US 4,621,053 AGrant

Process for the Production of Human Peptide Hormones

Issue Date:1986-11-04

•58 Claims

Abstract

Human peptide hormones, such as insulin, growth hormone, prolactin, adrenocorticotropic hormone, placental lactogen, calcitonin, parathyroid hormone and thyroid stimulating hormone, are produced by implanting a human.times.human hybridoma lymphoblastoid cell line capable of producing the human peptide hormone in a non-human warm-blooded animal. After a period of time, the resultant tumor is extracted and disaggregated and then cultured in vitro under conditions appropriate to accumulate the human peptide hormone. The human.times.human hybridoma lymphoblastoid cell line is preferably formed by fusing parent human cells inherently capable of producing the human peptide hormone with a human lymphoblastoid line, preferably of leukemic origin. This process permits a substantial increase in the amount of human peptide hormone which can be produced.

Metadata

Assignee

Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo

Inventor

Kaname Sugimoto

Application Information

Application Number:US 5840171

Filing Date:1984-02-27

Priority Date:1980-07-30

Art Unit:128

Classifications

IPC:

C12P 2100C12N 1500C12N 500C12N 502

Field of Search:

43542493543626053068;172.2;240;241;284-28685;86;8789;99;93;109;106548112 B;112 R399

Patent Drawings

This patent does not have any drawings.

Description

The following patents of the same inventor are related to the present application: U.S. Pat. Nos. 4,383,034, 4,383,035, 4,383,036. U.S. Pat. No. 4,377,513, of which the present inventor is a coinventor, is also related to the present application.

FIELD OF THE INVENTION

The present invention relates to a process for producing human peptide hormones, such as insulin, growth hormone (hGH), prolactin, adrenocorticotropic hormone (hACTH), placental lactogen (hPL), calcitonin (hCT), parathyroid hormone (hPTH), and thyroid stimulating hormone (hTSH).

DESCRIPTION OF THE PRIOR ART

All peptide hormones produced in the human body exert a physiological effect on the individual. Various undesirable diseases and conditions occur when there is a deficiency in any given peptide hormone. Accordingly, means to mass-produce such peptide hormones have been long sought so that they may be used in the treatment of such diseases or conditions.

While various of the peptide hormones produced in the human find their counterparts produced in animals, and are usually non-species-specific, it is less desirable to use such peptide hormones of animal origin as opposed to the corresponding peptide hormones of human origin. There is a particular risk as to antigen-antibody reaction of peptide hormones derived from animals when used as therapeutic agents for humans, as opposed to the corresponding peptide hormones from viable human cells.

All of these peptide hormones have been separated, in the laboratory, from the cells which naturally produce them and have been studied. It has not previously been posible to produce sufficient peptide hormone from the in vitro culturing of such human cells to serve therapeutic purposes. The techniques of chemical synthesis or cultivation of genetically recombined microorganisms have also been attempted, but the processes generally result in low yield and high production cost. Some of these peptide hormones, such as hACTH, can be recovered from human urine and others ay be recovered from human serum. However, these are expensive processes and the supply of starting product is limited.

Among the peptide hormones for therapeutic utility for which means of mass production have been particularly sought, are human growth hormone, human insulin, human prolactin, human adrenocorticotropic hormone, human placental lactogen, human calcitonin, human parathyroid hormone, and human thyroid stimulating hormone.

Human growth hormone is presently in commercial use as a therapeutic agent. It has been produced by processes such as chemical synthesis, in vitro tissue culture or cultivation of genetically recombined microorganisms, but all of these processes result in very low hGH yield and high production cost.

Human insulin is, of course, in general use for the treatment of diabetes. As is the case with human growth hormone, while production of human insulin has been attempted by means of chemical synthesis, in vitro tissue culture, and cultivation of genetically recombined microorganisms, these processes have resulted in a very low human insulin yield and high production cost. Accordingly, insulin from bovine or porcine sources has been utilized for therapeutic purposes in the past. A high yield - low cost method for producing human insulin is in great demand.

Human prolactin, also known as lactogenic hormone, is a protein secreted by human acidophile adenoma cells of the anterior pituitary which stimulates the growth of mammary gland, prostate and seminal vesicles. The compound has therapeutic utility in causing the stimulation of lactation. No process for the mass production of human prolactin has yet been established.

