FR2521588A1 - METHOD FOR PRODUCING HUMAN IMMUNE RESPONSE SUPPRESSOR - Google Patents

Abstract

METHOD FOR THE PRODUCTION OF A HUMAN IMMUNE RESPONSE SUPPRESSOR (SRIH). IT CONSISTS OF TRANSPLANTING HUMAN CELLS CAPABLE OF PRODUCING SRIH INTO THE BODY OF AN ANIMAL WITH HOT BLOOD, OR IN A DEVICE SUPPLIED BY THE BODY FLUID OF AN ANIMAL WITH HOT BLOOD, LET THESE CELLS MULTIPLY. , THEN TO PROMOTE THE RELEASE OF SRIH FROM THUS MULTIPLIED CELLS. THE PRODUCTION OF SRIH OBTAINED BY THIS PROCESS IS MUCH SUPERIOR THAN THAT OBTAINED BY CONVENTIONAL TISSUE CULTURE PROCESSES IN VITRO.

Description

The present invention relates to a method for producing a substance

biologically active,

  Human Immunity Response Suppressor, hereinafter

  abbreviated as SRIH, whose use in phar-

Macology is promising.

  SRIH means an Immunity Response Suppressor

  from viable human cells.

  As described by Carl Waltenbaugh in Biology of the Lymphokines, pp. 422-427 (1979), published by Academic Press, Inc. (New York), Immune Response Suppressor (IRS) is a proteinaceous substance found in the fluid. body, which inhibits the non-specific immune response of the species, regardless of the type of antigen It was therefore thought that SRI would be a prophylactic or therapeutic agent

  against hypersensitivity, for example allergy, shock

  naphylactic or hyper-auto-immunization, as well as against

  rejection during tissue or organ transplants.

  Although the non-specificity with regard to SRI species,

  suggests the possibility of using SRIs of non-hu-

  for the treatment of human diseases, the SRIH

  from viable human cells proves to be Cre and excel-

  slow because of its lower antigenicity.

  However, to date, there is no production on the scale

  the SRIH industry for the prevention or treatment of

of human diseases.

  The present invention makes it possible to remedy this

  disadvantage of the prior art, and to obtain on the scale

  and low cost, human cells capable of producing high titre SRIH, through an efficient method of multiplication of these cells, and with a higher yield of SRIH per cell, possibly by cell hybridization. The novel method of transplanting human cells into a warm-blooded animal, or into a device in which the body nutrient fluid of a warm-blooded animal circulates, and leaving these

  cells to multiply in vivo in the animal or in the

  positive, is characterized in that the cells are human cells, capable of producing SRIH, and in that the multiplied cells are allowed to release this factor,

  by their in vitro culture in a nutrient medium, possibly

  in the presence of a SRIH inducer.

  More particularly, this process involves transplanting human cells capable of producing SRIH into a warm-blooded animal to feed this animal, so that

  that the human cells can use its fluid cor-

  nutritive porel for their multiplication, to extract and

  to aggregate the resultant tumor that is formed in the animal, to obtain the multiplied human cells, to cultivate

  these cells in vitro in a nutrient medium in the presence of

  possibly induce a SRIH inducer, for a sufficient period of time

  to accumulate a significant amount of SRIH, and to

  harvest this accumulated in the culture We can also

  place a suspension of human cells capable of

  to produce SRIH, in a standard class diffusion chamber

  sique, designed to receive, or include in, the nutritive body fluid of a warm-blooded animal

  the said room, feed the animal, to allow the

  human beings to use their bodily fluid for their

  tiplication, collect human cells in the chamber

  multiplied, cultivate them in vitro, possibly in

  it induces a SRIH inducer for a sufficient period of time, to accumulate a significant amount of SRIH, then to harvest the

SRIH accumulated in culture.

  It is found that the production of SRIH is much greater if one uses human cells, obtained by in vivo multiplication using a warm-blooded animal of human cells capable of producing SRIH, instead of

  use the same cells multiplied in vitro.

