Title:  DNA encoding growth hormone antagonists

United States Patent:  6,787,336

Issued:  September 7, 2004

Inventors:  Kopchick; John J. (Athens, OH); Chen; Wen Y. (Athens, OH)

Assignee:  Ohio University/Edison Biotechnology Institute (Athens, OH)

Appl. No.:  488164

Filed:  June 7, 1995

Abstract

The present invention relates to DNA molecules which encode antagonists of vertebrate growth hormones obtained by mutation of at least the amino acid corresponding to Glu-119 in bovine growth hormone. The DNA molecules may be used to express the antagonists, either in cell culture, or in the cells of the patient of interest. The antagonist so expressed may be used to inhibit GH activity in a subject.

SUMMARY OF THE INVENTION

The present invention relates to proteins which are substantially homologous with a vertebrate GH but have growth-inhibitory activity.

We have discovered that mutation of Gly-119 in bGH to Arg ("G119R"), Pro ("G119P"), Lys ("G119K"), Trp ("G119W") or Leu ("G119L"), or the homologous Gly-120 in hGH to Arg ("G120R") or Trp ("G120W"), results in a mutein (mutant protein or peptide fragment thereof) which has growth-inhibitory activity in vertebrates, especially mammals. These novel hormones may be administered to mammals (or other vertebrates), in particular humans and bovines, when growth inhibition is desirable.

In one embodiment of the invention, the hormone is produced exogenously and administered to the subject. In view of the size of the hormone, it is preferably produced by expression in a suitable host of a gene coding for it. Such a gene is most readily prepared by site-specific mutagenesis of a bGH gene. However, the hormone may also be produced by other techniques, such as by condensation of fragments of native bGH with a synthetic peptide carrying the replacement of amino acid. If a peptide fragment has the desired growth-inhibitory activity, it may be prepared in toto by a Merrifield-type synthesis.

In a second embodiment of the invention, this gene is introduced into a prenatal form of a mammal by known techniques, and the prenatal form is developed into a transgenic mammal which expresses a reduced growth phenotype. Conceivably, a mammal could be genetically modified after birth, i.e., "gene therapy" and/or "gene/cell therapy".

Thus, growth-inhibited animals may be produced either by administration of the growth-inhibitory hormone of this invention in pharmaceutical form, or by genetic transformation of a prenatal or postnatal form of the animal.

The growth-inhibitory hormone, or the gene encoding it, is useful in the production of small animals for use in research facilities where space is restricted, as pets for pet lovers with limited quarters, and as livestock for farmers having small tracts.

The hormone may also be useful in the treatment of human gigantism, and in research on gigantism and dwarfism, in the treatment of diabetes and its sequelae, in the control of cholesterol, and in the prevention and treatment of certain cancers, particularly those whose growth is facilitated by GH or insulin-like growth factor-1 ("IGF-1").

In general, GH antagonists are therapeutically or prophylactically useful in countering the adverse effects of elevated levels of GHs, both endogenous hormones and hormones administered clinically.

In the course of our work, we have discovered a correlation between the ability of mouse L cells to secrete the protein and the protein having an effect (positive or negative) on growth rate in a transgenic animal. The use of an L cell secretion assay to identify growth-modulating proteins is also a part of this invention.

Another aspect of the invention is to provide methods for the treatment of various diseases involving the production of excess GH, wherein the methods comprise the step of administering an effective amount of a GH antagonist. Specifically, the invention provides methods of treating acromegaly, gigantism, cancer, diabetes, vascular eye diseases (diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, retinopathy of sickle-cell anemia, etc.) as well as nephropathy.

Another aspect of the invention is to provide pharmaceutical formulations for the treatment of diseases, wherein the formulation comprise at least one GH antagonist. The formulations may be adapted for the treatment of specific diseases and adapted for the administration to specific body sites.

More specifically, the present invention relates to a method for treatment of disorders or diseases which are wholly or partially regulated by GHRs using an antagonist to GHRs. One example of the antagonist used in the present invention are proteins which are substantially homologous with a vertebrate GH but have growth-inhibitory activity. However, any antagonist of GHRs can be used in the method of the present invention.

The disorders or diseases which can be treated by the method of the present invention are many. Any disorder or disease that is exacerbated by the action of an agonist on GHRs can be treated by the present invention.

In one embodiment of the present invention, the disorder is acromegaly or gigantism. Specifically, the invention includes methods for treating acromegaly and/or giantism by administering to patients having such disorders a therapeutically effective amount of a growth hormone antagonist together with a pharmaceutically acceptable carrier to reduce a pathological effect or symptom of acromegaly and or giantism and, in particular, to lower levels of IGF-1 in plasma and/or tissue. The pathological effects and symptoms of these disorders are discussed above.

In another embodiment, the disease is diabetes mellitus (DM). More specifically, the method of the present invention is used to prevent or reduce proliferative diseases associated with diabetes such as diabetic retinopathy and glomerulosclerosis in patients with DM. In particular, such methods include administering a growth hormone antagonist in a therapeutically effective amount to reduce a pathological effect or symptom of diabetes, such as nephropathy or retinopathy, and/or to lower blood glucose levels.

Additionally, GH is well known to possess anti-insulin or diabetogenic activities which involves the ability of GH to inhibit insulin's action on target tissue, especially muscle and fat. This diabetogenic activity may result in an increase in the dose of insulin taken by the type I or type II diabetic patient. A GH antagonist may be used in this scenario to inhibit GH's diabetogenic activity, thereby increasing a patient's sensitivity to insulin. Thus, treatment of a diabetic patient with a GH antagonist could ultimately decrease the patient's insulin requirement.

While GHs have not previously been implicated in hypercholesterolemia, in another embodiment, the method of the invention is used to lower serum cholesterol levels.

