FR2495938A1 - PROCESS FOR THE PRODUCTION OF HUMAN ADRENOCORTICOTROPIC HORMONE - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR THE PRODUCTION OF HUMAN ADRENOCORTICOTROPIC HORMONE; THE IN VIVO MULTIPLICATION OF HUMAN LYMPHOBLASTOID CELLS, CAPABLE OF PRODUCING THIS HORMONE, IS PERFORMED, USING A HOT BLOOD ANIMAL, WITH EXPOSURE OF THE HUMAN LYMPHOBLASTOID CELLS MULTIPLIED TO THE ACTION OF A HORM INDUCTOR; THERE IS SIMULTANEOUS PRODUCTION OF OTHER HUMAN HORMONES, SUCH AS THE STIMULATING MELANOCYTE HORMONE AND HUMAN LIPOTROPIC HORMONE; THE HACTH PRODUCTION RATE, ACCORDING TO THE INVENTION, IS VERY HIGHER THAN THAT OF THE CONVENTIONAL PROCESS; THE INVENTION THEREFORE MAKES AVAILABLE A SUFFICIENT QUANTITY OF HACTH FOR THE PREVENTION AND TREATMENT OF HUMAN DISEASES.

Description

The present invention relates to a method for

  the production of human adrenocorticotropic hormone.

  This hormone, hereinafter referred to as

  viation hACTH, is a hormone secreted by the anterior lobe of the pituitary gland, which stimulates the synthesis of hor-

  mones steroldes and secretion of human insulin.

  The Applicant has studied methods for the mass production of hACTH. These efforts have led to the unexpected discovery that lymphoblastoid cells, human, capable of producing this hormone, are suitable for the massive production of hACTH, due very high values of their multiplication speed and their hACTH production per cell. In particular the invention

  relates to a process for the production of hACTH, which

  helps to multiply lymphoblastold cells,

  human, capable of producing this hormone, by trans-

  planting the cells in question in the body of a warm-blooded animal or letting them multiply in

  a device providing them with a nutritious liquid of gold

  ganism of a warm-blooded animal; lymphoblas cells-

  toldes, human, thus multiplied, then release 1 '

hACTH that we collect.

  The method according to the invention, in addition to a pro-

  increased production of hACTH, does not require or requires much less nutrient medium containing expensive serum to

  cell multiplication; he makes the maintenance of the 4-

  equilibrium of the culture medium, during cell multiplication, much easier than in the case of cultures

  tissue in vitro. In particular, we can do multi-

  easily fold any lymphoblastold cell, human, -

  capable of producing hACTH, using a nu-

  tritive from the body of a warm-blooded animal, by trans-

  planting of cells in the animal's organism or

  their suspension in a class diffusion chamber

  sic, designed to receive the nutritive liquid from the organ-

  nism, the animal being fed in the usual way. In or-

  tre, the process is characterized by a multiplication

  more stable and abundant cell, with pro-

increased duction of hACTF per cell.

  As regards the human lymphoblastoid cells which can be used in the invention, any lymphoblastoid cells can be taken, provided that they produce hATCH and multiply in 1 '

  warm-blooded animal organism. The lympho- cells

  blastoids, human, which we prefer, are those in

  which we have introduced genetic sites,

  in the production of hACTH, human cells capable of

  to produce this hormone, such as naturally occurring hACTH cells, such as those in the anterior pituitary lobe, tumor cells

  pituitary or chromophobic adenoma, or other cel-

  human cells, which produce ectopic hACTH, such as

  the as the pancreatic carcinoma cells reaching

  lots of Langerhans, or of carcinoma cells pul-

  monaire. The introduction can be done by cellular fusion.

  re, using polyethylene glycol or Sendal virus, or alternatively using a genetic recombination technique with DNA! ligase, nuclease and DNA polymerase, or human lymphoblastold cells producing hACTH

ectopic.

  Like the use of lymphoblastoid cells,

  human, causes the formation of a massive tumor, faci-

  the to disaggregate when transplanting cells into 1 '

  organism of a warm-blooded animal, and that the mass tumor

  ve is hardly contaminated by the cells of the animal.

  host, human, living lymphoblastoid cells,

  multiplied, can be easily harvested.

