EP0441889A1 - Modified human growth hormone - Google Patents
Modified human growth hormoneInfo
- Publication number
- EP0441889A1 EP0441889A1 EP89913122A EP89913122A EP0441889A1 EP 0441889 A1 EP0441889 A1 EP 0441889A1 EP 89913122 A EP89913122 A EP 89913122A EP 89913122 A EP89913122 A EP 89913122A EP 0441889 A1 EP0441889 A1 EP 0441889A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- growth hormone
- human growth
- modified
- hgh
- treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
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- 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
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- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to a protein derived from human growth hormone (hereinafter abbreviated as hGH), which no longer has the first two amino acids at the amino terminus and which retains its main biological activity. stimulation of bone and somatic growth, but which no longer shows the transient and diabetogenic insulin effects generally observed during the administration of hGH.
- hGH human growth hormone
- growth hormone is a protein synthesized and secreted by the somatotropic cells of the anterior lobe of the pituitary gland.
- the important role of this hormone in human growth is well known and in therapy it is capable of filling the pituitary deficit responsible for certain forms of dwarfism.
- Human growth hormone is a polypeptide molecule of 191 amino acids and 22,000 daltons of molecular weight, having two disulfide bridges between residues 53 and 165 on the one hand, and 182 and 189 on the other hand ( Figure 1).
- Human growth hormone is characterized by the multiplicity of its biological activities. Its major property is to stimulate bone and somatic growth by increasing chondrogenesis, protein synthesis and cell proliferation. These somatogenic effects are mediated by growth factors ( somatomedins or IGF factors "Insulin-li e Growth Factors "), secreted under the influence of hGH mainly in the liver.
- growth hormone exerts a wide range of metabolic effects which we can summarize as follows: the" diabetogenic "effect, anti-insulin acting on the carbohydrate metabolism, corresponds to a reduction in the assimilation of glucose, associated with a decrease in insulin sensitivity.
- hGH in a subject with insufficient pituitary or growth hormone deficiency, the administration of human hGH exerts a transient insulin-like effect resulting in transient hypoglycemia (Merimee, 1972; Goodman, 1981). Like insulin, hGH is able in this case to increase the entry and use of glucose in the muscles (Park et al., 1952) and in fat cells (Goodman, 1967), as well as it shows a significant anti-lipolytic effect (Birnbaum and Goodman, 1976). All of these effects only last a few hours, after which the tissues become refractory to this insulin effect of growth hormone and, on the other hand, become sensitive to its anti-insulin effect described above.
- Figure 2 schematically summarizes the modes of action of hGH as well as the overall system for regulating its secretion from the antehypophysis.
- hGH hGH
- authentic recombinant hormone which is identical to the natural hormone isolated from pituitary extracts and et-hGH
- a recombinant hormone possessing an additional methionine at its amino terminal end a recombinant hormone possessing an additional methionine at its amino terminal end.
- These two recombinant proteins have properties identical to those of the natural hormone, indicating that the lipolytic and diabetogenic activity, and the transient insulin effect, are intrinsic properties of human growth hormone.
- the development of growth hormones devoid of diabetogenic activity would have very important therapeutic interests for the treatment of certain clinical cases for which the metabolic effects of hGH on tolerance to carbohydrates are to be avoided: cachexic patients, newborns, people elderly.
- a modification of the primary structure of hGH in particular by genetic means, results in a modified hGH reflecting different biological activities and improved properties compared to the natural hormone or to the recombinant hormones.
- the subject of the invention is a modified human growth hormone demonstrating a characterized somatid growth stimulation activity.
- n being greater than or equal to 2 and not going beyond that which causes a modification of the somatid growth stimulating activity compared to natural human growth hormone
- n is different from 13.
- the growth hormone is characterized in that the number n of amino acids deleted does not go beyond this which causes a modification of the natural three-dimensional configuration relative to the natural human growth hormone.
- the subject of the invention is a modified growth hormone characterized in that it comprises a deletion of the first n N-terminal amino acids of human growth hormone, n taking any of the values 2 to 24, and being different from 13.
- - human growth hormone is characterized in that n takes any one of the values from 2 to 15, for example 15.
- the human growth hormone is characterized in that n takes any one of the values from 2 to 14, for example 14. According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 12, for example 12.
- the human growth hormone is characterized in that n takes any one of the values from 2 to 9, for example 9.
- the human growth hormone is characterized in that n takes any one of the values from 2 to 7, for example 7.
- the human growth hormone is characterized in that n takes any one of the values from 2 to 5, for example 5.
- the human growth hormone is characterized in that n is equal to 2.
- the elimination of the first two amino acids from natural hGH was carried out by genetic engineering by planing the first two codons of the hGH gene. This results in a modified hGH protein retaining all of its growth-stimulating activity but devoid of side effects on the metabolism of carbohydrates.
- the invention also relates to the recombinant DNAs coding for the modified growth hormone of the invention defined above.
- the invention also relates to the process for producing a modified hGH protein according to the invention. It includes in particular: the culture of a competent cellular host, previously transformed with a nucleic acid containing a nucleotide sequence coding for the modified hGH protein itself placed under the control of regulatory elements, including in particular a promoter recognized by the polymerases of this competent cellular host, the elements of initiation and termination of translation, and the recovery of the modified protein produced from expression products of this cellular host.
- nucleic acids described above preceded by a nucleotide sequence comprising the regulatory elements including the promoter under whose control transcription of the modified hGH gene sequence and the initiating elements are carried out and translation terminators.
- the invention also relates to the recombinant vectors of this type, cosmids, plasmids, phages modified by a nucleic acid coding for the hGH modified in one of their sites which are not essential for their replication.
- the invention relates to vectors modified by an insert consisting of one of the recombinant DNAs defined above, under the control of a promoter and of appropriate regulatory elements allowing the expression of this recombinant DNA in a host. competent cell transformed by said recombinant vector.
- the above recombinant vector is an expression vector containing, at one of its sites not essential for its replication, elements necessary to promote expression, in a host cell, of a nucleic acid encoding the modified hGH of the invention, a promoter compatible with said cell host, in particular an inducible promoter.
- the invention also relates to cellular hosts transformed by a recombinant vector as described above, a vector capable of replicating in said microorganism and comprising the regulatory elements allowing the expression of the nucleic sequence coding for the modified protein of the invention in this host.
- a first preferred cellular host consists of E. coli transformed with a recombinant vector as will be described in the examples which follow.
- the invention relates more particularly to cell cultures transformed under the above-mentioned conditions and capable of synthesizing the modified hGH according to the invention.
- the invention relates to pharmaceutical compositions containing as active ingredient the modified human growth hormone of the invention.
- compositions of the invention are suitable:
- the invention also relates to the use of the growth hormone of the invention, in the preparation of medicaments intended for the treatment of pituitary deficiency or growth hormone deficiencies, in the treatment of growth retardation or growth retardation, in the treatment obesity, treatment of scars after accident or operation, treatment of the phenomena of aging, without showing a diabetogenic effect and / or an insulin effect, especially in cachexic patients, the elderly or newborns.
- the invention also relates to the use of growth hormone comprising a deletion of the first 13 N-terminal amino acids of natural human growth hormone, for the preparation of medicaments intended for the treatment of pituitary deficiency or hormone deficiencies of growth, to the treatment of stunted or insufficient growth, to the treatment of obesity, to the treatment of scars after, accident or operation, to the treatment of the phenomena of aging, without presenting a diabetogenic effect and / or insulin effect , in cachexic patients, the elderly or newborns.
- the invention also relates to a method for improving the physical appearance of a human being having scars, comprising the administration of the growth hormone of the invention, in an amount suitable for regenerating the tissues and the renewal of the dose. administered until attenuation or disappearance of scars, improving the aesthetics of the subject.
- the invention also relates to the application as a cosmetic product of the growth hormone of the invention.
- the invention also relates to the cosmetic compositions containing the growth hormone of the invention.
- the cosmetic application of the products of the invention results from the tissue regeneration properties of the modified growth hormone of the invention, whether in the reduction or disappearance of scars or the fight against the effects of aging, such as than wrinkles.
- hGH human growth hormone
- the cDNA library was screened using probes obtained from the plasmid pchGH ⁇ OO containing the hGH gene (Martial et al., 1979). The cDNAs of the positive clones were then cloned into the plasmid pBR322, and the nucleotide sequence of the cDNAs was determined.
- the expression system that was used to express the modified hGH gene is a prokaryotic system, in two cistrons: the first cistron consists of the cDNA coding for human Cu / Zn superoxide dismutase (hSOD), the second by the cDNA -hGH.
- the two genes are dependent on a single promoter, in this case the tac promoter.
- Figure 4a shows the pSOD vector used. This plasmid is derived from the vector ptac ⁇ in which was cloned the cDNA coding for human Cu / Zn superoxide dismutase or hSOD (Hallewell et al., 1985).
- the modified hGH gene was constructed from an Xba I - Sal I fragment of the hGH cDNA isolated from the plasmid pDMIOO-hGH. This cDNA fragment is 580 bp long and its 5 'end corresponds to the second nuleotide of the codon of amino acid 7 of hGH. It is represented in part b of FIG. 5.
- the modified hGH gene was constructed by adding to this fragment, on the 5 ′ side, a synthetic oligonucleotide represented in part a of FIG.
- the sequence of which comprises: a 5 'end compatible with an Nco I end; the AGGA sequence from Shine-Dalgarno; a TAA translation termination codon determining the end of the first cistron; a second messenger translation initiation ATG codon; codons for amino acids 3, 4, 5 and 6 of hGH; a 3 'end compatible with an Xba I end. Consequently, the resulting modified hGH gene has a deletion of the first two codons of the natural hGH gene, and an additional methionine initiator codon at the end
- the total proteins expressed in the transformed bacteria were analyzed by electrophoresis on polyacrylamide gel in the presence of SDS after induction of the tac promoter.
- the appearance of two additional proteins is observed in comparison with an uninduced sample: hSOD and a second protein having the size of that expected for the modified hGH.
- the second step in the construction of the modified hGH expression vector consisted in deleting the internal region of the hSOD gene while keeping its 5 'and 3' ends intact, in order not to affect the level of expression of hGH modified.
- 2 unique or infrequent restriction sites making it possible to delete a part of this cDNA, without modifying the reading phase: the Stu I site at the level of codon 41 of cDNA-hSOD, and the Nco I site at codon 153 of cDNA-hSOD.
- the plasmid was circularized by the action of DNA ligase and transformed into bacteria E. coli D1210 ( Figure 7).
- the plasmids present in the clones of transformed bacteria and among 12 were analyzed. clones taken at random, 10 contained the deleted plasmid, called pSGHD.
- the proteins expressed in the clones containing the plasmid pSGHD by electrophoresis of the total proteins expressed in these clones were analyzed. This electrophoresis test clearly shows that the bacteria which possess the plasmid pSGHD produce only one of the two proteins previously induced: the one whose size corresponds to the expected size for the modified human growth hormone (FIG. 8a). The rate was estimated expression of the hormone to 8% of total bacterial proteins ⁇ s. This high expression yield will allow a simplified procedure to be used to purify the expressed protein.
- the partial purification of the modified hGH protein expressed by the plasmid pSGHD was carried out in two stages: the isolation and the solubilization of the inclusion bodies on the one hand, and their purification by HPLC chromatography on the other hand.