It is expected that the GH antagonists of the invention can reverse the anti-insulin effects observed in both type I and type II diabetes patients and have substantial clinical effects on diabetic control in patients. In type I diabetics, a rise in the serum glucose-levels in the early hours of the morning while the patient remains asleep (the "dawn phenomena") has been linked to a nocturnal rise in GH levels. Treatment with a GH antagonist is expected to abolish this effect, leading to more consistent control and lower fasting serum glucose levels. In type II diabetics, the principal cause of elevated fasting glucose levels is unrestrained hepatic glucose production ("HGP"). In normal, non-diabetic subjects insulin effectively suppresses HGP to modest levels throughout the night. Type II patients, with insulin resistance at the level of the liver as well as in peripheral tissue, have greatly increased HGP despite normal or higher than normal insulin levels. Given the effects of GH in raising HGP and intensifying the insulin resistance of type II diabetic patients, it is expected that antagonism of GH action with a GH antagonist will have a significant effect in lowering HGP, resulting in a decrease in fasting glucose levels.

In another embodiment, the method of the present invention is used to treat or prevent cancers, including but not limited to, lymphoblastic leukemia, melanoma, lymphoma, adenocarcinoma, colorectalcarcinoma and lung, breast, ovarian, pancreatic and prostate cancer. Specifically, the methods of the invention involve administering a growth hormone antagonist together with a pharmaceutically acceptable carrier to reduce tumor load and/or reduce a pathological effect or symptom of the cancer. Additionally, such methods decrease the need (frequency) for and improve the efficacy of radiation therapy and chemotherapy and generally improve the quality of life.

In other embodiments, the GH antagonists of the invention are used in methods for treating vascular eye diseases to reduce a pathological effect or symptom of the disease and/or prevent the development or retard the progression of neovascularization as found, for example, in diabetic retinopathy, retinopathy of prematurity, retinopathy of sickle-cell anemia and age-related macular degeneration.

In yet another embodiment, a GH antagonist is used to prevent restenosis after coronary balloon angioplasty.

In another embodiment, the present invention is used to counter the adverse effects of endogenous GH or clinically administered GH. Adverse effects of endogenous GH include but are not limited to symptoms associated with acromegaly, gigantism, and diabetes mellitus. These symptoms have been described in detail in the section on BACKGROUND OF THE INVENTION.

DETAILED DESCRIPTION OF THE PREFERRED EMODIMENTS

The present invention relates to GH antagonists, especially growth inhibitors, which are peptides or proteins having a similarity in sequence and secondary structure to vertebrate GHs, especially hGH and bGH.

The present invention also relates to methods of using such antagonists for treating diseases and disorders which are regulated wholly or partly by GHRS. Such diseases and disorders would include (a) those in which hGH action was excessive due to normal sensitivity of the tissues to increased levels of hGH (such as acromegaly and diabetes) and (b) those in which hGH action was excessive due to increased sensitivity of the tissues (as might result from increased GHR density) to normal levels of hGH (as in cancer and restenosis). However, other diseases and disorders, such as vascular eye diseases, might not be associated with either situation but might nonetheless be amenable to treatment with GH antagonists.

Preferably, the GH antagonist comprises an alpha helix having an amino acid sequence homology of a least about 50% with the third alpha helix of a vertebrate GH, especially bGH or hGH. Other alpha helices of the native hormone may be omitted if this can be done without loss of growth-inhibitory and/or other GH antagonist activity. The use of the term "antagonist" is in a functional sense and is not intended to limit the invention to compounds having a particular mechanism of action.

The overall percentage homology of bGH with other mammalian GHs is high: porcine (92%), ovine (99%), human (66%) and rat (87%). Insofar as the third alpha helix (amino acid sequence homologous to bGH 109-126) is concerned, the percentage homology is comparable to the overall figure:

porcine (94%), ovine (94%;), human (66%), and rat (94%).

The secondary structure of a polypeptide is a regular arrangement of a linear segment of the polypeptide chain. The most commonly encountered secondary structures are the beta-sheets and the alpha helices. See Schulz and Schimer, Principles of Protein Structure 69 (Springer-Verlag: 1979). The alpha helix is stabilized by hydrogen bonding between peptide amide and carbonyl groups of residues separated by a single turn of the helix. Secondary structure predictions are based on observation of the frequency of occurrence of the amino acid in a beta-sheet, alpha helix, etc., in a protein having a known three-dimensional structure.

The three-dimensional structure of porcine GH has been determined by X-ray diffraction and compared to that of other GHs. Abdel-Meguid et al., Proc. Natl. Acad. Sci. USA 84:6434 (1987). Like the other GHs thus studied, it is a single domain protein arranged as a four helix bundle with the helices in an antiparallel relationship. Its four helices are made up of residues 7-34, 75-87, 106-127 and 152-183. For X-ray studies of bGH and hGH, see Bell et al., J. Biol. Chem. 260:8520-25 (1985) and DeVos et al., Science 255:306-312 (1992). The three-dimensional structures of other GHs may be deduced by comparison of the sequences with due regard for the secondary structure tendencies of substituted amino acids. Detailed structural models of GH may, in conjunction with the information provided in this application, be used by the person of ordinary skill in the art to introduce one or more mutations into GH antagonists without interfering with ability of a given GH antagonist to serve as a GH antagonist.

bGH is 92% homologous at the amino acid sequence level with porcine GH, and bGH's structure has been deduced by study of the two sequences and of the structure of porcine GH. Its four alpha helices have been reported to be assumed by amino acids 4-33, 66-80, 108-127 and 150-179. The third alpha helix of bGH is defined as amino acids 106-129. However, it will be noted that the ends of this helix have a less marked alpha helical secondary structure than does the central region, which is 109-126. The exact bounds of the third alpha helix may differ for other GHs, depending on the alpha helical tendencies of "end" amino acids. The conformation is reasonably consistent with the predictions made by Chen and Sonenberg, Biochemistry 16:2110 (1977) using the method of Chou and Fasman, Biochemistry 13:222 (1974) (amino acids 10-34, 66-87, 111-127, 186-191).