  As warm-blooded animals, one can use

  any animal if human lymphoblastoid cells become

  grow in his body. For example, it is advantageously possible to employ in the invention volatiles such as chicken or pigeon and mammals such as dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or naked mouse. As the transplantation of human lymphoblastoid cells to the animal causes an undesirable immune reaction, the use of a newborn or very young animal, preferably at the earliest possible stage, for example of egg, embryo or fetus, is desirable. To reduce as much as

  possible immune reaction, we can treat ani-

  poorly, before cell transplantation, by irradiation with X-rays or t at the rate of about 200 to 600 rems

  or by injection of an antiserum or an immunosup-

  presser prepared according to a conventional process. Like the naked mouse, 'when used as a warm-blooded animal

  has a weaker immune response even when exposed to

  as an adult, we can practically transplant all human lymphoblastoid lines provided that they multiply rapidly, without any pretreatment being

necessary.

  Stabilized cell multiplication and increased hACTH production can be performed

  by repeated transplantation with one or more combinations

  sounds of different warm-blooded animals; we can reach

  the objectives, by first setting up the cells

  human lymphoblastolds to hamsters, so that they

  multiply and then reimplant them into naked mice.

  Repeated transplantation can be performed with ani-

  evils of the same class or the same group or the same es-

pbce or the like.

  We can implant lymphoblastold cells

  humans at any site in the animal, solely

  dition that they multiply: for example we can e-

  implant in the allantoic cavity or by

  intravenously, intraperitoneally or subcutaneously.

  Apart from cell transplantation di-

  correct in the organism of an animal, we can make

  multiply any conventional human lymphoblastoid lines capable of producing hACTH, using the nutritive liquid supplied by the organism of an animal to

  warm-blooded, by inclusion, for example intraper-

  ritoneal, in the animal's body, a conventional diffusion chamber of appropriate shape and size, provided with a porous filter membrane, an ultrafilter or hollow fibers having pores of about 10- 7 to 10-5

  meter in diameter, preventing contamination of the cham-

  bre by the cells of the host animal. As the diffusion chamber can, if necessary, be designed so that it can be placed, for example, on the body of the host animal and allow the circulation of the liquid of the animal in the

  bedroom. allow observation of the cell suspension

  space contained in the room by one or more feng-

  very lateral, transparent, with one or more

  on the walls of the room and allow the replacement

  and exchange with a new room, cell production

  area per host can be increased over the lifetime of

  the animal, without there being any need for sacri-

  proud this one. In addition, when using such a room,

  broadcast bre, as no reaction occurs

  immune, thanks to the absence of direct contact of

  human cells with the cells of the host animal we can

  employ various warm-blooded animals as hosts, without

  pre-treat to reduce their immune reactions

  and you can easily harvest the lym-

  human phoblastoldes, multiplied.

  One can easily feed the host animal, in which human lymphoblastoid cells have been implanted at the

  usual way, even after a cell transplant

  re, and no special care is required.

  The multiplication of cells reaches general-

  a maximum in 1 to 20 weeks after transplanting

  cellular tion. According to the invention, the number of cells

  human lymphoblastold, obtained by host, is com-

  taken between about 107 and 1012 or more. In other words

  mes, the number of human lymphoblastold cells, implanted in the organism of the animal, increases by approximately 102 to 107 times cu or more by approximately 10 to 106 times or more

  compared to what we get by the cul-

  tissue culture in vitro, with a nutrient medium; therefore human lymphoblastoid cells are suitable for

hACTH production.

  For the induction of hACTH, any method can be used in which the lymphoblastold cells

  human, obtained according to the invention, release this hor-

  mone. For example, lym-

  human phoblastoldes, obtained by multiplication in suspension in the ascites fluid and the collection in this ascites fluid, or by the extraction of the massive tumor, formed under the skin, and the harvest after disintegration of

  massive tumor, to get a cell concentration

  milk of about 104 to 108 cells per ml, in a nutritive medium preheated to a temperature of about 20 to 40 C;

  then they are subjected to the action of an a-hormone inducer

  drenocorticotrope at this temperature, for about 1 to

  50 hours, to induce the production of this hormone.

