WO1994008611A1 - GROWTH HORMONE FRAGMENT hGH 108-129 - Google Patents

GROWTH HORMONE FRAGMENT hGH 108-129 Download PDF

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Publication number
WO1994008611A1
WO1994008611A1 PCT/US1993/010088 US9310088W WO9408611A1 WO 1994008611 A1 WO1994008611 A1 WO 1994008611A1 US 9310088 W US9310088 W US 9310088W WO 9408611 A1 WO9408611 A1 WO 9408611A1
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Prior art keywords
hgh
cells
polypeptide
amount
animal
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PCT/US1993/010088
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French (fr)
Inventor
Martin Sonenberg
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Sloan-Kettering Institute For Cancer Research
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Priority to AU54464/94A priority Critical patent/AU5446494A/en
Publication of WO1994008611A1 publication Critical patent/WO1994008611A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • growth hormone is composed of 4 helices.
  • bGH 96-133 has been well characterized chemically (2,5) and physically (6,7) including NMR spectroscopy (8,9).
  • the importance of residues within helix 3 in GH for growth promotion has been further emphasized by studies of site directed mutagenesis (10-16) .
  • hGH (1) 108-129, hGH 113-130, hGH 123-131, and hGH (d) 108-129 with d-amino acids were studied. These were assayed for mitogenic activity. In addition, binding and affinity labeling of target cells with biologically active peptides were studied. The result of the study produced the invention described herein, hGH (1) 108- 129, which has been shown to have mitogenic and tyrosine phosphorylating activities and binds to the GH receptor.
  • the improvement which this invention provides is a polypeptide with a 22 amino acid sequence, derived from human growth hormone, capable of being chemically synthesized and having properties similar to the 38 amino acid bovine growth hormone fragment, bGH 96-133, yet being unexpectedly 100 to 500 times more potent than the bovine growth hormone fragment.
  • This invention provides a polypeptide having the sequence from the amino terminal to the carboxy terminal:
  • FIGS. 1A, IB and 1C show [ 3 H]-Thymidine Incorporation in 3T3-F442A Cells
  • 3T3-F442A cells maintained in serum-free medium for 48 h were exposed to the indicated concentrations of peptides.
  • FIGS 2A-2D show Cell Cycle Analysis of 3T3-F442A Cells after Exposure to Peptides
  • 3T3-F442A cells were incubated with SFM (Figure 2A) , DMEM, 10% FCS ( Figure 2B) , 0.5 nM hGH (1) 108-129 ( Figure 2C) , or hGH 113-130 ( Figure 2D) in SFM at 37°C. Two days later, DNA content per cell was analyzed by FACS analysis.
  • Figure 3 shows Affinity Cross Linking of hGH (1) 108-129 and hGH in 3T3-F442A Cells
  • Figure 4 shows Binding Characteristics of hGH (1) 108- 129 .
  • 3T3-F442A cells were incubated in binding medium containing 0.04 nM of radiolabeled hGH (1) 108-129 in the presence of different concentrations of competitors.
  • Non-specific binding activity is defined as the percentage of ligand bound in the presence of a 1000 fold excess of competitor.
  • Figure 5 is a Scatchard Analysis of hGH (1) 108-129 Binding
  • Figure 6 shows Binding of [ 125 I]-hGH in the Presence of GHR Antibody
  • Binding studies were performed as described in the above description for Figure 4. 3T3-F442 A cells were incubated in binding buffer with increasing concentrations of MAb 263 for 2 hours at 4 °C. Cells were washed 3 times with the same buffer. [ 125 I]-hGH was incubated with increasing concentrations of GHR antibody, MAb 263 and incubation was continued at 4 °C for 2 hours.
  • Figure 7 shows Effect of Blocking by GHR Antibody on Binding of hGH and hGH (1) 108-129.
  • 3T3-F442A cells were suspended in binding buffer with various concentrations of MAb 263 for 2 h at 4°C, Cells were washed 3 times with the same buffer. Radiolabeled hGH or hGH (1) 108-129 was added to these cells and incubation was continued at 4°C for 2 h.
  • Figures 8A and 8B show Blocking of Phosphorylation with MAb 263
  • Samples for 10% SDS-PAGE under reducing conditions were obtained from extracts of 3T3-F442A cells incubated with hGH (1) 108-129 (0.5 nM) or hGH (1.0 nM) alone or after treatment with anti-GHR monoclonal antibody (MAb 263) for 2 h.
  • the MAb 263 concentration was 2 ⁇ g/ml.
  • the time of incubation with hGH 108-129 varied from 10 to 60 min. Pre-incubation with the MAb 263 monoclonal antibody is indicated by ⁇ . Extracts prepared as described in Methods.
  • Figure 9 shows the results of the Growth Hormone BioAssay of hGH 108-129 as described in Methods.
  • This invention provides a polypeptide having the sequence from the amino terminal to the carboxy terminal:
  • the polypeptide of the invention corresponds to hGH 108-129, the 1 form of amino acid residues 108 through 129 on helix 3 of human growth hormone.
  • the polypeptide of the invention can be synthetically derived or it can be derived from human growth hormone.
  • the polypeptide of the invention may contain one or two additional, or one or two less, amino acid residues within the sequence or on either the amino terminal or the carboxy terminal of the sequence.
  • the polypeptide of the invention is related to 38 amino acid polypeptide of bovine growth hormone (bGH 96-133) described in U.S. Patent No. 4,056,520, issued November 1, 1977 (Sonenberg et al.). Within bGH 96-133, 22 amino acid residues, residues 106 to 127, (bGH 106-127) have 15 amino acids identical to the peptide of the present invention: hGH 108-129 bGH 106-127
  • the peptide of this invention is similar to the bovine growth hormone fragment of U.S. Patent No.4,056,520, bGH 96-133, in that it is useful in humans and bovine in addition to other animals.
  • the uses of bovine growth hormone are well known to those skilled in the art.
  • the peptide of this invention differs from the bovine growth hormone fragment of U.S. Patent No. 4,056,520 in that it is of human origin, has a shorter sequence, and is unexpectedly 100 to 500 times more potent than bGH 96-133.
  • This invention also provides a composition comprising an amount of the polypeptide effective to promote the growth of an animal and a physiologically acceptable carrier.
  • physiologically acceptable carrier means any physiological composition generally accepted by those in the art as a carrier for administration of a substance to an animal. Examples include, but are not limited to, aqueous buffers, e.g., phosphate buffers, and may include other buffers known to those skilled in the art. Examples of animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
  • This invention also provides a method of treating a subject having a disease characterized by an insufficient amount of undifferentiated cells comprising administering to the subject an effective amount of the polypeptide.
  • Differentiation of cells in a body is the process of acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues, an example of which can be seen in the blood system.
  • the blood of an animal contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. However, all blood cells are generated ultimately from the same type of stem cell. Differentiation of this stem cell, the hematopoietic stem cell, gives rise to all of the different types of terminally differentiated blood cells.
  • the term "insufficient amount of undifferentiated cells” means having a greater amount of undifferentiated cells in a body of an animal than is needed for normal body functioning.
  • a body of an animal which has an "insufficient amount of undifferentiated cells” is characterized by abnormal body functioning. Such abnormal body function may result in disease. Diseases which are characterized by an insufficient amount of undifferentiated cells are well known to those skilled in the art.
  • compositions are administered topically.
  • Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams.
  • the composition is administered intranasally.
  • Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of an aerosol or mist are well known to those skilled in the art.
  • the composition is administered by injection.
  • Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
  • an "effective amount" of a composition is any amount of the composition effective to decrease the amount of undifferentiated cells in the body of an animal.
  • animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
  • This invention also provides a method of treating a subject having a disease characterized by an abnormally low amount of tissue growth comprising administering to the subject an effective amount of the polypeptide.
  • tissues in which growth is promoted by this invention include, but are not limited to, bone, cartiledge and muscle.
  • Diseases associated with an abnormally low amount of tissue growth are well known to those skilled in the art and include, but are not limited to, achondroplasia.
  • compositions are administered topically.
  • Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams.
  • the composition is administered intranasally.
  • Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of the aerosol or mist are well known to those skilled in the art.
  • the composition is administered by injection.
  • Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
  • an "effective amount" of the composition is any amount of the composition effective to promote the growth of tissues in the body of an animal.
  • animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
  • This invention also provides a composition comprising an amount of the polypeptide effective to inhibit the growth of transformed dedifferentiated cells in an animal and a physiologically acceptable carrier.
  • transformed dedifferentiated cells are cells which have regressed from a more specialized or complex form to a simpler state and have become malignant. Examples of malignant cells include, but are not limited to, breast cancer cells.
  • This invention also provides a method of inhibiting the growth of transformed dedifferentiated cells comprising contacting the transformed dedifferentiated cells with the polypeptide.
  • transformed dedifferentiated cells for which this invention is useful include, but are not limited to, cancer cells.
  • This invention also provides a method of treating an animal afflicted with a cancer which comprises administering to the animal an effective amount of the composition.
  • cancers against which this invention is useful include, but are not limited to, breast cancer.
  • compositions are administered topically.
  • Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams.
  • the composition is administered intranasally.
  • Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of an aerosol or mist are well known to those skilled in the art.
  • the composition is administered by injection.
  • Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
  • an "effective amount" of the composition is any amount of the composition effective to inhibit the growth of cancer cells in the body of an animal.
  • animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
  • This invention further provides a composition comprising an amount of the polypeptide effective to increase the ratio of weight gain to feed consumption in animals and a physiologically acceptable carrier.
  • a physiologically acceptable carrier means any physiological composition generally accepted by those skilled in the art as a carrier for administration of a substance to an animal. Examples include, but are , not limited to, aqueous buffers, e.g., phosphate buffers, and may include other buffers known to those skilled in the art. Examples of animals in which this invention is useful include, but are not limited to, bovine.
  • this invention also provides a method of increasing the ratio of weight gained to feed consumed in an animal which comprises administering to the animal an effective amount of the polypeptide.
  • compositions are administered topically.
  • Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams.
  • the composition is administered intranasally.
  • Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of the aerosol or mist are well known to those skilled in the art.
  • the composition is administered by injection.
  • Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
  • an "effective amount" of the composition is any amount of the composition effective to increase the ratio of weight gained to feed consumed of an animal.
  • animals in which this invention is useful include, but are not limited to, bovine.
  • Bovine serum albumin (BSA Fraction V) was purchased from ICN Biomedicals, Inc. (Lisle, IL) .
  • GHR antibody (MAb 363) was obtained from Agen, Inc. (Parsippany, NJ) .
  • Biosystem 431 A automatic peptide synthesizer. Peptides were purified to homogeneity by HPLC with a Waters 600 multi-solvent delivery system. Purity was established by amino acid analysis and N-terminal sequencing with an Applied Biosystems 477A automatic protein sequencer with a 120 PTH detector based on the published method (21) .
  • Peptides (1.5 ⁇ g of hGH and hGH (1) 108-129) were radio- iodinated by the chloramine T method (25) . Specific activities of radiolabeling were in the range of 400-500 ⁇ Ci/ ⁇ g. Unlabeled iodine was removed by chromatography on a Sephadex G50 column. Biological Methods
  • r 3 H1-thymidine incorporation assay Cells were inoculated in 6 well dishes (9.6 cm 2 area) in a volume of 3 ml at a density of 2 x 10 4 cells/well. The following day, medium was removed and cells were washed 3 times with HBSS (Hanks Buffered Salt Solution) , and SFM containing various concentrations of peptides were added to cells. Incubations were carried out for 2 days at 37°C, and 3 h prior to cell harvest, cells were labeled with [ 3 H]-thymidine (20 ⁇ Ci/ml) at 37°C. Cells were solubilized with 0.5% SDS at 37°C for 10 min.
  • Portions of cell lysates were precipitated with 10% TCA and counted by scintillation counting. Radioactivity of each sample was normalized by protein concentration based on A 562 using the micro-BCA protein assay. In addition, cells were harvested on day 5 and counted by Coulter counting.
  • Flow cvtometrv Cells (10 6 /ml) were detached by trypsin and were harvested by centrifugation at 1,000 x g for 10 min. Cell pellets were suspended in 0.5 ml PBS buffer and then an equal volume of 95% ethanol was added. After fixing overnight at 4°C, cells were harvested at 1,000 x g for 10 min and cell pellets were resuspended in staining solution containing 50 ⁇ g/ml propidium iodide and 50 ⁇ g/ml RNase A. The propidium iodide staining does not distinguish G 0 cells from G ⁇ cells. The staining reaction continued for at least 2 h at room temperature, and the DNA content of each sample was analyzed by an Epics profile II flow cytometer.
  • Binding assay Monolayer cultures of 3T3-F442A cells (2 x 10 5 cells/well) were washed 4 times with HBSS buffer, and then binding buffer (10 mM Hepes [pH 7.2], 0.1% BSA containing 1 nM of [ 125 I]-hGH (1) 108-129, and indicated amounts of hGH, hGH (1) 108-129, insulin, hGH 113-130, or bGH 7-34 were added. The binding reaction was carried out for 3 h at 4°C on a platform shaker. Unlabeled ligand was removed by washing monolayers with ice cold HBSS buffer.
  • Cell lysates were prepared by using extraction buffer (150 mM NaCl, 20 mM Tris-HCl [pH 8.0], 1 mM MgCl 2 , 0.1 mM ZnCl 2 , 0.5% NP40, 2 mM PMSF). Aliquots were saved for protein determination. Bound peptide was measured by counting samples in a gamma counter. Specific binding is defined as the difference between total binding and binding observed at a 1000 fold excess of hGH (1) 108-129.
  • Affinity cross-linking Monolayer cultures of 3T3-F442A cells (10 5 /well) were washed 3 times with HBSS buffer. Binding buffer (2 ml) containing [ 125 I]-hGH (1 nM) or [ 125 ]-hGH (1) 108-129 (1 nM) was added. The binding reaction was carried out for 3 h at 4°C. Binding was terminated by removing unbound ligand by washing 3 times with HBSS buffer. DSS (0.5 mM) in binding buffer was then added to the cells and the cross linking reaction was carried out at 4°C for 30 min.
  • Cell lysates were prepared using an extraction buffer (20 mM Hepes [pH 7.4], 1% triton X 100, 10% glycerol, 50 mM sodium fluoride, ImM sodium orthovanadate, 10 ⁇ g/ml leupeptin, 0.5 mM PMSF)and clarified by centrifugation at 30,000 g for 30 min. Supernatants were boiled for 5 min and were subjected to SDS-PAGE (7.5%). Gels were fixed, dried, and exposed on Kodak XAR-5 film at -70°C.
  • extraction buffer 20 mM Hepes [pH 7.4], 1% triton X 100, 10% glycerol, 50 mM sodium fluoride, ImM sodium orthovanadate, 10 ⁇ g/ml leupeptin, 0.5 mM PMSF
  • Nitrocellulose filter was incubated with blocking solution (1% BSA, 0.2% Tween 20 in TBS) at room temperature for 2 h, and washed 3 times with IX TBS buffer. Nitrocellulose filter was incubated with monoclonal anti-mouse phosphotyrosine Ab(2 ⁇ g/ml) for 2 h at room temperature and was washed with TBS buffer 3 times. Alkaline phosphatase conjugated mouse IgG was added to the nitrocellulose filter, and the reaction continued for 2 h at room temperature.
  • blocking solution 1% BSA, 0.2% Tween 20 in TBS
  • the nitrocellulose was incubated with alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl, Tris HCl (pH 9.5)) containing NBT (50 mg/ml in 70% N-N-dimethylformamide) and BCIP (50 mg/ml in 100% N-N-dimethylformamide) .
  • alkaline phosphatase buffer 100 mM NaCl, 5 mM MgCl, Tris HCl (pH 9.5)
  • NBT 50 mg/ml in 70% N-N-dimethylformamide
  • BCIP 50 mg/ml in 100% N-N-dimethylformamide
  • the mitogenicity of each peptide was evaluated by [ 3 H]- thymidine incorporation and by flow cytometric analysis of cells.
  • Native hGH was anti-mitogenic with an EC 50 about 2.0 nM in unstimulated cells (Table I and ref. 26).
  • hGH (1) 108-129 stimulated [ 3 H]-thymidine incorporation about five fold with a maximal effect noted at 0.5 nM (Fig. la).