- the modified hGH protein produced after induction with IPTG is not soluble, but is present in precipitated form in inclusion bodies.
- These inclusion bodies consist essentially of growth hormone (Figure 8b), the solubilization of which was carried out by incubating them at room temperature in an 8 M urea solution dissolved in a 50 mM phosphate buffer, pH 7.0. The solution is then dialyzed against a large volume of 50 mM NH4HC03, then lyophilized.
- the second purification step is HPLC chromatography on anion exchange resin, DEAE. This chromatography was carried out according to the conditions established for hGH of extractive origin. As the results shown in Figure 9 show, this procedure provides sufficiently purified modified hGH protein fractions. Characterization of the modified hGH:
- radioimmunoassays were performed in the presence of a polyclonal or monoclonal antibody specific for pituitary hGH 22K, (2) experiments of binding to preparations of hepatic receptors isolated from the liver of pregnant rabbits and (3) test of somatogenic activity ÎJ vivo.
- Radioimmunoassays three series of RIA assays were carried out, differing by the nature of the antibody used: a rabbit serum directed against the natural hormone, a monoclonal antibody directed against its NH2-terminal end, and a second monoclonal antibody directed against an internal region of the hormone.
- the assays were carried out on the basis of the competition between the modified hGH protein and the labeled pituitary growth hormone ( 125 I-hGH).
- modified hGH hormones necessary to shift the binding of the tracer to the antibody by 50% (ED50) were compared to the dose of pituitary hormone causing the same displacement. So we have. evaluated, for each antibody, the competition capacities of the modified hGH protein vis-à-vis the 125 I-hGH tracer, expressed as a percentage of the competition capacity of the pituitary hormone.
- RIA assays show that the purified modified hGH is a good competitor of the pituitary tracer for the antiserum directed against the hormone.
- pituitary and especially for the monoclonal directed against an internal region of this hormone: in the latter case, the modified hGH protein has in fact a capacity for competition vis-à-vis the tracer equivalent to 80% of that of the hGH standard.
- the modified hGH does not compete at all with the tracer for the NH2-terminal monoclonal antibody. This result is not surprising since the modified hGH protein is deleted from amino acids 1 and 2 of the natural hormone.
- Binding to hepatic receptors it is well established that growth hormone acts mainly in the liver, where it stimulates the synthesis of somatomedins and is involved in the regulation of metabolic systems such as gluconeogenesis and the metabolism of fatty acids.
- the liver is therefore an organ rich in specific receptors for growth hormone, and in particular the liver of a pregnant rabbit (Posner et al., 1974). Consequently, the hepatic receptors used for the binding experiments are prepared from the fraction enriched in plasma and microsomal membranes of the liver of the pregnant rabbit.
- the dosing by radio receivers of a given hormone consists in incubating a small fixed quantity of marked hormone (tracer) and increasing concentrations of cold hormone to be assayed.
- hGH has a relative capacity of 125% compared to that of the international standard hGH to displace by 50% (ED50) the initial binding of the tracer 125 I-hGH to hepatic receptors.
- the modified hGH therefore shows a capacity displacement greater than that of the standard pituitary hormone.
- Somatogenic activity in vivo to verify that the modified hGH protein which normally attaches to somatogenic hormone receptors in the liver, retains its main biological activity of somatogenic stimulation, a test was used: (1) the tibia test (Greenspan et al., 1974).
- modified hGH protein not only has a three-dimensional conformation very similar to that of the standard pituitary hGH hormone, but still retains its main biological activity to stimulate somatogenic growth at 100%. Insulin activity of modified hGH:
- the transient insulin activity of human growth hormone can be measured in vitro by a lipogenesis stimulation test, carried out on rat adipocytes originating from hGH deficient tissues.
- the presence of endogenous growth hormone in fact induces a resistance of adipose tissue to the insulin effects of hGH.
- the insulin activity test of the recombinant modified hGH is based on the measurement of the incorporation of tritiated flucose into the lipids, as a function of the addition of increasing doses of hGH, ⁇ and is based on the test, described ( Moody et al., 1975) for the stimulation of lipogenesis by insulin. Insulin activity was measured as follows:
- adipocytes Preparation of adipocytes: the rats are sacrificed by dislocation of the cervical vertebrae and the adipose tissue from the kidneys and epididymis removed. This tissue, cut into small pieces is immediately digested with collagenase (1 mg / ml) for 30 minutes at 37 ° C. with vigorous stirring, in the KRH pH 7.4 buffer containing 35 mg / ml dialyzed BSA, and 0.27 mM of glucose, using 3 ml of KRH buffer per rat. After filtration on gas, the cell suspension is centrifuged at 600 g for 30 seconds and the adipocytes, of lower density than the buffer, rise to the surface. The infranate is aspirated with a syringe with a long needle and the cells are resuspended in the fresh buffer. The cells are washed 5 times in KRH buffer pH 7.4 at 37 C containing 1% BSA.
- Lipogenesis stimulation the protocol which was used to measure insulin activity is adapted from the lipogenesis stimulation test with insulin, de Moody et al., (1975).
- the rat adipocytes are first reincubated for 4 hours at 37 ° C. in KRH buffer with a BSA concentration of 10 mg / ml, then washed and resuspended at a concentration of 0.8 x 10 5 cells / ml.
- the test is carried out in triplicate in 10 ml polyethylene vials to which are successively added: - 400 ⁇ l of the adipocyte suspension, - 50 ⁇ l of KRH buffer containing increasing doses of hGH (0.1 to 10,000 ng / l) or insulin (0.05 to 100 ng / ml), - 50 ⁇ l of tritiated glucose (D- [3- 3 H] glucose) containing 50,000 to 100,000 dpm, in a total volume of 0.5 ml.
- the final concentration of adipocytes is 1% (V / V) for the experiments on adipocytes from normal rats and 2% (V / V) for adipocytes from hypox rats.
- modified hGH does not induce an increase in basic lipogenesis, even at concentrations up to 10 ⁇ g / l. In other words, the modified hGH does not reveal any transient insulin activity, although we are in an endogenous hGH deficiency system.
- the test that has been used is to measure insulin resistance in dogs treated with the modified hGH hormone as described by ADER et al., 1987.
- the diabetogenic activity of the modified hGH was also tested on rat adipocytes.
- the stimulation of glucose transport by insulin in the presence or absence of the modified hGH was measured and compared with that of natural hGH.
- Modified hGH unlike natural hGH does not inhibit action insulin in this test, which further shows that the modified hGH has no diabetogenic activity.
- hGH Human growth hormone
- SOD DNA coding for human superoxide dismutase.
- cDNA-hSOD sequences belonging to the human superoxide dismutase gene.
- hGHM DNA coding for the modified hGH protein.
- cDNA-hSOP DNA coding for human superoxide dismutase.
- hGHM DNA coding for the modified hGH protein.
- cDNA-hSOD DNA coding for human superoxide dismutase.
- the tac promoter a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. 80, 21-25.
- Hu ang growth hormone co plementary DNA cloning and expression in bacteria. Science 205, 602-607.
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Abstract
L'invention concerne une protéine dérivée de l'hormone de croissance humaine, qui n'a plus les deux premiers acides aminés de l'hormone hypophysaire naturelle à l'extrémité amino terminale, retenant son activité biologique principale de stimulation de la croissance osseuse et somatique, mais ne témoignant plus des effets insuliniques transitoires et diabétogènes généralement observés lors de l'administration de hGH. Cette protéine d'hormone de croissance modifiée est utilisable en thérapeutique, en particulier pour le traitement de cas cliniques pour lesquels les effets sur le métabolisme des sucres de l'hormone de croissance sont à éviter: patients cachexiques, nouveau-nés et personnes âgées.The invention relates to a protein derived from human growth hormone, which no longer has the first two amino acids of the natural pituitary hormone at the amino terminal end, retaining its main biological activity of stimulating bone growth and somatic, but no longer reflecting the transient and diabetogenic insulin effects generally observed during the administration of hGH. This modified growth hormone protein can be used therapeutically, in particular for the treatment of clinical cases in which the effects on the metabolism of sugars of growth hormone are to be avoided: cachexic patients, newborns and the elderly.
Description
HORMONE DE CROISSANCE HUMAINE MODIFIEE MODIFIED HUMAN GROWTH HORMONE
L'invention concerne une protéine dérivée de l'hormone de croissance humaine (ci-après désignée sous l'abréviation hGH), qui n'a plus les deux premiers acides aminés à l'extrémité amino terminale et qui retient son activité biologique principale de stimulation de la croissance osseuse et somatique, mais qui ne témoigne plus des effets insuliniques transitoires et diabetogenes généralement observés lors de l'administration de hGH.The invention relates to a protein derived from human growth hormone (hereinafter abbreviated as hGH), which no longer has the first two amino acids at the amino terminus and which retains its main biological activity. stimulation of bone and somatic growth, but which no longer shows the transient and diabetogenic insulin effects generally observed during the administration of hGH.
A titre d'arrière plan on rappelle que l'hormone de croissance est une protéine synthétisée et sécrétée par les cellules somatotropes du lobe antérieur de l'hypophyse. Le rôle important de cette hormone dans la croissance humaine est bien connu et en thérapeutique elle est capable de combler le déficit hypophysaire responsable de certaines formes de nanisme. L'hormone de croissance humaine est une molécule polypeptidique de 191 acides aminés et de 22000 daltons de poids moléculaire, possédant deux ponts disulfure entre les résidus 53 et 165 d'une part, et 182 et 189 d'autre part (Figure 1).As a background, it is recalled that growth hormone is a protein synthesized and secreted by the somatotropic cells of the anterior lobe of the pituitary gland. The important role of this hormone in human growth is well known and in therapy it is capable of filling the pituitary deficit responsible for certain forms of dwarfism. Human growth hormone is a polypeptide molecule of 191 amino acids and 22,000 daltons of molecular weight, having two disulfide bridges between residues 53 and 165 on the one hand, and 182 and 189 on the other hand (Figure 1).
L'hormone de croissance humaine se caractérise par la multiplicité de ses activités biologiques. Sa propriété majeure est de stimuler la croissance osseuse et somatique par une augmentation de la chondrogenèse, de la synthèse des protéines et de la prolifération des cellules. Ces effets somatogéniques sont xnédiés par des facteurs de croissance (somatomédines ou facteurs IGF "Insulin-li e Growth
Factors"), sécrétés sous l'influence de hGH essentiellement au niveau du foie. De plus, l'hormone de croissance exerce une gamme étendue d'effets métaboliques que l'on peur résumer comme suit: l'effet "diabétogène", effet anti-insulinique agissant sur le métabolisme des hydrates de carbone, correspond à une réduction de l'assimilation du glucose, associée à une diminution de la sensibilité à l'insuline. Cet effet est observé soit dans le cas d'une sécrétion excessive de hGH (acromégalie) , soit lorsque celle-ci est administrée peu avant une surcharge en glucose QU de manière chronique à un sujet diabétique (Levine et Luft, 1964) . Cet effet anti-insulinique de la hGH se manifeste également au niveau du métabolisme des lipides, aboutissant à une stimulation de la lipolyse (effet lipolytique) : l'hormone de croissance favorise en effet la mobilisation des lipides vers le foie - sous forme d'acides gras - et stimule leur oxydation par rapport à celle des acides aminés et des sucres (Fain et al., 1965; Goodman, 1968).Human growth hormone is characterized by the multiplicity of its biological activities. Its major property is to stimulate bone and somatic growth by increasing chondrogenesis, protein synthesis and cell proliferation. These somatogenic effects are mediated by growth factors ( somatomedins or IGF factors "Insulin-li e Growth Factors "), secreted under the influence of hGH mainly in the liver. In addition, growth hormone exerts a wide range of metabolic effects which we can summarize as follows: the" diabetogenic "effect, anti-insulin acting on the carbohydrate metabolism, corresponds to a reduction in the assimilation of glucose, associated with a decrease in insulin sensitivity. This effect is observed either in the case of an excessive secretion of hGH (acromegaly), that is to say when it is administered shortly before an overload of QU glucose in a chronic manner to a diabetic subject (Levine and Luft, 1964) This anti-insulin effect of hGH is also manifested in lipid metabolism , leading to stimulation of lipolysis (lipolytic effect): growth hormone indeed promotes the mobilization of lipids towards the liver - in the form of fatty acids - and stimulates their oxidation compared to that of acids amines and sugars (Fain et al., 1965; Goodman, 1968).
