Novel Antimurine Thyroid-Stimulating Hormone Receptor Monoclonal Antibodies

Abstract

Autoantibodies against thyroid proteins are present in several thyroid diseases. Thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor (GPCR) that binds to thyroid-stimulating hormone (TSH) and stimulates production of thyroxine (T4) and triiodothyronine (T3). When agonized by anti-TSHR autoantibodies, aberrant production of thyroid hormone can lead to Graves' Disease (GD). In Hashimoto's thyroiditis (HT), anti-TSHR autoantibodies target the thyroid for immune attack. To better understand the role of anti-TSHR antibodies in thyroid disease, we generated a set of rat antimouse (m)TSHR monoclonal antibodies with a range of affinities, blocking of TSH, and agonist activity. These antibodies could be used to investigate the etiology and therapy of thyroid disease in mouse models and as building blocks in protein therapeutics that target the thyroid for treatment in either HT or GD.

Introduction

Several thyroid diseases are characterized by the presence of autoantibodies against the thyroid. Hashimoto's thyroiditis (HT) is an autoimmune disease wherein the thyroid is slowly destroyed over the course of a patient's lifespan.¹ It is most often observed in women of ages 30 to 50 years, and ∼5 in 100 Americans are estimated to have the disease.^2,3 HT is characterized more commonly by antithyroid peroxidase or thyroglobulin antibodies, and less commonly by antithyroid-stimulating hormone receptor (TSHR) antibodies.⁴

TSHR is a G-protein-coupled receptor that binds to its ligand thyroid-stimulating hormone (TSH, also known as thyrotropin) (Fig. 1).⁵ The pituitary gland in the brain releases TSH in a negative feedback loop reliant on levels of triiodothyronine (T3) and thyroxine (T4) present in the blood.⁶ TSHR relays signals in response to TSH and initiates a G-protein-coupled signaling cascade with cyclic adenosine monophosphate formation that results in the production of T3 and T4 (Fig. 1).⁷

FIG. 1.

TSH—TSHR—T3 pathway. TRH stimulates the anterior pituitary gland to produce TSH, which then stimulates TSHR. TSHR is a GPCR, which when stimulated activates the Gα and AC. AC generates cAMP to activate the transcription factor CREB. CREB is critical for T3 production. After T3 and T4 are produced, they travel through the circulation and are transported into the nuclei of peripheral tissue cells where they bind to TRs (thyroid hormone receptors) with its heterodimeric partner the RXR. This complex increases the transcription of genes with a TRE in their promoter. AC, adenyl cyclase; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; Gα, alpha G subunit; GPCR, G-protein coupled receptor; RXR, retinoid X receptor; T3, triiodothyronine; T4, thyroxine; TRE, thyroid response element; TRH, thyrotropin releasing hormone; TSH, thyroid-stimulating hormone; TSHR, thyroid-stimulating hormone receptor.

In Graves's disease (GD), autoantibodies against TSHR agonize TSHR, leading to the aberrant production of thyroid hormone. GD is characterized by increased metabolism as a consequence of higher levels of thyroid hormone, resulting in a rapid heartbeat, tremors, and a goiter.⁸

TSHR undergoes abundant glycosylation with six glycosylation sites in the extracellular domain that are highly conserved, and constitute a significant portion, ∼30%–40% of the receptor's molecular weight.⁹ TSHR expression is primarily restricted to the thyroid; however, it has limited expression in other organs such as the testis.¹⁰ TSHR is expressed on the basolateral membrane of thyroid epithelial cells and, therefore, drugs administered through the bloodstream have access to TSHR on intact thyroid follicles. In HT, lymphocytes infiltrate the thyroid, resulting in destruction of thyroid epithelial cells and the thyroid follicles of which they are a part. Much of this destruction is attributable to cytotoxic CD8⁺ T cells.

This destruction of thyroid follicles results in reduced T3 and T4 production in the thyroid, triggering hypothyroidism in the patient. In some cases, TSH production is increased to maintain levels of T3 and T4. In this case, the patient has fewer symptoms of hypothyroidism as this additional TSH signaling increases thyroid hormone production, allowing homeostasis to be temporarily maintained. Most patients are treated with levothyroxine, a thyroid hormone substitute.¹¹ However, T4 levels may fluctuate, and eventually decrease with age as the thyroid continues to be destroyed by immune cells.