  Unlike conventional in vitro tissue culture methods, the method using the in vivo procedure does not

  requires little or no nutrient medium containing

  expensive rum, and makes it easier to maintain the middle of

  during cell multiplication, while producing

  sant SRIH to a much higher title.

  In addition, the process according to the invention differs

  classical methods of cell multiplication in vi-

  with the following characteristics:

  much more stable and fast, cellular production

  superior and much higher production of SRIH by

read the.

  Human cells, usable according to the

  vention, are cells that produce a sen-

  sible for SRIH and who are able to multiply in a

  a warm-blooded animal, when transplanted there:

  for example, normal human cells, such as

  peripheral blood, spleen and tonsils; cells obtained by transformation of normal human cells using radiation or carcinogenic viruses;

  human cells derived from tonsillar abscess, car-

  cinoma of the liver or lung, of the spleen tumor; as well as established cell lines of the above human cells. In accordance with the invention, known human cell lines such as Ad-L, HL-4, L 84-Ly, NPC-501, and HB can be used. -7, as described in The

  Tissue Culture, Vol 6, pp 527-546 (1980).

  A much higher rate of cell multiplication can be

  to be achieved by using instead of human cells

  described above, of a human lymphoblastoid line, which can be much more readily subcultured, and into which human genes encoding SRIH production can be introduced, using a cell fusion technique using polyethylene

  glycol or Sendai virus, or by a recombinant

  genetic combination using nuclease, ligase and

DNA polymerase.

  There is thus a higher production of SRIH per cell, in particular about 2 to 10 times higher than that which one

  gets when using normal cells or tu-

moral, described above.

  The transplantation of these lymphoma lines

  Human blastoids in an animal, leads to the formation of tumors that are hardly contaminated by cells

  animal host In addition, their post-extraction and unpleasant

  is easy, and harvesting viable human cells can easily be done for these purposes.

  known lines of human lymphoblastoids, leukemia

  serum or lymphoma, eg Namalwa, BALL-1, NALL-1,

TALL-1 and JBL.

  As warm-blooded animals, usable in the method according to the invention, any animal can be taken, provided that the cell lines develop in its

  For example, poultry

  me chickens and pigeons, or mammals such as dog, cat, monkey, goat, pig, horse, rabbit, cow, guinea pig,

  naked rat, hamster, mouse, or nude mouse.

  Since the transplantation of human cells to the animal causes an undesirable immune reaction, the use of a very young, newborn animal or at the earliest possible stage, for example of an egg, embryo or fetus, is To reduce the immune response as much as possible, the animal can be treated, prior to cell transplantation, by irradiation with

  X-ray or Y-radiation at about 200 to 600 rems, or by

  injection of an antiserum or immunosuppressive agent.

  As the nude mouse, or the bare rat used as a host animal, has a weaker immune response even in the state

  adult, one can conveniently transplant

  established human cell systems to multiply

  fold quickly, without the need for pretreatment.

  Cellular multiplication, stabilization, and increased production of SRIH can be achieved by repeated transplantation with a combination of different warm-blooded animals; for example we can reach the

  objectives, by first implanting the human cells

  to hamsters, then re-implanting the cells and

  if multiplied in nude mice In this case repeat trans- plantation can be done with blood animals

  of the same class or group, of the same

species or the same kind.

  We can implant human cells into a

  site of the animal, provided that they multiply

  fold: for example, one can perform the implantation in

  the allantoic cavity, intravenously, intraperitoneally

neonatal or subcutaneous.

  Instead of transplanting animals into the

  Human cells can be easily multiplied one of the human cells described above, placing them in a conventional diffusion chamber of appropriate shape and size, provided with a porous filter membrane,

  ultrafilter or hollow fibers with pores

  10-7 to 10-5 m in diameter, preventing contamination

  of the chamber by the cells of the host animal and allow

  the intake of the nutritive liquid from the body of the animal

  evil to human cells This room is included, by

  example intraperitoneally, in the animal; we leave

  get the human cells grow there by using the

  nutrient body fluid provided by the animal.

