Classifications

• • 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 hormones [GH] (Somatotropin)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

• This invention relates to analogs of animal somatotropins or growth hormones. More specifically, the invention relates to novel bovine growth hormone analogs, particularly with changes in amino acid residue 99.
• Bovine somatotropin is a growth hormone that has been well studied (Paladini, A.C. et al., CRC Grit. Rev. Biochem. 15:25-56 (1983)). Somatotropins were originally discovered in pituitary gland extracts from various animals. In general, somatotropins are conserved molecules and similarities in amino acid sequences and structure are found between different species of animals.
• Somatostatins including bSt, are globular proteins comprising a single chain of about 200 amino acids with two intramolecular disulfide bonds.
• bSt has a single 190-191 amino acid chain, a globular structure with two intramolecular disulfide bonds and a molecular weight of about 22,000 daltons.
• Natural bSt extracted from pituitary glands is heterogeneous. At least six major forms of the protein have been described. The longest form has 191 amino acid residues and an ala-phe amino terminus. The second form has 190 amino acid residues and a phe amino terminus. The third form has 187 amino acid residues and a met amino terminus. The remaining three forms of bSt substitute valine for leucine at position 127. In addition to this heterogeneity, undefined heterogeneity of bovine somatotropin has also been described (Hart, I.C. et al., Biochem. J. 218:573-581 (1984); Wallace, M. and Dickson, H.B.F., Biochem. J. 100:593-600 (1965)).
• Bovine somatotropin produced by recombinant microorganisms is important commercially. It increases lactation in dairy cattle and increases size and meat production in beef cattle. It is estimated that upwards to 20 mg per animal per day will be needed to effect commercially acceptable improvements in production. Such a dosage will require efficient methods of administration. Improvements in the potency and stability of bSt such as described in this invention will be of benefit because of resulting reductions in the amount of drug administered to each animal per day.
• one of the problems in preparing recombinantly-produced bSt is that liquid processing and storage of rbSt at acid or alkaline pH results in the conversion of the asparagine residue at position 99 to isoaspartic acid.
• the resulting rbSt is referred to as "early eluting rbSt" because it elutes earlier than native rbSt on reversed phase HPLC.
• Isoaspartate is formed when the asparagine side chain condenses with the peptide backbone resulting in the elimina- tion of ammonia. Chain cleavage also occurs by a condensation reaction between the peptide backbone and the asparagine residue at position 99 of the rbSt molecule upon storage.
• the chain-cleaved product is covalently held together by the disulfide bond between cysteine residues 53 and 164 and has been called "early-early eluting rbSt" because of its eluting position relative to native and early eluting rbSt.
• P.Y. Chou and G.D. Fasman Ann. Rev. Biochem., 47, pp. 251-76 (1978) refer to the use of amino acid sequences to predict the secondary and tertiary structure of proteins.
• This invention relates to the enhancement of bioactivity or stability in liquid storage, or both, of bSt and analogs thereof, by substituting different amino acids for the asparagine corresponding to the residue at position 99 of native bSt. Similar changes can be made in somatotropins from other animals, particularly mammals, including porcine, fish, ovine, horse, rat, monkey, and human.
• bSt-like compounds wherein the asparagine located at amino acid residue 99 is replaced with a different amino acid residue including specifically glycine, serine, proline, aspartic acid, glutamic acid, serine-serine or serine-aspartic acid.
• animal somatotropin is selected from the group consisting of bovine, porcine, fish, ovine, horse, rat, monkey, and human somatotropins.
• the animal somatotropin is bovine somatotropin.
• Also provided is a method for increasing milk production in a female ruminant comprising administering to the female ruminant an effective amount of an animal somatotropin of the instant invention, and, in particular, where the animal is a bovine and the somatotropin is bSt.
• somatotropins Due to the molecular heterogeneity of somatotropins, the position numbers of amino acid residues of the various somatotropins may differ.
