FR2521164A1 - PROCESS FOR THE PRODUCTION OF HUMAN KALLIKREIN - Google Patents

Abstract

PROCESS FOR PRODUCING HUMAN KALLIKREIN; HUMAN CELLS CAPABLE OF PRODUCING HUMAN KALLIKREIN ARE MULTIPLIED IN VIVO AND HUMAN KALLIKREIN IS PRODUCED IN VITRO WITH THE HUMAN CELLS OBTAINED THUS; HUMAN CELLS ARE PREFERREDLY PANCREAS, SALIVARY GLAND OR LIVER CELLS OR NORMAL OR TUMORAL PULMONARY FIBROBLASTOID CELLS OR HUMAN LYMPHOBLASTOID CELLS TRANSFORMED BY HUMAN HUMBRID CELL FUSION OF HALLOWELLA; THE PRODUCTION OF HUMAN KALLIKREIN OBTAINED THANKS TO THE INVENTION IS VERY SUPERIOR THAN CONVENTIONAL PROCESSES USING IN VITRO TISSUE CULTURE.

Description

The present invention relates to a method for

  produce human kallikrein.

  Kallikrein (EC 3 4 21 8) consists of several enzymes from different sources and of different specificities having the common property of releasing vasomotor peptides (kinins) from their precursors

  inactive (kininogens) in plasma.

  The pharmacological actions of kallikrein, that is to say the vasodepressive and hypotonic actions, make it useful as a therapeutic agent in diseases of the circulatory organs, for example arteriosclerosis

  cerebral or cardiac or hypertension; so the be-

  kallikrein treatments are increasing rapidly.

  It is well known that kallikrein is obtained from mammalian organs, such as the liver or the pancreas, or from fluids of the mammalian organism, such as

than blood, saliva or urine.

  As described in patent applications, by

  example ballast Japanese patent applications published unexamined

  mined no 102 495/77 and N O 167 228/80, kallikrein has generally been produced to date with pig organs

  but in insufficient quantity to meet demand.

  As a result of the antigen-anti-

  body caused by kallikrein when used in human therapy, the use of kallikrein derived from living human cells is excellent and devoid

  of danger due to the slightest antigenicity.

  So we are looking for an inexpensive process to

  produce human kallikrein on an industrial scale.

  As the following description shows, the invention satisfies

does this need.

  The subject of the invention is an effective method for multiplying human cells capable of producing human kallikrein;

  a process for producing human kallikrein

  high titer with human cells so ob-

  outfits; a method for obtaining hybrid cells capable of producing human kallikrein, having an extremely high cell multiplication rate and a human kallikrein production rate; and

  a process to increase the production of kal-

human likrein per cell.

  In general, these objectives are achieved

  as well as others according to the method of the invention which comprises

  takes the multiplication of human cells capable of producing human kallikrein, by transplanting

  say cells in a warm-blooded animal other than human

  me or otherwise by letting the said cells multiply in a conventional diffusion chamber which provides

  say cells a nutrient fluid from the body of an

  evil to warm-blooded other than humans, and the production of

  human kallikrein with multi- human cells

  folded by one or other of the multiplication modes in

vivo above.

  More specifically, the objectives of the invention

  tion were achieved by the method which includes (1) transplanting human cells capable of producing

  human kallikrein in a warm-blooded animal

  be that man; feeding the animal to allow

  be for human cells to use a nutritive liquid from the organism supplied by the animal for their multiplication;

  extraction and disintegration of the tumor obtained for-

  mée in animals to obtain human cells multi-

  folded; in vitro culture of human cells in a

  nutrient medium with or without a kalli- inducer

  kreine for a period sufficient to accumulate a significant amount of human kallikrein; and harvesting human kallikrein accumulated from the culture or otherwise: (2) the establishment of a suspension of human cells capable of producing human kallikrein

  in a conventional diffusion chamber which provides

  nit to said human cells a nutritive liquid from the organism of a warm-blooded animal other than man; inclusion or placement of the chamber in or on the warm-blooded animal other than man; feeding the animal to allow human cells to use

  a nutritious liquid from the body provided for their multi-

  plication; harvesting multiplied human cells from the chamber; culturing human cells in vitro in a nutrient medium with or without an inducer

  kallikrein for a period sufficient to accumulate

  a significant amount of human kallikrein; and the re-

  colt of human kallikrein accumulated in the culture.