  • No other peptide on the basis of the hGH or bGH sequence i.e. bGH 7-34, hGH 113- 130, hGH (d) 108-129 or hGH itself was mitogenic based on [ 3 H]-thymidine incorporation in doses up to 1 ⁇ M (Fig. la).
  • hGH(l) 108-129 is a part of hGH, it was necessary to check whether hGH has mitogenic activity itself. Under the same assay conditions wild type hGH was anti-mitogenic with an EC 50 about 0.5 nM (Fig. lb) . This result is quite consistent with our previous report (26) which shows involvement of hGH in fat cell differentiation. It was reported (26) that hGH treated 3T3-F442A cells are resistant to the mitogenic effects of other growth factors including PDGF. The mitogenic effect of hGH (1) 108-129 was not as effective as TGF-/3 which had an EC 50 of about 1 pM and was maximally effective at 50 pM (Fig. 1C) .
  • IGF-1, 3T3-F442A cells were incubated with the peptide in the presence of hGH (1) 108-129 as well as IGF-1. Although both peptides individually gave a strong mitogenic response, the presence of an antibody to IGF-1 blocked only the IGF-1 response without effect on the mitogenesis induced by hGH (1) 108-129 (data not shown) .
  • hGH:hGH (1) 108-129 When incubated together in molar ratios (hGH:hGH (1) 108-129) of 2:1 and 20:1, the mitogenic response to 0.5 nM hGH (1) 108- 129 was completely offset and the cells manifested the prevailing anti-mitogenic response of hGH (Table I) . Although not intending to be bound by any theory it is believed that this might be due to possible competition between hGH and hGH (1) 108-129 for binding to the same receptor. In this assay it seems clear that hGH (1) 108-129 is not an antagonist because it does not affect the anti-mitogenic activity of hGH in 3T3-F442A cells.
  • [ 125 I]-hGH (1) 108-129 was cross-linked to a protein of the same size, and cross-linking was blocked by an excess of hGH (1) 108-129 (lanes 3 and 4 respectively) .
  • hGH and hGH (1) 108-129 were also able to inhibit cross-linking of the other peptide (lanes 5 and 6 respectively) .
  • hGH (1) 108-129 acts through GHR, it was of interest to determine whether a possible block in binding to GHR is associated with a defect in the mitogenic response to hGH (1) 108-129. Indeed in molar ratios of 2 to 1 to 20 to 1, the mitogenic response to 0.5 nM was completely blocked and the cells manifested the prevailing anti-mitogenic response of hGH (Table I) . Monoclonal mouse anti-GHR Ab(MAb 263) inhibited the mitogenic activity of hGH (1) 108-129 suggesting that the GHR is important in mediating the mitogenic activity of hGH (1) 108-129 (Table II) . MAb 263 also inhibited the anti-mitogenic activity of hGH when incubated together with the hormone indicating the importance of GHR in mediating the actions of hGH (data not shown) .
  • hGH had a binding affinity of approximately 0.2 nM and approximately 28,000 binding sites on 3T3-F442A cells (data not shown) .
  • hGH 108-129 bound with an affinity of approximately 5 nM and had 560,000 binding sites per cell (Fig. 5).
  • Affinity cross-linking of [ 125 I]-hGH to its receptor was not duplicated with [ 125 I]-hGH 108-129.
  • hGH has been known to cause tyrosine phosphorylation of GHR. Tyrosine phosphorylated GHR is believed to transduce its signal to the cells. This transduction of the GH signal by GHR is known to cause cellular tyrosine phosphorylation of several molecules such as MAPII kinases.
  • hGH and hGH (1) 108-129 both cause induction of tyrosine phosphorylation of cellular molecules.
  • Western blot analysis using monoclonal anti- mouse phosphotyrosine Ab was carried out. As is seen in Fig. 6, both hGH and hGH (1) 108-129 were shown to increase tyrosine phosphorylation of cellular molecules most notably the 42 kDa molecule.
  • Tyrosine phos ⁇ phorylation of cellular molecules became evident after 10 min. of stimulation with hGH or hGH (1) 108-129.
  • the fact that the 42 kDa molecule was phosphorylated by these two different peptides suggests that there might be different molecular events beyond tyrosine phosphorylation to explain the opposite effects displayed by these two peptides.
  • the optimal concentration of hGH (1) 108-129 for inducing tyrosine phosphorylation was about 1 nM, slightly higher than the optimal concentration for [ 3 H]-thymidine incorporation.
  • Figures 8A and 8B show that in the presence of MAb 263, tyrosine phosphorylation of the 42 kDa molecule induced by, hGH (1) 108-129 ( Figure 8A) or hGH ( Figure 8B) was completely inhibited. These results suggest that hGH and hGH (1) 108-129 act through GHR.
  • hGH (1) 108-129 is mediated through GHR in 3T3-F442A cells.
  • Affinity cross linking, competitive binding assays and antibody blocking reactions all implicate GHR in mediating the mitogenic activity of hGH (1) 108-129.
  • the binding was specific and saturable in the nanomolar (10-20 nM) range in contrast to the sub-nanomolar range for its mitogenic activity. This indicates the existence of spare receptors for hGH (1) 108-129 on 3T3- F442A cells.
  • hGH binds to 3T3- F442A cells with half saturating concentrations of 0.77 - 1.8 x 10 "9 (31) with an EC 50 for an adipogenic response of about 0.1 nM (32).
  • the anti-mitogenic effect occurs with an EC 50 of approximately 0.05 nM (26). This indicates that the half-maximal anti-mitogenic response occurs when approximately 5% of the sites are occupied.
  • hGH (1) 108-129 binding is 5.2 nM, and the EC 50 for mitogenesis is approximately 0.2 nM, when only 1% of the receptors are occupied, i.e., suggesting that there are spare receptors.
  • hGh (1) 108-129 has a K d of binding of 5.2 nM compound to a value of about 0.2 to 1 nM for hGH (32).
  • This lower affinity for the smaller peptide would be consistent with binding at site 1 and site 2 of hGH and only at site 1 residues of hGH present in hGH (1) 108- 129.
  • the lower affinity is not soley due to the size of the fragment since we have found that the 38 amino acid sequence bGH 96-133 binds with a K d of approximately 100 nM with corresponding mitogenic activity (unpublished results) .
  • the numbers of binding sites (Bmax) determined by Scatchard analysis of [ 125 I]-hGH and [ 125 ]-hGH (1) 108- 129 binding are 28 X 10 3 and 560 X 10 3 . There may also be several species of GHR , s to which hGH and hGH (1)
  • 108-129 binds to a receptor other than GHR. This would be consistent with the fact that hGH with a K d of about 0.2 to 1 nM is less effective than hGH (1) 108-129 with a K d of 5.2 nM in competing for binding of [ 125 I]- hGH (1) 108-129 (Fig. 4).
  • GHR itself does not have a consensus tyrosine kinase domain (33) it is difficult to attribute the mitogenic activity to tyrosine phosphorylation by GHR as a protein kinase.
  • GHR-mediated tyrosine phosphorylation involves an associated tyrosine kinase (34) .
  • This hypothesis is consistent with the fact that the receptors for NGF (35) , CD4 (36) , and CD8 (37) complex with a family of tyrosine kinase proteins. From our affinity labeling and binding studies it would appear that hGH (1) 108-129 binds to GHR.
  • hGH 95-133 and bGH 96-133 prepared by chemical synthesis or enzymatic digestion, have proliferative activity, albeit at lower doses (unpublished data) .
  • Rats were hypophysectomized and weighed on a daily basis. On the 14th day following surgery, rats which were 3 gm or less than original body weight or 12 gm or more than original body weight were discarded (less than 5% or less of total population of hypophysectomized rats) . The remaining rats were randomly assigned to the specified treatment groups (8 rats per group) . Rats were injected once daily for 10 days with either 1 ml saline, 24 mg hGH 108-129 in 1 ml saline or 0.16 mg human growth hormone (hGH) in 1 ml saline. Rats were sacrificed after 10 days of injection, 24 hours after last injection.
  • hGH human growth hormone
  • 3T3-F442A cells were incubated with hGH or hGH (1) 108- 129, either alone or in combination at the indicated concentrations. [ 3 H]-thymidine incorporation was done as detailed in Fig. lc legend. Results ( ⁇ S.E.) are compared to SFM control.
