strategies typically require the introduction of a recognition tag, into the biologic or at its termini, as substrate for enzymatic ligation reactions (e.g. ligations catalyzed by sortase A, transglutaminase, biotin ligase, formyglycine generating enzyme, lipoic acid ligase or tyrosinase). Due to the exceptionally high specificity of enzymes, faster kinetics and milder coupling conditions compared to chemical conjugation, enzymatic ligation provides advantages, especially for sensitive biologies and complex biomaterials. In particular, Sortase A (derived from Staphylococcus aureus) and engineered enzyme variants are frequently used to attach oligoglycine derivatives to proteins expressed with a LPXTG tag (Sortag). As schematically illustrated in Figure 4, in this system Sortase transpeptidase cleaves the bond between threonine and glycine and a new peptide bond is created between the N-terminus of the oligoglycine and the threonine. Sortase-mediated reactions have been used extensively for site-specific C-terminal and internal labelling of proteins (Guimaraes CP et al., Site-specific C-terminal and internal loop labeling of proteins using sortase- mediated reactions, Nat. Protoc. 8 (2013) 1787-1799) and bioconjugation in vitro and in living cells, especially for cell surface proteins expressing the LPXTG motif (Popp MW et al., Sortagging: a versatile method for protein labeling, Nat. Chem. Biol. 3 (2007) 707-708) for site-specific PEGylation (Popp MWL et al., Making and breaking peptide bonds: protein engineering using sortase, Angew. Chem. Int. Ed. 50 (2011) 5024-5032), protein immobilization on solid surfaces (Sinisi A. et al., Development of an influenza virus protein array using sortagging technology, Bioconjug. Chem. 23 (2012) 1119-1126), antigen coupling to virus-like particles (Tang S et al., A modular vaccine development platform based on sortase-mediated site-specific tagging of antigens onto virus-like particles, Sci. Rep. 6 (2016) 25741), or for other purposes (Antos JM et al., Recent advances in sortase- catalyzed ligation methodology, Current Opinion in Structural Biology 2016, 38: 111-118; Dai X et al., Broadening the scope of sortagging, RSC Adv., 2019, 9, 4700-4721).

In a preferred embodiment of the invention, the TSHR fusion proteins may further comprise a sortase recognition sequence including the motif LPXTG (Leu-Pro-any-Thr-Gly - SEQ ID NO: 13) (wherein the occurrence of X represents independently any amino acid residue). An exemplary amino acid sequence of a sortase recognition sequence suitable to be employed in the invention is LPETG (SEQ ID NO. 14). For example, the sortase recognition sequence may be located at the C-terminus of the TSHR fusion proteins according to the invention, preferably at the C-terminus of the fusion partner, more preferably at the C-terminus of the MBP protein or at the C-terminus of the monomeric Fc domain or fragment thereof. Other variant sortase recognition sequences, known in the art, can also be used for the purpose of the invention.

According to the aforementioned embodiment, as will be described in more detail below, a single functional moiety may be conjugated to a specific site of a TSHR fusion protein molecule through the enzymatic activity of a sortase, for example a Sortase A activity.

Exemplary TSHR fusion proteins according to the invention embodiments as above described comprise or consist of the amino acid sequences selected from the group consisting of SEQ ID NOs. 15-19 (as schematically depicted in Figure 5A-E), and SEQ ID NO. 20 (as schematically depicted in Figure 3).

The above-described embodiments of the invention may be used separately or in any combination, as appreciated by those skilled in the art in light of the above teachings.

In another aspect, the present invention is directed to an isolated nucleic acid sequence encoding any of the capture or detection TSHR fusion protein as above defined. Preferably, said nucleic acid sequence comprises or consists of a sequence selected from the group consisting of SEQ ID NO. 21 (encoding the TSHR fusion protein of SEQ ID NO. 9), SEQ ID NO. 22 (encoding the TSHR fusion protein of SEQ ID NO. 10), SEQ ID NO. 23 (encoding the TSHR fusion protein of SEQ ID NO. 11), SEQ ID NO. 24 (encoding the TSHR fusion protein of SEQ ID NO. 12), SEQ ID NO. 25 (encoding the TSHR fusion protein of SEQ ID NO. 15), SEQ ID NO. 26 (encoding the TSHR fusion protein of SEQ ID NO. 16), SEQ ID NO. 27 (encoding the TSHR fusion protein of SEQ ID NO. 17), and SEQ ID NO. 28 (encoding the TSHR fusion protein of SEQ ID NO. 20).

A further aspect of the present invention is an expression vector comprising the nucleic acid sequence as defined above, and optionally further comprising a promoter sequence and a polyadenylation signal sequence, as well as a host cell comprising the above expression vector.

Recombinant expression vectors for use in the manufacture of peptides or proteins are known and described in the state of the art, therefore the selection and use thereof are within the skills of those of ordinary skill in the art. Such vectors can be prokaryotic or eukaryotic vectors. By way of non-limiting example, eukaryotic vectors suitable for expression in mammalian cells include pcDNA3, pcDNA5, pcDNA3.1, and pcDNA3.4.

Preferably, the cell system used for the expression of the expression vector of the invention is selected from eukaryotic systems, for example mammalian cells, such as e.g. CHO, BHK, HeLa, Hek293 cells, or insect cells, such as e.g. sf9, sf21, HiFive cells.

The person skilled in the art is well aware of the standard methods for incorporation of a polynucleotide into a host cell, for example transfection, lipofection, electroporation, microinjection, viral infection, thermal shock, transformation after chemical permeabilisation of the membrane or cell fusion.

The present invention also relates to a method for the preparation of the TSHR fusion proteins according to the invention, according to which the transformed host cell is cultured under suitable conditions and for a time sufficient for the expression of the fusion protein of the invention. Typically, suitable culture conditions and times depend on the cell system used and may be related, for example, to the composition of the culture medium, the pH, the relative humidity, the gaseous component of O2 and CO2, as well as the temperature. The selection of the most suitable culture conditions and times to be used in the method of the invention is well within the skills of those of ordinary skill in the art. In a preferred embodiment, the method according to the invention additionally comprises the step of recovering the fusion proteins produced from the cell culture. The recovery step can be carried out by using protein purification methods belonging to the prior art, for example protein denaturation, solubilization and/or renaturation, or one or more chromatographic and/or desalting steps, or still by ultrafiltration, dialysis and/or freeze- drying.

In a yet another aspect, the present invention is directed to an in vitro method for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample, which employs a pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein as above defined and comprises the steps of:

(i) contacting the biological fluid sample with said capture fusion protein, thereby obtaining binding of the anti-TSHR autoantibodies possibly present in the biological fluid sample to the capture fusion protein, and

(ii) detecting the anti-TSHR autoantibodies bound to the capture fusion protein by means of said detection fusion protein.

A preferred capture TSHR fusion protein according to the invention is a TSHR fusion protein as above described wherein the TSHR ectodomain is fused to a monomeric form of a nonhuman immunoglobulin Fc domain or fragment thereof, preferably a monomeric Fc domain or fragment thereof of ovine origin, more preferably a monomeric sheep Fc domain or fragment thereof.