  In addition, the diffusion chamber can be designed to be

  be placed on the animal, for example, so that the nutritive fluid of the body and the nutrient solution

  can freely circulate in the room of this ma-

  However, culture can be observed in the chamber, during cell multiplication, by one or more windows.

  transparent sides, provided on the walls of the room.

  and / or we can replace this room with a bedroom

  new, both to continue the cell multiplication

  during the life of the animal, without the need to sacrifice it, and to increase further

  plus cell production per animal Moreover, by the

  use of such a diffusion chamber, the absence of direct contact of the transplanted human cells with the

  cells of the host animal, the result of which is not to provoke

  immune response, so that it is possible to

  bend various warm-blooded animals as hosts without pretreatment to reduce their reactions

  immune system, and the cells can be easily

the multiplied, viable humans.

  The animal is fed in a conventional manner

  that even after cell transplantation, and no care

particular is necessary.

  The multiplication of cells generally reaches

  maximum 1 to 20 weeks after transplantation.

  When the human cells, transplanted to the host animal, are of tumoral origin or lymphoblastoid, one can obtain

  their maximum multiplication 1 to 5 weeks after trans-

planting.

  The number of human cells thus obtained

  per host is about 107 to 1012 or more. In other words,

  my, the number of human cells transplanted to animals

  badly, increases from about 2 to 107 times or more

  presents about 10 to 10 times more compared to what the

  the multiplication method of the nutrient medium

  in vitro The human cells obtained can therefore be

  advantageously used for the production of SRIH.

  Cell generation of human cells,

  multiplied, can be regulated by subjecting these cells to

  them to an in vitro tissue culture of 1 to 4 days, before

  submit them at the introductory stage described below.

  According to the invention, any method for inducing SRIH can be used to produce SRIH.

  production in human cells multiplied; eg-

  For example, they are suspended in a nutrient medium

  heated between 20 to 400 C of human cells,

  obtained by multiplication in suspension in the liquid

  of ascites and harvest in this liquid, or by extraction of a massive tumor, formed under the skin, then harvesting

  after disintegration of the tumor, to obtain a

  centration of about 104 to 108 cells / ml; we put

  following incubation at the same temperature to produce SRIH.

  Human cells can then be exposed to

  inductor, in order to further increase the production of

from SRI.

  Suitable inducers are those

  the production of SRIH by human cells

  in the body of a warm-blooded animal; we can

  use, for example, mitogens such as phytohemoagglutin

  tinin, concanavalin A, lipopolysaccharide, endotoxin, polysaccharide, bacterium, virus, nucleic acid, or a

plant mitogen ("pokeweed").

  The culture medium can also, during the production of SRIH, be added together with a

  stabilization of the formed SRIH, which further increases

duction of it.

  SRIH can be easily harvested from the culture by purification and separation techniques using conventional procedures, such as

  salting out, dialysis, filtration, centrifugation, concentra-

  and / or lyophilization If a preparation is desired

  more purified, this can be achieved by combining

  mentioned above with one or more others, as

  sorption and desorption, with ion exchange, gel filtration, affinity chromatography, fractionation in point

  isoelectric and / or electrophoresis.

  SRIH according to the invention can be advantageously used i-

  solemnly or in combination with one or more

  vitamin, hormone, carcinostatic agent, by adminis-

  internal injection or injection, for the prevention or

treatment of human diseases.

  In the present invention, the SRIH titers are determined by the method mentioned by Robelt Rich R and Carl W Pierce in "Journal of Immunology", Vol 112, pp.

  1360-1368 (1974), with slight modifications, and are ex-

  awarded in the amount that suppresses the response of the plaque forming cells; 1 ml portions of a cell suspension containing 1 × 10 6 spleen cells from C57 BL / 6 mice are placed in Petri dishes and are each added with 0.1 ml portions of SRIH preparations. a series of dilutions The mixtures

  in the Petri dishes are incubated during a

  time, then we count the plates formed during this incubation The titre in SRIH corresponds to the dilution

  for which a plate reduction of 50% is obtained.