  • 3T3-F442A cells were incubated with hGH (1) 108-129 alone or in the presence of the anti-GHR monoclonal antibody (MAb 263) .
  • [ 3 H]-thymidine incorporation was performed as described in Fig. lc legend. Results are expressed as control of SFM.

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Abstract

This invention provides a polypeptide having the sequence from the amino terminal to the carboxy terminal: Ser Asp Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Lwu Met Gly Arg Leu Glu.

Description

GROWTH HORMONE FRAGMENT hGH 108-129
The invention described herein was made in the course of work under Grant Number DK 41931 from the National Institutes of Health. The United States Government has certain rights in this invention.
Background of the invention
Throughout this application various publications are referenced to by arabic numerals within parentheses. Full bibliographic citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures for the publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Based on x-ray crystallography (1) growth hormone (GH) is composed of 4 helices. A fragment of bovine growth hormone (bGH) isolated from enzymatic digests of bGH with the amino acid sequence 96 to 133, encompassing helix 3, has been reported (2,3,4) to have growth promoting activity in the hypophysectomized rat. bGH 96-133 has been well characterized chemically (2,5) and physically (6,7) including NMR spectroscopy (8,9). The importance of residues within helix 3 in GH for growth promotion has been further emphasized by studies of site directed mutagenesis (10-16) .
Based on the known growth promoting activity of helix 3 related peptides, a series of peptides including, bGH 7- 34, hGH (1) 108-129, hGH 113-130, hGH 123-131, and hGH (d) 108-129 with d-amino acids were studied. These were assayed for mitogenic activity. In addition, binding and affinity labeling of target cells with biologically active peptides were studied. The result of the study produced the invention described herein, hGH (1) 108- 129, which has been shown to have mitogenic and tyrosine phosphorylating activities and binds to the GH receptor.
The improvement which this invention provides is a polypeptide with a 22 amino acid sequence, derived from human growth hormone, capable of being chemically synthesized and having properties similar to the 38 amino acid bovine growth hormone fragment, bGH 96-133, yet being unexpectedly 100 to 500 times more potent than the bovine growth hormone fragment.
Summary of the Invention
This invention provides a polypeptide having the sequence from the amino terminal to the carboxy terminal:
Ser Asp Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly lie Gin Thr Leu Met Gly Arg Leu Glu.
Brief Description of the Figures
Figures 1A, IB and 1C show [3H]-Thymidine Incorporation in 3T3-F442A Cells
3T3-F442A cells maintained in serum-free medium for 48 h were exposed to the indicated concentrations of peptides. Levels of DNA synthesis were determined by measurement of [3H]-thymidine (10 μCi/ml) incorporation in a 3 h labeling 21 h after the addition of peptides. Data were normalized on the basis of protein concentration, compared to incorporation in serum-free medium, and are presented as the mean ± S.E.M.(n = 3)
1A. Effect of peptides IB. Effect Of GH
1C. Effect of growth factors
Figures 2A-2D show Cell Cycle Analysis of 3T3-F442A Cells after Exposure to Peptides
3T3-F442A cells were incubated with SFM (Figure 2A) , DMEM, 10% FCS (Figure 2B) , 0.5 nM hGH (1) 108-129 (Figure 2C) , or hGH 113-130 (Figure 2D) in SFM at 37°C. Two days later, DNA content per cell was analyzed by FACS analysis.
Figure 3 shows Affinity Cross Linking of hGH (1) 108-129 and hGH in 3T3-F442A Cells
SDS-PAGE gels after cells were cross-linked with [125I]- hGH in the absence (lane 1) or in the presence of unlabeled hGH (lane 2) or hGH (1) 108-129 (lane 3) ; after cross-linking with [125I]-hGH (1) 108-129 in the absence (lane 4) or presence of unlabeled hGH (1) 108- 129 (lane 5) or hGH (lane 6).
Figure 4 shows Binding Characteristics of hGH (1) 108- 129 .
3T3-F442A cells were incubated in binding medium containing 0.04 nM of radiolabeled hGH (1) 108-129 in the presence of different concentrations of competitors. Non-specific binding activity is defined as the percentage of ligand bound in the presence of a 1000 fold excess of competitor.
Figure 5 is a Scatchard Analysis of hGH (1) 108-129 Binding
Binding studies were performed as described in the above description for Figure 4. [125I]-hGH (1) 108-129 was incubated with increasing concentration of hGH (1) 108- 129 in the presence of 2 X 104 3T3-F442 A cells.
Figure 6 shows Binding of [125I]-hGH in the Presence of GHR Antibody
Binding studies were performed as described in the above description for Figure 4. 3T3-F442 A cells were incubated in binding buffer with increasing concentrations of MAb 263 for 2 hours at 4 °C. Cells were washed 3 times with the same buffer. [125I]-hGH was incubated with increasing concentrations of GHR antibody, MAb 263 and incubation was continued at 4 °C for 2 hours.
Figure 7 shows Effect of Blocking by GHR Antibody on Binding of hGH and hGH (1) 108-129.
3T3-F442A cells were suspended in binding buffer with various concentrations of MAb 263 for 2 h at 4°C, Cells were washed 3 times with the same buffer. Radiolabeled hGH or hGH (1) 108-129 was added to these cells and incubation was continued at 4°C for 2 h. Figures 8A and 8B show Blocking of Phosphorylation with MAb 263
Samples for 10% SDS-PAGE under reducing conditions were obtained from extracts of 3T3-F442A cells incubated with hGH (1) 108-129 (0.5 nM) or hGH (1.0 nM) alone or after treatment with anti-GHR monoclonal antibody (MAb 263) for 2 h. The MAb 263 concentration was 2 μg/ml. The time of incubation with hGH 108-129 varied from 10 to 60 min. Pre-incubation with the MAb 263 monoclonal antibody is indicated by ■. Extracts prepared as described in Methods.
8A. Incubation with hGH (1) 108-129 8B. Incubation with hGH
Figure 9 shows the results of the Growth Hormone BioAssay of hGH 108-129 as described in Methods.
Detailed Description of the Invention
This invention provides a polypeptide having the sequence from the amino terminal to the carboxy terminal:
Ser Asp Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly lie Gin Thr Leu Met Gly Arg Leu Glu.
Preferably, the polypeptide of the invention corresponds to hGH 108-129, the 1 form of amino acid residues 108 through 129 on helix 3 of human growth hormone. The polypeptide of the invention can be synthetically derived or it can be derived from human growth hormone. The polypeptide of the invention may contain one or two additional, or one or two less, amino acid residues within the sequence or on either the amino terminal or the carboxy terminal of the sequence.
The polypeptide of the invention is related to 38 amino acid polypeptide of bovine growth hormone (bGH 96-133) described in U.S. Patent No. 4,056,520, issued November 1, 1977 (Sonenberg et al.). Within bGH 96-133, 22 amino acid residues, residues 106 to 127, (bGH 106-127) have 15 amino acids identical to the peptide of the present invention: hGH 108-129 bGH 106-127
Ser = Ser Asp = Asp
Arg
Val = Val
Tyr = Tyr
Asp Gl Leu Lys
Leu = Leu
Lys = Lys
Asp — Asp
Leu = Leu Glu = Gl
Glu - Glu
Gly = Gly
He = He Gln Leu
Thr Ala
Leu - Leu
Met = Met Gly Arg
Arg Glu
Leu — Leu Glu
The peptide of this invention is similar to the bovine growth hormone fragment of U.S. Patent No.4,056,520, bGH 96-133, in that it is useful in humans and bovine in addition to other animals. The uses of bovine growth hormone are well known to those skilled in the art. However, the peptide of this invention differs from the bovine growth hormone fragment of U.S. Patent No. 4,056,520 in that it is of human origin, has a shorter sequence, and is unexpectedly 100 to 500 times more potent than bGH 96-133.