Preferably, the capture TSHR fusion protein is immobilized on a solid support.

In one embodiment of the method of the invention, the TSHR fusion protein employed as capture fusion protein is conjugated with one or more capture moieties, preferably with one or more biotin moieties and/or one or more hapten moieties.

According to this embodiment, the solid support is coated with one or more binding partners for binding said one or more capture moieties. Preferably, the one or more binding moieties are streptavidin and/or avidin moieties.

Thus, the capture TSHR fusion protein conjugated to one or more capture moieties, for example a biotin- or hapten-conjugated TSHR fusion protein according to the invention may be immobilised on an immobilizing phase, e.g. a solid support, coated with one or more binding partners of the capture moieties such as e.g a biotin- binding partner or a haptenbinding partner as above described, through the binding of said one or more capture moieties with the one or more binding partners onto the solid support.

Non-limiting examples of suitable solid supports are the wells of a microtitre plate, the surface of a microparticle such as a latex, polystyrene, silica, chelating sepharose or magnetic beads, membranes, strips or chips.

In the method of the present invention, the detection of the anti-TSHR autoantibodies bound to the capture TSHR fusion protein may be accomplished through a wide range of techniques. For example, a detectable signal may be generated directly by employing a detection TSHR fusion protein according to the invention, preferably a detection TSHR fusion protein conjugated with a signal generating moiety (i.e. a detectable label). According to the invention, said labeled TSHR fusion protein is capable of binding anti-TSHR autoantibodies captured by the capture TSHR fusion protein. Alternatively, a detectable signal may be generated indirectly via a labeled detector molecule that is capable of binding the detection TSHR fusion protein. Typically, the detector molecule is an antibody directed to an epitope on the detection TSHR fusion protein that is different from the epitope recognized by the anti-TSHR autoantibodies.

The detectable label may be any substance capable of producing a signal that is detectable by visual or instrumental means. Suitable labels for use in the present invention include for example fluorescent compounds, chemiluminescent compounds, radioactive compounds, enzymes and enzyme substrates, molecules suitable for colorimetric detection, binding proteins, epitopes, enzymes or substrates. In practice, any signal molecule or label known in the art may be incorporated in embodiments of the method and kit of the present invention. According to the invention, any TSHR fusion protein as above defined may be employed in the method of the invention or any combination thereof as above defined in connection with preferred embodiments of the invention.

According to a preferred embodiment of the invention, the capture TSHR fusion protein is conjugated to one single capture moiety, and/or the detection TSHR fusion protein is conjugated to one single detectable label. More preferably, the single capture moiety and/or the single detectable label are conjugated to the capture and/or detection TSHR fusion proteins, respectively. In this embodiment, the single capture moiety and/or the single detectable label conjugated per fusion protein molecule are preferably at a site located away from the TSHR extracellular domain.

As illustrated in the examples that follow, the present inventors have surprisingly observed that a higher signal intensity is achieved in the method of the present invention by using TSHR fusion proteins conjugated with a single detectable label per protein, i.e. a single chemiluminescent molecule introduced by site-directed conjugation, rather than TSHR fusion proteins labeled at multiple sites by conventional approaches. Conjugation of the TSHR fusion proteins according to the invention through conventional amine-targeting methods, relying on reaction of activated esters of the labeling molecules (biotin or ABEI) with the protein lysine side-chains, and resulting in multiple and random introduction of the labeling moiety on the protein, gave rise to unstable and poorly functional products, even at low degrees of labelling.

Thus, in the method of the present invention the unique features of the engineered proteins used for detecting anti-TSHR autoantibodies along with direct conjugation means advantageously allows to achieve a significantly improved assay sensitivity.

The assay formats that can be used by practicing the method of the present invention and that can be incorporated in kit form are many, and include, for example, enzyme-linked immunosorbent assays (ELISA) also referred to as enzyme immunoassays (EIA), Chemiluminescent immunoassays (CLIA), Fluorescence immunoassays, Enzyme-linked immunoassays (ELISA), Luminescense immunosorbent assays (LISA), radioimmunoassays (RIA), Western blot assays, immunoprecipitation assays, Luminex-based bead arrays, protein microarray assays as well as flow cytometric assays. According to a preferred embodiment, the method of the invention is a sandwich immunoassay.

The use of any type of immunoassay format, the selection of which falls within the skills of the person skilled in the art, is within the scope of the present invention.

Figures 1 and 2 illustrate, by way of example, sandwich immunoassays according to the invention, wherein autoantibodies to TSHR are captured by a capture biotinylated TSHR fusion protein according to the invention. In the examples of Figures 1 and 2 the TSHR fusion protein employed as capture fusion protein comprises a monomeric sheep Fc domain as fusion partner and is immobilized on a solid support. The solid support is a paramagnetic particle (PMP) coated with streptavidin.

In the specific embodiment of Figure 1, the detection step is accomplished by means of a detection TSHR fusion protein comprising a monomeric sheep Fc domain linked to the TSHR ectodomain and conjugated to an isoluminol derivative (Amino-Butyl-Ethyl- Isoluminol, ABEI). According to the embodiment of Figure 1, the immune complex formed from the binding of anti-TSHR autoantibodies to the capture THSR fusion protein is captured on streptavidin-coated paramagnetic particles through the interaction with the capture biotinylated TSHR fusion protein, and a chemiluminescence reaction is then performed by means of the ABEI-labeled detection TSHR fusion protein, to detect the immune complex containing the anti-TSHR autoantibody.

Figure 2 illustrates an alternative embodiment of the method of the invention, wherein the detection TSHR fusion protein comprises a TSHR ectodomain linked to a maltose binding protein. In this embodiment, the detection TSHR fusion protein binds the immune complex formed from the binding of anti-TSHR autoantibodies to the capture biotinylated TSHR fusion protein and captured on streptavidin-coated paramagnetic particles, and indirect detection is performed by means of a labeled antibody specific for MBP. The label is Amino- Butyl-Ethyl-Isoluminol (ABEI). In the context of the present invention, the biological fluid sample is preferably selected from the group consisting of whole blood, serum, plasma, and urine. The biological fluid sample may optionally include further components, such as for example: diluents, preservatives, stabilizing agents and/or buffers. If needed, dilutions of the biological fluid sample are prepared using any suitable diluent buffer known in the art.

Preferably, the biological fluid sample is from a patient affected by a thyroid or thyroid- related disease or disease condition, more preferably from a patient affected by Graves’ disease.

A yet further aspect of the present invention is a kit as defined above, for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample. A suitable pair of capture and detection TSHR fusion proteins to be used in the kit of the invention is as described above in connection with the method of the invention. In an embodiment of the kit of the invention, the capture and/or the detection TSHR fusion protein according to the invention comprises a TSHR ectodomain linked to a monomeric non-human Fc domain or fragment thereof.