  Several embodiments of the invention are described below, which, however, do not limit the scope thereof.

EXAMPLE 1

  Subcutaneous transplants in adult nude mice, an Ad-L human adenoid line, and animals

  are fed in the usual way for 3 weeks.

  This cell transplantation, and the nourishment

  followed by animals, result in the formation of subcutaneous massive tumors of approximately 12 g each, which are

  then extracted, chopped and disaggregated by

  pension in a saline solution containing trypsin. The human cells are then washed with serum-free RPMI 1640 medium (pH 7.2), and suspended in fresh medium of identical composition, so that

  have a concentration of the order of 1 x 10 cells / ml.

  To this cell suspension, we add concanavali-

  at a rate of 1 μg / ml, and this mixture is incubated

  370 C for 20 hours, to induce the production of SRIH.

  The culture is centrifuged at 8000 rpm for 30 minutes.

  minutes and the SRIH title of the supernatant is de-

  terminated as described above The production is of the order of 2400 units / 0.1 ml of culture. The control experiment proceeds as follows: Ad-L human adenoid line is cultured in vitro in medium

  Eagle (p H 7.2), supplemented with 1% by volume / volume of

  fetal veal and 20% by volume / volume of meat extract, with a view to its multiplication, and then treated as

  above to induce SRIH production.

  in the resultant culture is only 90 units / year.

ml approx.

EXAMPLE 2

  Suspended tumor cells are suspended together

  of human spleen, obtained by extraction and hashing

  tumor tissue from a patient, and a

  phoblastoid, human, Namalwa, in a saline solution containing 140 mM NaCl, 54 m MK Cl, 1 mM NaH 2 PO 4 and 2 mM Ca C 12, to obtain a concentration of each type of cells of about 1000 cells per ml is added to the cell suspension, while cooling with ice,

  a fresh preparation of the saline solution of the same

  sition, containing previously inactivated Sendai virus

  by ultraviolet irradiation; we transfer to a

  at 370 C after 5 minutes, after mixing, and

  stirred for 30 minutes to effect the cell fusion.

  and introduce the ability to produce SRIH from human cells of the splenic tumor in cells

de-Namalwa.

  The resulting hybrid Namalwa cells are then transplanted intraperitoneally into mice.

  naked, adults, who are fed habitually

  5 weeks massive tumors, formed intraperitoneally

  tonally, and weighing approximately 15 g each, are extracted and treated as in Example 1 to produce SRIH,

  except that concanavalin A is replaced by

  viron 10 μg / ml phytohaemagglutinin.

  The production of SRIH is of the order of 12 800 units / O, 1 mi of culture.

  The control, in which the hybridized cells are cultured

  in vitro for their multiplication, then treated as in Example 1 to induce the production of SRIH,

  gives only about 400 units of SRIH per 0.1 ml of cul-

ture.

EXAMPLE 3

  After injecting newborn hamsters with an antiseptic

  prepared from the rabbit according to a conventional method, to reduce their immune reaction, subcutaneous animals are implanted with cells of the lymphoblastoid cell line, hybridized human, JBL which was

  granted, as in Example 2, the production capacity

  tion of the SRIH, then the animals are usually fed

  For 3 weeks the massive, resulting tumors formed under the skin and weighing about 18 g each are extracted; the cells are treated as in Example 1

  to induce the production of SRIH, except that the

  place RPMI 1640, serum-free, is replaced by medium

  of Eagle (p H 7.2) supplemented with 20% in volume / volume of ex-

trait de v Lande.

  The production of SRIH is of the order of 8 500 units / O, 1

ml of culture.

  The control, in which the JBL hybridized cells are cultured

  in vitro for their multiplication, then are treated as in Example 1 to induce the production of SRIH,

  gives only about 240 units of SRIH / O, 1 ml of culture.

EXAMPLE 4

Intravenous infants were implanted with human hybrid BALL-1 delymphoblastoid lines, which, as in Example 2, were given the production capacity of SRIH, and the animals were then fed to the animals.

  usual for 4 weeks Mass tumors were removed

  resultant, weighing approximately 35 g each, and

  treats as in Example 2 to produce SRIH.