When the thyroid is first targeted by the immune system, it can release large amounts of T3 and T4 from the follicular lumen.¹² This may present as euthyroid or hyperthyroid initially in the patient; however, with the continued destruction of thyroid follicles, chronic hypothyroidism develops.

Although there are antihuman TSHR monoclonal and polyclonal antibodies available commercially, there are only polyclonal antibodies available for murine TSHR. Many of the antihuman TSHR mAbs such as KSAb1 and KSAb2^13,14 agonize TSHR. Some anti-mTSHR monoclonal antibodies were developed in the 1990s¹⁵ but are not commercially available. Antimouse TSHR antibodies are needed for developing mouse models where the thyroid is their target. For this reason, we developed a panel of rat antimouse TSHR monoclonal antibodies that can be used to study autoimmune thyroiditis and methods of drug delivery in thyroid cancer.

In addition, agonist antibodies against mouse TSHR could be used as a tool for studying GD.¹⁶ GD is characterized by the production of autoantibodies that stimulate TSHR, causing hyperthyroid symptoms in patients. Current treatments for GD such as radioactive iodine, antithyroid therapies, and surgery are all quite severe and can result in destruction or elimination of the thyroid gland. These antibodies could provide a path to studying alternative, less extreme, therapies for GD.

Materials and Methods

Cell lines and culture

300.19 cells are an Abelson leukemia virus transformed pre-B cell line from Swiss Webster mice. 300.19 cells were transfected with TSHR cDNA in the pEF-Puro expression vector and selected in medium with 5 μg/mL puromycin to generate 300.19-TSHR cells. These cells were cultured in R10 medium consisting of RPMI1640 medium (Life Technologies, Carlsbad, CA), 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamax, and 5 μg/mL puromycin. All cell lines tested negative for mycoplasma using the Venor GeM Mycoplasma Detection Kit (Sigma-Aldrich, St. Louis, MO).

Immunization of rats with TSHR

Female Lewis strain rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) were prepared for cDNA immunization by injecting 100 μL of 10 mM cardiotoxin (Sigma Chemical Company, St. Louis, MO) in 0.9% saline into the tibialis anterior muscle of each hind limb. Five days later, 100 μL of 1 mg/mL purified murine TSHR cDNA in the pEF-Puro mammalian expression vector in 0.9% saline was injected into each regenerating anterior tibialis anterior muscle of each rat. The cDNA immunization was repeated three times at 2- to 3-week intervals. Rats were then immunized with 1–5 × 10⁷ 293T-mTSHR transfectants four times at 2- to 5-week intervals.

Five days before fusion, the rat was immunized with both cDNA (200 μg) and cells (5 × 10⁷). Prebleed (before immunization) and postbleed rat sera were obtained and 300-mTSHR cells and untransfected 300.19 cells were stained to assess the antibody response against TSHR. Spleen cells from the immunized rat were fused with SP2/0 myeloma cells. Cells were distributed in ten 96-well plates in selective medium, and when hybridoma cells grew out, supernatants were assayed as described below, and highly positive clones were selected for further analysis and subcloned.

Identification of antimouse TSHR antibodies by flow cytometry screening

Hybridoma supernatants were incubated with 75,000 mouse TSHR transfected 300.19 cells, untransfected 300.19, or 300 cells transfected with an irrelevant gene (mPD-L2) in 96-well plates (3799; Corning) at 4°C for 30 minutes. After incubation, cells were washed twice in FACS wash buffer (PBS +2% FBS, 0.02% azide), and 10 μg/mL goat antirat IgG PE antibody was added. Cells were incubated for 30 minutes at 4°C, washed twice, and fixed in PBS with 2% neutral buffered formalin before being read on a BD Biosciences Fortessa X-20. All mAbs were of the rat IgG2b isotype except for 1B11 and 1G2, which were IgG2a. Antibodies were purified from culture supernatants by protein G affinity chromatography. All purified mAbs had endotoxin <2 EU/mg.

Antibody blockade of TSH binding to TSHR cells

TSH was conjugated to Alexa Fluor™ 647 using an Invitrogen AF647 Antibody Labeling Kit (No. A20186; ThermoFisher). Seventy-five thousand 300 cells expressing TSHR were added to wells of a round-bottomed 96-well plate (No. 3799; Corning), followed by 50 μL of each anti-TSHR antibody clone or ratIgG2a as an isotype control at 40 μg/mL. After incubating the plate for 30 minutes at 4°C, either the TSH-AF647 protein, the mIgG1-AF647 protein (negative control), or buffer alone was added to each well at 20 μg/mL. The plates were incubated for 30 minutes at 4°C, washed twice in FACS wash buffer, and then fixed in 2% neutral buffered formalin in PBS. Cells were then analyzed on a BD Biosciences Fortessa X-20.