In another embodiment of the kit of the invention, the capture TSHR fusion protein of the pair of the invention, comprises a TSHR ectodomain linked to a monomeric non-human Fc domain or fragment thereof, and the detection TSHR fusion protein of the pair of the invention, comprises a TSHR ectodomain linked to a bacterial MBP.

In a still another embodiment of the invention, the kit further comprises a labeled antibody capable of binding the detection TSHR fusion protein.

The kit of the invention may further comprise a solid support such as, without limitation, beads, microparticles, nanoparticles, super paramagnetic particles, a microtiter plate, a cuvette, a lateral flow device, a flow cell, or any surface to which the capture moiety can be passively or covalently bound. The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section reference is made to the appended drawings, wherein:

- Figure 1 shows a schematic representation of an embodiment of the method of the invention, wherein the capture and detection TSHR fusion proteins are a biotin- and ABEI conjugated TSHR fusion protein comprising a monomeric sheep Fc domain linked to the TSHR ectodomain, respectively;

- Figure 2 shows a schematic representation of another embodiment of the method of the invention, wherein the capture fusion protein is a biotinylated TSHR fusion protein comprising a monomeric sheep Fc domain linked to the TSHR ectodomain, and the detection fusion protein is a TSHR fusion protein comprising a TSHR ectodomain linked to a maltose binding protein;

- Figure 3 shows a schematic representation of an exemplary TSHR fusion protein according to the invention (designated as SEQ ID NO. 20, hereinafter referred to as mMBP-mTSHR) comprising, from the N-terminus to the C-terminus: (i) a MBP variant comprising the indicated amino acid substitutions (amino acids 1-367); (iii) a linker (368-370), (iv) a variant THSR domain comprising the indicated amino acid substitutions (amino acids 371-611); (v) a linker and a His affinity tag (amino acids 612-628);

- Figure 4 illustrates a general scheme of protein modification via Sortase-catalyzed transpeptidation (Antos JM et al., 2016);

- Figure 5A-C shows a schematic representation of exemplary TSHR fusion proteins according to the invention (designated as SEQ ID NO. 15-17, respectively) comprising, from the N-terminus to the C-terminus: (i) a variant THSR domain comprising the indicated amino acid substitutions (amino acids 1-241); (ii) a monomeric sheep Fc domain fragment comprising the indicated amino acid substitutions (amino acids 242-460); (iii) a linker and a sortase recognition sequence (Sortag, amino acids 461-472); (iv) a linker and a His affinity tag (amino acids 473-489). The TSHR fusion protein of SEQ ID NO. 15 is hereinafter referred to as mTSHR-mSFc.

- Figure 5D-E. Figure 5D shows a schematic representation of an exemplary fusion protein according to the invention (designated as SEQ ID No. 18) conjugated to a biotin molecule that comprises, from the N-terminus to the C-terminus: (i) a variant TSHR domain comprising the indicated amino acid substitutions (amino acids 1-241), (ii) a monomeric sheep Fc domain fragment comprising the indicated amino acid substitutions (amino acids 242-460), (iii) a linker and a sortase recognition sequence (amino acids 461-472), (iv) a linker carrying a biotin molecule, linked at the amino group of the C-terminal lysine side chain through amide bond (amino acids 473-477). Figure 5E shows a schematic representation of an exemplary fusion protein according to the invention (designated as SEQ ID No. 19) labeled with an ABEI molecule that comprises, from the N-terminus to the C- terminus: (i) a variant TSHR domain comprising the indicated amino acid substitutions (amino acids 1-241), (ii) a monomeric sheep Fc domain fragment comprising the indicated amino acid substitutions (amino acids 242-460), (iii) a linker and a sortase recognition sequence (amino acids 461-472), (iv) a linker carrying an ABEI molecule, linked at the thiol group of the C-terminal cysteine side chain through mal eimide (amino acids 473-482);

- Figure 6 shows the results of a comparative analysis of dose-response curves obtained according to the method embodiment depicted in Figure 1 (embodiment 1) on 10 serum samples with random vs site-specific conjugation of TSHR fusion proteins according to the invention, on a standard curve. The results are illustrated in the graph in the upper part (random conjugation, black line; site-specific conjugation, grey line) and summarized in the table in the lower part. The values are expressed in relative luminescence units (RLU);

- Figure 7 shows a comparative analysis of dose-response curves obtained according to the method embodiment depicted in Figure 2 (embodiment 2) on 12 serum samples with random vs site-specific biotinylation of mTSHR-msFc fusion proteins, on a standard curve. The results are shown in the graph in the upper part (random biotinylation, black line; sitespecific biotinylation, grey line) and summarized in the table in the lower part. The values are expressed in relative luminescence units (RLU);

- Figure 8 shows a comparative analysis of dose-response curves obtained according to the method embodiment depicted in Figure 2 (embodiment 2) on 12 serum samples with direct vs indirect labeling of mMBP-mTSHR fusion proteins, on a standard curve. The results are illustrated in the graph in the upper part (direct labeling, black line; indirect labeling, grey line) and summarized in the table in the lower part. The values are expressed in relative luminescence units (RLU);

- Figure 9 illustrates a comparative analysis of dose-response curves obtained according to the method embodiment depicted in Figure 1 (embodiment 1) on 12 serum samples using a pair of TSHR fusion proteins both comprising a mutant TSHR ectodomain vs a pair of TSHR fusion proteins both comprising wild type TSHR ectodomain, on a standard curve.. The results are shown in the graph in the upper part (mutant TSHR ectodomain, black line; wild type TSHR ectodomain, grey line) and summarized in the table in the lower part. The values are expressed in relative luminescence units (RLU);

- Figure 10A-B shows the signal values of anti-TSHR autoantibodies -positive (A) and - negative serum samples (B) tested according to the method embodiment depicted in Figure 1 (embodiment 1). Specifically, a pair of TSHR fusion proteins both comprising a mutant TSHR ectodomain (dark grey bars) vs a pair of TSHR fusion proteins both comprising wild type TSHR ectodomain (light grey bars) were used. The values are expressed in relative light units (RLU).

Declaration under Art 170 bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code

The present invention has been attained in accordance with the provisions established by Article 170-bis, paragraphs 2, 3 and 4 of the Italian Industrial Property Code concerning the obtainment of an informed consent.

EXAMPLES

1. Generation of the mTSHR-mSFc and mMBP-mTSHR fusion proteins

For their experiments, the present inventors made use of TSHR fusion proteins according to the invention, comprising (i) a mutant extracellular domain of a human TSHR (C4S, C9S, Y96S; SEQ ID NO. 3) linked at the C-terminal to a mutant monomeric sheep Fc domain (L121Y, T136Y, L138A, T167R, F177R, Y179M, R181A; SEQ ID NO. 5), hereinafter referred to as mTSHR-mSFc, or (ii) a mutant extracellular domain of a human TSHR as above defined linked at the N-terminal to a mutant E. coli maltose binding protein (I2T, D82A, K83A, E172A, N173A, K239A, A312V, 1317V, E359A, K362A, D363A, R367N; SEQ ID NO. 8), hereinafter referred to as mMBP-mTSHR. In particular, the fusion protein mTSHR-mSFc consists of the amino acid sequence of SEQ ID NO. 15, and the fusion protein mMBP-mTSHR consists of the amino acid sequence of SEQ ID NO. 20. Standard cloning techniques were used for preparing the vectors for the expression of the TSHR fusion proteins. Briefly, the synthetic genes encoding for mTSHR-mSFc and mMBP-mTSHR, optimized for the expression in CHO cells, were commissioned to GeneArt (Invitrogen - Thermo Fisher Scientific, Carlsbad, CA) and were cloned between Nhel and Xhol restriction sites in pcDNA3.4-TOPO (Invitrogen - Thermo Fisher Scientific).