  The preferred embodiments of the invention

  tion will now be described in detail.

  The invention is based on the discovery that the pro-

  duction of human kallikrein with human cells, obtained by in vivo multiplication of human cells

  capable of producing human kallikrein in use

  being a warm-blooded animal other than man, is extremely

  much higher than that obtained with

  In vitro propagated cells The invention provides a method

  to produce human kallikrein which consists of mul-

  multiply human cells capable of producing human kallikrein by transplanting said cells into a warm-blooded animal other than man or otherwise letting said cells multiply in a room

  of the conventional type which provides said cells with

  nutrient fluid from the body of a blood animal

  hot other than human and to produce human kallikrein

  not desired with the human cells thus obtained.

  Unlike conventional methods using an in vitro tissue culture, the in vivo method requires no less or much less nutrient medium containing expensive serum and makes maintenance much easier.

  culture during cell multiplication and pro-

  additionally contains a human kallikrein of much higher

  laughing. More particularly, in the process according to the invention which uses a warm-blooded animal other than man as host, it is easy to multiply certain

  human cells using a gold nutrient liquid

  organism provided by the animal by transplanting these cells into the animal or otherwise by placing them in a

  classic type diffusion designed to receive a liquid

  from the body and by including or placing the chamber in or on the animal and feeding the animal in the usual way.

  In addition, the process of the invention differs

  conventional methods of cell multiplication in vitro because of the fact that additional characteristics can be obtained, that is to say cell multiplication which is much more stabilized and rapid, increased production of

  cells and an extremely higher production of kalli-

human krein per cell.

  Human cells usable in the invention

  tion are those which produce a significant quantity of human kallikrein and which can be multiplied in a warm-blooded animal other than man after having been transplanted therein. Normal human cells of the pancreas can be favorably used in the invention. , from

  salivary glands, liver, kidney, bladder and fi-

  pulmonary broblasts; cells obtained by trans-

  formation of normal human cells using a

  carcinogenic or radiation virus; tumor cells

  human pancreatic cancer, gland tumor

  salivary, liver cancer, kidney cancer and can-

  bladder cer; and established cell lines of

human cells above.

  Instead of using human cells

  above, the use of a lymphoblastold line

  human, which is much easier to submit to

  tappings and into which one or more

  several human genes coding for the production of kal-

  likrein by cell fusion using polyethylene

  neglycol or the Sendai virus or by techniques of re-

  genetic combination using nuclease, ligase and DNA polymerase, leads to an extreme increase

  cell multiplication speed and / or pro-

  duction of human kallikrein per cell which are envi-

  ron two to ten times greater than what we get when

  whether using the normal or tumor cells described above.

  The transplantation of such a lymphoblas line-

  human coat to the animal tends to form one or more tu

  massive dies who are very little contaminated by cel-

  lules of the host animal In addition, their disintegration after extraction is very easy because the harvesting of cells

  living humans can be easily performed.

  Human lymphoblastold lines from

  leukemia or human lymphoma, such as li-

  Namalwa, BALL-1, NALL-1, TALL-1 and JBL are particularly

  duly suitable for these purposes The warm-blooded animal other than man which can be used in the invention may be one of those in which human cells can be multiplied, for example birds such as chicken and pigeon ; or mammals such as dogs, cats, monkeys, goats, pigs, horses, rabbits, cows, guinea pigs,

  rat, naked rat, hamster, mouse or naked mouse.

  As the transplantation of these human cells-

  the animal triggers an unwanted immune response

  ble, the use of a warm-blooded animal other than man at the earliest possible age, for example in the state of

  egg, embryo, fetus, newborn animal or animal

  evil very young is desirable to reduce as much as pos-

immune response.