The amino acid sequences of the GHs isolated from various vertebrate species are highly conserved. In a comparison of flounder GH with other GHs, including bGH, Watahiki et al., J. Biol. Chem. 264:312 (1989), which is incorporated herein by reference, identified five conserved regions. Watahiki's conserved region GD4 comprises the stretch LKDLEEGILALMRELED of bGH, i.e., residues 113 to 129. Watahiki's FIG. 3 identifies residues conserved among the GHs and residues predicted to be important for the manifestation of growth-promoting activity.

Studying Watahiki's GD4 consensus region, several families of GHs may be discerned. The first family (I) comprises cGH, pGH, oGH, bGH, and hGH. These begin with LKDLEEG. They then continue with IQA (cGH, rGH, pGH), ILA (oGH, bGH) or IQT (hGH). All members of family I then conclude GH4 with LMRELED (except for rGH, LMQELED, and hGH, LMGRLED). The second family (II) comprises fGH, yGH, tGH and sGH. These have the consensus sequence LS (E/D) LK (MIT) G(L/I) (L/G/H/N) (K/L) LI (E/T/R/I) (A/G) (N/S) QD.

Five amino acids in GD4 are conserved among all of the GHs noted by Watahiki: Leu-113, Leu-116, Gly-119, Leu-123 and Asp-129 (numbering according to the bGH sequence). Of the amino acids nearest Gly-119 on the face of the third alpha helix, Asp-115 is strongly conserved (replaced by Glu in the fish hormones); Leu-116 is invariant, Glu-118 is conserved among the mammals and birds, but replaced by Met, Thr or Val in fish; Ile-120 is almost invariant (replaced by Leu in fGH), and Ala-122 is well conserved, especially in mammals and birds (replaced by Thr in hGH and Leu or Lys in fish GHs). (It should be understood that the present invention is not limited to mutants in which these conservations are maintained).

It has been shown that a recombinant molecule containing a hGH (1-134) fragment linked to a human placental lactogen (141-191) fragment retained full hGH immunological activity and binding affinity to GH receptors isolated from rabbit liver. Russell et al., J. Biol. Chem. 256:296-300 (1981). By using the homolog-scanning mutagenesis technique, gene fragments of homologous hormones, i.e., human placental lactogen or human prolactin, were systematically substituted throughout the hGH gene, thus producing various chimeric hormones. Cunningham et al., Science 243:1330-36 (1989).

The dissociation constants (K_D) were calculated from Scatchard analysis of competitive binding of 125 labeled hGH to the cloned and purified soluble hGH receptor at 25^oC. (23). Standard errors in these measurements were generally at or below +20 percent. Mutants are named on the basis of the first and last residue mutated in the region. The specific mutations introduced are described by a nomenclature where the wild-type hGH residue is given first followed by its sequence position and then the mutant residue. The single letter code designates each amino acid. A comparison of the binding affinities of these mutant GHs and wild-type hGH to a cloned liver human GHR led to the conclusion that there were three discontinuous polypeptide determinants in hGH involved in receptor binding. They were located at the amino-terminus, carboxy-terminus, and within a loop between amino acid residues 54 and 74. These putative binding domains were further analyzed by an alanine-scanning mutagenesis technique in which alanine residues were systematically substituted throughout those regions. Amino acid residues at positions 10, 58, 64, 68, 172, 174, 175 and 176 of hGH were shown to be important for GH receptor binding. However, none of the mutant GHs were reported to inhibit growth. Cunningham et al., Science 244:1081-85 (1989).

The complete results are given in the table below:

Table B: Binding of Alanine Substitution (Residues 2-19, 54-74, and 167-191) Mutants of hGH to the soluble hGH receptor.

The present invention is not limited to the mutation of the third alpha helix of bGH or hGH. Rather, it encompasses the mutation of the third alpha helix of any mammalian or other vertebrate GH, including, but not limited to, the GHs whose sequences are given in Watahiki (1989): flounder, yellowtail, tuna, salmon, chicken, rat, porcine, ovine, bovine and human GHs. Expression of mutants of other GHs is facilitated by the availability of genes encoding the latter. See, e.g., Goeddel et al., Nature 281:544-548 (1979). In addition, the present invention is not limited to muteins of mammalian or vertebrate GHs comprising mutations only within the third alpha helix, but also encompasses muteins having mutations outside the third alpha helix in addition to those mutations within the third alpha helix such that the muteins exhibit GH antagonist activity.

The concept of a polypeptide which is substantially homologous to bGH is deemed to include (but is not limited to) any polypeptide which differs from bGH or hGH by (a) a substitution (or deletion) at an amino acid corresponding to amino acids 115 to 119 of bGH, (b) a substitution (or deletion) at an amino acid corresponding to an amino acid of bGH or hGH which is not conserved among the vertebrate GHs, especially the replacement of that amino acid by one found at the site in a different GH, and/or (c) truncation of amino acids 1-95 and/or 134-191. (Conserved amino acids are identified in Watahiki et al., 1979). Thus, all non-bovine vertebrate GHs are "substantially homologous" with bGH and/or hGH. Preferably, the polypeptide is a least about 50% homologous, more preferably at least 80% homologous, with bGH or hGH in the subsequence substantially corresponding to the third alpha helix (approximately, residues 106-129) of bGH, and more preferably over the entire length of the polypeptide (ignoring extraneous non-bGH-related fusions to the amino-terminus or carboxy-terminus).