• the term "native mammalian somatotropin" includes these naturally occurring species. Chart 1 illustrates the specific region of one species of bSt that corresponds to the position 99 residue modified by this invention. The numbering for other somatotropins may differ where other species or analogs are involved.
• the preferred genetic modifications rely upon single site specific mutation methods for insertion of various amino acid residues in replacement of the naturally occurring asparagine.
• animal somatotropin refers to somatotropins originating from animals, e.g., mammals, and includes somatotropins derived from either natural sources, e.g., pituitary gland tissue or from microorganisms transformed by recombinant genetics to produce a naturally-occurring form of somatotropin.
• a specific mammalian source such as a bovine somatotropin or a somatotropin of bovine origin
• the somatotropin includes those derived from either natural sources or from transformed microorganisms.
• microorganism is used herein to include both single cellular prokaryotic and eukaryotic organisms such as bacteria, yeast, actinomycetes and single cells from higher plants and animals grown in cell culture.
• somatotropins refers to naturally-occurring forms of somatotropins which may have been derived from either natural sources, e.g., pituitary gland tissue or from microorganisms transformed by recombinant genetics to produce a somatotropin having the same amino acid sequence as the naturally-occurring form of somatotropin.
• the mammalian somatotropins are very similar in amino acid sequence and physical structure. Although the processes described in the Examples are directed toward bSt, the processes are equally applicable to any animal, e.g., mammalian somatotropin having the requisite asparagine residue available for replacement particularly wherein similar liquid processing and storage problems are encountered.
• the high relative potency of the bSt analogs of the present invention is readily determined using hypophysectomized rats. Evans, H.M. and Long J.A., Anat. Rec. 21:61, 1921. Relative increases in total body weight are recorded using pituitary bSt, recombinant bSt (rbSt) and various bSt analogs of the invention.
• Site-Directed Mutagenesis Several techniques for site-directed mutagenesis have been developed for introducing specific changes in a DNA sequence and may be used to produce the compounds of the instant invention (Kramer, W., et al, Nucl. Acids Res., 12, pp. 9441-56 (1984); Mandecki, W., Proc. Natl. Acad. Sci. USA, 83, pp. 7177-81 (1986); Zoller, M.J. and Smith, M., Nucl. Acids Res., 10, pp. 6487-6500 (1982); Norrander, J., et. al., Gene, 26, pp. 101-106 (1983); Kunkel, T.A., Proc. Natl. Acad.
• Colony Filter Hybridization The screening technique of filter hybridization is based upon the ability of a single-stranded segment of DNA to locate its complementary sequence and hybridize to form a double-stranded segment, Hanahan, D. and Meselson, M., Meth. Enzymol., 100, pp. 333-42 (1983).
• the thermal stability of this binding is dependent upon the number of matches and mismatches contained within the double stranded region. The more mismatches it contains, the weaker the base-pair binding and the lower the temperature necessary to disrupt the DNA binding. This temperature differential is exploited during colony filter hybridization, Bryan, R., et. al., Microbiology (1986).
• nitrocellulose filters are overlayed onto plates and wetted. The filters and plates are marked for orientation and the filters are then carefully lifted off the plates. The master filter plates are incubated overnight at room temperature to allow re-growth of the colonies.
• the filters are denatured by laying them one by one onto Whatman paper soaked in 0.5 M NaOH, 1.5 M NaCl for 10 minutes and neutralized in two successive changes of Whatman paper soaked in 1 M Tris, pH 7.4, 1.5 M NaCl, for 10 minutes each and air dried on fresh Whatman paper for 30 minutes. They are then baked for 2 hours at 80oG in vacuum.