  Before transplantation, we can also treat

  ter such an animal by irradiation with X-rays or Y-rays at the rate of about 200 to 600 rems or inject

  an antiserum or immunosuppressant to reduce the reaction

  immune system at the lowest possible level -

  The use of a laboratory animal with an immune deficiency, such as the naked mouse or the naked rat, as a host animal, produces a less undesirable immune reaction even when the animal is adult and it is easy to transplant to the animal. any of

  types of human cells described above without effect

  kill such pretreatment and cells multiply there

  quickly with less risk of causing an im-

  community. We can obtain both a stabilization of

  cell multiplication and increased pro-

  duction of human kallikrein by repeated transplantation using a combination of different warm-blooded animals other than humans For example, the two objectives can be achieved by first transplanting human cells into the hamster and multiplying them, then by retransplanting the multiplied human cells in the naked mouse In this case, the repeated transplantation can be carried out with warm-blooded animals other than the man belonging to the same class or to the same order or

  belonging to the same species or the same genus.

  As a human cell transplant site

  in animals, you can choose any site as long as the cells multiply there: you can for example

  use the allantoic cavity or perform the apparation

  intravenously, intraperitoneally or subcation

tanned.

  Instead of transplanting human cells

  in animals, one can easily multiply any one

  than types of human cells previously described in

  placing them in a standard type diffusion chamber

  that of various shapes and sizes provided for example with a filter membrane, an ultrafilter or hollow fibers having a nominal pore size of approximately 10 7 to 105 m, preventing contamination of the chamber by the cells of the but feeding human cells

  in nutritive liquid of the body; by performing the

  clusion for example intraperitoneal of the room at

  the animal and leaving human cells there multi-

  fold using the nutrient liquid from the organism four-

nor by the animal.

  In addition, the diffusion chamber designed for this

  effect can be placed on the animal so that the nu-

  organism tritive and the nutrient solution in the

  room can flow freely through the room.

  In this way, you can observe the culture in the room

  during cell multiplication through one or more

  several transparent side windows arranged on a

  or more walls of the chamber and / or replace continuously-

  Lement the room by a new room to continue cell multiplication during the life of the animal without sacrificing it and increase considerably

  cell production per animal.

  Since the use of such a diffusion chamber reduces the undesirable immune reaction due to the absence of direct contact between human and animal cells, it is easy to use

  as a host a warm-blooded animal other than any man

  that without pretreatment and we can collect it easily

  living human cells multiplied.

  You can easily feed the animal in the usual way and no special care is necessary, even

after the transplant.

  The period required to obtain the multiplica-

  maximum cell tion is usually 1 to 20 weeks

  When human cells are tumor cells,

  or lymphoblastold, the maximum cell multiplication can usually be obtained in about 1 to 5 weeks. The number of human cells thus obtained can

  be about 107 to 1012 or more per animal Depending on the

  vention, the production of transplanted human cells

  is increased by about 102 to 107 or more times, which is

  about 10 to 106 times what you get according to a

  in vitro multiplication process using a naked medium

  tritive So multiplied human cells can be

  be used favorably for the production of

human kallikrein.

  We can regulate the reproduction of human cells.

  hands multiplied by subjecting them to a tissue culture-

  in vitro milk from 1 to 4 days before the induction stage

described below.

  Any method of inducing multiplied human cells to produce human kallikrein can be employed in the invention. For example, multiplied human cells obtained by harvesting from a

  suspension in ascites or extraction and de-

  aggregation of one or more massive tumors, formed for example under the skin, are suspended in a nutritive medium preheated to a temperature included in

  the range of about 20 to 40 'C to obtain a cel density

  about 104 to 1 o 8 cells per milliliter and in-

  cubed to obtain human kallikrein In this case,

  human cells can be exposed to the action of an

  kallikrein producer to further increase production

  tion of kallikrein The kallikrein inducer usable in the invention can consist of one or more of those which induce the production of human kallikrein in human cells obtained by multiplication of human cells by use of a warm-blooded animal

  other than man For example we can use as in-

  kallikrein producer of amino acids such as glycine or phenylalanine, proteins such as casein, saccharides such as glucose, inositol, ribose or deoxyribose or a hormone such as adrenaline. Stabilization of human kallikrein in

  cultivation and increased production of kal-

  human likrein can be obtained by addition to the

  culture of a kallikrein stabilizer or a stimulus

  kallikrein lator such as trypsin (EC 3 4 21 4)

or an alkaloid.