- chez un sujet insuffisant hypophysaire ou carence en hormone de croissance, l'administration de hGH humaine exerce un effet transitoire de type insulinique entraînant une hypoglycémie passagère (Mérimée, 1972; Goodman, 1981). Comme l'insuline, la hGH est capable dans ce cas d'augmenter l'entrée et l'utilisation de glucose dans les muscles (Park et al., 1952) et dans les cellules adipeuses (Goodman, 1967), de même qu'elle montre un effet anti-lipolytique important (Birnbaum et Goodman, 1976). L'ensemble de ces effets ne dure cependant que quelques heures, après quoi les tissus deviennent réfractaires à cet effet insulinique de l'hormone de croissance et deviennent en revanche sensibles à son effet anti-insulinique décrit précédemmen .
Bien que les effets diabetogenes et insuliniques se manifestent de manière différente, on peut supposer qu'ils ont un dénominateur biologique et moléculaire commun. La Figure 2 résume schématiquement les modes d'action de la hGH ainsi que le système global de régulation de sa sécrétion à partir de 1•antéhypophyse.- in a subject with insufficient pituitary or growth hormone deficiency, the administration of human hGH exerts a transient insulin-like effect resulting in transient hypoglycemia (Merimee, 1972; Goodman, 1981). Like insulin, hGH is able in this case to increase the entry and use of glucose in the muscles (Park et al., 1952) and in fat cells (Goodman, 1967), as well as it shows a significant anti-lipolytic effect (Birnbaum and Goodman, 1976). All of these effects only last a few hours, after which the tissues become refractory to this insulin effect of growth hormone and, on the other hand, become sensitive to its anti-insulin effect described above. Although the diabetogenic and insulin effects manifest themselves differently, it can be assumed that they have a common biological and molecular denominator. Figure 2 schematically summarizes the modes of action of hGH as well as the overall system for regulating its secretion from the antehypophysis.
Actuellement, deux types d'hormones de croissance recombinantes, produites à partir de gènes clones et exprimés dans des microorganismes, sont utilisées en thérapeutique pour le traitement de différentes formes de déficiences hypoph^saires : la hGH, hormone recombinante authentique, qui est identique à l'hormone naturelle isolée d'extraits hypophysaires et la et-hGH, hormone recombinante possédant une méthionine additionnelle à son extrémité amino terminale. Ces deux protéines recombinantes ont des propriétés tout à fait identiques à celles de l'hormone naturelle, indiquant que l'activité lipolytique et diabétogène, et l'effet insulinique transitoire, sont des propriétés intrinsèques de l'hormone de croissance humaine. Le développement d'hormones de croissance dépourvues d'activité diabétogène aurait des intérêts thérapeutiques très importants pour le traitement de certains cas cliniques pour lesquels les effets métaboliques de la hGH sur la tolérance aux carbohydrates sont à éviter: patients cachexiques, nouveaux-nés, personnes âgées.Currently, two types of recombinant growth hormones, produced from cloned genes and expressed in microorganisms, are used therapeutically for the treatment of different forms of pituitary deficiencies: hGH, authentic recombinant hormone, which is identical to the natural hormone isolated from pituitary extracts and et-hGH, a recombinant hormone possessing an additional methionine at its amino terminal end. These two recombinant proteins have properties identical to those of the natural hormone, indicating that the lipolytic and diabetogenic activity, and the transient insulin effect, are intrinsic properties of human growth hormone. The development of growth hormones devoid of diabetogenic activity would have very important therapeutic interests for the treatment of certain clinical cases for which the metabolic effects of hGH on tolerance to carbohydrates are to be avoided: cachexic patients, newborns, people elderly.
Dans cette invention, on démontre qu'une modification de la structure primaire de la hGH notamment par voie génétique, résulte en une hGH modifiée témoignant d'activités biologiques différentes et de propriétés améliorées par rapport à l'hormone naturelle ou aux hormones recombinantes.
L'invention a pour objet une hormone de croissance humaine modifiée témoignant d'une activité de stimulation de croissance somatidique caractériséeIn this invention, it is demonstrated that a modification of the primary structure of hGH, in particular by genetic means, results in a modified hGH reflecting different biological activities and improved properties compared to the natural hormone or to the recombinant hormones. The subject of the invention is a modified human growth hormone demonstrating a characterized somatid growth stimulation activity.
- en ce qu'elle comporte une délétion des n premiers acides aminés N-terminaux de l'hormone de croissance humaine naturelle, n étant supérieur ou égal à 2 et n'allant pas au-delà de ce qui entraîne une modification de l'activité de stimulation de croissance somatidique par rapport à l'hormone de croissance naturelle humaine,- in that it comprises a deletion of the first n N-terminal amino acids of the natural human growth hormone, n being greater than or equal to 2 and not going beyond that which causes a modification of the somatid growth stimulating activity compared to natural human growth hormone,
- et en ce qu'elle est dépourvue soit d'activité diabétogène, soit d'activité insulinique, agissant au niveau du métabolisme des carbohydrates, ou des deux activités à la fois,- and in that it is devoid of either diabetogenic activity or insulin activity, acting at the level of carbohydrate metabolism, or of both activities at the same time,
- sous réserve que n soit différent de 13.- provided that n is different from 13.
Selon un mode de réalisation préféré de l'invention, l'hormone de croissance est caractérisée en ce que le nombre n d'acides aminés délétés ne va pas au-delà de ce qui entraîne une modification de la configuration tridimensionnelle naturelle par rapport à l'hormone de croissance humaine naturelle.According to a preferred embodiment of the invention, the growth hormone is characterized in that the number n of amino acids deleted does not go beyond this which causes a modification of the natural three-dimensional configuration relative to the natural human growth hormone.
L'invention a pour objet une hormone de croissance modifiée caractérisée en ce qu'elle comporte une délétion des n premiers acides aminés N- terminaux de l'hormone de croissance humaine, n prenant l'une quelconque des valeurs 2 à 24, et étant différent de 13.The subject of the invention is a modified growth hormone characterized in that it comprises a deletion of the first n N-terminal amino acids of human growth hormone, n taking any of the values 2 to 24, and being different from 13.
Selon un mode de réalisation préféré de l'invention, - l'hormone de croissance humaine est caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 15, par exemple 15.According to a preferred embodiment of the invention, - human growth hormone is characterized in that n takes any one of the values from 2 to 15, for example 15.
Selon un autre mode de réalisation préféré de l'invention, l'hormone de croissance humaine est caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 14, par exemple 14.
Selon un autre mode de réalisation préféré de l'invention, l'hormone de croissance humaine est caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 12, par exemple 12.According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 14, for example 14. According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 12, for example 12.
Selon un autre mode de réalisation préféré de l'invention, l'hormone de croissance humaine est caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 9, par exemple 9.According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 9, for example 9.
Selon un autre mode de réalisation préféré de l'invention, l'hormone de croissance humaine est caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 7, par exemple 7.According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 7, for example 7.
Selon un autre mode de réalisation préféré de l'invention, l'hormone de croissance humaine est caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 5, par exemple 5.According to another preferred embodiment of the invention, the human growth hormone is characterized in that n takes any one of the values from 2 to 5, for example 5.
Selon un autre mode de réalisation préféré de l'invention, l'hormone de croissance humaine est caractérisée en ce que n est égal à 2.According to another preferred embodiment of the invention, the human growth hormone is characterized in that n is equal to 2.
Dans la mise en oeuvre préférée de l'invention, l'élimination des deux premiers acides aminés de la hGH naturelle a été réalisée par génie génétique en rabottant les deux premiers codons du gène hGH. Ceci résulte en une protéine hGH modifiée retenant toute son activité de stimulation de croissance mais dépourvue d'effets secondaires sur le métabolisme des carbohydrates.In the preferred implementation of the invention, the elimination of the first two amino acids from natural hGH was carried out by genetic engineering by planing the first two codons of the hGH gene. This results in a modified hGH protein retaining all of its growth-stimulating activity but devoid of side effects on the metabolism of carbohydrates.
L'invention concerne également les ADN recombinants codant pour l'hormone de croissance modifiée de l'invention définie ci-dessus.The invention also relates to the recombinant DNAs coding for the modified growth hormone of the invention defined above.
L'invention concerne aussi le procédé de production d'une protéine hGH modifiée conforme à l'invention. Il comprend notamment : la culture d'un hôte cellulaire compétent, auparavant transformé avec un acide nucléique contenant une séquence de nucléotides codant pour la protéine hGH modifiée
elle-même placée sous le contrôle d'éléments de régulation, dont notamment un promoteur reconnu par les polymérases de cet hôte cellulaire compétent, les éléments d'initiation et de terminaison de la traduction, et la récupération de la protéine modifiée produite à partir des produits d'expression de cet hôte cellulaire.The invention also relates to the process for producing a modified hGH protein according to the invention. It includes in particular: the culture of a competent cellular host, previously transformed with a nucleic acid containing a nucleotide sequence coding for the modified hGH protein itself placed under the control of regulatory elements, including in particular a promoter recognized by the polymerases of this competent cellular host, the elements of initiation and termination of translation, and the recovery of the modified protein produced from expression products of this cellular host.
Appartiennent également à l'invention, les acides nucléiques décrits ci-dessus, précédés d'une séquence nucléotidique comprenant les éléments de régulation y inclus le promoteur sous le contrôle duquel s'effectue la transcription de la séquence du gène hGH modifiée et les éléments initiateurs et terminateurs de la traduction.Also belonging to the invention, the nucleic acids described above, preceded by a nucleotide sequence comprising the regulatory elements including the promoter under whose control transcription of the modified hGH gene sequence and the initiating elements are carried out and translation terminators.
L'invention concerne aussi les vecteurs recombinants de ce type, cosmides, plasmides, phages modifiés par un acide nucléique codant pour la hGH modifiée en l'un de leurs sites non essentiels pour leur réplication.The invention also relates to the recombinant vectors of this type, cosmids, plasmids, phages modified by a nucleic acid coding for the hGH modified in one of their sites which are not essential for their replication.