  As inducers of adrenocorticotropic hormone, which are useful according to the invention, any inducer capable of inducing the release of this hormone by the

  human lymphoblastold cells. Hor inducers

  My favorite adrenocorticotropic moneys are vasopres-

  sine, atropine methylnitrate, insulin, glucagon,

  a bacterial endotoxin, serotonin and propanolol.

  The Applicant has discovered that after the induction of hACTH, a large quantity of human melanocyte stimulating hormone, hereinafter designated by the abbreviation hMSH,

  and human lipotropin (hLPH) is produced simultaneously - -

  is lying. We can easily collect hACTH, hMSH

  and hLPH using purification and separation techniques

  paration using conventional operations, such as

  salting out, dialysis, filtration, centrifugation, concentration

  tration and lyophilization. If a more purified preparation

  reliable is desirable, it can be obtained by combining the above techniques with other conventional operations, such as adsorption and desorption with ion exchange,

  gel filtration, affinity chromatography, fraction-

  iscelectric point and electrophoresis.

  The hACTH preparation thus obtained can 4-

  be used advantageously alone or in combination

  sound with one or more agents for administration by injection, externally, internally or in a

  diagnostic goal in the prevention and treatment of my-

  human ladies, such as pituitary insufficiency.

  In the present description, the production of

  hACTH is determined according to a radioimmunological method as described by S. Mataukura et al., in J. Lab. Clin. Med., Vol. 77, ppo 490-500 (1971), it is expressed in

  weight per ml of cell suspension.

  Also the productions of hMSH and hLPH are

  determined by a radioimmunological method, such as

  written by K. Abe et al. in J. Clin. Invest., Vol. 46,

  pp. 1906-1916 (1967). We first determine the quanti-

  total hormones with a human P-MSH standard, then the weight ratio of the hormones is calculated from the quantities of human p-MSH and PLPH, isolated by

gel filtration.

  Non-limiting embodiments of the information

vention are described below.

EXAMPLE 1

  Human cells are suspended together

  nes of pulmonary carcinoma obtained by extraction at a

  sick, with pulmonary carcinoma, followed by ha-

  chage, and leukemic lymphoblastoid cells, hu-

  maines, from the Namalwa line, in a container, with a saline solution 140 mM in NaCI, 54 mM in KC1, 1 mM in NaH2PO4 and 2 mM in CaC12, to obtain concentrations of each cell type of approximately 1C5 cells per mli

  The frozen cell suspension is mixed with a pre-

  fresh paration of the same salt solution containing Sendat virus previously inactivated by ultraviolet irradiation; the whole is placed in an oven at 370C for 5 minutes, after mixing, then stirred for

  minutes, to perform cell fusion; the capa-

  cited for the production of hACTH from human lung carcinoma cells is thus introduced into the human leukemic lymphoblastoid line. After cloning, by a conventional method, of the hybridoma cell strain capable of producing hACTH, this strain is implanted in naked adult mice, which are then fed with

  usual way, for 5 weeks. We extract the tu-

  massive dies formed under the skin, each weighing about

  ron 15 g, and they are disaggregated by chopping and additionally

  board in physiological saline, containing trypsin. After washing the cells with Earle's medium 199 (pH 7.2) added with 10% by volume of fetal calf serum, the cells are resuspended at a

  concentration of approximately 106 cells per ml, in a pre-

  fresh paration of the same medium containing 0.1 pq of glu-

  cagon per ml as an inducer, then incubate-at 350C,

  20 hours, to induce the production of hormones.

  Then the cells are subjected to a treatment with ul-

  and the hACTH, hMSH and hLPH are determined in the supernatant. The production of hACTH is 25OQug per ml of cell suspension, that of hMSH 580 "g per ml and that of

1'hLPH of 40 / Ug per ml.

  Control cells are obtained by implanting human lung carcinoma cells into mice

  naked, adults, feeding animals in the usual way-

  for 5 weeks, extraction of massive tumors,

  formed under the skin and weighing about 5 g each, and

  aggregation of these tumors. These control cells, obtained, are treated as above, to induce the production of hormones. The production of hACTH is then only 600 ng per ml of cell suspension, that of hMSH from

  1.2 g per ml and that of hLPH 350 ng per ml.