The compound is considered to be growth-inhibitory if the growth of test animals of at least one vertebrate species which are treated with the compound (or which have been genetically engineered to express it themselves) is significantly (at a 0.95 confidence level) depressed with respect to the growth of control animals (the term "significant" being used in its statistical sense). Preferably, it is growth-inhibitory in a plurality of species, or at least in humans and/or bovines. GHs have considerable interspecies cross-reactivity. Gill et al., biotechnology 3:643 (1985) reported that recombinant chicken and bovine GHs accelerate growth in juvenile pacific salmon.

It is known that certain fragments of GHs also have growth-promoting activity, and it is expected that the growth-inhibitory peptides (the term "peptides" is used herein to include proteins) of the present invention need not be as large as bGH. Preferably, the peptides are at least 11 amino acids long (three turns of an alpha helix) and more preferably at least 50 amino acids long. These peptides may retain the growth-inhibiting action of, e.g., bGH (G119R), yet lack other, undesirable biological activities of the native size mutant. They may also have more desirable pharmacokinetic characteristics.

The growth-inhibitory peptides of the present invention may also be larger than bGH, provided that the additional amino acids do not result in the compound being unable to reduce the growth rate of a vertebrate.

While the mechanism of action of the growth-inhibitory peptides is not known, it is believed that they function as antagonists to wild-type GHs endogenously produced by the target animal. We have shown that, e.g., bGH (G119R) and bGH (G119R,E117L,A122D), both competitively inhibit the binding of wild-type bGH to liver membrane preparations. Thus, it is believed that the compound has a net result of inhibiting growth because its growth-promoting activity is substantially less than that of wild-type GHs (and perhaps is negligible), yet it can displace from GHR sites the endogenous native GH (whose stimulation of growth would have been more pronounced). However, applicants are not bound by this theory.

DeVos et al., Science 255:306 (1992) examined the complex of hGH and the extracellular domain of its receptor by X-ray diffraction. The first receptor-binding region of hGH is concave and is formed mainly by residues on exposed faces of helix 4, but also by exposed residues of helix 1 and residues in the region connecting helices 1 and 2. The second receptor-binding region comprises the exposed sides of helices 1 and 3 and is relatively flat. The role of helix 3 is shown best in DeVos' FIG. 5; there is a significant decrease in solvent accessibility around hGH E119 upon complex formation. The complex had the form hGH-(hGHR)₂ ; that is, the receptor dimerizes to interact with hGH. It is possible that our GH antagonists interfere with this dimerization.

Preferably, the compounds of the present invention have an ED50 which is less than about 10 times the ED50 of wild-type bGH in an assay of the ability of the compound to displace radiolabeled wild-type bGH from a liver membrane preparation made as described below. More preferably, the compounds have an ED50 at least comparable to that of wild-type bGH. Most preferably, the compounds have a higher affinity for GHRs than does the GH native to the animal receiving the compound. For purification and characterization of a human GHR, see Leung et al., Nature 330:537-43 (1987).

A GH mutein may be considered an antagonist, even if it lacks growth-inhibitory activity, if it antagonizes another GH-mediated activity, e.g., diabetogenic, glomerulosclerotic, hypercholesterolemic, tumorigenic or retinopathic activities.

The preferred growth-inhibitory peptides are characterized by a modification of the surface topography of the third alpha helix. It will be seen from FIG. 3 that in the third alpha helix of "wild-type" bGH, there is a surface cleft or depression beginning at the Asp-115, deepening at the Gly-119, and ending with the Ala-122. All of the mutants prepared so far, both those which retain the wild-type growth-promoting activity and those which do not, are consistent with the theory that growth-promoting activity requires the presence of this cleft or depression and that, if the center of this cleft is "filled in" by substitution of amino acids with bulkier side chains, the mutein inhibits the growth of the subject.

Mutations which substantially destabilize the alpha helix are undesirable since they may result in the loss of all growth-related activity. we have observed such loss in the case of several mutations which were expected to disrupt the alpha helix.

For a discussion of alpha helix formers and breakers, see Chou and Fasman, supra. Glu, Ala and Leu are the preferred alpha helix formers while Pro and Gly are characterized as strong helix breakers. Substitutions which introduce strong alpha helix breakers are less desirable, but may be tolerated in a particular case, such as at the end of the helix. The secondary structures of our analogues have been predicted using the "Micro Genie" computer program, which uses the algorithm of Ganier et al., J. Biol. Chem. 120:97-120 (1978).

With respect to amino acid 119, Gly is both the smallest amino acid residue and the one least favorable to alpha helix formation. Thus, it is believed that any other amino acid may be substituted for it without destabilizing the alpha helix, while at the same time filling in the aforementioned cleft. Alternatively, the replacement amino acid may be chemically modified to have a chemical moiety that effectively "fills" the cleft, e.g., an amino acid such as lysine that has been chemically substituted with polyethylene glycol (see the discussion below). All of the G119 bGH substitutions tested resulted in a "small animal" phenotype. These substitutions were Arg (a large, aromatic amino acid), Pro (a cyclic aliphatic amino acid), Lys (a large, positively charged amino acid), Trp (a large aromatic amino acid) and Leu (a large, nonpolar, aliphatic amino acid). In hGH, the homologous Gly is at position 120. Substitution of Arg or Trp resulted in an antagonist, however, hGH G120A retained growth-promoting activity. Consequently, it is presently believed that this Gly, which is conserved in all vertebrate GHs, may be replaced by any amino acid other than Ala (the second smallest amino acid), and more preferably by any amino acid which is at least as large as Pro (the smallest replacement amino acid known to result in a "small" animal phenotype). The deletion of bGH Gly-119 and hGH Gly-120 is also known to result in a "small" animal phenotype and would be a GH antagonist.