• the kinase reaction to radiolabel the mutant oligonucleotide for use as a probe is as follows: 2 ⁇ g of oligo, 2 ⁇ l of 10X kinase buffer, 100 ⁇ Ci ⁇ 32-P ATP, 2 ⁇ l T4 kinase and 4 ⁇ l water are mixed and incubated for 1 hour at 37oC. A 1 ml column is packed with DEAE-Sephacel in a 10 ml disposable column and equilibrated with 2-3 ml of high salt buffer (1.5M NaCl in TE) and then 2-3 ml of low salt buffer (0.2M NaCl in TE).
• the kinase reaction is diluted with 200 ⁇ l of low salt buffer and loaded directly into the column.
• the column Is washed with 10 ml of low salt buffer until no further counts elute from the column.
• the probe is eluted in 4 ml of high salt buffer.
• the filters are placed in a crystallization dish and batch pre-hybridized in 5X Denhardts (1% BSA, 1% Ficoll and 1% PVP) , 5X SSC (0.75 M NaCl, 0.075 M sodium citrate) and 0.1% SDS for 1 hour at 40oC.
• the hybridization solution is changed and the probe is added.
• the dish is covered and the hybridization done overnight with gentle agitation.
• the filters are then rinsed with several changes of 5X SSC, 0.1% SDS.
• the filters sit in this solution while the water bath and wash solution (5X SSC, 0.1% SDS) Is heated up to washing temperature (46oC).
• the filters are transferred one by one to a fresh crystallization dish and washed 3 ⁇ 20 minutes, changing dishes after each wash. They are then air dried on Whatman paper, wrapped in Saran wrap and exposed as necessary.
• Vector DNA Preparation DNA for sequencing is obtained according to the method of L. Agellon and T. Chen, Gene Anal. Techn., 3, pp. 86-89 (1986) except that only one combined phenol/chloroform extraction is performed and the DNA is not spin-dialyzed through a Sephadex G-50 column.
• Double-stranded sequencing is performed according to the following protocol: 3 ⁇ l 2N NaOH, 2 mM EDTA is added to 12 ⁇ l of DNA (2 ⁇ g) and incubated for 15 minutes. 6 ⁇ l 3 M NaOAc, 1 ⁇ l primer and 100 ⁇ l 95% ethanol are added and the - DNA precipitated on dry ice for 20-30 minutes. The pellet is collected, washed and vacuum dried. It is dissolved in 13 ⁇ l water and 4 ⁇ l RT buffer (0.3 M Tris-HCl, pH 8.3, 0.375 M NaCl, 37.5 mM MgCl 2 , 2.5 mM DTT), 2 ⁇ l ⁇ 32P dCTP and 1 ⁇ l reverse transcriptase are added.
• 4 ⁇ l of this mix is pipetted into 4 eppendorf tubes, each containing 1 ⁇ l of G mix, A mix, T mix or C mix. The tubes are incubated for 10 minutes at 42°C. 1 ⁇ l of chase mix (0.25 mM dNTPs) is added and they are incubated for an additional 5 minutes. 10 ⁇ l stop solution (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol and 0.1% bromphenol blue) is added, the reactions are boiled 3 minutes and 3 ⁇ l of each is loaded onto a sequencing gel.
• 1 ⁇ l of chase mix (0.25 mM dNTPs
• 10 ⁇ l stop solution 80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol and 0.1% bromphenol blue
• a site-directed mutagenic technique for double-stranded primer extension is used to introduce altered codons for serine and proline at amino acid position 99 in the rbSt cDNA m4 gene (PCT patent application PCT/US 88/00328, filed 27 January 1988 and incorporated herein by reference).
• the target sequence is cloned into a suitable plasmid and plasmid DNA is prepared.
• the plasmid DNA is denatured by treatment with NaOH which causes "nicks" in the DNA molecule deoxyribose-phosphate backbone. This relaxes the DNA and permits an oligomer containing the desired sequence changes to hybridize to the plasmid sequence containing the position 99 residue of bSt.
• the 3' end of the oligomer generates a primer for the DNA polymerase activity of the reverse transcriptase which extends the primer, synthesizes a new DNA strand containing the mutagenic oligomer and displaces the normal complementary strand.
