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Design of a Long-Lived Thyrotropin Antagonist from Derivatives of Human Chorionic Gonadotropin^*

R. HOERMANN, S. M. AMIR, T. NOMURA and S. H. INGBAR

Charles A. Dana Research Institute, Harvard Thorndike Laboratory, and the Department of Medicine, Beth Israel Hospital and Harvard Medical School Boston, Massachusetts 02215

Address requests for reprints to: Dr. Sidney H. Ingbar, Harvard Medical School, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215

Abstract

Previous studies have revealed that desialylated forms of hCG have a high potency to inhibit both the binding of bovine TSH (bTSH) to human thyroid membranes and the bTSH-stimulated adenylate cyclase activity therein. The biological activities of these desialylated forms, however, are greatly reduced in vivo due to the high affinity of asialo-glycoproteins for hepatic receptors and their consequent rapid clearance from the circulation. Intact purified hCG (hCG_p), on the other hand, has little affinity for hepatic receptors and has a relatively long half-life in vivo, but displays only negligible inhibitory potency in the human thyroid. In the present studies, we have sought to obtain one or more compounds derived from hCG that are able to inhibit binding to TSH to the high affinity receptor in human thyroid membranes, and, consequently, could inhibit the stimulatory effect of TSH and Graves' immunoglobulin G and which also display a relatively low affinity for the hepatic asialoglycoprotein receptor and, as a consequence, have a reasonable survival in the circulation.

Two hCG forms isolated by sequential chromatography of crude hCG on DEAE-52 and Sephadex G-100 were of interest, since they displayed some degree of selectivity in binding to thyroid and liver receptors. The first form (hCG_v), whose isolation we described previously, was comprised of partially desialylated variant forms of intact hCG. The second material, as judged from its immunoreactivity, elution behavior on Bio-Gel P-100, and migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contained a fragment of the β-subunit (BF), similar to the -core fragment described by others. However, BF differed from the β-core fragment in having a higher sialic acid content. Interestingly, we found that BF as well as the enzymatically desialylated form of BF displayed a much lower affinity for mouse liver receptors than did asialo-hCG_p or the free asialo- β- and asialo--subunits. Further, the activity of BF to inhibit the binding of [125I]bTSH to human thyroid membranes exceeded that of the desialylated subunits of hCG_p as well as that of intact hCG_p, but was only exerted at the low affinity binding site and was not accompanied by inhibition of TSH-stimulated adenylate cyclase.

In an attempt to shift the locus of BF action from the low to the high affinity TSH receptor, we recombined BF with either an intact -subunit of hCG or an asialo-a-subunit. This led to the creation of two novel forms of hCG with properties of the type that we were seeking. First, they did inhibit the binding of [¹²⁵I]bTSH to human thyroid membranes, and unlike the effect of BF itself, this inhibition was now primarily directed at the high affinity class of TSH receptors. Second, the compounds proved to be inhibitory to the adenylate cyclase response in human thyroid membranes to both bTSH and Graves' immunoglobulin. Third, with respect to binding to liver membranes, the recombined a-BF was nearly as inactive as hCG_P) whereas asialo-a-BF had a much higher activity, though it was still only about 2% that of asialo-hCG_p. Fourth, the uptake of these compounds by the liver of the mouse after iv injection correlated well with the results of in vitro binding studies to liver membranes, and both new compounds did survive in the circulation of the mouse for a much longer period than did asialo-hCG_p. Thus, in the present studies we have designed two novel forms of hCG. Both possess a particular combination of properties that are those of relatively long-lived TSH antagonists. Because of these properties, the new compounds may offer some potential to block the TSH receptor in vivo, particularly in the study and treatment of Graves' disease (Endocrinology 124: 223–232, 1989)

Footnotes

^* This work was supported in part by Grant AM-18416 from the NIDDK, NIH (Bethesda, MD).

Supported by a fellowship from the Deutsche Forschungsgemeinschaft, West Germany.

Received August 29, 1988.