Modification of position 115 is suggested by our "cleft" theory. The Asp at position 115 may be replaced by a bulkier amino acid which does not destroy the alpha helix. Preferably, the replacement amino acid has a size greater than that of Asp. The amino acids His, Met, Ile, Leu, Lys, Arg, Phe, Tyr and Trp are substantially larger than Asp. Of these, His, Met, Leu and Trp are more preferred because they combine the advantages of bulk with a reasonably strong alpha-helical propensity. Note, however, that Glu is the strongest alpha helix former of all of the amino acids. The D115A mutant of bGH is not a GH antagonist, but Ala is smaller than Asp, so this is not probative of the value of replacing Asp-115 with a bulkier amino acid.

It is possible that G119A might lead to a "small" phenotype if coupled with other mutations, e.g., at positions 115 and 112.

It is possible to systemically screen for the effect of all possible amino acid substitutions at positions 115 and 119. (There are 20² -1 or 399 combinatorial possibilities). DNA which encodes bGH and is degenerate at these positions, so as to encode all possible amino acids, or only those with acceptable alpha-helical propensities, is prepared, e.g., by a "dirty bottle" synthesis. Phage are prepared, as taught by Ladner et al., PCT/US89/03731, WO90/02809, which display the mutant bGHs as a domain of a chimeric coat protein. The phage are incubated with a chromatographic support bearing a GHR. (For the techniques of isolating GHRs, see Leung et al., Nature 330:537 (1987) and Spencer et al., J. Biol. Chem. 263:7862 (1988).) Native bGH is also incubated with the support, before, during or after the phage incubation. Bound phage are recovered, amplified and examined to determine the sequence of the mutant bGH (usually by sequencing the corresponding gene in the phage genome). These mutants have demonstrated the ability to compete with wild-type bGH for a GHR. Their ability to antagonize GH activity in vivo is then confirmed by, e.g., administering them directly to an animal or by preparing a suitable transgenic animal, or by the in vitro assay described in Example 7. This approach may be extended, if desired, to other amino acid positions in the third alpha helix. Amino acids which are particularly preferred for screening are the six amino acids spatially nearest bGH's Gly-119, that is, Ala-122, Leu-123, Ile-120, Leu-116, Asp-115 and Glu-118. It should be noted that Bass et al., Proteins: Structure, Function and Genetics, 8:309-314 (1990) prepared "hormone phage" which express and display hGH-geneIII fusion proteins and which were bound by anti-hGH monoclonal antibodies. Moreover, it was possible to separate phage bearing wild-type hGH from phage bearing the low affinity hGH mutant R64A by means of affinity chromatography (using the extracellular domain of the hGH receptor bound to nonporous oxirane beads).

Besides the mutations at position 119, which is deemed necessary to impart the desired growth-inhibitory activity, additional mutations are possible which will leave the growth-inhibitory activity or other antagonist activity intact. These mutations may take the form of single or multiple substitutions, deletions, or insertions, in nonessential regions of the polypeptide. For example, it is possible to alter another amino acid in the alpha helix provided that the substitution does not destroy the alpha helix. Preferably, such alterations replace an amino acid with one of similar size and polarity. It may be advantageous to modify amino acids flanking the primary mutation site 119 in order to increase the alpha-helical propensities of the sequence, particularly if the mutation at position 119 is one expected to destabilize the helix.

The following table may be helpful in identifying candidate mutants:

In addition to any mutations in the third alpha helix that are deemed necessary to impart the desired growth-inhibitory activity, additional mutations are possible outside of the third alpha helix that will leave the growth-inhibitory activity or other antagonist activity intact. These additional mutations may take the form of single or multiple substitutions, deletions, or insertions, in regions of the polypeptide outside of the third alpha helix. For example, the invention encompasses a polypeptide having the amino acid sequence of a vertebrate GH in which an amino acid corresponding to Gly-119 of bGH is deleted or substituted with an amino acid, and in which at least one other position outside the third alpha helix of the polypeptide is deleted or substituted with an amino acid, as long as the resulting polypeptide retains GH antagonist activity. The mutation outside the third alpha helix may be anywhere that does not result in loss of GH antagonist activity. Preferably, such mutation outside the third alpha helix should be at a position corresponding to a non-conserved amino acid of a vertebrate GH. However, as described below, mutations at conserved amino acid positions, if carefully chosen, may be tolerated. More preferably, the mutation at a non-conserved amino acid position outside the third alpha helix should be at a position corresponding to a non-essential region of a vertebrate GH.

Several of the cited references provide guidance as to where and where not the polypeptide will tolerate mutagenesis. Watahiki et al. (1989) compared the sequence of flounder, yellowtail, tuna, salmon, chicken, rat, porcine, ovine, bovine and human GHs. They identified five conserved domains which were labeled GD1-GD5. Mutations in these conserved domains are more likely to affect activity; GD4 corresponds to the third alpha helix of bGH. In mutating a known GH antagonist with the desire to retain inhibitory activity, mutations outside the conserved domains are more prudent. However, mutations in these conserved regions, if carefully chosen, may be tolerated; for example, the mutation E117L does not modify the activity of either wild-type bGH or a bGH G119R mutant. Note that not only substitutions, but also insertions and deletions, are suggested by the example of the cognate hormones.

Abdel-Meguid et al. (1987) determined the three-dimensional structure of recombinant methionyl porcine GH, and suggested that it revealed the "general three-dimensional fold" of the GHs. The three-dimensional structure can be used to identify interior and surface residues; generally speaking, proteins mutated at surface residues (other than the receptor binding site) are more likely to remain functional. However, Creighton and Chothia, Nature 339:14 (1989) discuss the toleration of mutations at buried residues. The structure may also be used to determine flexible surface "loops"; proteins are more tolerant of deletions and insertions in such regions.

Cunningham et al. (1989) used homolog-scanning mutagenesis to identify the epitopes of hGH for its cloned liver receptor. Only variant hormones having mutations in regions C(54-74), F(164-190), and, to a lesser extent, A(11-33), exhibited reduced binding affinity. Cunningham and Wells, Science 244:1081 (1989) used a related technique, alanine-scanning mutagenesis, to further study these regions. Note, however, that binding to the receptor utilized by Cunningham is not necessarily critical to the growth-promoting or growth-inhibitory activity of the mutant.