• the extension reaction increases the probability of the incorporation of the oligomer-directed change.
• the DNA is transformed into competent cells and the resultant colonies are screened by colony filter hybridization. Plasmid DNA is isolated and sequenced from positive candidates.
• the oligomers used to construct the position 99 serine and proline changes in the rbSt m4 gene are produced by techniques previously described (PCT/US 88/00328) .
• An oligonucleotide so produced and designated CST-88 (Chart 3) contains the change on the DNA sequence asparagine AAC to serine TCT and another designated CST-89 (Chart 3) contains the asparagine AAC to proline CCG change. They are both designed with a Tm of 53oC thus allowing for hybridization at 40oC and stringent washing at 46oC as set forth above.
• the serine and proline oligomers are hybridized to the pBR322 derived vector pTrp-BStm4.
• This vector contains the trp promoter and the m4 cDNA for rbSt (PCT/US 88/00328).
• the DNA is used to transform competent cells of MC1000 (available in the Experiments with Gene Fusion Strain Kit, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
• the transformed cells are plated to give 100-200 colonies on ten plates. The colonies are then transferred onto nitrocellulose.
• the cells are lysed and their DNA affixed to the filter.
• Radiolabeled oligomer probes are prepared from CST-88 and CST-89 by the oligomers with kinase and 32P-ATP. Details of the colony filter hybridization probing are set forth above.
• the m4-99ser and m4-99pro genes are excised from the parental vector as an EcoRI-Hindlll fragment and cloned into the EcoRI-Hindlll restriction sites of the pURA-m4 vector (PCT/US 88/00328).
• Chart 2 shows the cloning of the m4-99ser gene into the pURA-m4 vector. The identical construction is carried out for the m4-99pro gene.
• the vectors Upon sequence confirmation of the clonings, the vectors are designated pURA-99Ser and pURA-99Pro. These vectors are transformed into fermentation expression strain BST-1C (PCT/US 88/00328).
• Transformed cells from each of the clonings are induced and samples analyzed by SDS-PAGE to assess the ability of the cells to express the modified rbSt genes under non-optimized conditions.
• Results of SDS-PAGE analysis showed that pURA-99Ser produced rbSt in three individual inductions at 13.8%, 14.6% and 26.4% of total cellular protein.
• Results of SDS-PAGE analysis showed pURA-99Pro in four separate inductions expressed rbSt at 28.2%, 29.6%, 34.0% and 41.9% of total cellular protein.
• the mutant oligomer Because of poor mutagenic efficiency, the mutant oligomer must be constructed so that there is at least a 5°C temperature difference between its Tm and that of the native sequence. Without this difference, effective screening of the candidates cannot be accomplished.
• Analogs having two amino acids substituted in place of the position 99 asparagine were constructed by following the site directed protocol of Kramer et al, Nucl. Acids Res., 12, p. 9441-56 (1984) as described in the "Site Directed Mutagenesis Kit", commercially available from Boehringer Mannheim Biochemicals, PO Box 50816, Indianapolis, IN 46250 (see also, Kramer, W. and H-J Fritz, Meth. Enz., 154, pp. 350-67 (1987)). The procedure requires cloning the DNA sequence which is to be modified into the M13mp9 vector.
• Single stranded DNA is isolated using the derived vector M13mp9-m4 (Messing, supra) and used in the mutagenesis procedure as previously described. Cloning of these analogs into the pURA expression vector was done as described in Example 1, in which the double-stranded DNA of the M13 vector, supra, is substituted for the pBR322 vector.
• a position 99 analog having glycine substituted for asparagine was also constructed using the techniques described in Example 1.
• the mutagenesis oligonucleotide used is designated C-ST84 (Chart 3). It encodes the change to glycine.