EXAMPLE 2

  As in Example 1, the fusion of

  human chromophobic adenoma cells, obtained by

  traction to a patient with chromophobic adenoma of the hy-

  pophysis and hash, with lymphoblastold cells leu-

  cemic, human, from the JBL line, to introduce the hACTH production capacity of human cells

  chromophobic adenoma in the leuko- lymphoblastold line

  human mique. After cloning, by a conventional method, of the strain of hybridoma cells capable of producing hACTH, these hybridoma cells are implanted, subcutaneously, in newborn hamsters, to which have been previously injected an antiserum prepared on the rabbit to reduce their immune responses; the animals are fed in the usual way for 3 weeks. We extract

  massive tumors, formed under the skin and weighing about

  ron 10 g each, they are broken up by chopping and

  suspension in a saline, physiological solution, containing

  collagenase. After washing the cells with essential, minimal Eagle medium (pH 7.2), supplemented with 5% by volume of human serum, the cells are resuspended, at a concentration of approximately 105 cells per ml, in a preparation fralche of the same medium, containing 3 U of vasopressin per ml as inducer; incubated at 370C for 15 hours, to induce the production of hormones. Production, determined as in the example

  1, is for hACTH 3801Jg per ml of cell suspension

  re; for the hMSH of 740 g per ml and for the hLPH of 510 μg per mi. The control cells obtained by implantation of human chromophobic adenoma cells in newborn hamsters, feeding animals in the usual way, are treated in a similar manner, to induce the production of hormones.

  for 3 weeks, extraction of massive tumors,

  under the skin and weighing about 3 g each, and

  aggregation of these tumors. The production of hACTH per ml is only 750 ng per ml; that of hMSH of 560 ng and

that of the hLPH of 430 ng.

EXAMPLE 3

  Rats are implanted intravenously

  newborn leukemia lymphoblastold cells, hu-

  maine, of the BALL-1 line, to which the ability to produce hACTH of human cells of human carcinoma has been conferred, as in Example 1; the rats are fed in the usual way for 4 weeks. Massive, formed tumors weighing approximately 30 g each are extracted and disaggregated. After washing with RPMI 1640 medium (pH 7.4) added with 10% by volume of fetal calf serum, the cells are resuspended, at a concentration of approximately 1 cells per ml, in a fresh preparation

  6mM mnme / ail in serotonin (inducer), then we in-

  cube at 300C for about 40 days. The production of hACTH is 730.4 g / ml of cell suspension and that of hMSH is 940 μg per ml. Control cells obtained by implantation of human cells of pulmonary carcinoma in newborn rats are treated as above. , feeding animals so

  usual for 4 weeks, removal of mass tumors

  sives, formed, weighing about 5 g each, and disaggregated

  tion of these tumors. The production of hACTH is then only 650 ng per ml of cell suspension and that of

i'hMSH of 1.8 / g per ml.

EXAMPLE 4

  After irradiating adult mice with an X-ray dose equivalent of approximately 400 rems, to reduce their immune responses,

  animals, subcutaneously, lymphoblas cells-

  tota leukemia, human, of NALL_1 line, to which

  we have given the production capacity of the hACTH of

  human chromophobic adenoma cells, as in the example

  ple 2, and they are fed in the usual way for 3 weeks. Massive tumors, formed under the skin and

  weighing approximately 15 g each, are extracted and disaggregated.

  The cells are treated as in Example 2, to induce the production of hormone. The production of hACTH is 480 pg per ml of cell suspension and that of hLPH of

350 / Ag per ml.

  The control cells obtained by implantation of human chromophobic adenoma cells are treated as above.

  the mouse. Animal feeding takes place in a habit-

  tuelle for 3 weeks, after which we proceed to the ex-

  traction of massive tumors, formed, weighing about 5 g each, / to the disintegration of these tumors. The production

  of hACTH is only 520 ng per ml of cell suspension

  re; that of hLPH is 790 ng per ml.