For example, it seems likely that major amino-terminal and carboxy-terminal truncations can be made without adverse effects on growth-inhibitory activity, since the 96-133 fragment of bGH (K112L) is understood to retain bioactivity. Truncations may be generated by gene modifications or by peptidase treatment.

In terms of the kinds of substitutions which may be made, one may look first to analyses of the frequencies of amino acid changes between homologous proteins of different organisms, such as those presented in Table 1-2 of Schulz and Schimer, supra and FIGS. 3-9 of Creighton, supra. Based on such analyses, we define conservative substitutions as exchanges within the groups set forth below:

I small aliphatic, nonpolar or slightly polar residues--Ala, Ser, Thr, (Pro), (Gly)

II negatively charged residues and their amides--Asn, Asp, Glu, Gln

III positively charged residues--His, Arg, Lys

IV large aliphatic nonpolar residues--Met, Ile, Leu, Val, (Cys)

V large aromatic residues--Phe, Tyr, Trp

Three residues are parenthesized because of their special roles in protein architecture. Gly is the only residue without a side chain and therefore imparts flexibility to the chain. Pro has an unusual geometry which tightly constrains the chain. Cys can participate in disulfide bonds which hold proteins into a particular folding; the four Cysteines of bGH are highly conserved. Note that Schulz and Schimer would merge I and II above. Note also that Tyr, because of its hydrogen bonding potential, has some kinship with Ser, Thr, etc.

Within the GH family itself, we see a wide variety of substitutions of other amino acids for the residues of bGH. For example, among the vertebrate GHs set forth in Watahiki et al., (1989), Pro appears 6 times in bGH. The first Pro is not substituted, but is absent from sGH. The second is replaced by Leu in fGH; Thr in yGH and tGH. The third is replaced by Leu in fGH, Ile in yGH and tGH, Val in sGH. The fourth Pro is conserved. The fifth Pro is replaced by Phe in fGH; Ser in yGH. The Sixth Pro is replaced by Phe in fGH, yGH and tGH, and Leu in sGH. Overall, Pro is replaced 4 times by Phe, 3 times by Leu, 2 times each by Thr and Ile, and once each by Val and Ser. When this analysis is extended to all amino acids of bGH, we obtain the following tallies:

Ala (14; 2 conserved).fwdarw.

Thr (14), Ser (11), Asp (7), Ile (4), Glu (4), Gly (4),

Val (3), Gln (3), Leu (3), Lys (2), Phe (1), Asn (1)

Asp (10; 2 conserved).fwdarw.

Tyr (6), Asn (4), Val (4), Glu (3), Lys (2), Arg (2),

Gly (2), Thr (1), Ser (1), Phe (1), Ala (1)

Glu (13; 2 conserved).fwdarw.

Asi (14), Lys (10), Gln (4), Ala (3), Pro (2), Thr (2),

Asn (1), Ser (1), Val (1), Met (1), Arg (1), Gly (1)

Phe (13; 3 conserved).fwdarw.

Tyr (7), Leu (6), Asn (6), Ser (5), Gln (3), Ile (2),

Gly (2), His (1), Thr (1), Val (1)

Gly (10; 1 conserved).fwdarw.

Arg (9), Glu (8), Asp (7), Val (4), Pro (4), Ser (4),

Asn (3), Phe (3), Asp (2), His (1), Thr (1), Tyr (1),

Ala (1)

His (3; 1 conserved).fwdarw.

Ara (1), Asn (1), Tyr (1), Asp (1)

Ile (7).fwdarw.

Gln (7), Asn (5), Leu (4), Phe (4), Val (4), Ala (2),

Ser (1), Arg (1)

Lys (12; 2 conserved).fwdarw.

Ser (11), Ara (7), Gly (4), Gln (2), Leu (2), Asn (1)

Leu (27; 11 conserved).fwdarw.

Ser (11), Val (9), Asn (7), Met (7), Gln (7), Arg (4),

Glu (4), Phe (3), Tyr (3), Gly (1), Pro (1), His (1)

Met (4).fwdarw.

Ile (7), Ala (4), Thr (3), Ser (3), Leu (1), Asn (1),

Val (1)

Asn (6).fwdarw.

Ile (8), His (4), Asp (3), Gln (3), Glu (2), Ser (1)

Pro (6; 1 conserved).fwdarw.

Phe (4); Leu (3), Thr (2), Ile (2), Val (1), Ser (1)

Gln (11; 1 conserved).fwdarw.

Leu (13), Arg (6), Lys (5), Ser (4), Glu (4), His (1),

Gly (1), Asp (1), Pro (1)

Arg (13; 1 conserved).fwdarw.

Lys (11), Ser (9), Thr (7), Ile (3), Glu (2), Gly (2),

Asn (2), His (2), Val (1), Gln (1), Asp (1), Ala (1)

Ser (13; 3 conserved).fwdarw.

Ala (8), Asn (8), Gln (4), Leu (4), Gly (3), Glu (2)

Asp (2), Thr (2), Arg (1), Val (1)

Thr (12; 1 conserved).fwdarw.

Ser (14), Ala (13), Val (7), Tyr (5), Phe (4), Ile (4),

Met (3), Leu (3), Pro (2), Asn (2), Gly (1)

Val (6).fwdarw.

Ala (4), Ser (4), Ile (3), Thr (2), Gln (6), Gly (2),

Met (2), Leu (1), Lys (1)

Tyr (6).fwdarw.

Leu (5), Pro (4), Gln (3), Phe (2), Glu (1), Ser (1)

Note that the above figures are not normalized to adjust for the relative frequencies of occurrence of the various amino acids. We further note that in our own mutagenesis experiments, changing Lys-112 to Leu or Lys-114 to Trp (M1), Glu to Gly (E126G) or Leu (M4), or Ala to Thr (A122T) did not alter activity, while changing Lys, Glu or Leu to Pro abolished activity.