Human adrenocorticotropic hormone, also known as adrenocorticotropin, is a polypeptide secreted by the anterior pituitary and has therapeutic utility in stimulating the adrenocortex to produce cortical hormones. It may be isolated from pituitaries or from female human urine, but no means of mass production of this hormone is yet known.

Human placental lactogen, also known as human chorionic somatomammotropin, is a polypeptide secreted by the human syncytiotrophoblast of the chorionic villi in the human placenta. This hormone has many physiochemical and immunological similarities to hGH. It has some metabolic effects which are qualitatively similar to those of growth hormone, including inhibition of glucose uptake, stimulation of free fatty acid and glycerol release, enhancement of nitrogen and calcium retention (despite increased urinary calcium excretion), reduction in the urinary excretion of phosphorus and potassium, and an increase in the turnover of hydroxyproline, as reflected by increased urinary excretion of that amino acid. No process for the mass production of low cost human placental lactogen has yet been established.

Human calcitonin is a peptide secreted by human parafollicular cells in the thyroid gland which suppresses the release of calcium from bones into the blood, and also the secretion of insulin. Again, no effective process to produce a large amount of human calcitonin at low cost has heretofore been established.

Human parathyroid hormone, also known as parathormone, is a hormone secreted by the parathyroid gland that regulates the synthesis of activated vitamin D.sub.3, stimulates the release of calcium from bone, and leads to an increase in the blood level of calcium. As with the other hormones discussed, no process for the mass production of low cost human parathyroid hormone has yet been established.

Human thyroid stimulating hormone, also known as human thyrotropin, is a glycoprotein secreted by the pituitary gland which stimulates the growth of the thyroid gland and the intake of iodine into other human hormones. It further activates ATPase. The mass production of low cost human thyroid stimulating hormone has also been long sought.

The present inventor is the inventor or co-inventor of prior U.S. Pat. Nos. 4,276,282, 4,285,929 and 4,328,207. U.S. Pat. No. 4,276,282 is drawn to a process for the production of human interferon (HuIFN), while U.S. Pat. No. 4,285,929 is drawn to a similar process for the specific production of human Type II interferon (HuIFN-.gamma.). The U.S. Pat. No. 4,328,207 is drawn to a related process for the production of mouse interferon (MuIFN). The processes disclosed in the first two of these patents comprise multiplying an established human cell line by transplanting it into a non-human warm-blooded animal body, or into a diffusion chamber through which the nutrient body fluids of the animal passes, in which multiplication of the transplanted cells takes place. The multiplied cells are then exposed to an interferon inducer, either in vivo or in vitro to induce the production of interferon, and the interferon produced is then purified and separated from the cells. These patents disclose that by growing a human lymphoblastoid line in or on a non-human warm-blooded animal, it multiplies more stably and rapidly to obtain a larger production of cells than can be obtained in -vitro, and to unexpectedly obtain a higher interferon yield per cell than is obtainable when the cells are multiplied in vitro. U.S. Pat. No. 4,328,207 discloses a similar process for producing mouse interferon.

Human interferon is not a peptide hormone. It is produced only in extremely minute quantities, and then only after being induced by an interferon inducing agent. In addition, IFNs are strongly species-specific while peptide hormones are substantially non-species-specific. The fact that the yield of human interferon per cell can be increased by first multiplying a human lymphoblastoid cell line in or on a non-human warm-blooded animal would not make obvious the subsequent discovery by the present inventor that all human peptide hormones can be mass-produced by first fusing parent human cells inherently capable of producing the human peptide hormone with a human lumphoblastoid line and then multiplying the obtained hybridoma line in or on a non-human warm-blooded animal, obtaining in every case a higher yield of the substance being produced per milliliter of cell suspension that obtainable from in vitro production. The prior Sugimoto interferon patents disclose nothing whatsoever about the use of hybridomas, nor does the experience with interferon make obvious the results obtained with human peptide hormones.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to eliminate the deficiencies of the prior art as discussed hereinabove.

It is a further object of the present invention to provide a process for the low cost mass production of human peptide hormones.

It is another object of the present invention to provide a process for obtaining hybridoma cells capable of human peptide hormone production higher than that of the parent cells used to obtain the hybridoma.

It is still another object of the present invention to provide a process to augment the per cell human peptide hormone production of cell lines capable of producing such a human peptide hormone.