  Human kallikrein can be easily

  collected in the culture according to purification and separation methods which use conventional techniques, for example concentration, salting out, dialysis, filtration, centrifugation and / or lyophilization If a more purified human kallikrein preparation is

  desirable, a preparation having the pu-

  maximum possible restraint using the techniques described

  above in combination with other conventional techniques

  such as absorption and desorption with an ion exchanger, gel filtration, electrophoresis, affinity chromatography and / or fractionation at

isoelectric point.

  The present human kallikrein is identical

  immunologically to that from human urine

  maine and it contains neither pyrogen nor hepatic virus

  tite: we can therefore use it advantageously isolated-

  ment or in combination with one or more agents such as

  a steroid hormone, an anticoagulant or a car-

  kinostatic for internal administration or for injection

  tion for the prevention or treatment of human diseases

maines.

  Throughout this description, the titles

  of human kallikrein were determined according to the

  bioassay method indicated by Moriya et al, Journal of Biochemistry, Vol 58, page 201 (1965) with a slight modification and they are expressed in biological units (UK) determined by measuring the lowering of blood pressure in a dog after administration of

human kallikrein.

  Several embodiments of the invention will be described without the scope of the invention being

limited in any way.

EXAMPLE 1

  We transplant subcutaneously at

  adult naked ris, a human cell line of can-

  cer of the pancreas, COLO 357, and we feed them in a habit-

tuelle for 4 weeks.

  This cell transplant and food

  subsequent animals cause sub- formation

  of massive tumors each weighing approximately 10 g

  that we extract, fragment and disaggregate by adding

  board in physiological saline solution containing

trypsin.

  Then we wash the human cells with

  Earle medium 199 (p H 7.2) added with 10% v / v of se-

  calf fetal rum and suspended in medium

  fresh with the same composition and containing more

  ron 1 mg / ml of casein, to obtain a cell density of approximately 1 x 105 cells per milliliter then incubated

  culture for 10 days at 37 ° C to induce pro-

duction of human kallikrein

  Then, the culture obtained is subjected to a treatment.

  by ultrasound and the titer is determined in kalli-

human krein from the supernatant.

  Human kallikrein production is around

ron 600 UK per ml of culture.

  1 61 The control is carried out as follows: Human pancreatic cancer cells of the COLO 357 line are cultured in vitro in Parker medium 199 (p H 7.2) supplemented with 10% v / v of fetal serum of calf and the multiplied human cells are subjected to an induc-

  tion similar to that above Production of kalli-

  human kreine is only 20 UK per ml of culture.

EXAMPLE 2

  Cells of the human pancreatic cancer line COLO 357 and of a human lymphoblastoid line Namalwa are put together in suspension in a container with a saline solution 140 m M in Na Cl, 54 m M in KC 1,

  1 m M in Na H 2 PO 4 and 2 m M inri Ca C 12, to obtain densi-

  respective cell tees of approximately 103 cells / ml To the cell suspension is added, cooling by

  ice, a fresh salt solution with the same composition

  additionally containing Sendai virus previously inactivated by ultraviolet irradiation. About 5 minutes after the addition, the mixture is transferred into a

* incubator at 37 ° C and stirred there for about 30 minutes

  to perform cell fusion and introduce the

  human kallikrein production capacity of li-

  human pancreatic cancer cell genus in the

  human lymphoma of lymphoblastoid cells.

  The hybrid cells obtained from adult naked mice are transplanted intraperitoneally.

  one feeds in the usual way for 5 weeks.

  After extraction of the massive tumors obtained, each weighing approximately 15 g, the cell suspension is subjected to an induction of human kallikrein production as in Example 1 except that the casein is replaced by 0.1% w / v of phenylalanine and 0.1% w / v of glucose.

  Human kallikrein production is about

approximately 1,300 UK per ml of culture.

  The control for which human cells hybridized in vitro are cultivated for their multiplication and then

  treated as in Example 1 to produce kalli-

  human kreine, provides only about 100 UK kallikre-

human ine / ml of culture.