Plus particulièrement, l'invention concerne les vecteurs modifiés par un insérât consistant en l'un des ADN recombinants définis ci-dessus, sous le contrôle d'un promoteur et des éléments de régulation appropriés permettant l'expression de cet ADN recombinant dans un hôte cellulaire compétent transformé par ledit vecteur recombinant.More particularly, the invention relates to vectors modified by an insert consisting of one of the recombinant DNAs defined above, under the control of a promoter and of appropriate regulatory elements allowing the expression of this recombinant DNA in a host. competent cell transformed by said recombinant vector.
Dans un mode de réalisation particulier de l'invention, le vecteur recombinant ci-dessus est un vecteur d'expression contenant, en l'un de ses sites non essentiels pour sa réplication, des éléments nécessaires pour promouvoir l'expression, dans un hôte cellulaire, d'un acide nucléique codant pour la hGH modifiée de l'invention, un promoteur compatible avec ledit hôte cellulaire, en particulier un promoteur inductible.
L'invention se rapporte encore aux hôtes cellulaires transformés par un vecteur recombinant tel que décrit plus haut, vecteur capable de se répliquer dans ledit microorganisme et comprenant les éléments de régulation permettant l'expression de la séquence nucléique codant pour la protéine modifiée de l'invention dans cet hôte.In a particular embodiment of the invention, the above recombinant vector is an expression vector containing, at one of its sites not essential for its replication, elements necessary to promote expression, in a host cell, of a nucleic acid encoding the modified hGH of the invention, a promoter compatible with said cell host, in particular an inducible promoter. The invention also relates to cellular hosts transformed by a recombinant vector as described above, a vector capable of replicating in said microorganism and comprising the regulatory elements allowing the expression of the nucleic sequence coding for the modified protein of the invention in this host.
Un premier hôte cellulaire préféré est constitué par E.coli transformé par un vecteur recombinant tel qu'il sera décrit dans les exemples qui suivent.A first preferred cellular host consists of E. coli transformed with a recombinant vector as will be described in the examples which follow.
L'invention concerne encore plus particulièrement des cultures cellulaires transformées dans les conditions sus-indiquées et aptes à synthétiser la hGH modifiée selon l'invention.The invention relates more particularly to cell cultures transformed under the above-mentioned conditions and capable of synthesizing the modified hGH according to the invention.
L'invention concerne les compositions pharmaceutiques contenant comme principe actif l'hormone de croissance humaine modifiée de 1'invention.The invention relates to pharmaceutical compositions containing as active ingredient the modified human growth hormone of the invention.
Les compositions pharmaceutiques de l'invention sont appropriées :The pharmaceutical compositions of the invention are suitable:
- au traitement du déficit hypophysaire ou des carences en hormones de croissance,- treatment of pituitary deficiency or growth hormone deficiencies,
- au traitement des retards ou des insuffisances de croissance,- the treatment of growth delays or insufficiencies,
- au traitement de l'obésité,- in the treatment of obesity,
- au traitement de cicatrices après accident ou opération,- to the treatment of scars after accident or operation,
- au traitement des phénomènes de vieillissement. L'invention concerne également l'utilisation de l'hormone de croissance de l'invention, à la préparation de médicaments destinés au traitement du déficit hypophysaire ou des carences en hormone de croissance, au traitement des retards ou des insuffisances de croissance, au traitement de l'obésité, au traitement de cicatrices après accident ou opération, au traitement des phénomènes de
vieillissement, sans présenter d'effet diabétogène et/ou d'effet insulinique, notamment chez les patients cachexiques, les vieillards ou les nouveaux-nés.- to the treatment of aging phenomena. The invention also relates to the use of the growth hormone of the invention, in the preparation of medicaments intended for the treatment of pituitary deficiency or growth hormone deficiencies, in the treatment of growth retardation or growth retardation, in the treatment obesity, treatment of scars after accident or operation, treatment of the phenomena of aging, without showing a diabetogenic effect and / or an insulin effect, especially in cachexic patients, the elderly or newborns.
L'invention concerne également l'utilisation de l'hormone de croissance comportant une délétion des 13 premiers acides aminés N-terminaux de l'hormone de croissance humaine naturelle, à la préparation de médicaments destinés au traitement du déficit hypophysaire ou des carences en hormone de croissance, au traitement des retards ou des insuffisances de croissance, au traitement de l'obésité, au traitement de cicatrices après ,accident ou opération, au traitement des phénomènes de vieillissement, sans présenter d'effet diabétogène et/ou d'effet insulinique, chez les patients cachexiques, les vieillards ou les nouveaux-nés.The invention also relates to the use of growth hormone comprising a deletion of the first 13 N-terminal amino acids of natural human growth hormone, for the preparation of medicaments intended for the treatment of pituitary deficiency or hormone deficiencies of growth, to the treatment of stunted or insufficient growth, to the treatment of obesity, to the treatment of scars after, accident or operation, to the treatment of the phenomena of aging, without presenting a diabetogenic effect and / or insulin effect , in cachexic patients, the elderly or newborns.
L'invention concerne également une méthode pour améliorer l'aspect physique d'un être humain présentant des cicatrices, comprenant l'administration de l'hormone de croissance de l'invention, en quantité propre à régénérer les tissus et le renouvellement de la dose administrée jusqu'à l'obtention de l'atténuation ou de la disparition des cicatrices, améliorant l'esthétique du sujet.The invention also relates to a method for improving the physical appearance of a human being having scars, comprising the administration of the growth hormone of the invention, in an amount suitable for regenerating the tissues and the renewal of the dose. administered until attenuation or disappearance of scars, improving the aesthetics of the subject.
L'invention concerne également l'application à titre de produit cosmétique de l'hormone de croissance de l'invention.The invention also relates to the application as a cosmetic product of the growth hormone of the invention.
L'invention concerne également les compositions cosmétiques contenant l'hormone de croissance de l'invention.The invention also relates to the cosmetic compositions containing the growth hormone of the invention.
L'application cosmétique des produits de 1'invention résulte des propriétés de régénération tissulaire de l'hormone de croissance modifiée de l'invention, que ce soit dans l'atténuation ou la disparition des cicatrices ou la lutte contre les effets du vieillissement, tels que les rides.
RESULTATSThe cosmetic application of the products of the invention results from the tissue regeneration properties of the modified growth hormone of the invention, whether in the reduction or disappearance of scars or the fight against the effects of aging, such as than wrinkles. RESULTS
Clonage et expression du gène hGH codant pour une protéine hGH modifiéeCloning and expression of the hGH gene coding for a modified hGH protein
On clone le gène codant pour 1'hormone de croissance humaine (hGH) à partir de tumeurs hypophysaires de patients acromégales, qui sont riches en RNA messager hGH. Après avoir extrait les RNA totaux de tumeurs individuelles, on a purifié lés RNA messagers par chromatographie d'affinité sur oligo [dT]-cellulose. Les échantillons les plus riches en messagers codant pour la hGH, identifiés par des tests de traduction acellulaiire, ont ensuite été utilisés pour la synthèse de DNA complémentaire (cDNA) , d'après la méthode de Goodman et MacDonnald (1979) . Le cDNA bicaténaire, fractionné sur Sepharose 4B, a été inséré dans la site EcoRI du vecteur lambda 641 en utilisant des oligonucléotides synthétiques contenant le site EcoRI. Après encapsidation in vitro du DNA recombinant, la bibliothèque de cDNA a été criblée à l'aide de sondes obtenues à partir du plasmide pchGHδOO contenant le gène hGH (Martial et al., 1979). Les cDNA des clones positifs ont ensuite été clones dans le plasmide pBR322, et la séquence des nucléotides des cDNA a été déterminée. Le cDNA du plasmide pDM 100 hGH dont la séquence est présentée dans la Figure 3, codant pour une hormone de croissance naturelle intacte a été utilisé dans la construction du gène hGH modifié.The gene encoding human growth hormone (hGH) is cloned from pituitary tumors of acromegalic patients, which are rich in hGH messenger RNA. After extracting the total RNA from individual tumors, the messenger RNA was purified by affinity chromatography on oligo [dT] -cellulose. The samples richest in messengers coding for hGH, identified by cell-free translation tests, were then used for the synthesis of complementary DNA (cDNA), according to the method of Goodman and MacDonnald (1979). The double-stranded cDNA, fractionated on Sepharose 4B, was inserted into the EcoRI site of the lambda 641 vector using synthetic oligonucleotides containing the EcoRI site. After in vitro packaging of the recombinant DNA, the cDNA library was screened using probes obtained from the plasmid pchGHδOO containing the hGH gene (Martial et al., 1979). The cDNAs of the positive clones were then cloned into the plasmid pBR322, and the nucleotide sequence of the cDNAs was determined. The cDNA of the plasmid pDM 100 hGH, the sequence of which is presented in FIG. 3, coding for an intact natural growth hormone was used in the construction of the modified hGH gene.
Le système d'expression que on a utilisé pour exprimer le gène hGH modifié est un système procaryote, en deux cistrons: le premier cistron est constitué par le cDNA codant pour la Cu/Zn superoxyde dismutase humaine (hSOD) , le deuxième par le cDNA-hGH. Les deux gènes sont sous la dépendance d'un seul promoteur, en l'occurrence le promoteur tac.
La Figure 4a représente le vecteur pSOD utilisé. Ce plasmide est dérivé du vecteur ptacδ dans lequel a été clone le cDNA codant pour la Cu/Zn superoxyde dismutase humaine ou hSOD (Hallewell et al., 1985). Il permet l'expression de cette protéine hSOD, sous la dépendance du promoteur tac (de Boer et al., 1983). La séquence originale du cDNA-hSOD a été modifiée à son extrémité 3' par l'insertion d'un polylinker dont la séquence est présentée dans la Figure 4b, et qui contient les sites de restriction Nco I et Sal I qui seront utilisés pour le clonage du cDNA hGH dans le vecteur pSOD. _The expression system that was used to express the modified hGH gene is a prokaryotic system, in two cistrons: the first cistron consists of the cDNA coding for human Cu / Zn superoxide dismutase (hSOD), the second by the cDNA -hGH. The two genes are dependent on a single promoter, in this case the tac promoter. Figure 4a shows the pSOD vector used. This plasmid is derived from the vector ptacδ in which was cloned the cDNA coding for human Cu / Zn superoxide dismutase or hSOD (Hallewell et al., 1985). It allows the expression of this hSOD protein, under the dependence of the tac promoter (de Boer et al., 1983). The original sequence of cDNA-hSOD was modified at its 3 ′ end by the insertion of a polylinker whose sequence is presented in Figure 4b, and which contains the Nco I and Sal I restriction sites which will be used for the cloning of cDNA hGH into the vector pSOD. _
On a construit le gène hGH modifié à partir d'un fragment Xba I - Sal I du cDNA hGH isolé du plasmide pDMIOO-hGH. Ce fragment de cDNA est long de 580 bp et son extrémité 5' correspond au deuxième nuléotide du codon de l'acide aminé 7 de la hGH. Il est représenté dans la partie b de la Figure 5. Le gène hGH modifié a été construit en ajoutant à ce fragment, du côté 5', un oligonucléotide synthétique représenté dans la partie a de la Figure 5, et dont la séquence comprend: une extrémité 5' compatible avec une extrémité Nco I; la séquence AGGA de Shine-Dalgarno; un codon TAA de terminaison de la traduction déterminant la fin du premier cistron; un codon ATG d'initiation de la traduction du deuxième messager; les codons pour les acides aminés 3, 4, 5 et 6 de la hGH; une extrémité 3' compatible avec une extrémité Xba I. Par conséquent, le gène hGH modifié résultant présente une délétion des deux premiers codons du gène hGH naturel, et un codon initiateur méthionine additionnel à l'extrémitéThe modified hGH gene was constructed from an Xba I - Sal I fragment of the hGH cDNA isolated from the plasmid pDMIOO-hGH. This cDNA fragment is 580 bp long and its 5 'end corresponds to the second nuleotide of the codon of amino acid 7 of hGH. It is represented in part b of FIG. 5. The modified hGH gene was constructed by adding to this fragment, on the 5 ′ side, a synthetic oligonucleotide represented in part a of FIG. 5, and the sequence of which comprises: a 5 'end compatible with an Nco I end; the AGGA sequence from Shine-Dalgarno; a TAA translation termination codon determining the end of the first cistron; a second messenger translation initiation ATG codon; codons for amino acids 3, 4, 5 and 6 of hGH; a 3 'end compatible with an Xba I end. Consequently, the resulting modified hGH gene has a deletion of the first two codons of the natural hGH gene, and an additional methionine initiator codon at the end
5'.5 '.