EXAMPLE 5

  Human leukemic lymphoblastot cells of the TALL-1 line are suspended in physiological saline, to which the ability to produce hACTH of human lung carcinoma cells has been conferred, in the same manner as in 'example 1;

  the solution is introduced into a cy-

  lindrique, in plastic material, of interior volume-

  about 10 ml, provided with a filter membrane having pores

  about 0.5 & year. After inclusion of the room in the

  riton of an adult rat, we feed the animal in a way

  usual for 4 weeks, then we remove the room.

  The density of the human cells in the chamber, obtained in the above operation, is approximately 7 x 10 cells per ml which is approximately at least 1 time higher than that obtained by in vitro culture with a C02 incubator. The human cells thus obtained are treated,

  as in Example 3, to induce the formation of hormone

  nes. The production of hACTH is 280 mg per ml of suspension.

  cell ion; that of hMSH of 770 μg per ml; and this-

16Oag lhLPH per ml.

  Control cells are treated as above.

  obtained by suspending human cells from

  pulmonary cinome in physiological saline,

  transfer of the cell suspension obtained in a chamber

  cylindrical plastic diffusion bre having an internal volume of approximately 10 ml and provided with a filtering membrane having pores of approximately 0.5 μm, inclusion of the chamber in the peritoneum of an adult rat, feeding

  of the animal in the usual way for 4 weeks and

  collects human cells, multiplied, ie approximately 107 cells per ml. The production of hACTH is only 480 ng per ml of cell suspension; that of 2.3 hg hMSH

  per ml and that of hLPH 250 ng per mi.

EXAMPLE 6

  Is implanted in the allantolic cavities of embryonated eggs, preincubated at 370C for 5 days, leukemic lymphoblastoid cells, human, of the JBL line, to which we have conferred, as in Example 1, the production capacity of hACTH human lung carcinoma cells. After incubating the eggs at this temperature,

  for another 1 week, the lympho- cells are collected

  human blastoids, multiplied. We treat these cells

  as in Example 2, to induce the production of hACTH.

  The production of hACTH is 520 μg per ml of cell suspension. In a control experiment, where human lung carcinoma cells are implanted in the allantoic cavities of embryonated eggs, we do not observe

cell multiplication.

Claims (9)

Claims   1. Process for the production of the hormone adrenocort-   human ticotrope, hACTH, characterized in that it consists   to multiply human lymphoblastot cells   nes, capable of producing this hormone, in a nutritive liquid of the organism of a warm-blooded animal, by transplantation of these cells in the organism itself of the animal, or in a device receiving the nutritive liquid of this animal, and then leave the cells multiplied release the hormone.   2. Method according to claim 1, characterized in   that the hormone produced is accompanied by the hormone sti-   mulante of melanocyte, hMSH, and / or lipotropin, hLPH.   3. Method according to one of claims 1 or 2,   characterized in that the human lymphoblastot cells   nes are hybridoma cells obtained by cell fusion   of a human lymphoblastoid line with cells   human cells capable of producing hACTH.   4. Method according to claim 3, characterized in that the hybridoma cells are obtained by cell fusion of a human lymphoblastold line with   human chromophobic adenoma cells.   5. Method according to claim 3, characterized in that said hybridoma cells are obtained by cell fusion from a human lymphoblastold line   with human lung carcinoma cells.   6. Method according to claim 3, characterized in   that said lymphoblastotde line is a lym- line leukemic phoblastoid, human.   7. Method according to claim 3, characterized in   that the human lymphoblastoid line is one of the   Namalwa, BALL-1, TALL-1, NALL-1 and JBL.   8. Method according to one of the preceding claims,   characterized in that the release of hACTH is induced in human lymphoblastoid cells, multiplied, by means of one or more substances: vasopressin,   atropine methylnitrate, insulin, glucagon, an endoto-   bacterial xine, serotonin, propanolol.   9. Method according to one of the preceding claims -   tes, characterized in that the warm-blooded animal is a chicken, pigeon, dog, cat, monkey, goat, pig, cow,   horse, rabbit, guinea pig, hamster spleen, mouse or naked mouse.