Assay for agonist activity of anti-TSHR mAbs (T3 production)

Cohorts of five C57BL/6 mice were injected intravenously with either TSH (0.3 mg/kg), anti-TSHR antibody clone 1F8 (200 μg), or PBS. Fifty microliters of blood was taken at 4 and 24 hours. Blood was spun down in a microfuge tube and serum was analyzed for T3 content using ThermoFisher T3 Competitive ELISA Kit (No. EIAT3C; ThermoFisher).

Quantitation of antibody concentration

Antibody concentrations of antimouse TSHR antibodies in hybridoma supernatants were determined by indirect ELISA. ELISA plates (No. 3369; Costar) were coated with 2 μg/mL unlabeled goat antirat Ig in PBS overnight. The next day, plates were blocked for 1 hour with BSA. After the plates were blocked, hybridoma supernatants were added to individual wells, incubated at 37°C for 1 hour, and then washed three times. HRP-conjugated goat antirat IgG secondary antibody was added to the wells, incubated at 37°C for 1 hour, and then washed three times.

3,3′,5,5′-Tetramethylbenzidine substrate was added to develop the color and plates were analyzed using a SpectraMax 190. Purified rat IgG was used to construct a standard curve. The determined values were used to calculate the antibody concentration used in FACS analyses (1:10, 1:100, 1:1000, and 1:10,000).

Results and Discussion

We immunized rats with mouse TSHR cDNA and transfected cells and generated a series of 25 rat antimouse TSHR mAbs, defined by their reactivity with mouse TSHR-transfected cells and lack of reactivity with untransfected cells. The mAbs show a range of affinities and maximal mean fluorescence intensity (MFI) for mouse TSHR (Fig. 2).

FIG. 2.

Characterization of a panel of anti-mTSHR antibodies. 300.19 cells expressing murine TSHR were stained with a panel of antimurine TSHR mAbs as hybridoma supernatants followed by goat antirat IgG-PE. mAbs, monoclonal antibodies; MFI, mean fluorescence intensity.

We tested the set of anti-TSHR antibodies for blockade of TSH binding to TSHR (Fig. 3A). Cells expressing TSHR were incubated with antibody, followed by TSH conjugated to AlexaFluor 647. Clones including 1B11, 1E12, 1G2, 6G1, 9H2, 11E13, and 11H3 blocked TSH from binding to TSHR (Fig. 3B, C). Other antibodies such as 1F8, 3G12, 5F6, 8H10, and 11A3 did not block TSH from binding to TSHR (Fig. 3B, C). Blockers of TSH would be expected to alter thyroid metabolism.

FIG. 3.

Functional characterization of anti-TSHR antibodies. (A) Antibody that binds outside of the TSH binding site permits TSH binding to TSHR (left). Antibody that binds to the TSH binding site blocks TSH from binding to TSHR (right). (B) 300.19-TSHR cells incubated with the indicated antibodies, then stained with fluorescently labeled TSH-AF647. (C) 300.19-TSHR cells incubated with the indicated antibodies and then stained with fluorescently labeled TSH-AF647. (D) 300.19 cells expressing murine TSHR were stained with the three indicated purified antimurine TSHR mAbs followed by goat antirat IgG-PE. Purified rat IgG2b was used as an isotype control.

Antibodies that do not block TSH would be candidates for targeting the thyroid without affecting thyroid metabolism. These results provide a guide for the design of biologicals targeting the thyroid. It is important to ensure that therapies targeting the thyroid do not block TSH unless the goal is to reduce hyperthyroidism.

We prepared purified mAb from three nonblocking clones, 1F8, 3G12, and 8H10, and found they had similar high apparent affinities (0.58, 0.4, and 0.55 nM, respectively) but different maximal MFI (7460, 5026, and 9583, respectively) (Fig. 3D). Their high affinities and low blocking of TSH are ideal characteristics for future therapies targeting the thyroid but avoiding metabolic pathway disturbance. The sequences of the variable regions of these mAbs are given in Table 1.

Table 1.