Après la ligation du DNA synthétique et du fragment CDNA-hGH, illustrée dans la Figure 5, on a obtenu un fragment de cDNA aux extrémités Nco I - Sal I, long de 610 bp, qui comprend la séquence codant
pour une protéine hGH délétée de ses deux premiers acides aminés et possédant une méthionine additionnelle à son extrémité NH2 terminale. Ce fragment de cDNA a été inséré entre les sites Nco I et Sal I du vecteur pSOD à l'aide de la T4-DNA ligase, (Figure 6) . Les clones recombinants obtenus après transformation des bactéries E.coli D1210, ont été détectés par hybridation avec une sonde spécifique radioactive dérivée du cDNA-hGH larqué au 32P par transfert de coupure.After ligation of the synthetic DNA and of the CDNA-hGH fragment, illustrated in FIG. 5, a fragment of cDNA with Nco I - Sal I ends, 610 bp long, which comprises the coding sequence, was obtained. for an hGH protein deleted from its first two amino acids and having an additional methionine at its NH2 terminal end. This cDNA fragment was inserted between the Nco I and Sal I sites of the pSOD vector using T4-DNA ligase, (Figure 6). The recombinant clones obtained after transformation of the bacteria E.coli D1210, were detected by hybridization with a specific radioactive probe derived from cDNA-hGH lacquered at 32 P by cleavage transfer.
On a analysé les protéines totales exprimées dans les bactéries transformées par électrophorèse sur gel de polyacrylamide en présence de SDS après induction du promoteur tac. Dans les extraits de bactéries contenant le plasmide pSOD-hGH, on observe en comparaison avec un échantillon non induit, l'apparition de deux protéines supplémentaires: la hSOD et une deuxième protéine ayant la taille de celle attendue pour la hGH modifiée.The total proteins expressed in the transformed bacteria were analyzed by electrophoresis on polyacrylamide gel in the presence of SDS after induction of the tac promoter. In the extracts of bacteria containing the plasmid pSOD-hGH, the appearance of two additional proteins is observed in comparison with an uninduced sample: hSOD and a second protein having the size of that expected for the modified hGH.
La deuxième étape dans la construction du vecteur d'expression de la hGH modifiée consistait à déléter le région interne du gène hSOD tout en gardant ses extrémités 5' et 3' intactes, ceci afin de ne pas affecter le niveau d'expression de la hGH modifiée. Pour cela, on a recherché, dans la séquence du cDNA- hSOD, 2 sites de restriction uniques ou peu fréquents permettant de déléter une partie de ce cDNA, sans en modifier la phase de lecture : le site Stu I au niveau du codon 41 d.u cDNA-hSOD, et le site Nco I au niveau du codon 153 du cDNA-hSOD. Après digestion du plasmide pSOD-hGH avec les enzymes Stu I et Nco I, suivi d'un traitement au fragment de Klenow de la DNA polymérase, le plasmide a été circularisé par l'action de la DNA ligase et transformé dans des bactéries E.coli D1210 (Figure 7). On a analysé les plasmides présents dans les clones de bactéries transformées et parmi 12
clones pris au hasard, 10 contenaient le plasmide délété, dénommé pSGHD.The second step in the construction of the modified hGH expression vector consisted in deleting the internal region of the hSOD gene while keeping its 5 'and 3' ends intact, in order not to affect the level of expression of hGH modified. For this, we sought, in the sequence of cDNA-hSOD, 2 unique or infrequent restriction sites making it possible to delete a part of this cDNA, without modifying the reading phase: the Stu I site at the level of codon 41 of cDNA-hSOD, and the Nco I site at codon 153 of cDNA-hSOD. After digestion of the plasmid pSOD-hGH with the enzymes Stu I and Nco I, followed by treatment with the Klenow fragment of DNA polymerase, the plasmid was circularized by the action of DNA ligase and transformed into bacteria E. coli D1210 (Figure 7). The plasmids present in the clones of transformed bacteria and among 12 were analyzed. clones taken at random, 10 contained the deleted plasmid, called pSGHD.
Les protéines exprimées dans les clones contenant le plasmide pSGHD par électrophorèse des protéines totales exprimées dans ces clones ont été analysées. Ce test d'électrophorèse montre clairement que les bactéries qui possèdent le plasmide pSGHD ne produisent qu'une seule des deux protéines induites précédemment : celle dont la taille correspond à la taille attendue pour l'hormone de croissance humaine modifiée (Figure 8a) . On a estimé le taux d'expression de l'hormone à 8% de^s protéines bactériennes totales. Ce rendement élevé d'expression permettra d'utiliser une procédure simplifiée pour purifier la protéine exprimée.The proteins expressed in the clones containing the plasmid pSGHD by electrophoresis of the total proteins expressed in these clones were analyzed. This electrophoresis test clearly shows that the bacteria which possess the plasmid pSGHD produce only one of the two proteins previously induced: the one whose size corresponds to the expected size for the modified human growth hormone (FIG. 8a). The rate was estimated expression of the hormone to 8% of total bacterial proteins ^ s. This high expression yield will allow a simplified procedure to be used to purify the expressed protein.
La purification partielle de la protéine hGH modifiée exprimée par le plasmide pSGHD a été réalisée en deux étapes : l'isolement et la solubilisation des corps d'inclusion d'une part, et leur purification par chromatographie HPLC d'autre part. En effet, on a observé que la protéine hGH modifiée produite après induction à l'IPTG, n'est pas soluble, mais est présente sous forme précipitée dans des corps d'inclusion. Ces corps d'inclusion sont constitués essentiellement d'hormone de croissance (Figure 8b) dont la solubilisation a été réalisée en les incubant à température ambiante dans une solution d'urée 8 M dissoute dans un tampon phosphate 50 mM pH 7.0. La solution est .ensuite dialysée contre un grand volume de NH4HC03 50 mM, puis lyophilisée. La deuxième étape de purification est une chromatographie HPLC sur résine échangeuse d'anions, DEAE. Cette chromatographie a été réalisée suivant les conditions établies pour la hGH d'origine extractive. Comme le montrent les résultats présentés dans la Figure 9,
cette procédure permet d'obtenir des fractions de protéine hGH modifiée suffisamment purifiée. Caractérisation de la hGH modifiée:The partial purification of the modified hGH protein expressed by the plasmid pSGHD was carried out in two stages: the isolation and the solubilization of the inclusion bodies on the one hand, and their purification by HPLC chromatography on the other hand. Indeed, it has been observed that the modified hGH protein produced after induction with IPTG, is not soluble, but is present in precipitated form in inclusion bodies. These inclusion bodies consist essentially of growth hormone (Figure 8b), the solubilization of which was carried out by incubating them at room temperature in an 8 M urea solution dissolved in a 50 mM phosphate buffer, pH 7.0. The solution is then dialyzed against a large volume of 50 mM NH4HC03, then lyophilized. The second purification step is HPLC chromatography on anion exchange resin, DEAE. This chromatography was carried out according to the conditions established for hGH of extractive origin. As the results shown in Figure 9 show, this procedure provides sufficiently purified modified hGH protein fractions. Characterization of the modified hGH:
Pour démontrer que la délétion des deux premiers acides aminés n'a pas d'influence sur la structure tridimensionnelle et l'activité biologique principale de stimulation de croissance somatogénique de la protéine hGH modifiée, on a effectué (1) des dosages radioimmunologiques en présence d'un anticorps polyclonal ou monoclonal spécifique de la hGH 22K hypophysaire, (2) des expériences de liaison à des préparations de récepteurs hépatiques isolés de foie de lapine gestante et (3) test d'activité somatogénique ÎJ vivo. Dosages radioimmunologiques (RIA) : trois séries de dosages RIA ont été réalisées, différant par la nature de l'anticorps utilisé : un sérum de lapin dirigé contre l'hormone naturelle, un anticorps monoclonal dirigé contre son extrémité NH2- terminale, et un second anticorps monoclonal dirigé contre une région interne de l'hormone. Les dosages ont été réalisés sur base de la compétition entre la protéine hGH modifiée et l'hormone de croissance hypophysaire marquée (125I-hGH) .To demonstrate that the deletion of the first two amino acids has no influence on the three-dimensional structure and the main biological activity of somatogenic growth stimulation of the modified hGH protein, radioimmunoassays were performed in the presence of a polyclonal or monoclonal antibody specific for pituitary hGH 22K, (2) experiments of binding to preparations of hepatic receptors isolated from the liver of pregnant rabbits and (3) test of somatogenic activity ÎJ vivo. Radioimmunoassays (RIA): three series of RIA assays were carried out, differing by the nature of the antibody used: a rabbit serum directed against the natural hormone, a monoclonal antibody directed against its NH2-terminal end, and a second monoclonal antibody directed against an internal region of the hormone. The assays were carried out on the basis of the competition between the modified hGH protein and the labeled pituitary growth hormone ( 125 I-hGH).
Les doses d'hormones hGH modifiées nécessaires pour déplacer de 50 % la fixation du traceur à l'anticorps (ED50) ont été comparées à la dose d'hormone hypophysaire provoquant le même déplacement. On a ainsi . évalué, pour chaque anticorps, les capacités de compétition de la protéine hGH modifiée vis-à-vis du traceur 125I-hGH, exprimées en pourcentage de la capacité de compétition de l'hormone hypophysaire.The doses of modified hGH hormones necessary to shift the binding of the tracer to the antibody by 50% (ED50) were compared to the dose of pituitary hormone causing the same displacement. So we have. evaluated, for each antibody, the competition capacities of the modified hGH protein vis-à-vis the 125 I-hGH tracer, expressed as a percentage of the competition capacity of the pituitary hormone.