• FCM fat corrected milk yield
• cows were assigned randomly in replicates, based upon milk yield, to 9 experimental groups: no injection (Control), 5 mg and 20 mg Gly-99 rbSt daily, 5 ag and 20 mg Ser-99 rbSt daily, 5 mg and 20 mg Asp-99 rbSt daily, and 5 mg and 20 mg native rbSt daily.
• Cows were injected imtramuscularly in the semitendinosus muscle once daily for 21 days. Twice daily milk weights were recorded for three days prior to initiation of injection, during the 21 days of injections, and for five days after the last injection. Concentration of rbSt was expected to be 10 mg/ml.
• the concentration of residual post-injection rbSt solutions measured by HPLC, averaged 11.5 for native rbSt, 12.1 for Asp-99 rbSt, 11.6 for Gly-99 rbSt, and 11.6 for Ser-99 rbSt.
• the area percent was greater than 99% normal rbSt for Gly-99 rbSt (by "normal rbSt" Is meant the amino acid sequence encoded by a particular gene, i.e., no degradation products, etc.), for Ser-99 rbSt, and for Asp-99 rbSt, but averaged 90.5 normal rbSt for native rbSt.
• Native rbSt averaged 2% early-early eluting rbSt and 7.6% early eluting rbSt.
• cows were assigned randomly in replicates, based upon milk yield, to 7 experimental groups: no injection (Control), 5 mg and 15 mg Pro-99 rbSt daily, 5 mg and 15 mg Glu-99 rbSt daily, and 5 mg and 15 mg native rbSt daily.
• Cows were injected imtramuscularly in the semitendinosus muscle once daily for 7 days. Twice daily milk weights were recorded for three days prior to initiation of injection, during the 7 days of injections, and for five days after the last injection.
• Cows assigned to receive 15 mg rbSt received 15.12 mg, 14.64 mg, and 15.10 mg rbSt for cows of the Pro-99, Glu-99, and native rbSt respectively.
• the percentage of early-early eluting rbSt, early eluting rbSt, oxidized rbSt, and post-oxidized rbSt appeared to be similar for native rbSt, Glu-99 rbSt, and Pro-99 rbSt.
• FCM was significantly greater for cows of the 15 mg rbSt group than for cows of the 5 mg rbSt group.
• FCM of rbSt previously injected cows decreased at similar rates during the 5 days after cessation of rbSt injections.
• the rats were monitored for a 12-day preliminary period to allow for adaptation to environmental and feeding conditions. Body weights were obtained on four occasions between days one and twelve. Rats that showed weight losses of one gram per day or less or weight gains of 2 grams per day or less during the preliminary period were selected for injection with rbSt. The rats were ranked according to ascending magnitude of average daily gain (ADG), and seven blocks of 25 rats were created. Treatments within each block were randomly assigned.
• ADG average daily gain
• rbSt treatments lasted 10 days. During this time, test compounds were injected twice daily for nine days, and body weights were monitored using a Mettler Model 3600 top loading balance equipped with lab-Pac programming which determines the weight of the animals while taking into account their movements.
• Stock solutions of rbSt at 2 mg/ml were prepared in a buffer of 0.03 M sodium bicarbonate and 0.15 M NaCl at pH 9.5. To facilitate suspension of rbSt, the lyophilized preparations were first dissolved in this buffer at pH 10.8, then the pH was adjusted to 9.5 using 2 N HCl and brought to final volume using the stock buffer at pH 9.5 and filtered if necessary.
• the stock solutions were diluted using stock buffer (pH 9.5) to solutions of 37.5, 75, 150, and 300 mg of protein/ml.
• the rats were injected subcutaneously twice daily with 100 ⁇ l of the respective solutions, and controls received 100 ⁇ l of buffer.
• the experiment lasted 10 days with average daily weight gain monitored.
• the first set of incubations was carried out in 50 mM sodium carbonate, pH 10.0 at ambient temperature (protein concentration 20 mg/ml) to simulate conditions similar to those encountered during the Isolation and formulation of rbSt.