Sequences of Variable Regions of Selected Antithyroid-Stimulating Hormone Receptor Antibodies

3G12:

Variable Heavy (VH):

EVQLQQSGPELQRPGASVKLSCKASGYTFTEYYMYWVKQRPKQGLELIGRIDPEDGNTDYVEKFKNKATLTADTSSNTAYMQLSSLTSEDTATYFCARSGRYNLYYWYFDFWGPGTMVTVSS

Variable Light (VL):

DIVLTQSPVLAVSLGQRATISCRASQSVSVSSINLMHWYQQKPGQQPKLLIYRASNLASGIPARFSGSGSGTDFTLTIDPVQADDIVAYYCQQTRESPWTFGGGTKLELK

1F8:

Variable Heavy (VH):

EVRLLESGGGLVQPGGSMRLSCAASGFTFTDFYMNWIRQPAGKAPEWLGFIRNRANGYTTEYNPSVKGRFTISRDNTQNMLYLQMNTLRAEDTATYYCARSYYYGRYIYYVMDAWGQGASVTVSS

Variable Light (VL):

DIVMTQGTLPNPVPSGESVSITCRSNKSLLHSDGKTYLNWYLQRPGQSPQFLIHWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGVYYCQQGLEFPLTFGSGTKLEIK

When selecting an anti-TSHR mAb for use in modeling therapeutic antibodies or thyroid drug delivery, it is also important to understand whether it agonizes TSHR and could potentially induce GD. We developed a method to detect mAb-mediated agonism of mTSHR by measuring the capacity to induce production of T3 in vivo since the only commercially available kits are for human TSHR. C57BL/6 mice were injected i.p. with purified antibody or with TSH as a positive control, and T3 levels in the circulation were measured at 4 and 24 hour.

TSH rapidly induced production of T3 at 4 hour that declined to homeostatic levels by 24 hour. The 1F8 antibody induced T3 production at 4 hour that was approximately half the level induced by TSH (Fig. 4). The T3 levels for the 1F8 antibody cohort remained elevated at the 24-hour mark, even after the T3 levels for the TSH-treated mice had subsided. This is likely due to the longer half-life of an antibody relative to the TSH protein. Owing to its agonism of TSHR, the 1F8 clone is likely not a good candidate for in vivo studies unless the goal was to model GD.

FIG. 4.

T3 production by C57BL/6 mice in response to intraperitoneal TSH injection. C57BL/6 mice were injected with PBS, TSH, or 1F8 antibody clone (five mice per group). Blood samples from 4 and 24 hours were assayed to quantify T3 levels.

Conclusions

In conclusion, we characterized a set of 25 mAbs against mouse TSHR to identify those that block and do not block TSH. The 3G12, 3C10, 5F6, 8H10, and 11A3 antibody clones do not block TSH, making them promising candidates for biological therapeutics that target the thyroid in mice to model treatments for GD or HT. 1F8 is not a good candidate due to its agonism of TSHR in vivo. Through the agonist assay that detected T3 production in response to antibody injection in vivo, we show that the antibodies can recognize physiological TSHR in living mice and have potential to provide selective access to the thyroid in future mouse studies.

Authors' Contributions

A.N.K. contributed to conceptualization (equal), methodology (equal), investigation (lead), formal analysis (lead), visualization, and writing—original draft (lead). J.A.T. was involved in conceptualization (supporting), methodology (supporting), writing—review and editing (equal), formal analysis (supporting), and writing—review and editing (equal). G.J.F. was in charge of conceptualization (equal), methodology (equal), supervision (lead), formal analysis (supporting), writing—review and editing (equal), resources (lead), and funding acquisition (lead).

Author Disclosure Statement

G.J.F. has patents/pending royalties on the PD-L1/PD-1 pathway from Roche, Merck MSD, Bristol-Myers-Squibb, Merck KGA, Boehringer-Ingelheim, AstraZeneca, Dako, Leica, Mayo Clinic, Eli Lilly, and Novartis. G.J.F. has served on advisory boards for Roche, Bristol-Myers-Squibb, Origimed, Triursus, iTeos, NextPoint, IgM, Jubilant, Trillium, GV20, IOME, and Geode. G.J.F. has equity in Nextpoint, Triursus, Xios, iTeos, IgM, Trillium, Invaria, GV20, and Geode.

Funding Information

This study was supported by grants from the National Institutes of Health P01 AI56299 to G.J.F.