Ces dosages RIA montrent que la hGH modifiée purifiée est un bon compétiteur du traceur hypophysaire pour l'antisérum dirigé contre l'hormone
hypophysaire, et surtout pour le monoclonal dirigé contre une région interne de cette hormone : dans ce dernier cas, la protéine hGH modifiée possède en effet une capacité de compétition vis-à-vis du traceur équivalente à 80 % de celle du standard hGH. Par contre, la hGH modifiée n'entre pas du tout en compétition avec le traceur pour l'anticorps monoclonal NH2-terminal. Ce résultat n'est- pas surprenant étant donné que la protéine hGH modifiée est délétée des acides aminés 1 et 2 de l'hormone naturelle. Liaison à dés récepteurs hépatiques: il est bien établi que l'hormone de croissance agit principalement au niveau du foie, où elle stimule la synthèse des somatomédines et est impliquée dans la régulation de systèmes métaboliques tels que la gluconéogenèse et le métabolisme des acides gras. Le foie est donc un organe riche en récepteurs spécifiques de l'hormone de croissance, et particulièrement le foie de lapine gestante (Posner et al., 1974). Par conséquent, les récepteurs hépatiques utilisés pour les expériences de liaison sont préparés à partir de la fraction enrichie en membranes plasmiques et microsomiales du foie de lapine gestante. Le dosage par radiorécepteurs d'une hormone donnée consiste à incuber une faible quantité fixe d'hormone marquée (traceur) et de concentrations croissantes en hormone froide à doser. La quantité de traceur lié aux récepteurs est mesurée à l'équilibre, et est d'autant plus réduite que la concentration et l'affinité des hormones froides sont plus élevées. Les résultats obtenus montrent que la hGH a une capacité relative de 125 % par rapport à celle du standard international hGH à déplacer de 50 % (ED50) la liaison initiale du traceur 125I-hGH aux récepteurs hépatiques. La hGH modifiée montre donc une capacité
de déplacement supérieure à celle de l'hormone hypophysaire standard. Activité somatogénique in vivo: pour vérifier que la protéine hGH modifiée qui s'attache normalement aux récepteurs d'hormones somatogéniques du foie, retient bien son activité biologique principale de stimulation somatogénique on a utilisé un test : (1) le test du tibia (Greenspan et al., 1974).These RIA assays show that the purified modified hGH is a good competitor of the pituitary tracer for the antiserum directed against the hormone. pituitary, and especially for the monoclonal directed against an internal region of this hormone: in the latter case, the modified hGH protein has in fact a capacity for competition vis-à-vis the tracer equivalent to 80% of that of the hGH standard. In contrast, the modified hGH does not compete at all with the tracer for the NH2-terminal monoclonal antibody. This result is not surprising since the modified hGH protein is deleted from amino acids 1 and 2 of the natural hormone. Binding to hepatic receptors: it is well established that growth hormone acts mainly in the liver, where it stimulates the synthesis of somatomedins and is involved in the regulation of metabolic systems such as gluconeogenesis and the metabolism of fatty acids. The liver is therefore an organ rich in specific receptors for growth hormone, and in particular the liver of a pregnant rabbit (Posner et al., 1974). Consequently, the hepatic receptors used for the binding experiments are prepared from the fraction enriched in plasma and microsomal membranes of the liver of the pregnant rabbit. The dosing by radio receivers of a given hormone consists in incubating a small fixed quantity of marked hormone (tracer) and increasing concentrations of cold hormone to be assayed. The amount of tracer linked to receptors is measured at equilibrium, and is reduced the higher the concentration and the affinity of cold hormones. The results obtained show that hGH has a relative capacity of 125% compared to that of the international standard hGH to displace by 50% (ED50) the initial binding of the tracer 125 I-hGH to hepatic receptors. The modified hGH therefore shows a capacity displacement greater than that of the standard pituitary hormone. Somatogenic activity in vivo: to verify that the modified hGH protein which normally attaches to somatogenic hormone receptors in the liver, retains its main biological activity of somatogenic stimulation, a test was used: (1) the tibia test (Greenspan et al., 1974).
Dans ce test du tibia, on a comparé de façon quantitative l'épaississement des cartilages de conjugaison induit par la hGH modifiée et le standard hGH international chez des jeunes rats hypophysectomes. Les résultats obtenus démontrent le même rapport linéaire entre l'épissage et le logarithme de la dose d'hormone pour les deux hormones. Activité lactoqénique in vivo:In this tibia test, the thickening of the conjugation cartilage induced by the modified hGH and the international hGH standard was quantitatively compared in young hypophysectoma rats. The results obtained demonstrate the same linear relationship between splicing and the logarithm of the hormone dose for the two hormones. Lactoqene activity in vivo:
Dans le test cellulaire NB2 (Tanaka T., Shiu R.P.C., Goût P. ., Béer C.T., Noble R.L., Friesen H.G. 1980 - Journal of Clinical Endocrinology and metabolism, vol. 51, p. 1058-1063), on a mesuré la stimulation de la croissance des cellules NB2 de rats en absence de sérum sous 1'influence de la hGH modifiée et du standard international hGH. Les résultats ont clairement démontré que la hGH modifiée induisait une multiplication des cellules NB2 après 12h, 24h et 36h tout à fait comparable au standard hGH international..In the NB2 cell test (Tanaka T., Shiu RPC, Goût P.., Béer CT, Noble RL, Friesen HG 1980 - Journal of Clinical Endocrinology and metabolism, vol. 51, p. 1058-1063), the stimulation of the growth of rat NB2 cells in the absence of serum under the influence of the modified hGH and of the international standard hGH. The results clearly demonstrated that the modified hGH induces multiplication of NB2 cells after 12 h, 24 h and 36 h which is entirely comparable to the international hGH standard.
En conclusion, ces tests démontrent que la protéine hGH modifiée a non seulement une conformation tridimensionnelle très similaire à celle de l'hormone hGH hypophysaire standard, mais retient toujours à 100% son activité biologique principale de stimulation de la croissance somatogénique.
Activité insulinique de la hGH modifiée:In conclusion, these tests demonstrate that the modified hGH protein not only has a three-dimensional conformation very similar to that of the standard pituitary hGH hormone, but still retains its main biological activity to stimulate somatogenic growth at 100%. Insulin activity of modified hGH:
L'activité insulinique transitoire de l'hormone de croissance humaine peut être mesurée in vitro par un test de stimulation de la lipogenèse, réalisé sur des adipocytes de rat provenant de tissus carences en hGH. La présence d'hormone de croissance endogène induit en effet une résistance du tissu adipeux aux effets insuliniques de la hGH. Le test de l'activité insulinique de la hGH modifiée recombinante est basé sur la mesure de l'incorporation de flucose tritié dans les lipides, en fonction de l'addition de doses croissantes en hGH , ^et est basé sur le test, décrit (Moody et al., 1975) pour la stimulation de la lipogenèse par l'insuline. L'activité insulinique a été mesurée comme suit:The transient insulin activity of human growth hormone can be measured in vitro by a lipogenesis stimulation test, carried out on rat adipocytes originating from hGH deficient tissues. The presence of endogenous growth hormone in fact induces a resistance of adipose tissue to the insulin effects of hGH. The insulin activity test of the recombinant modified hGH is based on the measurement of the incorporation of tritiated flucose into the lipids, as a function of the addition of increasing doses of hGH, ^ and is based on the test, described ( Moody et al., 1975) for the stimulation of lipogenesis by insulin. Insulin activity was measured as follows:
Préparation des adipocytes: les rats sont sacrifiés par dislocation des vertèbres cervicales et le tissu adipeux des reins et des épididymes prélevé. Ce tissu, découpé en petits morceaux est immédiatement digéré à la collagénase (1 mg/ml) pendant 30 minutes à 37*C sous violente agitation, dans le tampon KRH pH 7.4 contenant 35 mg/ml BSA dialysée, et 0.27 mM de glucose, en utilisant 3 ml de tampon KRH par rat. Après filtration sur gazé, la suspension cellulaire est centrifugée à 600 g pendant 30 secondes et les adipocytes, de densité plus faible que le tampon remontent à la surface. L'infranageant est aspiré avec une seringue munie d'une longue aiguille et on remet les cellules en suspension dans le tampon frais. Les cellules sont lavées 5 fois dans du tampon KRH pH 7.4 à 37 C contenant 1 % BSA.Preparation of adipocytes: the rats are sacrificed by dislocation of the cervical vertebrae and the adipose tissue from the kidneys and epididymis removed. This tissue, cut into small pieces is immediately digested with collagenase (1 mg / ml) for 30 minutes at 37 ° C. with vigorous stirring, in the KRH pH 7.4 buffer containing 35 mg / ml dialyzed BSA, and 0.27 mM of glucose, using 3 ml of KRH buffer per rat. After filtration on gas, the cell suspension is centrifuged at 600 g for 30 seconds and the adipocytes, of lower density than the buffer, rise to the surface. The infranate is aspirated with a syringe with a long needle and the cells are resuspended in the fresh buffer. The cells are washed 5 times in KRH buffer pH 7.4 at 37 C containing 1% BSA.
Stimulation de la lipogenèse : le protocole qui a été utilisé pour mesurer l'activité insulinique est adapté du test de stimulation de la lipogenèse par l'insuline, de Moody et al., (1975). Les adipocytes de rat sont d'abord réincubés pendant 4 heures à 37*C
dans le tampon KRH avec une concentration de BSA de 10 mg/ml, puis lavés et remis en suspension à une concentration de 0.8 x 105 cellules/ml. Le test est mené en triple dans des fioles de 10 ml en polyéthylène où l'on ajoute successivement : - 400 μl de la suspension d'adipocytes, - 50 μl de tampon KRH contenant des doses croissantes d'hGH (0.1 à 10.000 ng/ l) ou d'insuline (0.05 à 100 ng/ml) , - 50 μl de glucose tritié (D-[3-3H]glucose) contenant 50.000 à 100.000 dpm, dans un volume total de 0.5 ml. La concentration finale d'adipocytes est de 1 % (V/V) pour les expériences^ sur adipocytes de rats normaux et 2 % (V/V) sur adipocytes de rats hypox. Cette concentration est ramenée à 0.5 % (V/V) pour l'insuline. Les fioles sont incubées 2 heures à 37'C, sous agitation douce. L'incubation est interrompue par l'addition de 5 ml par tube d'une phase organique scintillante (1 L. de toluène + 0.3 gr dimethyl POPOP + 5 g PPO) , les fioles sont fermées et agitées violemment pendant 30 secondes pour rompre les cellules. On laisse reposer 1 heure pour permettre l'extraction des lipides dans la phase organique. La radioactivité extraite dans la phase organique est mesurée dans un compteur béta (Beckman LS1880) . Le rendement de comptage est mesuré individuellement sur chaque échantillon par un programme de standardisation interne calibré sur des standards tritiés et le résultat est donné en dpm. La lipogenèse basale est obtenue en effectuant le test complet sans addition d'hGH ou d'insuline dans les tubes. La radioactivité non spécifique est obtenue en effectuant le test complet sans adipocytes et est soustraite de tous les résultats.Lipogenesis stimulation: the protocol which was used to measure insulin activity is adapted from the lipogenesis stimulation test with insulin, de Moody et al., (1975). The rat adipocytes are first reincubated for 4 hours at 37 ° C. in KRH buffer with a BSA concentration of 10 mg / ml, then washed and resuspended at a concentration of 0.8 x 10 5 cells / ml. The test is carried out in triplicate in 10 ml polyethylene vials to which are successively added: - 400 μl of the adipocyte suspension, - 50 μl of KRH buffer containing increasing doses of hGH (0.1 to 10,000 ng / l) or insulin (0.05 to 100 ng / ml), - 50 μl of tritiated glucose (D- [3- 3 H] glucose) containing 50,000 to 100,000 dpm, in a total volume of 0.5 ml. The final concentration of adipocytes is 1% (V / V) for the experiments on adipocytes from normal rats and 2% (V / V) for adipocytes from hypox rats. This concentration is reduced to 0.5% (V / V) for insulin. The flasks are incubated for 2 hours at 37 ° C., with gentle shaking. The incubation is interrupted by the addition of 5 ml per tube of a scintillating organic phase (1 L. of toluene + 0.3 gr dimethyl POPOP + 5 g PPO), the flasks are closed and shaken violently for 30 seconds to break the cells. It is left to stand for 1 hour to allow the extraction of the lipids in the organic phase. The radioactivity extracted in the organic phase is measured in a beta counter (Beckman LS1880). The counting yield is measured individually on each sample by an internal standardization program calibrated on tritiated standards and the result is given in dpm. Basal lipogenesis is achieved by performing the full test without adding hGH or insulin to the tubes. Nonspecific radioactivity is obtained by performing the complete test without adipocytes and is subtracted from all results.