It is another object of the present invention to provide an efficient process for multiplying human hybridoma cells capable of producing human peptide hormones.

It is yet a further object of the present invention to provide a process for producing high titered human peptide hormones with the human hybridoma cells thus obtained.

It is still another object of the present invention to provide a process for the low cost mass production of human growth hormone.

It is still another object of the present invention to provide a process for the low cost mass production of human insulin.

It is still another object of the present invention to provide a process for the low cost mass production of human prolactin.

It is still another object of the present invention to provide a process for the low cost mass production of human adrenocorticotropic hormone.

It is still another object of the present invention to provide a process for the low cost mass production of human placental lactogen.

It is still another object of the present invention to provide a process for the low cost mass production of human calcitonin.

It is still another object of the present invention to provide a process for the low cost mass production of human parathyroid hormone.

It is still another object of the present invention to provide a process for the low cost mass production of human thyroid stimulating hormone.

Broadly, these and other objects, as may become apparent hereinafter, have been obtained by the process which comprises multiplying a human lymphoblastoid line capable of producing the specific desired human peptide hormone by transplanting said cell line to a non-human warm-blooded animal or, alternatively, allowing said cell line to multiply in a conventional-type diffusion chamber by which the nutrient body fluid of a non-human warm-blooded animal is supplied to the cell line. The human peptide hormone is then produced with the human cells multiplied by either of the above in vivo multiplication procedures.

More particularly, these and other objects as may become apparent have been obtained by the process comprising either (1) implanting the human cell line capable of producing the desired human peptide hormone to a non-human warm-blooded animal; feeding the animal to allow the human cell line to utilize the nutrient body fluids supplied from the animal for their multiplication; extracting and disaggregating the resultant tumor, formed in the animal, to obtain the multiplied human cells; culturing the human cells in an in vitro nutrient medium for a period sufficient to accumulate a significant amount of the desired human peptide hormone; and recovering the accumulated human peptide hormone from the culture, or alternatively, (2) placing a suspension of such human cell line in a conventional type diffusion chamber by which the nutrient body fluid of a non-human warm-blooded animal is supplied to the human cell line; embedding or placing the chamber in or on the non-human warm-blooded animal feeding the animal to allow the human cell to utilize the nutrient body fluid supplied for their multiplication; and then collecting and culturing the cells in vitro and recovering the accumulated human peptide hormone as described in the first alternative procedure.

The feature of the present invention which allows even greater multiplication of the human cells and larger production of the desired human peptide hormone, comprises using as the human cells which are implanted into the non-human warm-blooded animal, or into the diffusion chamber, a human.times.human hybridoma line which comprises a human lymphoblastoid line in which the genes coded for the production of the desired peptide hormone have been introduced by a suitable technique. The use of such a hybridoma line results in an extreme increase in cell multiplication rate and human peptide hormone production per cell, i.e. about 2-10-fold higher than that obtained by means of the process of the present invention using non-hybridoma cells productive of the human peptide hormone.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The outstanding increase in human peptide hormone production obtainable by means of the present invention is made possible due to the combination of two discoveries by the present inventor. The first is that the rate of cell multiplication and the amount of human peptide hormone obtainable per cell from a cell line which is otherwise capable of producing the human peptide hormone in question, can be greatly increased by first multiplying the cell line in or on a non-human warm-blooded animal, i.e. by either implanting the cells directly into the animal, or by growing them in a diffusion chamber which is attached to the animal so as to allow the nutrient body fluids of the living animal to be supplied to the cells.

The second discovery is that human cells inherently capable of producing the human peptide hormone in question, can be fused to a human lymphoblastoid cell line, preferably of leukemic origin, in order to obtain a faster growing cell line with increased human peptide hormone productivity.

The combination of these discoveries synergistically provided the result that when such hybridoma cells are multiplied in or on a non-human warm-blooded animal, the productivity of human peptide hormone per cell increases to such a large extent that it could not be predictable either from the productivity of the hybridoma in vitro or from the productivity of the parent hormone-producing cell line multiplied in or on a non-human warm-blooded animal.