The subject invention also provides for various GH antagonist conjugates. The GH antagonists conjugates of the invention comprise a GH antagonist described herein (and in U.S. Pat. No. 5,350,836) covalently linked one or more water soluble polymers. Water soluble polymers, especially polyethylene glycol, have been conjugated to proteins so as to provide additional desirable properties while retaining, at least in part, the GH antagonist properties of the GH antagonist. These desirable properties include increased solubility in aqueous solutions, increased stability in storage, reduced immunogenicity, increased resistance to proteolytic degradation, and increased in vivo half-life. Water soluble polymers suitable for use in the subject GH antagonists include polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, polyoxethylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, and .alpha.,.beta.-poly[(2-hydroxyethyl)-DL-aspartamide]. Polyethylene glycol is particularly preferred. Methods of making water-soluble polymer conjugates of proteins are described in, among other places, U.S. Pat. Nos. 4,179,337; 4,609,546; 4,261,973; 4,055,635; 3,960,830; 4,415,665; 4,412,989; 4,002,531; 4,414,147; 3,788,948; 4,732,863; 4,745,180; EP No. 152,847; EP No. 98,110 (published Jan. 11, 1984); JP No. 5,792,435.

Another aspect of the invention is formulations that provide for the sustained release of GH antagonist. Examples of such sustained release formulations include composites of biocompatible polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including, A. Domb et al., Polymers for Advanced Technologies 3:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds.), "Biodegradable Polymers as Drug Delivery Systems," Vol. 45 of "Drugs and the Pharmaceutical Sciences," M. Dekker, New York, 1990. Liposomes may also be used to provide for the sustained release of GH antagonists. Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places, U.S. Pat. Nos. 4,944,948; 5,008,050; 4,921,706; 4,927,637; 4,452,747; 4,016,100; 4,311,712; 4,370,349; 4,372,949; 4,529,561; 5,009,956; 4,725,442; 4,737,323; 4,920,016. Sustained release formulations are of particular interest when it is desirable to provide a high local concentration of GH antagonist, e.g., in an eye chamber for diabetic or proliferative retinopathy, near a tumor, etc.

The present invention is not limited to any particular method of producing the desired GH antagonists. Preferably, these antagonists are produced by first altering a gene encoding a vertebrate GH (e.g., bGH or hGH) having the "native" third alpha helix by site-specific mutagenesis, and then cloning and expressing the altered gene in a suitable host. Molecular biology techniques are described in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab Press; 2nd ed., 1989). The gene may be of genomic origin, it may be cDNA prepared from bGH messenger RNA, it may be synthetic, or it may be a combination thereof. For the amino acid sequence of bGH and for the cDNA sequence of the bGH gene, see Miller et al., J. Biol. Chem. 255:7521-24 (1980). For the genomic bGH sequence, see Woychick et al., Nucleic Acids Res. 10:7197-7120 (1982). The cDNA sequence for hGH is given by Chang et al., Gene 55:189 (1987) and DeNoto et al., Nucleic Acid Res. 9:3719 (1981), and the genomic hGH sequence is in Robbins et al., Cell 29:623 (1982).

The host may be any convenient organism, including a bacterial, yeast, insect, or mammalian cell. The gene is operably linked to a promoter functional in the host. A constitutive promoter would activate gene expression in a general manner, i.e., in many tissue and at all times during development. A regulatable promotor may be activated in a tissue or cell specific manner, at a precise time during development, or in response to changes in the environment. A constitutive promoter is usually employed when larger amounts of gene product are required or when the gene product is required in many cells or in many tissues. A regulatable promoter is utilized when one gene product is required in a small number of cells of a particular tissue or at a given time during development.

The expression system may be engineered so that the antagonist is secreted into the culture medium, or the host cells may be grown to a high cell density and then lysed to release the compound.

One method suitable for the purification of bGH (G119R) and the like is described in Leung et al., Endocrinology 119:1489-1496 (1986). Essentially, this procedure involves purification by (a) ammonium sulfate precipitation, (b) fractionation on DEAE-cellulose (or any equivalent ion-exchange column), and (c) gel filtration (e.g., on a Sephadex G-25 and/or Sephacryl S-200 column). Other procedures applicable to purification of GH-related compounds are set forth in Reichert, Jr., "Purification of Anterior Pituitary Hormones: Bovine, Rat and Rabbit," Meth. Enzymol. 37:360 et seq. (Academic Press, N.Y.:1975). Polyclonal or monoclonal antibodies which specifically recognize the protein of interest may also be used in the purification process.

The invention provides for methods for the treatment of several diseases wherein the method comprises the step of administering an effective amount of one or more GH antagonists. The diseased tissues or systems which can be treated by the present invention all express GHRs. In humans, since the expression of GHRs is ubiquitous, i.e., found in nearly all human tissues, the range of diseased tissues and organ systems which can be treated by the present invention is very broad. Mercado et al., J. Clin. Endocrinol. and Metabol. 78:731-735 (1993).

The term "treatment" as used herein with reference to a disease is used broadly and is not limited to a method of curing the disease. The term "treatment" includes any method that serves to reduce one or more of the pathological effects or symptoms of a disease or to reduce the rate of progression of one or more of such pathological effects or symptoms. Diseases that may be treated by the methods of the invention are diseases characterized by one or more of the following criteria: elevated levels of GH production, elevated levels of GHR production, and elevated cellular response of GHRs to GH. The term "elevated" as used herein is used with respect to the normal levels of GH production, GHR production, or GH-mediated cellular response in a tissue (or tissues) of a diseased person (or animal) as compared to level in a normal individual. Diseases that may be treated by the methods of the invention include, but are not limited to, acromegaly, gigantism, cancer, diabetes, vascular eye diseases (diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration, retinopathy of sickle-cell anemia, etc.) as well as nephropathy.