New England Biolabs (NEB, Beverly, Massachusetts) was the supplier for the restriction enzymes used in the cloning step.

2, Expression of the fusion proteins mTSHR-mSFc and mMBP-mTSHR

The expression of mTSHR fusion proteins according to the invention was obtained by transient transgene expression in the CHO-S cell line following standard procedures of transient transfection, i.e. transfection with chemical agent, in particular cationic lipids- based transfection reagents, and in mild hypothermia culture condition (ExpiCHO expression system kit - Thermo Fisher Scientific; Waltham, MA). Transient fusion protein productions were performed in flasks both in small and large scale and maintained in shaking incubators. The fusion proteins were designed to be secreted in the supernatant, therefore at the day of harvest the supernatants were collected, clarified and stored at -80°C.

3, Purification of mMBP-mTSHR Fusion Proteins

Cell supernatant containing the mMBP-mTSHR fusion protein was thawed and then underwent a buffer exchange using a dialysis membrane with a molecular weight cut-off (MWCO) of 10 kDa against phosphate buffer. After that, the whole sample was clarified by centrifugation and purified by Immobilized-Metal Affinity Chromatography (IMAC) using a HisTrap excel column (Cytiva). Unbound proteins were eliminated flowing phosphate buffer through the IMAC column. Bound mMBP-mTSHR fusion protein was eluted by flowing an imidazole solution through the IMAC column (phosphate 50 mM, NaCl 500 mM, Imidazole 500 mM).

The chromatographic fractions were analyzed by SDS-PAGE and the most concentrated eluted fractions were pooled together. The resulting IMAC pool was then subjected to a gel filtration chromatographic step (GFC) by means of a HiLoad 26 600 Superdex 200 prep grade column (Cytiva) that was previously equilibrated in the storage buffer (phosphate 50 mM, NaCl 150 mM, pH 7.5). The chromatographic fractions were analyzed by SDS-PAGE and those containing the monomeric form of mMBP-mTSHR fusion protein were pooled together. The concentration of the purified mMBP-mTSHR was determined spectrophotometrically.

4, Purification of mTSHR-msFc Fusion Proteins

The cell supernatant containing the mTSHR-msFc fusion protein was thawed and then underwent a buffer exchange using a dialysis membrane with a molecular weight cut-off (MWCO) of 10 kDa against phosphate buffer. Afterwards, the sample was clarified by centrifugation and purified by Immobilized-Metal Affinity Chromatography (IMAC) using a HisTrap excel resin (Cytiva). Unbound proteins were eliminated by flowing phosphate buffer through the IMAC column. Bound mTSHR-msFc fusion protein was eluted by flowing imidazole through the IMAC column (phosphate 50 mM, NaCl 500 mM, Imidazole 500 mM).

The chromatographic fractions were analyzed by SDS-PAGE and the most concentrated fractions were pooled together. The resulting IMAC pool was then subjected to a gel filtration chromatographic step (GFC) by means of a HiLoad 26/600 Superdex 200 prep grade column (Cytiva) that was previously equilibrated in final storage buffer (HEPES 50 mM, NaCl 150 mM, pH 7,5). The chromatographic fractions were analyzed by SDS-PAGE and those containing the monomeric form of mTSHR-msFc fusion protein were pooled together.

The concentration of the purified mTSHR-msFc protein was determined spectrophotometrically.

5, Random conjugation of the TSHR fusion proteins Biotinylation

The mTSHR-msFc fusion protein according to the invention was reacted with a 5-fold molar excess of EZ-Link NHS-PEG4-Biotin (Thermo Scientific). The labeling reaction was performed in a phosphate buffer at pH 7.4 for two hours at room temperature. The biotinylated mTSHR-msFc product was isolated from unreacted NHS-PEG4-Biotin by means of a desalting chromatographic step on a PD-10 desalting column (Cytiva). The concentration of the purified biotinylated protein was determined spectrophotometrically.

Chemiluminescent labeling

The mTSHR-msFc fusion protein according to the invention was reacted with a 5-fold molar excess of an isoluminol derivative (ABEI, Palmioli A. et al., A new isoluminol reagent for chemiluminescence labeling of proteins. Tetrahedron Letters. (2009) Vol. 54, Issue 33, 4446-4450). The labeled product was isolated from unreacted ABEI by means of a desalting chromatographic step on a HiPrep™ 26/10 desalting column (Cytiva). The concentration of the purified labeled protein was determined spectrophotometrically.

The relative degree of chemiluminescent labeling (Fh) was determined as the ratio of the absorbance values at 280 and at 329 nm, as specific absorbances of proteins and isoluminol, respectively, taken at the chromatographic peak top corresponding to the mTSHR-msFc- ABEI conjugate. This parameter allows the comparison of the labeling efficacy on the mTSHR-msFc protein. The lower this value is, the higher is the number of ABEI molecules covalently introduced on the protein.

For the random labeling of mTSHR-msFc, the Fh value was 3.2.

6, Site-directed conjugation of mTSHR-msFc fusion protein

Biotinylation

The mTSHR-msFc fusion protein was biotinylated exploiting the Sortase A enzyme, which ligated the amino acids LPETG (SEQ ID NO. 14) at the C-terminus of the mTSHR-msFc fusion protein to a peptide carrying a biotin molecule, as already described above (Figure 4). The resulting product, mTSHR-msFc-Biotin (SEQ ID NO. 18), was characterized by comprising a single biotin molecule introduced at its C-terminus.

Experimentally, the mTSHR-msFc fusion protein was reacted with a 20-fold molar excess of a synthetic peptide carrying the biotin molecule in the presence of an equimolar amount of Sortase A enzyme for 3 hours at room temperature in Hepes 50mM, NaCl 150mM, CaCh 5mM, pH 7.4. After the conjugation reaction, the protein mixture underwent a desalting step using a HiPrep 26/10 Desalting column (Cytiva) that had previously been equilibrated in phosphate buffer (100 mM phosphate, 300 mM NaCl, pH 7.5). This desalting phase allowed the removal of the biotinylated peptide excess.

The desalted sample was loaded on an HisTrap Excel IMAC column (Cytiva). The mTSHR- msFc-biotin product flowed through the IMAC column, having lost its C-terminal histidine tag, whereas the starting mTSHR-msFc fusion protein bound the IMAC resin, having the original C-terminal histidine sequence. The target mTSHR-msFc-Biotin was therefore found in the unbound sample and isolated from the unreacted mTSHR-msFc protein precursor. The eluted fractions were analyzed by SDS-PAGE. The fractions containing the mTSHR-msFc- Biotin were pooled together and the concentration of the purified pool was determined spectrophotometrically.

