Title:  Mutant human growth hormones and their uses

United States Patent:  6,238,915

Assignee:  JCR Pharmaceuticals Co., Ltd. (Hyogo, JP)

Filed:  February 12, 1997

Foreign Application Priority Data:  Feb 13, 1996[JP] (8-050940);  Jun 18, 1996[JP] (8-178643)

In accordance with the present invention, there are provided mutant human growth hormone proteins which exhibit enhanced affinity for growth hormone but lowered hormone activity, base sequences encoding the same and their production processes as well as uses of said proteins. The proteins according to the present invention, with their enhanced affinities for the growth hormone receptor, can inhibit the binding of growth hormone to its receptor, while they retain lowered growth hormone activities, thus finding application as a medicament for the treatment of acromegaly and gigantism.

SUMMARY OF THE INVENTION

The present inventor found that a 5-years old boy with dwarfism having a delayed bone age showed a high serum concentration of growth hormone and, in the induction test, retained a lowered level of IGF-1, though he exhibited an increased serum concentration of growth hormone, and this finding, followed by further subsequent research, culminated into the present invention.

It seemed likely that this endocrinological finding is consistent with the phenomena noted in the growth hormone insensitivity syndrome (Rosenbloom, A. L., Acta Pediatr. Scand. (Suppl), 383: 117, 1992).

However, consecutive administration of growth hormone brought about a significant improvement in growth of the patient, which excluded the possibility of diagnosing it as the Laron type syndrome, because Laron-type dwarfism is caused by the disorders of growth hormone receptors.

The present inventor, using the Nb2 bioassay method, discovered that the serum growth hormone found in the children suffering from this sort of disorders is an inactive type growth hormone, unlike the one secreted by normal children, and also identified the hormone as a mutant growth hormone by use of isoelectric focusing.

The mutant growth hormone was found to undergo replacement of the arginine residue with the cysteine residue (R.fwdarw.C) at codon 77 of growth hormone. The site of replacement is located in the second .alpha.-helix of growth hormone, behind a site 1 of binding to the receptor (Cunningham, B. C. et al., Science, 254:821, 1991). The substituted cysteine is assumed to form a new disulfide bond and cause the resultant molecule to change the charge, and this brings about conformational alterations, resulting in generation of a mutant growth hormone with reduced growth-hormone activity.

In the intracellular signal transduction of growth hormone, dimerization of the growth hormone receptors through ligand bonding and phosphorylation of the tyrosine residue in their proteins are considered crucially important (Argetsinger, L. S. et al., Cell, 74: 237, 1993: Silva, C. M. et al.: J. Biol. Chem., 269: 27532, 1994).

The growth-hormone binding protein is located in the extracellular domain and functions as a growth hormone reservoir in serum in vivo (Herington, A. C. et al., Acta Endocrinol. (Copenh), 124: 14, 1991).

The affinity of the mutant growth hormone for the growth-hormone binding receptor was found to be about 6 times greater than that of the wild-type one, suggesting that the domains 1 and 2 in the mutant growth hormone show different affinities for the receptor from those in the wild-type one. The biological characteristic of the mutant growth hormone lies in markedly lowered activity of cellular signal transduction developed through phosphorylation of the receptor, despite its greater affinity for the receptor protein.

Wild-type growth hormone, after administered to the patient consecutively for 3 days, did not give rise to conspicuous response to IGF-1, whereas it, when given to the patient over a prolonged period of time, acting as an antagonist to suppress the secretion of endogenous mutant growth hormone as well as its binding to the receptor, was found to be effective in increasing the plasma concentration of IGF-1 and in improving the growth and development.

Consequently, these findings led the present inventor to the conclusion that the mutant growth hormone, when administered to patients with gigantism or acromegaly caused by oversecreted growth hormone, may act as an antagonist to suppress their excessive growth.

The present invention has been completed on the basis of the above novel findings and relates to (1) a mutant human growth hormone protein, (2) a deoxyribonucleotides showing a base sequence which encodes said amino acid sequence, (3) mutant human growth hormone proteins each showing an amino acid sequence which has its amino acid residue moiety subjected to partial replacement, insertion or depletion to such an extent as may not cause loss of its characteristic features that enhanced affinity for the growth hormone receptor is exhibited and that decreased growth hormone activity is retained, and (4) uses thereof.

Claim 1 of 8 Claims

What is claimed:

1. A protein having an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

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