EXAMPLE 3

  Human cells are suspended

  kidney tumor that was obtained by tissue extraction

  su of kidney tumor of a patient, fragmentation and disa-

  gregation and cells of the human lymphoblastold line BALL-1, in a container with a salty solution mif in Na Cl, 54 m M in K Cl, 1 m M in Na H 2 PO 4 and 2 m M in Ca Cl 2 for to obtain respective cell densities of approximately 103 cells per milliliter. To the cell suspension is added while cooling with ice, a

  fratche salt solution having the same composition and

  additionally of the Sendal virus previously inactivated by ultraviolet irradiation. The mixture is then transferred to an incubator at 370 ° C. about 5 minutes after the addition and is stirred there for about 30 minutes to effect cell fusion and introduce the capacity.

  of human kallikrein production of human cells

  hands of renal tumor in lym- line cells

  phoblastolde. After injection of an antiserum prepared in the usual way in rabbits, in newborn hamsters for

  reduce their immune response, we transplant them

  hybrid cells subcutaneously to these animals that

  feeds in the usual way for 3 weeks.

  We treat as in Example 1, to induce

  the production of human kallikrein, the cell suspension

  lular obtained from massive tumors formed under

  the skin and each weighing about 10 g.

  Human kallikrein production is around

ron 2 100 UK / ml of culture.

  The control, for which the hybridized BALL-1 cells are cultivated in vitro as in Example 1 to multiply them, then they are treated as in Example 1 to produce human kallikrein, provides only about

  UK human kallikrein / ml culture.

EXAMPLE 4

  We transplant intravenously to

  newborn rats, cells of a lymphoblas lines-

  hybrid human Namalwa in which we have in-

  produces the production capacity of human kallikrein

  as in Example 2 and the animals are fed in a

usual lesson for 4 weeks.

  After extraction of the massive tumors obtained,

  each weighing approximately 40 g, the suspension is treated

  as in Example 2 to induce production

of human kallikrein.

  Human kallikrein production is around

ron UK 1,400 / ml of culture.

EXAMPLE 5

  After irradiating adult mice with X-rays at a dose of approximately 400 rems to reduce their

  immune reactions, we transplant subcutaneously

  born to animals of the cells of the human cancer line

  COLO 357 and we feed them in the usual way for

4 weeks.

  We extract the massive tumors obtained for-

  més under the skin each weighing approximately 15 g and they are treated as in Example 1 to induce the production of

human kallikrein.

  Human kallikrein production is around

ron 900 UK / ml of culture.

EXAMPLE 6

  Cells of the hybridized BALL-1 human lymphoblastoid line are suspended in which

  we introduced the ability to produce kallikrein hu-

  maine in the same way as in Example 3, in a cham-

  cylindrical plastic diffusion bre having an internal volume of approximately 10 ml provided with a filter membrane having a nominal pore size of approximately 0.5 Dam with physiological saline solution and the chamber is placed in the peritoneum an adult rat Then we feed the animal in the usual way for 4 weeks

and we take out the room.

  The density of human cells in the chamber is approximately 2 x 109 cells / ml which is approximately 10 times higher than that obtained by a

  tissue culture in vitro with a C 02 incubator.

  The human cells are then treated as in Example 2 to induce the production of human kallikrein.

  Human kallikrein production is around

ron 1,800 UK / ml of culture.

EXAMPLE 7

  We transplant into the allantoic cavities-

  nes of embryonated eggs which have been preincubated for 5 days at 370 ° C., cells of the hybridized human lymphoblastoid line BALL-1 into which the capacity to produce human kallikrein has been introduced, as in

  Example 3 and the eggs are incubated at this temperature for

for about 1 week.

  Human cells are collected from the eggs and treated as in Example 2 to induce

  production of human kallikrein.

  Human kallikrein production is around

ron 1,200 UK / ml of culture.