This invention also provides a composition comprising an amount of the polypeptide effective to promote the growth of an animal and a physiologically acceptable carrier. For the purposes of this invention, the term "physiologically acceptable carrier" means any physiological composition generally accepted by those in the art as a carrier for administration of a substance to an animal. Examples include, but are not limited to, aqueous buffers, e.g., phosphate buffers, and may include other buffers known to those skilled in the art. Examples of animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
This invention also provides a method of treating a subject having a disease characterized by an insufficient amount of undifferentiated cells comprising administering to the subject an effective amount of the polypeptide. Differentiation of cells in a body is the process of acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues, an example of which can be seen in the blood system. The blood of an animal contains many types of cells with very different functions, ranging from the transport of oxygen to the production of antibodies. However, all blood cells are generated ultimately from the same type of stem cell. Differentiation of this stem cell, the hematopoietic stem cell, gives rise to all of the different types of terminally differentiated blood cells.
For the purposes of this invention, the term "insufficient amount of undifferentiated cells" means having a greater amount of undifferentiated cells in a body of an animal than is needed for normal body functioning. In other words, a body of an animal which has an "insufficient amount of undifferentiated cells" is characterized by abnormal body functioning. Such abnormal body function may result in disease. Diseases which are characterized by an insufficient amount of undifferentiated cells are well known to those skilled in the art.
Examples of methods of administration of this invention include, but are not limited to, injection, intranasal or topical administration. In one embodiment of this invention the composition is administered topically. Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams. In another embodiment of this invention, the composition is administered intranasally. Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of an aerosol or mist are well known to those skilled in the art.
In the preferred embodiment of this invention, the composition is administered by injection. Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
For the purposes of this invention, an "effective amount" of a composition is any amount of the composition effective to decrease the amount of undifferentiated cells in the body of an animal. Examples of animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
This invention also provides a method of treating a subject having a disease characterized by an abnormally low amount of tissue growth comprising administering to the subject an effective amount of the polypeptide. Examples of tissues in which growth is promoted by this invention include, but are not limited to, bone, cartiledge and muscle. Diseases associated with an abnormally low amount of tissue growth are well known to those skilled in the art and include, but are not limited to, achondroplasia.
Examples of methods of administration of this invention include, but are not limited to, injection, intranasal or topical administration. In one embodiment of this invention the composition is administered topically. Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams. In another embodiment of this invention, the composition is administered intranasally. Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of the aerosol or mist are well known to those skilled in the art.
In the preferred embodiment of this invention, the composition is administered by injection. Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
For the purposes of this invention, an "effective amount" of the composition is any amount of the composition effective to promote the growth of tissues in the body of an animal. Examples of animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
This invention also provides a composition comprising an amount of the polypeptide effective to inhibit the growth of transformed dedifferentiated cells in an animal and a physiologically acceptable carrier. For the purpose of this invention, "transformed dedifferentiated cells" are cells which have regressed from a more specialized or complex form to a simpler state and have become malignant. Examples of malignant cells include, but are not limited to, breast cancer cells.
This invention also provides a method of inhibiting the growth of transformed dedifferentiated cells comprising contacting the transformed dedifferentiated cells with the polypeptide. Examples of transformed dedifferentiated cells for which this invention is useful include, but are not limited to, cancer cells.
This invention also provides a method of treating an animal afflicted with a cancer which comprises administering to the animal an effective amount of the composition. Examples of cancers against which this invention is useful include, but are not limited to, breast cancer.
Examples of methods of administration of this invention include, but are not limited to, injection, intranasal or topical administration. In one embodiment of this invention the composition is administered topically. Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams. In another embodiment of this invention, the composition is administered intranasally. Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of an aerosol or mist are well known to those skilled in the art.
In the preferred embodiment of this invention, the composition is administered by injection. Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
For the purposes of this invention, an "effective amount" of the composition is any amount of the composition effective to inhibit the growth of cancer cells in the body of an animal. Examples of animals in which this invention is useful include, but are not limited to, mammals such as humans, bovine, rats and mice.
This invention further provides a composition comprising an amount of the polypeptide effective to increase the ratio of weight gain to feed consumption in animals and a physiologically acceptable carrier. For the purposes of this invention, the "ratio of weight gain to feed consumption" will vary for each species of animal to which the composition is administered and such ratio is well known to those skilled in the art. For the purposes of this invention, the term "physiologically acceptable carrier" means any physiological composition generally accepted by those skilled in the art as a carrier for administration of a substance to an animal. Examples include, but are , not limited to, aqueous buffers, e.g., phosphate buffers, and may include other buffers known to those skilled in the art. Examples of animals in which this invention is useful include, but are not limited to, bovine.
Lastly, this invention also provides a method of increasing the ratio of weight gained to feed consumed in an animal which comprises administering to the animal an effective amount of the polypeptide.
Examples of methods of administration of this invention include, but are not limited to, injection or intranasal or topical administration. In one embodiment of this invention the composition is administered topically. Administration topically can be effected by any method commonly known to those skilled in the art and include, but are not limited to, transdermal patches and creams. In another embodiment of this invention, the composition is administered intranasally. Administration intranasally can by effected by any method commonly known to those skilled in the art and include, but are not limited to, incorporation of the composition into an aerosol or mist. Methods of preparation of the aerosol or mist are well known to those skilled in the art.
In the preferred embodiment of this invention, the composition is administered by injection. Administration by injection may be effected by any method commonly known to those skilled in the art. Methods of injection include, but are not limited to, intramuscular, intravenous, subcutaneous and interperitoneal.
For the purposes of this invention, an "effective amount" of the composition is any amount of the composition effective to increase the ratio of weight gained to feed consumed of an animal. Examples of animals in which this invention is useful include, but are not limited to, bovine.
This invention is further illustrated in the Experimental Procedures section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow.
Experi ental Procedures
Materials
General biochemicals and chemicals of required high grade (tissue culture, ultra pure and sequencing grade) were from Sigma Chemical Co. (St. Louis, MO) , Boehringer Mannheim Biochemicals (Indianapolis, IN) , J.T. Baker Chemical (Phillipsburg, NJ) and Pierce Chemical Co. (Rockford, IL) . [3H]-thymidine (20 Ci/ mol) was from New England Nuclear (Boston, MA). Na[125I] (2176 Ci/mmole) was from Amersham (Arlington Heights, IL) . Solvents for HPLC of chromatography grade were from J.T. Baker Chemical Co. (Phillipsburg, NJ) and Pierce Chemical Co.
Stocks of Swiss 3T3-F442A and 3T3-C2 fibroblasts were generously supplied by Dr. Howard Green (Boston, MA) and passed in this laboratory as previously described (17) . Tissue culture plasticware was purchased from Corning Glass Works (Corning, NY) . Calf serum, fetal calf serum, trypsin, glutamine and 6-well plates were obtained from GIBCO Lab. (Grand Island, NY) . Recombinant hGH (met-HGH) was a gift from Genentech (South San Francisco, CA) and Eli Lilly (Indianapolis, IN) . Bovine transferrin, bovine insulin, bovine fetuin, and T3 were obtained from Sigma Chemical Co. (St. Louis, MO) . Bovine serum albumin (BSA Fraction V) was purchased from ICN Biomedicals, Inc. (Lisle, IL) . Murine epidermal growth factor (EGF) and human insulin¬ like growth factor I (IGF-I) were obtained from Collabo¬ rative Research (Waltham, MA) . GHR antibody (MAb 363) was obtained from Agen, Inc. (Parsippany, NJ) . General Methods
All HPLC analyses were carried out on a Waters 600 multisolvent delivery system with U6K injector and Model 481 UV detector. All the calorimetric procedures were carried out on a Gilford Spectrophotometer Model 260. Cells were counted with an electronic particle counter (Coulter Electronics, Hialeah, FL) . Protein was assayed by the published method (19) using a micro-BCA kit from Pierce Chemical Co. (Rockford, IL) .
Chemical Methods
Peptides were prepared by the solid phase synthesis method developed by Merrifield (20) using an Applied
Biosystem 431 A automatic peptide synthesizer. Peptides were purified to homogeneity by HPLC with a Waters 600 multi-solvent delivery system. Purity was established by amino acid analysis and N-terminal sequencing with an Applied Biosystems 477A automatic protein sequencer with a 120 PTH detector based on the published method (21) .
Synthesis, amino acid analysis and sequencing were performed in the Microchemistry Core Facility of the
Sloan-Kettering Institute. Laemmli SDS-PAGE gels were run as described (22) . The silver staining method for proteins (23) with formaldehyde fixation to make staining of small peptide fragments more efficient (24) was also employed.