Chemiluminescent labeling

The mTSHR-msFc fusion protein according to the invention was covalently linked to ABEI, exploiting the Sortase A enzyme, which ligated the amino acids LPETG (SEQ ID NO. 14) at the C-terminus of the mTSHR-msFc fusion protein to a peptide carrying an ABEI molecule, as already described above (Figure 4). The resulting product, mTSHR-msFc- ABEI (SEQ ID NO. 19), was characterized by comprising a single ABEI molecule introduced at its C-terminus.

Experimentally, the mTSHR-msFc fusion protein was reacted with a 20-fold molar excesses of a synthetic peptide carrying the ABEI molecule, in the presence of an equimolar amount of Sortase A enzyme for 3 hours at room temperature, in Hepes 50mM, NaCl 150mM, CaCh 5mM, pH 7.4. After the conjugation reaction, the protein mixture underwent a desalting step using a HiPrep 26/10 Desalting column (Cytiva) previously equilibrated in a phosphate buffer. This desalting phase allowed the removal of excess of the synthetic peptide carrying the ABEI molecule. The desalted sample was loaded on an HisTrap Excel IMAC column (Cytiva). The mTSHR-msFc-ABEI product flowed through the IMAC column, having lost its C-terminal histidine tag, whereas the starting mTSHR-msFc fusion protein bound the IMAC resin, having the original C-terminal histidine sequence. The target mTSHR-msFc- ABEI was therefore found in the unbound sample and isolated from the unreacted mTSHR- msFc protein precursor. The eluted fractions were analyzed by SDS-PAGE. The fractions containing the mTSHR-msFc- ABEI were pooled and the concentration of the purified pool was determined spectrophotometrically.

The resulting relative degree of labeling (Fh) was 6.2.

7, Detection of Autoantibodies against the TSH Receptor

The present inventors carried out a set of experiments on an in vitro method for the detection of autoantibodies to the TSH receptor in human serum and plasma samples. Particularly, the inventors set up a chemiluminescent immunoassay based on a bridge format suitable to be performed on the LIAISON® XL platform, a fully automated chemiluminescence analyser. In the following, experiments relating to different embodiments of the method of the invention are described.

7.1 First Embodiment of the method of the invention

In a first embodiment, the method of the invention employed a pair of recombinant mTSHR- msFc fusion proteins, mTSHR-msFc-Biotin (capture agent) and mTSHR-msFc- ABEI (detection agent), in a one-step assay protocol with three incubation times, in which Biotin and ABEI had been site-specifically conjugated to the mTSHR-msFc, respectively. Sample and standards were initially incubated with the assay diluent and then, in a second incubation step, with the TSHR fusion proteins as above indicated. The anti-TSHR autoantibodies present in the biological sample bound the two TSHR fusion proteins and the resulting immune complex was then captured on streptavidin paramagnetic particles. Unbound reagents were then removed with a wash cycle. Subsequently, the starter reagents were added and a flash chemiluminescence reaction was thus induced. The light signal, and hence the amount of mTSHR-msFc-ABEI conjugate, was measured by a photomultiplier as relative light units (RLU) and is indicative of autoantibodies to TSHR present in standard curve, samples or controls. A schematic representation of the first embodiment of the method of the invention as above described is provided in Figure 1.

Assay protocol

A total of 100 pl of sample were incubated for 13 minutes with 100 pl of assay diluent (1^st incubation time); 50 pl of mTSHR-msFc-Biotin plus 50 pl of mTSHR-msFc-ABEI were then added and incubated for 23 minutes (2^nd incubation time). Afterwards, 20 pl of streptavidin paramagnetic particles were added and incubated for 13 minutes to capture the immune complex (3^rd incubation time). The unbound material was then removed with a washing cycle. Finally, the starter reagents were added, and the emitted light was measured.

Reagents and formulations

A, Solid phase

Streptavidin paramagnetic particles (Dynal M-280 - Ref 35137) were diluted at a final concentration of 0.375% (w/v) in 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na₂HPO₄*H₂O, 0.24 g/L KH₂PO₄, 1 g/L BSA, 1 g/L NaN₃ pH 7.4.

B, Assay diluent

Assay diluent was composed as follows: 1.63 g/L Na2HPO4, 1.16 g/L KH2PO4, 29.22 g/L NaCl, 0.366 g/L EDTANa4 + lOg/L BSA frac V, 1.5 ml/L SDS, 4.5 g/L Tween 20, 0.25 g/L Kollidon 25 (PVP), 0.05 ml/L Anti foam 204, 0.10 g/L Gentamicin sulphate, 2 ml/L Proclin 300, 100 ml/L Sheep serum, pH 7.

C. Fusion protein mTSHR-msFc-Biotin

TSHR fusion proteins according to the invention having the amino acid sequence SEQ ID NO. 15, which comprise a mutant human TSHR extracellular domain of SEQ ID NO. 3 C- terminally fused to a mutant sheep monomeric Fc domain of SEQ ID NO.5, were labeled with biotin through random conjugation (mTSHR-msFc-Biotin 5x) or by means of sitespecific conjugation via Sortase A (mTSHR-msFc-Biotin via Sortase A). The labeled TSHR fusion proteins thus obtained were diluted at a final concentration of 800 ng/ml in the antigen diluent.

D. Fusion protein mTSHR-msFc-ABEI

TSHR fusion proteins according to the invention having the amino acid sequence SEQ ID NO. 15, which comprise a mutant human TSHR extracellular domain of SEQ ID NO. 3 C- terminally fused to a mutant sheep monomeric Fc domain of SEQ ID NO.5, were labeled with ABEI through random conjugation (mTSHR-msFc-ABEI 5X) or by means of sitespecific conjugation (mTSHR-msFc-ABEI via Sortase A). The labeled TSHR fusion proteins thus obtained were diluted at a final concentration of 800 ng/ml in the antigen diluent.

E, Antigens diluent

Antigen diluent was composed as follows: 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na2HPO4*H2O, 0.24 g/L KH2PO4, 1 g/L BSA, 0.5 g/L tween 20, pH 7.4.

F, Standard curve

A standard curve was prepared by diluting the commercial recombinant human antibody M22 (Creative Biolabs Cat.nr. PABW-172) in human negative universal serum (Golden West Diagnostic cat. UN1000). Ten serial dilutions were prepared with nominal concentrations ranging from 0 to 26.7 IU/L. The standard curve was standardized against the WHO 2nd International Std for Thyroid Stimulating Antibody, NIBSC (Code: 08/204).