The human.times.human hybridoma cell line which is used in accordance with the present invention, may conveniently be obtained by any of the techniques of cell fusion which may be known to the art. References which disclose methods of cell fusion include Yamanaka, T. et al, Biken Journal, vol. 9, pp. 159-175 (1966), or Pontecorvo, G., Somatic Cell Genet., Vol. 1, No. 4, pp. 397-400 (October 1975), the contents of which are hereby incorporated by reference. The parent human cells inherently capable of producing a human peptide hormone may be fused with a human lymphoblastoid line, preferably of leukemic origin, by suspending the parent human cells together with the human lymphoblastoid line in a salt solution containing an effective amount of a cell fusion inducing agent and allowing the resultant cell suspension to stand for a period of time sufficient to effect the cell fusion. The hybridoma lines obtained are then tested for human peptide hormone producibility. Those having a higher human peptide hormone producibility than that of the parent human cells are selected and cloned. It is these lines which will have a great increase in human peptide hormone producibility after being allowed to multiply in or on a non-human warm-blooded animal.

The fusion inducing agent which may be used in this process may be any agent which will induce cell fusion, preferably Sendai virus or polyethylene glycol.

Implantation of such a human.times.human hybridoma lymphoblastoid line into the animal body results in the formation of a massive tumor which is not contaminated in any substantial way with host animal cells. The massive tumors are easily disaggregatable and can thus be easily harvested. Such human.times.human hybridoma lymphoblastoid lines also reproduce very rapidly in a diffusion chamber attached to a non-human warm-blooded animal in such a manner that the nutrient body fluids of the animal is permitted to contact the cells within the chamber.

As long as the "hybridoma" cell line is basically a lymphoblastoid line, preferably of leukemic origin, which contains the genes governing the production of the human peptide hormone in question, it may be used in the process of the present invention. For example, the human peptide hormone production governing genes may be introduced into the lymphoblastoid line, preferably of leukemic origin, by means of genetic engineering techniques, such as recombinant DNA techniques, using enzymes such as DNA ligase, nuclease, and DNA polymerase. Thus, the term "human.times.human hybridoma lymphoblastoid line capable of producing human peptide hormone" as used throughout the present specification and claims is intended to include not only lymphoblastoid lines produced by cell fusion between parent human cells inherently capable of producing the human peptide hormone and the human lymphoblastoid line, but also to human lymphoblastoid lines which have been altered in any manner, such as by genetic engineering, so as to be capable of producing human peptide hormone.

The human lymphoblastoid line, into which the human peptide hormone-producing ability is introduced in order to obtain the human human hybridoma line used in the process of the present invention, may be any lymphoblastoid line of human origin which forms a tumor implanted in an immuno-deficient or immuno-suppressed non-human warm-blooded animal. Such human lymphoblastoid lines are obtainable by establishing in a suitable manner lymphoblastoid cells from a patient suffering from a leukemia, e.g. acute lymphatic leukemia, acute myelogeneous leukemia or chronic myelogenous leukemia, malignant lymphoma, Burkitt lymphoma or infectious mononucleosis. Human lymphoblastoid lines usable in the present invention may also be obtained by transforming normal human lymphocytes by use of a suitable carcinogenic virus, agent or irradiation, such as Epstein-Barr virus (EB virus), mitogen or x-ray irradiation, and establishing the obtained lymphoblastoid cells. Examples of such lines are B-Ta, Q-Ta, B-Ue, Q-Ue, B-Ke, and Q-Ku, reported in Sato, J., Protein, Nucleic Acid and Enzyme, 20, 6, 616-643 (1975). Preferably, the human lymphoblastoid line is of leukemic origin as, for example, Namalva, reported by Strander, H. et al, Journal of Microbiology, 1, 116-117 (1975), BALL-1, TALL- 1, or NALL-1, reported by Miyoshi, I. et al, Nature, 267, 4614, 843-844 (1977), or JBL, reported by Miyoshi, I. et al, Cancer, 40, 2999-3003 (1977). Other lymphoblastoid cell lines usable include the other human lymphoblastoid cell lines listed in the above cited Strander et al publication, including Akuba, PeHR-1 and LY-46. Others include the cell lines M-7002 and B-7101 as described in Journal of Immunology, 113, 1334-1345 (1974); EBV-Sa, EBV-Wa, MOLT-3 and EBV-HO, as described in The Tissue Culture, 6, 13, 527-536 (1980); CCRF-SB (ATCC CCL 120); BALM 2, DND-41; etc.

While these cell lines are specifically mentioned, it should be understood that any lymphoblastoid line which forms a tumor after being implanted in an immuno-deficient or immuno-suppressed non-human warm-blooded animal may be used in accordance with the present invention.

