Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-02-12
2001-05-29
Saoud, Christine J. (Department: 1647)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069400, C514S012200, C530S399000, C930S120000
Reexamination Certificate
active
06238915
ABSTRACT:
The present invention relates to a mutant human growth hormone protein having an amino acid sequence given in
FIG. 1
, to a deoxyribonucleotides showing a base sequence which encodes said amino acid sequence, to mutant human growth hormone proteins each having an amino acid sequence subjected to partial replacement, insertion or deletion to such an extent as may not cause loss of its characteristic features such as enhanced affinity for the growth hormone receptor is exhibited and decreased growth hormone activity, and to their uses in the manufacture of medicaments for the treatment of gigantism and acromegaly.
BACKGROUND OF THE INVENTION
The biologically inactive human growth hormones according to the present invention act as an antagonist of normal growth hormone for its receptor to thereby inhibit the disturbances and excessive growth caused by oversecretion, and can be utilized as a medicament with improved safety for the treatment of gigantism and acromegaly.
As the genetic disorders brought about by growth hormone, there are known growth retardation due to a deficiency of growth hormone as well as gigantism and acromegaly owing to excessive expression. For the growth hormone deficiency, supplementation therapy with growth hormone has been in wide use, but no effective drug has been developed so far for the treatment of gigantism and acromegaly.
In 1978, Kowarski et al. reported for the first time the discovery of a biologically inactive growth hormone (a mutant growth hormone) (Kowarski, A. A. et al., J. Clin. Endocrinol. Metab., 47: 461, 1978). However, understanding of the mutant growth hormone at the molecular level has not yet been elucidated up to now, although there was published a report that an abnormal polymer of growth hormone was identified in the blood from a child with dwarfism (Valena, L. J. et al., N. Engl. J. Med., 312:214, 1985). A child, who was found to contain a biologically inactive growth hormone in the circulatory blood, showed a high blood level of a mutant growth hormone but a low blood concentration of insulin-like growth factor (IGF-1), thereby causing retarded growth and development. However, such growth retardation is characterized by good response to normal growth hormone administered (Hayek A. et al., Pediatr. Res., 12: 413, 1973; Rudman, D. et al., N. Engl. J. Med., 305: 123, 1981; Plotnick, L. P. et al., Pediatrics 71: 324, 1983; Bright, G. M. et al., 71:576, 1983).
In recent years, progresses in protein engineering and genetic engineering have enabled structural research to be conducted on the binding of hormones to their receptors as well as the elicitation of their activities, and as a result, the causes for various genetic diseases have been clarified.
Cunningham et al. prepared a number of human growth hormone variants by using protein engineering procedures to conduct investigation on their binding sites for the growth hormone receptor, and as a result, identified the region being involved in the binding of growth hormone to the receptor, which constitutes a region consisting of the amino-terminal (2-19) amino acid residue, the carboxy-terminal (54-74) amino acid terminal and the carboxy-terminal (167-191) amino acid residue (Cunningham, B. C. et al., Science 243: 1330, 1989).
Furthermore, Uchida et al. prepared growth hormone variants having amino acid residues subjected to different replacements to thereby measure their differentiating activities for 3T3-F 442A cells, leading to the suggestion that the amino acid sequence 62 to 67 region is of critical importance to the development of biological activity (Uchida et al., Biochem. Biophys. Res. Commun., 172: 352, 1990).
Recently, a crystallographic study yielded a remarkable finding on the mode of binding of human growth hormone to its binding protein (a portion of the receptor protein) (De Vos A. M. et al., Science 255: 306, 1992); it is assumed that growth hormone binds consecutively to the growth hormone receptor in a manner where the domain 1 of growth hormone in the first place binds to the first growth hormone receptor and then the second domain 2 of growth hormone binds to the second growth hormone receptor, resulting in the formation of a dimer of the growth hormone receptor, whereupon signals of growth hormone are transmitted into cells.
Interesting among others is the fact that although the domain 1 of human growth hormone differs in amino acid residue from the domain 2, the binding sites of the receptor protein show the common amino acid residue. It was also recognized that growth hormone variants produced by protein engineering techniques binds competitively to the receptor (Fuh G. et al, Science, 256: 1677, 1992).
Recent progresses in gene analysis have made it feasible to identify the abnormal genes being contributed to a large number of genetic diseases. This is the case with the gene for growth hormone which brings about dwarfism, as well. Since growth hormone develops its physiological activity as mediated by the receptor on the cellular membrane, genetic abnormalities associated with growth hormone can roughly be divided into two groups, abnormality in receptor gene and the one in growth hormone itself.
Because growth hormone gene exists on the autosome, furthermore, its abnormalities are known to assume the form of recessive inheritance. In order to allow phenotypic expression of such abnormalities, consequently, it is required that abnormalities are brought about simultaneously in the alleles of the parent.
In the past, there have been reported many cases of growth retardation resulting from the complicated combination of mutations in the parent's growth hormone genes, such as whole depletion, partial depletion and base replacement. When either of the parent is normal, the mutant growth hormone is known to stay inside the intracellular secretory granules.
However, detailed investigation has not yet been conducted on the analysis at the molecular level of mutant growth hormones generated by missense mutation in the living body, as well as its role to be played in the living body. Neither known has been any effective method to suppress the overaction of growth hormone.
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→C) at codon 77 of growth hormone (FIG.
1
). The site of replacement is located in the second &agr;-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 b
Jacobson Price Holman & Stern PLLC
JCR Pharmaceuticals Co. Ltd.
Saoud Christine J.
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