Assay results

Comparison of random vs site-specific conjugation

As outlined above, the mTSHR-msFc fusion proteins were conjugated with biotin or ABEI by employing two different techniques: (i) multiple random conjugation (mTSHR-msFc- Biotin 5X and mTSHR-msFc-ABEI 5X, respectively) and (ii) site-specific conjugation (mTSHR-msFc-Biotin or mTSHR-msFc-ABEI via sortase A, respectively). Of particular note, random conjugation with ABEI resulted in a higher number of chemiluminescent molecules introduced on the fusion protein, compared to the site-specific method, as indicated by the relative degrees of labeling (Fh=3.2 and 6.2, respectively). Figure 6 shows the comparison of the dose-response curves expressed in RLUs and obtained with the two conjugation methods, respectively. Site-specific conjugations, although resulting in the incorporation of a single labeling molecule (Biotin and ABEI) onto the TSHR fusion protein and therefore in a lower total signal in reaction compared to the random conjugates (Figure 6), surprisingly provides a higher response than the non-specific random labeling. Moreover, the background signal (point zero of standard curve, in absence of target analyte) is lower for the site-specific conjugate compared to the random conjugates. A reduced background is also observed on negative samples (Table 1 below). As result, surprisingly, site-specific conjugation of TSHR fusion proteins according to the invention provides higher sensitivity than multiple random conjugation.

Table 1 Comparison of random (5-fold molar excess) vs site-specific (via Sortase)

Comparative analysis with the Immulite 2000 TSI assay by Siemens

A set of serum samples from 107 unselected blood donors and 30 TRAb positive serum samples were tested according to the first embodiment of the method of the invention. The results obtained were compared with the output of a commercial reference system, Immulite 2000 TSI Siemens. Serum samples were purchased from the following suppliers: Etablissement Frangais du Sang Centre- Atlantique, collection August 2017 (blood donors), Invent, Boca Biolistics and Cerba. Based on the results collected from negative and positive samples, the cut off value was fixed at 20,000 RLU (0.82 IU/L) and an agreement of 100% with the reference method was found, as reported in Table 2 below.

Table 2 Comparison of Embodiment 1 of the method of the invention versus Immulite 2000

TSI Siemens on 137 serum samples

IMMULITE

Neg Pos

-

107 30 137 w

7.2 Second Embodiment of the method of the invention

In a second embodiment, the method of the invention employed a pair of recombinant TSHR fusion proteins, mTSHR-msFc-Biotin (capture fusion protein) and mMBP-mTSHR (detection fusion protein), in a two-step reaction. During the first step, the anti-TSHR autoantibodies present in samples or standards formed a bridge between the TSHR ectodomains in the fusion proteins of the pair of the invention, and the immune complex thus formed was then captured by the streptavidin-coated paramagnetic particles (solid phase). The unbound material was removed with a wash cycle. During the second step, an anti-MBP monoclonal antibody, labeled with ABEI, was added, and bound the immune complex previously formed. The unbound reagent was then removed with a second wash cycle. Afterwards, the starter reagents were added and a flash chemiluminescence reaction was thus induced. The light signal, and hence the amount of isoluminol-antibody conjugate, was measured by a photomultiplier as relative light units (RLU) and was indicative of anti-TSHR autoantibodies concentration present in samples and standards. A schematic representation of the second embodiment of the method of the invention as above described is provided in Figure 2.

Assay protocol

A total of 50 pl of sample were incubated with 100 pl of assay diluent, 45 pl of mMBP- mTSHR and 50 pl of mTSHR-msFc-Biotin for 23 min, followed by addition of 20 pl of streptavidin-paramagnetic particles for 13 min. The unbound material was then removed with a washing cycle. Afterwards, 200 pl of the antibody tracer, mouse monoclonal antibody anti-MBP labeled with ABEI, were added and incubated for 13 min, followed by a second washing step. Finally, the starter reagents were added and the emitted light was measured.

Reagents and formulations

A, Solid phase

Streptavidin paramagnetic particles (Dynal M-280 - Ref 35137) were diluted at a final concentration of 0.375% (w/v) in 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na2HPO4*H2O, 0.24 g/L KH2PO4, 1 g/L BSA, 1 g/L NaN3 pH 7.4.

B, Assay diluent

Assay diluent was composed as follows: 1.63 g/L Na2HPO4, 1.16 g/L KH2PO4, 29.22 g/L NaCl, 0.366 g/L EDTANa₄ + lOg/L BSA frac V, 1.5 ml/L SDS, 4.5 g/L Tween 20, 0.25 g/L Kollidon 25 (PVP), 0.05 ml/L Anti foam 204, 0.10 g/L Gentamicin sulphate, 2 ml/L Proclin 300, 100 ml/L Sheep serum, pH 7.

C. Fusion protein mTSHR-msFc-Biotin

TSHR fusion proteins according to the invention having the amino acid sequence SEQ ID NO. 15, which comprise a mutant human TSHR extracellular domain of SEQ ID NO. 3 C- terminally fused to a mutant sheep monomeric Fc domain of SEQ ID NO.5, were site- specifically labeled with biotin molecules (mTSHR-msFc-Biotin via sortase A). The biotinylated fusion proteins thus obtained were diluted at a final concentration of 800 ng/ml in the antigen diluent.

D. Fusion protein mMBP-mTSHR

TSHR fusion proteins according to the invention having the amino acid sequence SEQ ID NO. 20, which comprise a mutant E. coli maltose binding protein (MBP) of SEQ ID NO.8 C-terminally linked to a mutant human TSHR extracellular domain of SEQ ID NO. 3 through a short peptide were diluted at a final concentration of 1066 ng/ml in a stabilizing dilution buffer (antigen diluent).

E, Antigens diluent

Antigen diluent was composed as follow: 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na2HPO4*H2O, 0.24 g/L KH2PO4, 1 g/L BSA, 0.5 g/L tween 20, 2.5 M TMAO (Sigma cod.92277), pH 7.4.

F, Tracer

Mouse monoclonal antibody anti -MBP was labeled with 10-fold molar excess of ABEI. The antibody was diluted at a final concentration of 200 ng/ml in 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na2HPO4*H2O, 0.24 g/L KH2PO4, 1 g/L BSA, 0.5 g/L tween 20 pH 7.4. G. Standard curve

A standard curve was prepared by diluting the commercial recombinant human antibody M22 (Creative Biolabs Cat.nr. PABW-172) in human negative universal serum (Golden West Diagnostic cat. UNI 000). Twelve serial dilutions were prepared with nominal concentrations ranging from 0 to 40 IU/L. The standard curve was standardized against the WHO 2nd International Std for Thyroid Stimulating Antibody, NIBSC (Code: 08/204). Figure 7 shows the dose-response curve, in RLUs, obtained with the method embodiment as above described (embodiment 2).