Les résultats obtenus illustrés dans la Figure 10, montrent l'effet insulinique que l'on obtient pour l'hormone de croissance hypophysaire naturelle, et
pour la hGH modifiée. Contrairement à l'hormone de croissance hypophysaire, la hGH modifiée n'induit aucune augmentation de la lipogenèse de base, même à des concentrations allant jusqu'à 10 μg/ l. Autrement dit, la hGH modifiée ne révèle aucune activité insulinique transitoire, bien que l'on se trouve dans un système carence en hGH endogène.The results obtained, illustrated in FIG. 10, show the insulin effect which is obtained for the natural pituitary growth hormone, and for modified hGH. Unlike pituitary growth hormone, modified hGH does not induce an increase in basic lipogenesis, even at concentrations up to 10 μg / l. In other words, the modified hGH does not reveal any transient insulin activity, although we are in an endogenous hGH deficiency system.
Comme cette activité biologique de ' type insulinique a été mise en évidence pour d'autres hGH recombinantes, notamment la Met-hGH "Somatonorm" (Rapport KabiVitrum, 1985; Goodman, 1984), et la "Genotropin", dont jLa séquence est identique à celle de l'hormone naturelle (Flash d'informations médicales de KabiVitrum, 1987), on peut conclure que l'absence d'activité insulinique est le résultat direct de la délétion des deux premiers acides aminés. Activité diabétogène de la hGH modifiée:As this biological activity 'type insulin has been demonstrated for other recombinant hGH, including Met-hGH "Somatonorm" (KabiVitrum Report, 1985; Goodman, 1984), and "Genotropin" whose sequence is identical jThe to that of the natural hormone (Flash medical information from KabiVitrum, 1987), it can be concluded that the absence of insulin activity is the direct result of the deletion of the first two amino acids. Diabetogenic activity of modified hGH:
On a testé pour vérifier si la hGH modifiée ne présente pas d'activité diabétogène. Le test que l'on a utilisé consiste à mesurer la résistance contre l'insuline dans des chien traités à l'hormone hGH modifiée comme décrit par ADER et al., 1987. Les résultats ont démontré que contrairement aux effets de résistance à l'insuline observé avec le standard hGH international, la hGH modifiée n'induisait aucune diminution de la sensibilité à l'insuline, durant une infusion chronique de 15 jours à basse dose (0.025 g/kg/jour) , ce qui prouve que l'hGH modifiée n'a pas d'activité diabétogène.It was tested to see if the modified hGH exhibits diabetogenic activity. The test that has been used is to measure insulin resistance in dogs treated with the modified hGH hormone as described by ADER et al., 1987. The results demonstrated that, unlike the effects of resistance to insulin observed with the international hGH standard, the modified hGH did not induce any reduction in insulin sensitivity, during a chronic infusion of 15 days at low dose (0.025 g / kg / day), which proves that hGH modified has no diabetogenic activity.
L'activité diabétogène de la hGH modifiée a aussi été testée sur adipocytes de rat. La stimulation du transport du glucose par l'insuline en présence ou en absence de la hGH modifiée a été mesurée et comparée à celle de la hGH naturelle. La hGH modifiée, contrairement à la hGH naturelle n'inhibe pas l'action
de l'insuline dans ce test, ce qui montre encore que la hGH modifiée n'a pas d'activité diabétogène. LEGENDE DES FIGURES:The diabetogenic activity of the modified hGH was also tested on rat adipocytes. The stimulation of glucose transport by insulin in the presence or absence of the modified hGH was measured and compared with that of natural hGH. Modified hGH, unlike natural hGH does not inhibit action insulin in this test, which further shows that the modified hGH has no diabetogenic activity. LEGEND OF THE FIGURES:
- Figure 1;- Figure 1;
Séquence peptidique de l'hormone de croissance humaine (hGH) .Human growth hormone (hGH) peptide sequence.
NH2 : extrémité aminoterminale. COOH : extrémité carboxy terminale.NH2: amino terminal end. COOH: carboxy terminal end.
- Figure 2:- Figure 2:
Schéma de la régulation et des activités biologiques de l'hormone de croissance humaine.Diagram of the regulation and biological activities of human growth hormone.
- Figure 3: ^- Figure 3: ^
La séquence du cDNA hGH clone dans le plasmide pDM 100 - hGH. Les codons des acides aminés de la hGH mature sont numérotés.The sequence of the cDNA hGH cloned in the plasmid pDM 100 - hGH. The amino acid codons of mature hGH are numbered.
- Figure 4 (a) :- Figure 4 (a):
Schéma de la carte physique et de la structure génétique du plasmide d'expression pSOD.Diagram of the physical map and of the genetic structure of the expression plasmid pSOD.
SOD : DNA codant pour la superoxyde dismutase humaine.SOD: DNA coding for human superoxide dismutase.
- Figure 4 (b) :- Figure 4 (b):
La séquence du polylinker inséré à l'extrémité 3' du gène hSOD dans le vecteur pSOD.The sequence of the polylinker inserted at the 3 'end of the hSOD gene in the vector pSOD.
- Figure 5:- Figure 5:
Schéma de la construction du gène hGH modifié.Diagram of the construction of the modified hGH gene.
(a) La séquence de l'oligonucléotide synthétique utilisé pour modifier la séquence amino-terminale du gène hGH.(a) The sequence of the synthetic oligonucleotide used to modify the amino-terminal sequence of the hGH gene.
(b) La séquence des extrémités du fragment Xba I(b) The sequence of the ends of the Xba I fragment
- Sal I du cDNA hGH.- Sal I of cDNA hGH.
(c) La séquence des extrémités du fragment Nco I(c) The sequence of the ends of the Nco I fragment
- Sal I contenant le gène hGH modifié. cDNA-hSOD : séquences appartenant au gène de la superoxyde dismutase humaine.- Sal I containing the modified hGH gene. cDNA-hSOD: sequences belonging to the human superoxide dismutase gene.
SD : séquence Shine-Delgarno.
- Figure 6 :SD: Shine-Delgarno sequence. - Figure 6:
Schéma de la construction du plasmide d'expression pSOD-hGH contenant le gène hGH modifié. hGHM : DNA codant pour la protéine hGH modifiée. cDNA-hSOP : DNA codant pour la superoxyde dismutase humaine.Diagram of the construction of the expression plasmid pSOD-hGH containing the modified hGH gene. hGHM: DNA coding for the modified hGH protein. cDNA-hSOP: DNA coding for human superoxide dismutase.
- Figure 7 :- Figure 7:
Schéma de la construction du plasmide d'expression pSGHD contenant un gène hSOD délété. hGHM : DNA codant pour la protéine hGH modifiée. cDNA-hSOD : DNA codant pour la superoxyde dismutase humaine. ^Diagram of the construction of the expression plasmid pSGHD containing a deleted hSOD gene. hGHM: DNA coding for the modified hGH protein. cDNA-hSOD: DNA coding for human superoxide dismutase. ^
- Figure 8(a) :- Figure 8 (a):
Analyse par PAGE-SDS 15 % des protéines totales, après induction [+] ou sans induction [-] à l'IPTG de l'expression du gène hGHM dans les souches E.coli D1210 (pSGHD) . La flèche indique la protéine hGHM.Analysis by PAGE-SDS of 15% of the total proteins, after induction [+] or without induction [-] at IPTG of the expression of the hGHM gene in the E.coli D1210 strains (pSGHD). The arrow indicates the hGHM protein.
- Figure 8(b) :- Figure 8 (b):
Analyse par PAGE-SDS des protéines contenues dans les corps d'inclusion obtenus après induction à l'IPTG des souches E.coli D1210 (pSGHD) . La flèche indique la protéine hGHM.Analysis by PAGE-SDS of the proteins contained in the inclusion bodies obtained after induction with IPTG of the E.coli D1210 strains (pSGHD). The arrow indicates the hGHM protein.
1. Extrait total des protéines bactériennes.1. Total extract of bacterial proteins.
2. Extrait des protéines des corps d'inclusion purifiés.2. Extract proteins from purified inclusion bodies.
- Figure 9:- Figure 9:
Purification de la protéine hGHM solubilisée à partir des corps d'inclusion purifiés par chromatographie HPLC.Purification of the solubilized hGHM protein from the inclusion bodies purified by HPLC chromatography.
(a) Profil d'élution obtenu avec un gradient linéaire en NH4HC03, de 0.02 à 0.4 M.(a) Elution profile obtained with a linear gradient in NH4HC03, from 0.02 to 0.4 M.
(b) PAGE-SDS 15 % de chacun des pics élues par la chromatographie. La flèche indique la protéine hGHM.(b) SDS-PAGE 15% of each of the peaks eluted by chromatography. The arrow indicates the hGHM protein.
- Figure 10:- Figure 10:
Comparaison de l'activité insulinique du standard hGH international et de la hGH modifiée. Mesure de la
stimulation de la lipogenèse par la hGH dans 1'adipocyte de rat préincubé pendant 4 heures dans un milieu sans hormone de croissance et incubé pendant 2 heures à 37*C en présence de D-[3-3H] glucose et de concentrations croissantes en hGH modifiée (*) ou hypophysaire (# ) . L'incorporation du glucose tritié (exprimée en pourcents de la lipogenèse de base) est mesurée en fonction de la dose d'hormone ajoutée*
Comparison of the insulin activity of the international hGH standard and of the modified hGH. Measure of stimulation of lipogenesis by hGH in the pre-incubated rat adipocyte for 4 hours in a medium without growth hormone and incubated for 2 hours at 37 ° C. in the presence of D- [3- 3 H] glucose and increasing concentrations of modified hGH (*) or pituitary (#). The incorporation of tritiated glucose (expressed in percent of basic lipogenesis) is measured according to the dose of hormone added *
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- Ader M., Agajanian T., Finegood D.T., and Bergman R.N. (1975). Endocrinology 120, 725- 730.
- Ader M., Agajanian T., Finegood D.T., and Bergman R.N. (1975). Endocrinology 120, 725-730.