Claims (8)

Claims   1 Process for producing human kallikrein,   characterized in that it comprises the stages which consist in: (1) transplanting human cells capable of producing human kallikrein in a warm-blooded animal other than man;   let human cells use a bare liquid   tritive from the animal's body for their multiplication; and allow the multiplied human cells to release human kallikrein, or if not, (2) place human cells capable of producing   human kallikrein in a device that provides them   a nutritious liquid from the body of a warm-blooded animal other than man; include or place the device in or on the animal;   let human cells use the bare liquid   tritive of the organism for their multiplication; and let the multiplied human cells release the human kallikrein.   2 Method according to claim 1, characterized in that it comprises the stages consisting in:   (1) transplant human cells capable of pro-   to kill human kallikrein in a warm-blooded animal other than man; feeding the animal to allow human cells to use a nutritive liquid from the animal's body for their multiplication;   extract and disintegrate the tumor obtained formed in A.   nimal to obtain multiplied human cells; culturing human cells in a nutritive medium in vitro in the presence or not of a kallikrein inducer for a period sufficient to accumulate a significant amount of human kallikrein; and collect the human kallikrein accumulated from the culture, or otherwise,   (2) place a suspension of human cells   to produce human kallikrein in a conventional diffusion chamber to supply the   say human cells into a nutrient liquid of gold-   ganism of a warm-blooded animal other than man; include or place the chamber in or on a warm-blooded animal;   feed the animal to allow human cells   nes to use the body fluid for their multi-   plication; collect the multiplied human cells from the chamber; cultivating human cells in a nutritive medium in vitro whether or not a kallikrein inducer is present for a period sufficient to accumulate a significant amount of human kallikrein; and collect the accumulated human kallikrein from of the culture.   3 Method according to claim 1, characterized in that said human cells capable of producing human kallikrein are normal human cells   chosen from the group made up of human cells   nes of pancreas, the human cells of salivary glands   res, human liver cells, human cells   kidney and human lymphoblastold-pulmonary cells   res. 4 Method according to claim 1, characterized in   that said human cells are human cells   nes selected from the group consisting of   human pancreatic cancer cells, human cells   salivary gland cancer cells, human liver cancer cells, human kidney cancer cells, human bladder cancer cells and those obtained by transformation of the human cells described in claim 3.   Method according to claim 1, characterized in that said human cells are hybrid human cells into which the capacity has been introduced.   to produce human kallikrein.   6 Method according to claim 1, characterized in that said human cells are cells of a   human lymphoblastoid line hybridized with one   conch of human cells described in the claims   3 and 4 to introduce production capacity   of human kallikrein from the second to the first.   7 Method according to claim 6, characterized in that said human lymphoblastoid line is part   of the group formed by the Namalwa, BALL-1, NALL- lines 1, TALL-1 and JBL.   8 Method according to claim 1, characterized in that said warm-blooded animal other than man is a mammal or bird.   9 Method according to claim 1, characterized in that said kallikrein inducer is one or more elements from the group consisting of amino acids,   proteins, sugars and hormones.   A process for producing human kallikrein comprising multiplying human cells capable   to produce human kallikrein and cell culture   humans multiplied in the presence or absence of an inducer   kallikrein for a period sufficient to accumulate   mulate a significant amount of human kallikrein, in which an extreme increase in speed is obtained   cell multiplication and kalli- production   human kreine, characterized in that it consists in: (1) transplanting human cells capable of producing human kallikrein in a warm-blooded animal other than man;   feed the animal to allow human cells   nes to use a nutritious liquid from the animal's organism   bad for their multiplication; extracting and disintegrating the tumor obtained formed in animals, to obtain multiplied human cells; cultivating human cells in a nutritive medium in vitro in the presence or not of a kallikrein inducer   for a period sufficient to accumulate a quantity   notable t of human kallikrein; and collect the human kallikrein accumulated from the culture, or otherwise (2) place a suspension of human cells capable of producing human kallikrein in a   conventional type diffusion to feed said cells   them with a nutritious liquid from the body; include or place the chamber in or on a warm-blooded animal other than man; feed the animal to allow human cells   to use the body fluid for their multiplica-   tion; collect multiplied human cells from from the room; -   culturing human cells in an in vitro nutrient medium in the presence or absence of a kallikrein inducer for a period sufficient to accumulate a significant amount of human kallikrein; and collect the accumulated human kallikrein from of the culture.