Assay results

Comparison of random vs site-specific conjugation

As outlined above, the capture mTSHR-msFc fusion protein was conjugated with biotin by employing two different techniques: (i) multiple random conjugation (mTSHR-msFc-Biotin 5X) and (ii) site-specific conjugation (mTSHR-msFc-Biotin via sortase A). Figure 7 shows the comparison of the dose-response curves expressed in RLUs and obtained with the two conjugation methods, respectively. Site-specific biotinylation provides a higher response than the non-specific random conjugation, thus conferring higher assay sensitivity. On the other side, detection was achieved by employing two different methods: (i) direct, multiple, random conjugation using 10-fold molar excess of ABEI (mMBP-mTSHR-ABEI lOx) over the detection fusion protein and (ii) indirect detection, through an anti-MBP antibody randomly labelled with ABEI. Figure 8 shows the comparison of the dose-response curves obtained with the two detection methods, respectively. Surprisingly, although the two methods had comparable total signal in reaction, the indirect detection, achieved through the addition of a labelled anti-MBP antibody, outperformed the direct one, obtained by direct labelling of the antigen. In particular, a lower background and much higher signals were observed on standard curve.

Comparative analysis with the Immulite 2000 TSI assay by Siemens A panel of serum samples from 115 unselected blood donors and 30 TRAb positive serum samples were tested according to the second embodiment of the method of the invention. The results obtained were compared with the output of a commercial reference system, Immulite 2000 TSI Siemens. Samples were purchased from the following suppliers: Etablissement Frangais du Sang Centre- Atlantique, collection August 2017 (blood donors), Invent, Boca Biolistics, Cerba. Based on data collected from negative and positive samples, a cut off value was fixed at 22,500 RLU (0.62 IU/L). As shown in Table 3 below, the second embodiment of the method of the invention shows 100% of negative agreement and 96.67% of positive agreement with the reference method.

Table 3 Comparison of Embodiment 2 of the method of the invention versus Immulite 2000 TSI Siemens on 145 serum samples

IMMULITE 115 30 145

20

8. Assay performance using a pair of wild-type TSHR fusion proteins vs a pair of mutant TSHR fusion proteins

The present inventors carried out a set of experiments employing the in vitro method of the invention for the detection of anti-TSHR autoantibodies in human serum samples, as outlined in Paragraph 7.1 of the Example section. Particularly, the one-step immunoassay protocol with three incubation times was performed using a pair of recombinant mTSHR- msFc fusion proteins, mTSHR-msFc-Biotin (capture fusion protein) and mTSHR-msFc- ABEI (detection fusion protein), both comprising a mutant human TSHR ectodomain according to the invention, or a pair of recombinant wtTSHR-msFc fusion proteins, wtTSHR-msFc-Biotin (capture fusion protein) and wtTSHR-msFc-ABEI (detection fusion protein), both comprising the wild type human TSHR ectodomain. In both experimental setups, Biotin and ABEI had been site-specifically conjugated to the TSHR fusion proteins, respectively.

The light signal, and hence the amount of mTSHR-msFc-ABEI or wtTSHR-msFc-ABEI conjugate, was measured by a photomultiplier as relative light units (RLU) and is indicative of autoantibodies to TSHR present in standard curve, samples or controls.

Assay protocol

A total of 100 pl of sample were incubated for 13 minutes with 100 pl of assay diluent (1^st incubation time); 50 pl of mTSHR-msFc-Biotin or wtTSHR-msFc-Biotin plus 50 pl of mTSHR-msFc-ABEI or wtTSHR-msFc-ABEI, respectively, were then added and incubated for 23 minutes (2^nd incubation time). Afterwards, 20 pl of streptavidin paramagnetic particles were added and incubated for 13 minutes to capture the immune complex (3^rd incubation time). The unbound material was then removed with a washing cycle. Finally, the starter reagents were added, and the emitted light was measured.

Reagents and formulations

A, Solid phase

Streptavidin paramagnetic particles (Dynal M-280 - Ref 35137) were diluted at a final concentration of 0.375% (w/v) in 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na₂HPO₄*H₂O, 0.24 g/L KH₂PO₄, 1 g/L BSA, 1 g/L NaN₃ pH 7.4.

B, Assay diluent

Assay diluent was composed as follows: 1.63 g/L Na2HPO4, 1.16 g/L KH2PO4, 29.22 g/L NaCl, 0.366 g/L EDTANa4 + lOg/L BSA frac V, 1.5 ml/L SDS, 4.5 g/L Tween 20, 0.25 g/L Kollidon 25 (PVP), 0.05 ml/L Anti foam 204, 0.10 g/L Gentamicin sulphate, 2 ml/L Proclin 300, 100 ml/L Sheep serum, pH 7.

C. Fusion proteins mTSHR-msFc-Biotin and mTSHR-msFc-ABEI TSHR fusion proteins according to the invention having the amino acid sequence SEQ ID NO. 15, which comprise a mutant human TSHR extracellular domain of SEQ ID NO. 3 C- terminally fused to a mutant sheep monomeric Fc domain of SEQ ID NO.5, were labeled with biotin by means of site-specific conjugation via Sortase A (mTSHR-msFc-Biotin via Sortase A). The same technique was employed to label the TSHR fusion proteins with ABEI (mTSHR-msFc-ABEI via Sortase A). The labeled TSHR fusion proteins thus obtained were diluted at a final concentration of 800 ng/ml in the antigen diluent.

D, Fusion proteins wtTSHR-msFc-Biotin and wtTSHR-msFc-ABEI

TSHR fusion proteins according to the invention having the amino acid sequence SEQ ID NO. 10, which comprise the wild type human TSHR extracellular domain of SEQ ID NO. 1 C-terminally fused to a mutant sheep monomeric Fc domain of SEQ ID NO.5, were labeled with biotin by means of site-specific conjugation via Sortase A (wtTSHR-msFc-Biotin via Sortase A). The same technique was employed to label the TSHR fusion proteins with ABEI (wtTSHR-msFc-ABEI via Sortase A). The labeled TSHR fusion proteins thus obtained were diluted at a final concentration of 800 ng/ml in the antigen diluent.

E, Antigens diluent

Antigen diluent was composed as follows: 8 g/L NaCl, 0.2 g/L KC1, 1.44 g/L Na2HPO4*H2O, 0.24 g/L KH2PO4, 1 g/L BSA, 0.5 g/L tween 20, pH 7.4.

F, Standard curve

A standard curve was prepared by diluting the commercial recombinant human antibody M22 (Creative Biolabs Cat.nr. PABW-172) in human negative universal serum (Golden West Diagnostic cat. UNI 000). Twelve serial dilutions were prepared with nominal concentrations ranging from 0 to 40 IU/L. The standard curve was standardized against the WHO 2nd International Std for Thyroid Stimulating Antibody, NIBSC (Code: 08/204). Figure 9 shows the dose-response curve, in RLUs, obtained with the method embodiment as above described, using the TSHR fusion protein pairs according to the invention, comprising mutant or wild type human TSHR ectodomains.

