SAT-557 Functional Thyrotropin Receptor Antibodies Confirm a Novel Graves' Disease Animal Model

Background / Objectives: A novel long-term animal model for human Graves’ disease (GD) was recently established in mice (Endocrinology 2015; 156: 1577-1589) using nine immunizations with recombinant adenovirus (Ad) expressing the extracellular A-subunit of the human thyrotropin receptor (TSHR) over 10 months. The aim of the present study is to detect and measure TSHR-Ab in the animal model of Ad-TSHR immunized mice. Methods: Female BALB/c mice received either 10(10) plaque-forming units of adenovirus carrying the A-subunit of the TSHR gene or the green fluorescent protein (GFP) gene (injected into the femoral muscles). In addition, age-matched immunologically naïve mice were studied. Blood was withdrawn out of the left or right tail vein or intracardially (end of the study). The study protocol used three three-weekly immunizations (initiation), followed by a maintenance phase with further regular four-weekly boosts until the ninth immunization. Serum TSHR stimulating (TSAb) and -blocking (TBAb) Ab were measured with reporter bioassays. TSAb was reported as percentage of specimen-to-reference ratio (cut-off SRR% <140). Blocking activity was defined as percent inhibition of luciferase expression relative to induction with bovine TSH alone (% inhibition <34). Results: A total of twenty-two BALB/c mice were studied (ten, eight, and four Ad-TSHR, control, and Ad-GFP-immunized mice, respectively). All twelve control- and Ad-GFP-immunized mice were negative for both thyrotropin-binding inhibitory immunoglobulins (TBII) as well as for TSAb and TBAb measured with the cell-based bioassays. In contrast, baseline TBII were highly positive in all Ad-TSHR-immunized mice (median 40 IU/L, 25%-75% percentiles 40-40 IU/L, normal cut-off 0.55 IU/L) All these mice also developed hyperthyroidism with elevated free T4 (fT4) levels (median 4.5 ng/dl, 25-75 percentile 4.1-6 ng/dl; normal range 0.9-1.8 ng/dl). All control (0.1, 0.1-0.1 IU/L) and Ad-GFP-immunized (0.2, 0.1-0.3 IU/L) mice were TBII negative and the corresponding fT4 levels were 2.2 ng/dl (2.1-2.4 ng/dl) and 2 ng/dl (1.9-2.1 ng/dl), respectively (p<0.001 for both TBII and fT4 versus Ad-TSHR mice). The serum positivity for TSAb and TBAb in a total of 28 consecutive samples obtained from ten Ad-TSHR mice was 12 (43%) and 21 (75%), respectively. Especially, serum samples of two (20%) Ad-TSHR mice were highly positive in the TSAb bioassay only with a SRR% value of 397 (329-465, p<0.001 vs. control mice). In comparison, sera of four Ad-TSHR mice (40%) were highly positive when measured with the TBAb bioassay only showing a % inhibition value of 69 (63-76). Furthermore, serum samples of four Ad-TSHR mice (40%) demonstrated dual TSAb/TBAb positivity. Conclusions: Functional TSHR-Ab are highly prevalent in Ad-TSHR immunized mice, thus confirming the successful establishment of a novel, long-term animal model for GD.