  The production of SRIH is of the order of 9 400 units / O, 1 ml

of culture.

  The control, in which the hybridized BALL-1 cells are cultured in vitro for their multiplication, and then treated as in Example 1 to induce the production of SRIH,

  gives only about 350 units / of SRIH per 0.1 ml of culture.

re.

EXAMPLE 5

  After irradiating adult rats with an equivalent of

  dose of about 400 rems to reduce their reactions.

  immunity is implanted with the animals by subcutaneous

  dermal a cell line derived from lymph nodules.

  human phages, HL-4, and then feed the animals

  usual way for 4 weeks.

  Massive tumors, resulting, formed under the skin and

  weighing approximately 15 g each, are extracted and processed

  example 3 to induce SRIH production.

  the production is of the order of 3 600 units / O, 1 ml of cul-

ture.

  The control, for which the human cell line is cul-

  in vitro for its multiplication, and then abandoned

  to leave the SRIH free, only about 30

of SRIH / O, 1 ml of culture.

EXAMPLE 6

  A human lymphoblastoid line, Ad-L, is suspended in physiological saline, in a cylindrical diffusion chamber, made of plastic, having an internal volume of about 10 ml and provided with a filtering membrane.

  whose pores are about 0.5 μm in diameter.

  intraperitoneal injection of the chamber into an adult rat,

  the animal in the usual way for 4 weeks and then

  the chamber is removed and the human cells are collected.

  The density of human cells in the chamber reaches up to about 10 9 cells per ml, which is about 100 times greater than that obtained by tissue culture in vitro, with a C 02 incubator. The human cells are treated as in Example 3 to induce the production of SRIH, which is about 4,200

units / 0.1 ml of culture.

EXAMPLE 7

  It is implanted in the allantoic cavities of embryonic eggs.

  born, preincubated at 370 C for 5 days, a line of

  human phoblastolts, hybrid, BALL-1, which was conferred as in example 4 the production capacity of SRIH After incubation of the eggs at this temperature

  for another week, the human cells are harvested

  multiplied, and treated as in Example 1 to induce SRIH production. This production is

  of the order of 3,200 units / l, 1 ml of culture.

Claims (8)

  1 Application of the method of transplanting human cells into a warm-blooded animal or   a device in which circulates the nutritive fluid cor-   porel of a warm-blooded animal, and to let these cells multiply there, characterized in that the cells chosen   are those who are able to produce the Suppres-   Human Immunity Response (SRIH), and that the   we let the multiplied cells release this factor.   2 Application according to claim 1, characterized in that the human cells, capable of producing   SRIH, are normal cells derived from the peri-   pheumatic, spleen or amygdala.   3 Application according to claim 1, characterized in that the human cells capable of producing the SRIH are tumor cells, originating from tonsillar or splenic tumors, or carcinoma of the liver or lung, or cells obtained by transformation of the cells.   human cells according to claim 3.   4 Application according to claim 1, characterized in that the human cells are hybrid cells,   which has been conferred the ability to produce SRIH.   Application according to Claim 1, characterized   in that the human cells are lymphocyte lines   human blasto, hybridized with one of the human cells   according to claims 3 or 4, in order to introduce 1 '   ability to produce SRIH from the last in the first.   6 Application according to claim 5, characterized   in that the human lymphoblastoid line is the   Namalwa, BALL-1, NALL-1, TALL-1 or JBL.   7 Application according to one of claims 1 to 6,   characterized in that the multiplied cells are subject   to the action of a SRIH inducer, to promote the duction of it.   8 Application according to claim 7, characterized   in that the inducer is a mitogen, a virus, or an nucleic acid.   Application according to one of Claims 1 to 8,   in which the warm-blooded animal is a chicken, pigeon,   dog, cat, monkey, goat, pig, cow, horse, rabbit, co   baye, rat, hamster, mouse, naked rat or naked mouse.