Assay results

As illustrated in Figure 9, similar dose-response curves expressed in RLUs were obtained with the method of the invention using the TSHR fusion protein pairs comprising the mutant or wild type human TSHR ectodomains. Moreover, a panel of 6 human serum samples positive for anti-TSHR autoantibodies was tested using the method of the invention employing either one of said TSHR fusion protein pairs. Of note, comparable luminescence signals were observed, suggesting that both the mutant and wild type human TSHR extracellular domains comprised in the fusion proteins of the pairs provide a reliable detection of anti-TSHR autoantibodies, as shown in Table 4 below and Figure 10A. Table 4 Comparison of anti-TSHR autoantibodies detection using TSHR fusion proteins comprising mutant vs wild type TSHR ectodomains, on positive sera. Remarkably, the same experiment performed on six anti-TSHR autoantibody-negative human serum samples highlighted two elevated signals obtained using the pair of capture and detection TSHR fusion proteins both comprising the wild type TSHR ectodomain. These samples were correctly detected as low employing the pair of TSHR fusion proteins both comprising a mutant TSHR ectodomain (Table 5 below).

Table 5 Comparison of anti-TSHR autoantibodies detection using a pair of TSHR fusion proteins both comprising a mutant TSHR ectodomain vs a pair of TSHR fusion proteins both comprising wild type TSHR ectodomain, on negative sera.

Collectively, these data show an improved assay consistency and precision with the method of the invention employing the pair of capture and detection TSHR fusion proteins both comprising a mutant TSHR ectodomain (mTSHR-msFc; SEQ ID NO. 15) because of the correct detection of negative samples thereby achieved (Fig. 10B).

Claims

1. A pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein, wherein said capture fusion protein and said detection fusion protein both comprise an extracellular domain of a human TSHR and a fusion partner selected from:

(i) a monomeric Fc domain of an immunoglobulin and

(ii) a bacterial maltose binding protein (MBP), wherein the TSHR extracellular domain consists of the amino acid sequence of SEQ ID NO. 1 or a variant thereof comprising up to five amino acid substitutions relative to the amino acid sequence of SEQ ID NO. 1, wherein the monomeric Fc domain is a non-human Fc domain or a fragment thereof, the monomeric Fc domain or the fragment thereof comprising up to fifteen amino acid substitutions relative to the wild type Fc domain, wherein said amino acid substitutions result in the Fc domain or fragment thereof not being capable of Fc domain dimerization, wherein the bacterial MBP consists of the amino acid sequence of SEQ ID NO. 2 or a variant thereof comprising up to fifteen amino acid substitutions relative to the amino acid sequence of SEQ ID NO. 2, wherein the human TSHR extracellular domain is linked to the fusion partner, optionally through a peptide linker.

2. The fusion protein pair according to claim 1, wherein the amino acid substitutions within the TSHR extracellular domain variant are at one or more amino acid positions selected from the group consisting of C4, C9, Y96, and any combination thereof, relative to the amino acid sequence of SEQ ID NO. 1.

3. The fusion protein pair according to claim 2, wherein the amino acid substitutions are selected from the group consisting of C4S, C9S, Y96S, and any combination thereof.

4. The fusion protein pair according to any of claims 1 to 3, wherein the monomeric non- human Fc domain is a monomeric ovine Fc domain.

5. The fusion protein pair according to claim 4, wherein the amino acid substitutions within the monomeric ovine Fc domain are at one or more amino acid positions selected from the group consisting of L121, T136, L138, T167, F177, Y179, R181 and any combination thereof, relative to a wild type ovine Fc domain of SEQ ID NO. 4.

6. The fusion protein pair according to claim 5, wherein the amino acid substitutions within the monomeric ovine Fc domain are selected from the group consisting of L121Y, L121S, L121K, T136Y, T136R, T136S, L138A, L138H, T167R, T167K, T167V, F177R, F177E, Y179M, Y179K, Y179A, R181A, R181Y, and any combination thereof

7. The fusion protein pair according to any of claims 1 to 6, wherein the amino acid substitutions within the bacterial MBP variant are at one or more amino acid positions selected from the group consisting of 12, D82, K83, E172, N173, K239, A312, 1317, E359, K362, D363, R367, and any combination thereof, relative to the amino acid sequence of SEQ ID NO. 2.

8. The fusion protein pair according to claim 7, wherein the amino acid substitutions within the bacterial MBP variant are selected from the group consisting of I2T, D82A, K83A, E172A, N173A, K239A, A312V, 1317V, E359A, K362A, D363A, R367N, and any combination thereof.

9. The fusion protein pair according to any of claims 1 to 8, wherein the TSHR extracellular domain consists of the amino acid sequence of SEQ ID NO. 3, the monomeric Fc domain consists of an amino acid sequence selected from SEQ ID NO. 5, 6 or 7, and/or the bacterial MBP consists of the amino acid sequence of SEQ ID NO. 8.

10. The fusion protein pair according to claim 9, wherein said capture fusion protein and/or said detection fusion protein comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs. 8-12 and 15-20.

11. An isolated nucleic acid sequence encoding a capture fusion protein or a detection fusion protein according to any of claims 1 to 10.

12. An expression vector comprising a nucleic acid sequence according to claim 11.

13. A host cell comprising an expression vector according to claim 12.

14. An in vitro method for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample, wherein the method employs a pair of anti- TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein as defined in any of claims 1 to 10 and comprises the steps of:

(i) contacting the biological fluid sample with said capture fusion protein, thereby obtaining binding of the anti-TSHR autoantibodies possibly present in the biological fluid sample to the capture fusion protein, and

(ii) detecting the anti-TSHR autoantibodies bound to the capture fusion protein by means of said detection fusion protein.

15. The method according to claim 14, wherein the capture fusion protein is immobilized on a solid support.

16. The method according to claim 15, wherein the capture fusion protein is conjugated to one or more capture moi eties, wherein the solid support is coated with one or more binding partners for binding said one or more capture moieties, the capture fusion protein being immobilized on the solid support through the binding of said one or more capture moieties with the one or more binding partners coated onto the solid support.

17. The method according to claim 16, wherein the capture fusion protein is conjugated to one single capture moiety.

18. The method according to claim 16 or 17, wherein the one or more capture moieties are selected from biotin and hapten molecules and/or wherein the one or more binding partners are selected from streptavidin, avidin and hapten-binding molecules.

19. The method according to claim 18, wherein the detection fusion protein is conjugated to one or more detectable labels, the one or more detectable labels being preferably selected from the group consisting of enzymatic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes, alkaline phosphatase (AP) labels, biotin labels, and any combination thereof.

20. The method according to claim 19, wherein the detection fusion protein is conjugated to one single detectable label.

21. The method according to any of claims 14 to 18, further comprising step (iii) of detecting the detection fusion protein by means of a labeled antibody capable of binding said detection fusion protein.

22. The method according to any of claims 14 to 21, which is a sandwich immunoassay.

23. The method according to any of claims 14 to 22, wherein the biological fluid sample is whole blood, plasma, serum or urine.

24. A kit for detecting autoantibodies to the thyroid stimulating hormone receptor (TSHR) in a biological fluid sample, the kit comprising a pair of anti-TSH receptor (TSHR) autoantibody-binding fusion proteins consisting of a capture fusion protein and a detection fusion protein as defined in any of claims 1 to 10.

25. The kit according to claim 24, wherein the detection fusion protein is conjugated to a detectable label.

26. The kit according to any of claims 24 to 25, further comprising a labeled antibody capable of binding the detection fusion protein.