Peptides (1.5 μg of hGH and hGH (1) 108-129) were radio- iodinated by the chloramine T method (25) . Specific activities of radiolabeling were in the range of 400-500 μCi/μg. Unlabeled iodine was removed by chromatography on a Sephadex G50 column. Biological Methods
The procedure for cell culture employing SFM has been described (17) . Cells were grown in a humidified atmosphere of 95% air-5% C02 at 37° C in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% calf serum. For all experiments, exponentially growing cells were employed for inoculation. SFM consisted of basal medium F-12/DMEM (2:1, vol/vol) supplemented with transferrin (10 μg/ml) , fetuin (50 μg/ml) , T3 (100 pg/ml) , EGF (50 ng/ml) , and BSA (1 mg/ml) .
r3H1-thymidine incorporation assay: Cells were inoculated in 6 well dishes (9.6 cm2 area) in a volume of 3 ml at a density of 2 x 104 cells/well. The following day, medium was removed and cells were washed 3 times with HBSS (Hanks Buffered Salt Solution) , and SFM containing various concentrations of peptides were added to cells. Incubations were carried out for 2 days at 37°C, and 3 h prior to cell harvest, cells were labeled with [3H]-thymidine (20 μCi/ml) at 37°C. Cells were solubilized with 0.5% SDS at 37°C for 10 min. Portions of cell lysates were precipitated with 10% TCA and counted by scintillation counting. Radioactivity of each sample was normalized by protein concentration based on A562 using the micro-BCA protein assay. In addition, cells were harvested on day 5 and counted by Coulter counting.
Flow cvtometrv: Cells (106/ml) were detached by trypsin and were harvested by centrifugation at 1,000 x g for 10 min. Cell pellets were suspended in 0.5 ml PBS buffer and then an equal volume of 95% ethanol was added. After fixing overnight at 4°C, cells were harvested at 1,000 x g for 10 min and cell pellets were resuspended in staining solution containing 50 μg/ml propidium iodide and 50 μg/ml RNase A. The propidium iodide staining does not distinguish G0 cells from Gλ cells. The staining reaction continued for at least 2 h at room temperature, and the DNA content of each sample was analyzed by an Epics profile II flow cytometer.
Binding assay: Monolayer cultures of 3T3-F442A cells (2 x 105 cells/well) were washed 4 times with HBSS buffer, and then binding buffer (10 mM Hepes [pH 7.2], 0.1% BSA containing 1 nM of [125I]-hGH (1) 108-129, and indicated amounts of hGH, hGH (1) 108-129, insulin, hGH 113-130, or bGH 7-34 were added. The binding reaction was carried out for 3 h at 4°C on a platform shaker. Unlabeled ligand was removed by washing monolayers with ice cold HBSS buffer. Cell lysates were prepared by using extraction buffer (150 mM NaCl, 20 mM Tris-HCl [pH 8.0], 1 mM MgCl2, 0.1 mM ZnCl2, 0.5% NP40, 2 mM PMSF). Aliquots were saved for protein determination. Bound peptide was measured by counting samples in a gamma counter. Specific binding is defined as the difference between total binding and binding observed at a 1000 fold excess of hGH (1) 108-129.
Affinity cross-linking: Monolayer cultures of 3T3-F442A cells (105/well) were washed 3 times with HBSS buffer. Binding buffer (2 ml) containing [125I]-hGH (1 nM) or [125]-hGH (1) 108-129 (1 nM) was added. The binding reaction was carried out for 3 h at 4°C. Binding was terminated by removing unbound ligand by washing 3 times with HBSS buffer. DSS (0.5 mM) in binding buffer was then added to the cells and the cross linking reaction was carried out at 4°C for 30 min. Cell lysates were prepared using an extraction buffer (20 mM Hepes [pH 7.4], 1% triton X 100, 10% glycerol, 50 mM sodium fluoride, ImM sodium orthovanadate, 10 μg/ml leupeptin, 0.5 mM PMSF)and clarified by centrifugation at 30,000 g for 30 min. Supernatants were boiled for 5 min and were subjected to SDS-PAGE (7.5%). Gels were fixed, dried, and exposed on Kodak XAR-5 film at -70°C.
Western blot analysis of tyrosine phosphorylated proteins.
106 cells were plated out into 60 mm plates. The next day, medium was removed and washed three times with SFM and resuspended in SFM. Either hGH or hGH (1) 108-129 was added to these cells. Cells were stimulated for various times, and cell lysates were prepared by extraction buffer. Cell lysates were cleared by centrifugation at 30,000 g for 30 min. Sample buffer was added to supernatants and 200 μg of cell lysates were loaded onto SDS-PAGE (10%) . Electrophoresed proteins were transferred onto nitrocellulose filter using transfer buffer (20 mM Tris-HCl, 192 mM glycine, 1.5 g/1 SDS, 20% methanol) . Nitrocellulose filter was incubated with blocking solution (1% BSA, 0.2% Tween 20 in TBS) at room temperature for 2 h, and washed 3 times with IX TBS buffer. Nitrocellulose filter was incubated with monoclonal anti-mouse phosphotyrosine Ab(2 μg/ml) for 2 h at room temperature and was washed with TBS buffer 3 times. Alkaline phosphatase conjugated mouse IgG was added to the nitrocellulose filter, and the reaction continued for 2 h at room temperature. To visualize tyrosine phosphorylated proteins, the nitrocellulose was incubated with alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl, Tris HCl (pH 9.5)) containing NBT (50 mg/ml in 70% N-N-dimethylformamide) and BCIP (50 mg/ml in 100% N-N-dimethylformamide) .
RESULTS
After solid phase peptide synthesis all the peptides were homogeneous by HPLC analysis. The amino acid composition and sequences were the same as the published sequences (27-30) (data not shown) .
Mitogenic activity
The mitogenicity of each peptide was evaluated by [3H]- thymidine incorporation and by flow cytometric analysis of cells. Native hGH was anti-mitogenic with an EC50 about 2.0 nM in unstimulated cells (Table I and ref. 26). hGH (1) 108-129 stimulated [3H]-thymidine incorporation about five fold with a maximal effect noted at 0.5 nM (Fig. la). No other peptide on the basis of the hGH or bGH sequence i.e. bGH 7-34, hGH 113- 130, hGH (d) 108-129 or hGH itself was mitogenic based on [3H]-thymidine incorporation in doses up to 1 μM (Fig. la). Since hGH(l) 108-129 is a part of hGH, it was necessary to check whether hGH has mitogenic activity itself. Under the same assay conditions wild type hGH was anti-mitogenic with an EC50 about 0.5 nM (Fig. lb) . This result is quite consistent with our previous report (26) which shows involvement of hGH in fat cell differentiation. It was reported (26) that hGH treated 3T3-F442A cells are resistant to the mitogenic effects of other growth factors including PDGF. The mitogenic effect of hGH (1) 108-129 was not as effective as TGF-/3 which had an EC50 of about 1 pM and was maximally effective at 50 pM (Fig. 1C) . hGH (1) 108-129 had potency similar to IGF-I (Fig. 1C) . Insulin was less potent in stimulating [3H]-thymidine incorporation, although IGF-I was more potent than insulin (Fig. 1C) . These mitogenicity studies under SFM conditions were also the same with 10% FCS.
To investigate the possibility that the mitogenic response to hGH (1) 108-129 was mediated by secretion of
IGF-1, 3T3-F442A cells were incubated with the peptide in the presence of hGH (1) 108-129 as well as IGF-1. Although both peptides individually gave a strong mitogenic response, the presence of an antibody to IGF-1 blocked only the IGF-1 response without effect on the mitogenesis induced by hGH (1) 108-129 (data not shown) .