Claims
1. Hormone de croissance humaine modifiée témoignant d'une activité de stimulation de croissance somatidique caractérisée1. Modified human growth hormone demonstrating a characterized somatid growth stimulation activity
- en ce qu'elle comporte une délétion des n premiers acides aminés N-terminaux de l'hormone de croissance humaine naturelle, n étant supérieur ou égal à 2 et n'allant pas au-delà de ce qui entraîne une modification de l'activité de stimulation de croissance somatidique par rapport à l'hormone de croissance naturelle humaine,- in that it comprises a deletion of the first n N-terminal amino acids of the natural human growth hormone, n being greater than or equal to 2 and not going beyond that which causes a modification of the somatid growth stimulating activity compared to natural human growth hormone,
- et en ce qu'elle est dépourvue soit d'activité diabétogène, soit d'activité insulinique, agissant au niveau du métabolisme des carbohydrates, ou des deux activités à la fois,- and in that it is devoid of either diabetogenic activity or insulin activity, acting at the level of carbohydrate metabolism, or of both activities at the same time,
- sous réserve que n soit différent de 13.- provided that n is different from 13.
2. Hormone de croissance humaine modifiée selon la revendication 1, caractérisée en ce que le nombre n d'acides aminés délétés ne va pas au-delà de ce qui entraîne une modification de la configuration tridimensionnelle naturelle par rapport à l'hormone de croissance humaine naturelle.2. Modified human growth hormone according to claim 1, characterized in that the number n of deleted amino acids does not go beyond what causes a modification of the natural three-dimensional configuration compared to human growth hormone natural.
3. Hormone de croissance modifiée caractérisée en ce qu'elle comporte une délétion des n premiers acides aminés N-terminaux de l'hormone de croissance humaine, n prenant l'une quelconque des valeurs 2 à 24, et étant différent de 13.3. Modified growth hormone characterized in that it comprises a deletion of the first n N-terminal amino acids of human growth hormone, n taking any one of the values 2 to 24, and being different from 13.
4. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 15.4. Modified human growth hormone according to claim 3, characterized in that n takes any one of the values from 2 to 15.
5. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 14. 5. Modified human growth hormone according to claim 3, characterized in that n takes any one of the values from 2 to 14.
6. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 12.6. Modified human growth hormone according to claim 3, characterized in that n takes any one of the values from 2 to 12.
7. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 9.7. Modified human growth hormone according to claim 3, characterized in that n takes any one of the values from 2 to 9.
8. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 7.8. Modified human growth hormone according to claim 3, characterized in that n takes any one of the values from 2 to 7.
9. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n prend l'une quelconque des valeurs de 2 à 5.9. Modified human growth hormone according to claim 3, characterized in that n takes any one of the values from 2 to 5.
10. Hormone de croissance humaine modifiée selon la revendication 3, caractérisée en ce que n est égal à 2.10. Modified human growth hormone according to claim 3, characterized in that n is equal to 2.
11. ADN recombinant codant pour la protéine modifiée selon l'une quelconque des revendications 1 à 3.11. Recombinant DNA coding for the modified protein according to any one of claims 1 to 3.
12. Vecteur modifié par un insérât consistant en l'ADN recombinant selon la revendication 11, sous le contrôle d'un promoteur et des éléments de régulation appropriés permettant l'expression de cet ADN recombinant dans un hôte cellulaire compétent transformé par ledit vecteur- recombinant.12. Vector modified by an insert consisting of the recombinant DNA according to claim 11, under the control of a promoter and appropriate regulatory elements allowing the expression of this recombinant DNA in a competent cellular host transformed by said recombinant vector. .
13. Culture cellulaire transformée par le vecteur recombinant selon la revendication 12.13. Cell culture transformed by the recombinant vector according to claim 12.
14. Procédé de production de l'hormone de croissance humaine modifiée selon l'une quelconque des revendications 1 à 10 comprenant :14. Method for producing the modified human growth hormone according to any one of claims 1 to 10 comprising:
- la culture d'un hôte cellulaire compétent auparavant transformé avec un acide nucléique contenant une séquence de nucléotides codant pour la susdite protéine modifiée, elle-même placée sous le contrôle d'éléments de régulation, dont notamment un promoteur reconnu par les polymérases de cet hôte cellulaire compétent, les éléments d'initiation et de terminaison de la traduction etthe culture of a competent cellular host previously transformed with a nucleic acid containing a nucleotide sequence coding for the above-mentioned modified protein, itself placed under the control of regulatory elements, including in particular a promoter recognized by the polymerases of this host competent cell, elements of initiation and termination of translation and
- la récupération de la protéine modifiée produite à partir des produits d'expression de cet hôte cellulaire.- recovery of the modified protein produced from the expression products of this cellular host.
15. Composition pharmaceutique contenant comme principe actif l'hormone de croissance humaine modifiée selon l'une des revendications 1 à 10.15. Pharmaceutical composition containing as active ingredient the modified human growth hormone according to one of claims 1 to 10.
16. Utilisation de l'hormone de croissance selon l'une des revendications 1 à 10, à la préparation de médicaments destinés au traitement du déficit hypophysaire ou des carences en hormone de croissance, au traitement des retards ou des insuffisances de croissance, au traitement de l'obésité, au traitement de cicatrices après accident ou opération, au traitement des phénomènes de vieillissement, sans présenter d'effet diabétogène et/ou d'effet insulinique, notamment chez les patients cachexiques, les vieillards ou les nouveaux-nés.16. Use of the growth hormone according to one of claims 1 to 10, in the preparation of medicaments intended for the treatment of pituitary deficiency or growth hormone deficiencies, in the treatment of stunted or insufficient growth, in the treatment obesity, to the treatment of scars after accident or operation, to the treatment of the phenomena of aging, without presenting a diabetogenic effect and / or insulinic effect, in particular in cachexic patients, the elderly or newborns.
17. Utilisation de l'hormone de croissance comportant une délétion des 13 premiers acides aminés N-terminaux de l'hormone de croissance humaine naturelle, à la préparation de médicaments destinés au traitement du déficit hypophysaire ou des carences en hormone de croissance, au traitement des retards ou des insuffisances de croissance, au traitement de l'obésité, au traitement de cicatrices après accident ou opération, au traitement des phénomènes de vieillissement, sans présenter d'effet diabétogène et/ou d'effet insulinique, chez les patients cachexiques, les vieillards ou les nouveaux-nés. 17. Use of growth hormone comprising a deletion of the first 13 N-terminal amino acids of natural human growth hormone, for the preparation of medicaments intended for the treatment of pituitary deficiency or growth hormone deficiencies, for the treatment delayed or insufficient growth, in the treatment of obesity, in the treatment of scars after accident or operation, in the treatment of the phenomena of aging, without exhibiting a diabetogenic effect and / or an insulin effect, in cachexic patients, old people or newborns.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8814514 | 1988-11-07 | ||
| FR8814514 | 1988-11-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP0441889A1 true EP0441889A1 (en) | 1991-08-21 |
Family
ID=9371636
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP89913122A Withdrawn EP0441889A1 (en) | 1988-11-07 | 1989-11-07 | Modified human growth hormone |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0441889A1 (en) |
| JP (1) | JPH04503154A (en) |
| WO (1) | WO1990005185A1 (en) |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5534617A (en) * | 1988-10-28 | 1996-07-09 | Genentech, Inc. | Human growth hormone variants having greater affinity for human growth hormone receptor at site 1 |
| US5688666A (en) * | 1988-10-28 | 1997-11-18 | Genentech, Inc. | Growth hormone variants with altered binding properties |
| WO1992021029A1 (en) | 1991-05-10 | 1992-11-26 | Genentech, Inc. | Selecting ligand agonists and antagonists |
| US6429186B1 (en) | 1991-05-10 | 2002-08-06 | Genentech, Inc. | Ligand antagonists for treatment of breast cancer |
| WO1994022469A1 (en) * | 1993-04-02 | 1994-10-13 | Ohio University | Enhancement of learning and/or memory by a human somatotropin |
| AU693478B2 (en) * | 1994-11-10 | 1998-07-02 | Metabolic Pharmaceuticals Limited | Treatment of obesity |
| WO1997011178A1 (en) * | 1995-09-21 | 1997-03-27 | Genentech, Inc. | Human growth hormone variants |
| SE0000837D0 (en) | 2000-03-13 | 2000-03-13 | Pharmacia & Upjohn Ab | New use |
| WO2001096565A2 (en) * | 2000-06-16 | 2001-12-20 | Asterion Limited | Binding agents: chimeric ligand/receptor proteins |
| AU2002325712C1 (en) | 2001-08-30 | 2008-07-31 | Stem Cell Therapeutics Inc. | Differentiation of neural stem cells and therapeutic use theeof |
| JP4906231B2 (en) | 2001-09-14 | 2012-03-28 | ステム セル セラピューティクス インコーポレイテッド | Prolactin-induced increase in neural stem cell number and its therapeutic use |
| AU2003250705A1 (en) | 2002-07-31 | 2004-02-16 | Stem Cell Therapeutics Inc. | Method of enhancing neural stem cell proliferation, differentiation, and survival using pituitary adenylate cyclase activating polypeptide (pacap) |
| AU2003258388C1 (en) * | 2002-09-13 | 2010-03-18 | The University Of Queensland | Gene expression system based on codon translation efficiency |
| DK1569680T3 (en) | 2002-10-22 | 2009-05-18 | Waratah Pharmaceuticals Inc | Treatment of diabetes |
| ATE556716T1 (en) | 2004-02-13 | 2012-05-15 | Stem Cell Therapeutics Corp | USE OF LUTEINIZING HORMONE (LH) AND CHORIONIC GONADOTROPHIN (HCG) FOR NEURAL STEM CELL PROLIFERATION AND NEUROGENesis |
| WO2007036033A1 (en) | 2005-09-27 | 2007-04-05 | Stem Cell Therapeutics Corp. | Oligodendrocyte precursor cell proliferation regulated by prolactin |
| JP2009530235A (en) | 2006-03-17 | 2009-08-27 | ステム セル セラピューティクス コーポレイション | Continuous administration regimen of neural stem cell proliferating agent and neural stem cell differentiation agent |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0103395A3 (en) * | 1982-08-17 | 1985-05-22 | Biogen N.V. | Dna sequences, recombinant dna molecules and processes for producing bovine growth hormone-like polypeptides in high yield |
| CA1341012C (en) * | 1982-09-27 | 2000-06-06 | Biogen, Inc. | Dna sequences, recombinant dna molecules and processes for producing swine growth hormone-like polypeptides |
| BG49718A3 (en) * | 1983-07-15 | 1992-01-15 | Bio- Technology General Corp | Method for preparing of polypeptid with superoxiddismutasne activitty |
| DK151585D0 (en) * | 1985-04-03 | 1985-04-03 | Nordisk Gentofte | DNA sequence |
| PT83613B (en) * | 1985-10-28 | 1988-11-21 | Lilly Co Eli | Process for the selective chemical removal of a protein amino-terminal residue |
-
1989
- 1989-11-07 EP EP89913122A patent/EP0441889A1/en not_active Withdrawn
- 1989-11-07 JP JP2500041A patent/JPH04503154A/en active Pending
- 1989-11-07 WO PCT/EP1989/001399 patent/WO1990005185A1/en not_active Application Discontinuation
Non-Patent Citations (1)
| Title |
|---|
| See references of WO9005185A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO1990005185A1 (en) | 1990-05-17 |
| JPH04503154A (en) | 1992-06-11 |
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