Cancers that may be treated by the subject method include, but are not limited to, cancers comprising tumor cells that express GHRs. Cancers that may be treated by the methods of the invention include, but are not limited to:

Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma

Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma

Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma)

Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma)

Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom, angiosarcoma, hepatocellular adenoma, hemangioma

Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors

Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma)

Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid tumors, celioblastoma, clear cell carcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopian tubes (carcinoma)

Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]

Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles, dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis

Adrenal glands: neuroblastoma

Another aspect of the subject invention is the discovery that many cancerous cells produce GH. The production of GHs from cancerous cells may have the same adverse effects as excess GH produced by the pituitary gland. Although not wishing to be bound by any particular theory of operation, GH secreted by cancerous cells may manifest pathological effects by means of autocrine stimulation of GHRs on the cancerous cells that secrete GH. GH produced by cancerous cells has a high effective concentration near the site of production. This high effective concentration of GH may serve to stimulate GHRs on cancerous cells at or near the site of GH production. Stimulation of GH receptors on cancerous cells may cause proliferation of the cancer.

The autocrine model of cancer cell GHR stimulation described above suggests that various cancers may be treated by GH antagonists that can interfere with the autocrine stimulation of GHRs on cancer cells. The GH antagonists described herein may be used to treat cancers in which at least some of the tumor cells produce GH. In addition to providing methods for the treatment of cancer with the subject GH antagonists, the invention also contemplates the treatment of various types of cancers by methods comprising the step of administering an effective amount of GH antagonists not specifically described herein.

Furthermore, it is preferable to use GH antagonists derived from a vertebrate species that is the same as the species that is being treated. Alternatively, the methods of treatment may use other compounds that have GH antagonist properties in the treatment methods.

The purified antagonist may be combined with compatible, nontoxic pharmaceutical excipients and administered to an animal, e.g., to treat a condition characterized by an excessive growth rate. (The term "animal" is intended to include humans.) In the case of administration to nonhuman animals, it may be preferable to incorporate the drug into the animal's feed, possibly in a prepared combination of drug and nutritional material ready for use by the farmer. The antagonist may be administered orally, rectally, transdermally, by pulmonary infiltration, insufflation or parenterally (including intravenously, subcutaneously and intramuscularly) to humans, in any suitable pharmaceutical dosage form. In the case of treatment of retinopathy, it may be administered directly onto or into the eye by means of a conventional ocular pharmaceutical form.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Primary among these is the amount of GH secreted by the pituitary, which is on the order of 0.5 mg/day for healthy adult humans. Additional factors include the size of the patient, the age of the patient, the general condition of the patient, the particular disease being treated, the severity of the disease, the presence of other drugs in the patient, the in vivo activity of the antagonist and the like. The trial dosages would be chosen after consideration of the results of animal studies and the clinical literature with respect to administration of GHs, and of somatostatin (a GH release inhibitor). It will be appreciated by the person of ordinary skill in the art that information such as binding constants and Ki derived from in vitro GH binding competition assays may also be used in calculating dosages.

A typical human dose of a GH antagonist would be from about 0.1 mg/day to about 10 mg/day, preferably from about 0.5 mg/day to about 2 mg/day, and most preferably about 1 mg/day.

The invention also provides pharmaceutical formulations for use in the subject methods of treating disease. The formulations comprise at least one GH antagonist, preferably a GH antagonist specifically provided for herein, and a pharmaceutically acceptable carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like. The pharmaceutical formulations may also comprise additional components that serve to extend the shelf-life of pharmaceutical formulations, including preservatives, protein stabilizers, and the like. The formulations are preferably sterile and free of particulate matter (for injectable forms). These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The formulations of the invention may be adapted for various forms of administration, including intramuscularly, subcutaneously, intravenously, intraocularly, and the like. The subject formulations may also be formulated so as to provide for the sustained release of GH antagonist. Actual methods for preparing parenterally administrable compositions and adjustments necessary for administration to subjects will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science. 17th Ed., Mack Publishing Company, Easton Pa. (1985), which is incorporated herein by reference.

In another embodiment, the gene is introduced into a host cell which is developed into genetically transformed cells of a transgenic animal. Linearized DNA bearing the GH antagonist gene may be introduced into a gamete, or microinjected into the pronuclei of fertilized eggs, into the cytoplasm, into the nuclei of two-cell embryos, into individual cells of a blastocyst, or into the blastocoel cavity. (Some of these targets may be reached by electroporation instead of microinjection.) Alternatively, a retrovirus bearing the gene may be constructed and used to infect preimplantation embryos or tissue culture cells (e.g., embryonic stem cells) which may be aggregated with such embryos. In either case, the genetically modified zygote, after a brief in vitro cultivation, is implanted into a foster mother and carried to term. For "gene therapy" post partum, see Cline et al., Nature 284:422-425 (1980); Williamson, Nature 298:416-18 (1982). Again the gene is operably linked to a promoter functional in the host, and the promoter may be constitutive or regulatable. Preferably, expression is regulated so abnormal embryonic or fetal development is avoided.

Claim 1 of 50 Claims

We claim:

1. A non-naturally occurring DNA molecule comprising a coding sequence encoding a vertebrate growth hormone variante comprising an amino acid substitution of an amino acid, other than glycine or alanine, for the amino acid of said vertebrate growth hormone at the position corresponding to the glycine at position 119 of bovine growth hormone, wherein the growth hormone variant has vertebrate growth hormone inhibitory activity, with the proviso that said variant does not correspond to human growth hormone with all of the following substitutions and no others: Y111V, L113I, K115E, D116Q, E118K, E119R, G120L, Q122E, T123G, G126L, R127I and E129S.

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