Since hGH and hGH (1) 108-129 show opposite effects on cellular growth, it was interesting to check whether hGH treated 3T3-F442A cells are resistant to the mitogenic effects of hGH (1) 108-129. Table 1 shows [3H]- thymidine incorporation assays in 3T3-F442A cells treated with hGH and/or hGH (1) 108-129. As expected both hGH and hGH (1) 108-129 show opposite effects when incubated separately with cells. When incubated together in molar ratios (hGH:hGH (1) 108-129) of 2:1 and 20:1, the mitogenic response to 0.5 nM hGH (1) 108- 129 was completely offset and the cells manifested the prevailing anti-mitogenic response of hGH (Table I) . Although not intending to be bound by any theory it is believed that this might be due to possible competition between hGH and hGH (1) 108-129 for binding to the same receptor. In this assay it seems clear that hGH (1) 108-129 is not an antagonist because it does not affect the anti-mitogenic activity of hGH in 3T3-F442A cells. This also confirms the antimutagenic effect of hGH since it prevents the mitogenic effect of hGH (1) 108-129. To examine whether the mitogenic effects of hGH (1) 108- 129 are accompanied by a cell cycle transition in 3T3- F442A cells, cell cycle analysis was carried out by using a FACS analyzer. As expected, cells maintained in SFM are primarily in the G1/G0 phase (Figure 2A) . Cells grown with 10% FCS showed 45% of the cells in S/G2/M phases (Figure 2B) . hGH (1) 108-129 (0.5 nM) increased the number (28%) of cells in S/G2/M phases (Figure 2C) . hGH (d) 113-130 did not cause any shift in the cell cycle phases in 3T3-F442A cells as is seen in Figure 2D. Interaction of hGH (1) 108-129 with GH receptor of 3T3- F442A cells
Since hGH and hGH (1) 108-129 show opposite effects on cellular growth, it was necessary to check whether these two bind to the same receptor. Affinity cross linking of [125I]-hGH and [125I]-hGH (1) 108-129 was used to study further the relationship of the binding sites for the peptides (Fig.3). [125I]-hGH was cross-linked to a protein of 115 kDa, identified as the GHR by Western analysis with a GHR monoclonal antibody (lane 1) . Cross-linking was completely inhibited by an excess of hGH(lane 2). [125I]-hGH (1) 108-129 was cross-linked to a protein of the same size, and cross-linking was blocked by an excess of hGH (1) 108-129 (lanes 3 and 4 respectively) . hGH and hGH (1) 108-129 were also able to inhibit cross-linking of the other peptide (lanes 5 and 6 respectively) . These results suggest that hGH and hGH (1) 108-129 share the same receptor.
Binding characteristics of hGH (1) 108-129
A series of experiments was carried out to determine the binding characteristics of the synthetic peptide hGH (1) 108-129 to 3T3-F442A cells (Figure 4). The IC50 (concentration of 50% inhibition of binding) of [125I]- hGH (1) 108-129 was between 10-20 nM. Binding was effectively competed for by an excess of hGH and hGH (1) 108-129, but not by bGH 7-34, hGH 113-130 or insulin. hGH (1) 108-129 binding also showed saturation kinetics (data not shown) . The optimal concentrations of hGH and hGH (1) 108-129 for their effect on cellular growth were lower than those of the Kd values suggesting GHR does not have to be fully occupied i.e. there are spare receptors.
Since it is apparent that hGH (1) 108-129 acts through GHR, it was of interest to determine whether a possible block in binding to GHR is associated with a defect in the mitogenic response to hGH (1) 108-129. Indeed in molar ratios of 2 to 1 to 20 to 1, the mitogenic response to 0.5 nM was completely blocked and the cells manifested the prevailing anti-mitogenic response of hGH (Table I) . Monoclonal mouse anti-GHR Ab(MAb 263) inhibited the mitogenic activity of hGH (1) 108-129 suggesting that the GHR is important in mediating the mitogenic activity of hGH (1) 108-129 (Table II) . MAb 263 also inhibited the anti-mitogenic activity of hGH when incubated together with the hormone indicating the importance of GHR in mediating the actions of hGH (data not shown) .
By Scatchard analysis, hGH had a binding affinity of approximately 0.2 nM and approximately 28,000 binding sites on 3T3-F442A cells (data not shown) . Under similar conditions hGH 108-129 bound with an affinity of approximately 5 nM and had 560,000 binding sites per cell (Fig. 5). Affinity cross-linking of [125I]-hGH to its receptor was not duplicated with [125I]-hGH 108-129.
To determine whether hGH and hGH 108-129 bound to the GHR, competitive binding studies were performed in the absence and presence of the anti-GHR antibody, MAb 263.
It was noted (Fig. 6) that MAb 263, in a dose related fashion from 10~12 to 10"6 μg ml inhibited binding of
[125I]-hGH to 3T3-F442A cells with complete inhibition of binding at concentrations of MAb 263 of 10~6 μg/ml and greater (Fig. 6) . In contradistinction there was no dose related inhibition of binding of [125I]-hGH 108-129 in any concentration range of MAb 263 between 10"7 and
10"3 μg/ml and the change in binding was generally less than 50% (Fig. 7) . Induction of tyrosine phosphorylation in 3T3-F442A cells treated with hGH and hGH (1) 108-129
hGH has been known to cause tyrosine phosphorylation of GHR. Tyrosine phosphorylated GHR is believed to transduce its signal to the cells. This transduction of the GH signal by GHR is known to cause cellular tyrosine phosphorylation of several molecules such as MAPII kinases. To check whether hGH and hGH (1) 108-129 both cause induction of tyrosine phosphorylation of cellular molecules, Western blot analysis using monoclonal anti- mouse phosphotyrosine Ab was carried out. As is seen in Fig. 6, both hGH and hGH (1) 108-129 were shown to increase tyrosine phosphorylation of cellular molecules most notably the 42 kDa molecule. Tyrosine phos¬ phorylation of cellular molecules became evident after 10 min. of stimulation with hGH or hGH (1) 108-129. The fact that the 42 kDa molecule was phosphorylated by these two different peptides suggests that there might be different molecular events beyond tyrosine phosphorylation to explain the opposite effects displayed by these two peptides. The optimal concentration of hGH (1) 108-129 for inducing tyrosine phosphorylation was about 1 nM, slightly higher than the optimal concentration for [3H]-thymidine incorporation.
Based on affinity cross linking and binding assays, it was apparent that hGH and hGH (1) 108-129 act through GHR. Therefore it was necessary to check whether tyrosine phosphorylation of cellular molecules induced by these two peptides is mediated through the action of GHR. To do this, a blocking experiment using monoclonal mouse anti-GHR Ab was carried out. Briefly 3T3-F442A cells were pre-incubated with MAb 263 for two hours in binding buffer at 4°C, and either h'GH or hGH (1) 108-129 was added to these cells. Western blot analysis using monoclonal anti-phosphotyrosine Ab was carried out according to standard methods. Figures 8A and 8B show that in the presence of MAb 263, tyrosine phosphorylation of the 42 kDa molecule induced by, hGH (1) 108-129 (Figure 8A) or hGH (Figure 8B) was completely inhibited. These results suggest that hGH and hGH (1) 108-129 act through GHR.
DISCUSSION
It would appear that the mitogenic activity of hGH (1) 108-129 is mediated through GHR in 3T3-F442A cells. Affinity cross linking, competitive binding assays and antibody blocking reactions all implicate GHR in mediating the mitogenic activity of hGH (1) 108-129. The binding was specific and saturable in the nanomolar (10-20 nM) range in contrast to the sub-nanomolar range for its mitogenic activity. This indicates the existence of spare receptors for hGH (1) 108-129 on 3T3- F442A cells.
It is of note to compare the binding to 3T3-F442A cells with different biological responses. hGH binds to 3T3- F442A cells with half saturating concentrations of 0.77 - 1.8 x 10"9 (31) with an EC50 for an adipogenic response of about 0.1 nM (32). The anti-mitogenic effect occurs with an EC50 of approximately 0.05 nM (26). This indicates that the half-maximal anti-mitogenic response occurs when approximately 5% of the sites are occupied. In the present study, hGH (1) 108-129 binding is 5.2 nM, and the EC50 for mitogenesis is approximately 0.2 nM, when only 1% of the receptors are occupied, i.e., suggesting that there are spare receptors. It is of note that hGh (1) 108-129 has a Kd of binding of 5.2 nM compound to a value of about 0.2 to 1 nM for hGH (32). This lower affinity for the smaller peptide would be consistent with binding at site 1 and site 2 of hGH and only at site 1 residues of hGH present in hGH (1) 108- 129. The lower affinity is not soley due to the size of the fragment since we have found that the 38 amino acid sequence bGH 96-133 binds with a Kd of approximately 100 nM with corresponding mitogenic activity (unpublished results) .