• the second set of incubations was carried out in a pH 7.4 Ringer's solution at 37oC (protein concentration 5 mg/ml) to simulate exposure to physiological conditions. All incubation samples were prepared aseptically. Samples were removed from the incubations over a three week period and stored at -20oC prior to analysis by isoelectric focusing, SDS-PAGE, and reversed phase HPLC.
• the glycine 99 and serine 99 rbSt analogs had IEF patterns that were virtually indistinguishable from native rbSt prior to incubation while the IEF pattern of native asparagine 99 rbSt was shifted approximately 1.2 pH units lower due to the introduction of the negative charge at position 99.
• the major difference noted between the IEF patterns of incubated samples of native rbSt and the position 99 analogs was the rate at which more acidic rbSt species were formed.
• the IEF pattern of native rbSt degraded into more acidic rbSt species at a faster rate under both sets of incubation conditions than did the IEF patterns of the position 99 analogs.
• Reversed phase HPLC was used to measure the amount of isoaspartate formation and chain cleavage which occurred at position 99 of the rbSt molecule since these species elute earlier upon reversed phase HPLC than native rbSt.
• rbSt which contained a chain cleavage between positions 99 and 100 of the rbSt molecule accounted for 8.5 area percent of native rbSt at the conclusion of the pH 10.0 incubation, and for 11.1 area percent of native rbSt at the conclusion of the pH 7.4 incubation.
• the amount of the rbSt analogs which eluted in this position was less than 0.5 area percent.
• the asparagine residue corresponding to bSt asparagine 99 is likely the third residue in a ⁇ -turn in each of these other somatotropins thus leading to isoaspartic acid formation and chain cleavage as with bSt. If this is found to be the case for other somatotropins by analysis of the products as set forth above, substituting an appropriate amino acid for the asparagine 99 would obviate the isoaspartate formation and chain cleavage.
• Administration of the bSt analogs into dairy cattle is according to known methods using any route effective to deliver the required dosage to the animal's circulatory system.
• Modes of administration include oral, intramuscular injections, subcutaneous injections and the use of timed-release implants.
• the preferred mode of administration is by subcutaneous injection using a timed-release implant.
• Appropriate vehicles for injections include physiologically compatible buffers such as sodium bicarbonate, sodium phosphate, or ammonium phosphate solutions.
• Timed-release implants are known in the art, e.g., U.S. patent 4,333,919.
• the effective dosage range is from 1.0 to 200 milligrams per animal per day.
• Mammalian growth hormones are very similar in their amino acid sequences and hormones originating from one animal source can enhance the growth of other unrelated species of animals.
• the analogs of the present invention can be used to produce increased growth in the same animal species in which native bSt has been shown to have growth-related bioactivity such as bovines, sheep, rats, salmon and chickens.
• the preferred animals are bovine used for beef cattle such as bulls, heifers or steers.
• Beef cattle are slaughtered just prior to reaching full maturity and size.
• the bSt analogs of the instant invention can be used to produce increased growth rates in beef cattle by administration any time between weaning until slaughter.
• the bSt are administered to beef cattle for a minimum of 30 days and for a maximum of 450 days depending upon desired time of slaughter.
• Animals used for veal are typically slaughtered at approximately 6 months of age and 10 to 30 mg/day of the bSt analog is administered up until the age of slaughter to effectuate desired increases in growth rate.
• the bSt analog Is administered between 30 and 90 days postpartum and continued for up to 300 days.
• the bSt analog will also increase lactation in other commercial milk-producing animals such as goats and sheep.
• CHART 1 Amino Acid Sequence Of Bovine Somatotropin
• a plasmid containing the m4-99ser gene is digested with EcoRI and Hindlll to produce Fragment 1.

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• 1989
  • 1989-12-11 JP JP2502084A patent/JPH04502762A/ja active Pending
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