The numbers of binding sites (Bmax) determined by Scatchard analysis of [125I]-hGH and [125]-hGH (1) 108- 129 binding are 28 X 103 and 560 X 103. There may also be several species of GHR,s to which hGH and hGH (1)
108-129 bind differently. It is also possible that hGH
(1) 108-129 binds to a receptor other than GHR. This would be consistent with the fact that hGH with a Kd of about 0.2 to 1 nM is less effective than hGH (1) 108-129 with a Kd of 5.2 nM in competing for binding of [125I]- hGH (1) 108-129 (Fig. 4).
Based on competition assays, it would appear that non- mitogenic peptides do not bind to GHR because these non- mitogenic peptides were not able to block binding of hGH (1) 108-129. The mechanism of mitogenicity by this synthetic peptide is not clear yet since the immediate consequence(s) of ligand binding to GHR in 3T3-F442A cells is not known. It is possible that mitogenic activity is preceded by the induction of early response genes in 3T3-F442A cells as suggested by the cell cycle analysis (Fig. 2). The effect of hGH (1) 108-129 on cell cycle phases was shown by FACS analysis. Since GHR itself does not have a consensus tyrosine kinase domain (33) it is difficult to attribute the mitogenic activity to tyrosine phosphorylation by GHR as a protein kinase. However GHR-mediated tyrosine phosphorylation involves an associated tyrosine kinase (34) . This hypothesis is consistent with the fact that the receptors for NGF (35) , CD4 (36) , and CD8 (37) complex with a family of tyrosine kinase proteins. From our affinity labeling and binding studies it would appear that hGH (1) 108-129 binds to GHR. This has been identified by Western analysis with a GHR monoclonal receptor antibody MAb 263 as a 115 kDa protein. When affinity labeled with radioiodinated GH the molecular weight increases to 130 kDa. The difference in the molecular weight (115 kDa) of GHR identified by Western analysis with GHR MAb 263 and that deduced by the GHR transcript as 70 kDa can not be accounted for by deglycosylation (MR-95kDa) (38) .
Binding of [125I]-hGH (1) 108-129 to GHR is presumably through site 2 of hGH (16,39,40). We have no data that suggest that hGH (1) 108-129 can induce dimerization of GHR as occurs with one molecule of wild type hGH which contains both site 1 and site 2 (16,39,40). This would suggest that the biological effects of hGH (1) 108-129 are due to hGH site 2 binding to GHR without dimerization of the latter. The affinity labeling and binding with cells in the present study reflect binding to GHR in the plasma membrane and not GHBP where dimerization was noted (16,39,40). Others have noted (41) SDS-PAGE patterns suggestive of dimerization after affinity labeling of GHR in 3T3-F442A cells. Further studies are necessary to establish this possibility.
We have previously demonstrated that native bGH and hGH are anti-mitogenic in 3T3-F442A cells (26) . We attributed this activity as important in establishing Gp, a differentiation-primed state. The present study is consistent with the fact that hGH (1) 108-129 is mitogenic in 3T3-F442A cells and does not induce a primed state, as we have demonstrated by its inability to promote adipogenesis with insulin.
There are no reports from this study or elsewhere to indicate tht hGH 108-129 is derived from natural sources either by de novo biosynthesis or biodegradation of hGH. There is, however, reason to study the biological and biochemical properties of helix 3 of GH. Sequences related to this helicogenic peptide are of interest as isolated peptides or within the larger polypeptide of GH. In addition to chemically synthesized hGH 108-129, hGH 95-133 and bGH 96-133, prepared by chemical synthesis or enzymatic digestion, have proliferative activity, albeit at lower doses (unpublished data) . In addition, modification of sequences within helix 3 of GH by site directed mutagenesis is associated with changes in growth promoting (11, 14, 42, 43) and binding activity (13). Our system affords the unique opportunity for the study of GH structure and function since native hormone and helix 3 of GH elicit opposite effects on cellular proliferation.
hGH 108-129 Bioassay
Rats (55-62 gm) were hypophysectomized and weighed on a daily basis. On the 14th day following surgery, rats which were 3 gm or less than original body weight or 12 gm or more than original body weight were discarded (less than 5% or less of total population of hypophysectomized rats) . The remaining rats were randomly assigned to the specified treatment groups (8 rats per group) . Rats were injected once daily for 10 days with either 1 ml saline, 24 mg hGH 108-129 in 1 ml saline or 0.16 mg human growth hormone (hGH) in 1 ml saline. Rats were sacrificed after 10 days of injection, 24 hours after last injection. Both left and right tibial were harvested and stained for epiphyseal plate width measurement under low power magnification. Tibiae were measured in a random order. Means were evaluated statistically by one way analysis of variance (ANOVA) . Results shown in Figure 9 demonstrate that hGH 108-129 is growth promoting. Table I [3H]-Thymidine Incorporation with hGH and hGH (1) 108-129 alone or in Combination
Substance Cone (nM) % of Control
SFM - 100 hGH 1 64 ± 14 5 40 ± 8
10 16 ± 9.7
hGH (1) 108-129 0.5 520 ± 100
hGH/hGH (1) 108-109 1/0.5 63 ± 21
5/0.5 21 ± 11
10/0.5 13 ± 2
3T3-F442A cells were incubated with hGH or hGH (1) 108- 129, either alone or in combination at the indicated concentrations. [3H]-thymidine incorporation was done as detailed in Fig. lc legend. Results (± S.E.) are compared to SFM control.
Table II Effect of Anti-GHR Monoclonal Antibody on Stimulation of [3H]-thymidine Incorporation by hGH (1) 108-129
[3H] -thymidine Incorporation
Substance Cone % of Control SFM 100 ± 130 hGH ( 1) 108-129 0. 1 nM 540 ± 150
1. 0 nM 590 ±
hGH (1) 108-129/MAb 263 0.1 nM/1 μg/ml 112 ± 20
1 nM/lkμg/ml 125 ± 40
3T3-F442A cells were incubated with hGH (1) 108-129 alone or in the presence of the anti-GHR monoclonal antibody (MAb 263) . [3H]-thymidine incorporation was performed as described in Fig. lc legend. Results are expressed as control of SFM.
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Claims

What is claimed is:
1. A polypeptide having the sequence from the amino terminal to the carboxy terminal:
Ser Asp Val Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly He Gin Thr Leu Met Gly Arg Leu Glu.
2. A composition comprising an amount of the polypeptide of claim 1 effective to promote the growth of an animal and a physiologically acceptable carrier.
3. A method of treating a subject having a disease characterized by an insufficient amount of undifferentiated cells comprising administering to the subject an amount of the polypeptide of claim 1 effective to decrease the amount of undifferentiated cells in the subject.
4. A method of treating a subject having a disease characterized by an abnormally low amount of tissue growth comprising administering to the subject an amount of the polypeptide of claim 1 effective to promote the growth of tissue in the subject.
5. A composition comprising an amount of the polypeptide of claim 1 effective to inhibit the growth of transformed dedifferentiated cells in an animal and a physiologically acceptable carrier.
6. A method of inhibiting the growth of transformed dedifferentiated cells comprising contacting the transformed dedifferentiated cells with the polypeptide of claim 1.
7. A composition comprising an amount of the polypeptide of claim 1 effective to increase the ratio of weight gain to feed consumption in animals and a physiologically acceptable carrier.
8. A method of increasing the ratio of weight gained to feed consumed in an animal which comprises administering to the animal an amount of the polypeptide of claim 1 effective to increase the ratio of weight gained to feed consumed by the animal.
9. A method of treating an animal afflicted with a cancer which comprises administering to the animal an effective amount of the composition of claim 5.
10. A method of claim 9, wherein the cancer is breast cancer.
PCT/US1993/010088 1992-10-22 1993-10-21 GROWTH HORMONE FRAGMENT hGH 108-129 WO1994008611A1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4056520A (en) * 1972-03-31 1977-11-01 Research Corporation Clinically active bovine growth hormone fraction
US4342832A (en) * 1979-07-05 1982-08-03 Genentech, Inc. Method of constructing a replicable cloning vehicle having quasi-synthetic genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4056520A (en) * 1972-03-31 1977-11-01 Research Corporation Clinically active bovine growth hormone fraction
US4342832A (en) * 1979-07-05 1982-08-03 Genentech, Inc. Method of constructing a replicable cloning vehicle having quasi-synthetic genes

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