The human parent cells which are fused with the lymphoblastoid cell line, in order to obtain the human.times.human hybridoma line used in the present invention, may be any human cell which inherently produces the human peptide hormone to be produced. For example, in the case of hGH, the cells may be intact human acidophile cells from the anterior pituitary or such cells which have first been transformed with EB virus or x-ray irradiation. Also usable are acidophile adenoma cells from a patient suffering from acidophile adenoma of the pituitary gland, or human lung carcinoma cells which produce ectopic hGH.

In the case of human insulin, cells may be intact human pancreas Langerhans island .beta.-cells, or such cells which have first been transformed by EB virus or x-ray irradiation. Also usable are insuloma cells from patients suffering from insuloma, or human lung carcinoma cells which produce ectopic human insulin.

In the case of prolactin, the cells may be intact human acidophile cells from the anterior pituitary, or such cells which have first been transformed with EB virus or x-ray irradiation. Also usable are acidophile adenoma cells from a patient suffering from acidophile adenoma of the pituitary gland, or human lung carcinoma cells which produce ectopic human prolactin.

For hACTH, the cells may be those from the anterior lobe of the pituitary gland, pituitary tumor cells or chromophobe adenoma cells. Also usable are other human cells which produce ectopic hACTH such as pancreas carcinoma cells from Langerhans island or lung carcinoma cells.

In the case of placental lactogen, the cells may be syncytiotrophoblast cells of the chorionic villi, or chorionic epithelioma cells. Also usable are lung carcinoma cells which produce ectopic hPL.

In the case of hCT. the cells may be human parafollicular cells from the thyroid gland or thyroidea adenoma cells. Also usable are human tumor cells capable of producing ectopic hCT, such as human carcinoid cells or human lung tumor cells.

In the case of hPTH, cells may be normal or transformed parathyroid cells, or cells which produce ectopic hPTH, such as lung carcinoma cells, ovarian tumor cells, kidney carcinoma cells, or liver carcinoma cells.

In the case of hTSH, cells may be basophile cells from the anterior lobe of the pituitary gland, pituitary tumor cells, or chromophobe adenoma cells from the pituitary gland. Also usable are human cells which produce ectopic hTSH, such as lung carcinoma cells.

The above list is not intended to be complete, and any cell line which inherently produces the human peptide hormone being sought may be used as the parent cells which are fused with the lymphoblastoid cell line to obtain the human.times.human hybridoma used in accordance with the present invention.

The human.times.human hybridoma cells which are capable of producing human peptide hormone may be directly implanted into, or inserted into a diffusion chamber which is connected to any non-human warm-blooded animal which will support cell multiplication. Examples of usable animals include poultry, such as chickens and pigeons, or mammalians, such as dogs, cats, monkeys, goats, pigs, cows. horses, rabbits, guinea pigs, rats, hamsters, mice, and nude mice.

Since direct cell implantation often elicits undesirable immuno-reaction, the use of immuno-deficient or immuno-suppressed animals is preferable. Examples of immuno-deficient animals are newborn or infant animals, or those in the youngest possible stage, for example, the use of eggs, embryos, or fetuses is desirable. Since the nude mouse, when used as the non-human warm-blooded animal, exhibits weaker immuno-reaction, even when in its adulthood, they may be used directly as the immuno-deficient animal without need of any immuno-suppression. Alternatively, immune reaction may be suppressed prior to cell implantation by treatment of the animal with x-ray or .gamma.-ray irradiation, for example about 200-600 rem, or injection of antiserum or immuno-suppressive agent prepared according to conventional methods.

When the diffusion chamber embodiment of the present invention is used, since no immuno-reaction is elicited due to the absence of direct contact of the hybridoma cells with the host animal cells, any non-human warm-blooded animal can be used without any pretreatment to suppress immuno-reaction.

A particularly advantageous method to stabilize cell multiplication and augment human peptide hormone production involves repeated transplantation, using a combination or combinations of different non-human warm-blooded animals. For example, the hybridoma cells may first be implanted into a hamster and multiplied therein, and then re-implanted in a nude mouse. The repeated transplantation may be carried out with animals of different species within the same class or division, as well as with those of the same species or genus.

The hybridoma cells can be implanted at any site of the animal, as long as the cells multiply thereat. For example, the cells may be implanted in the allantoic cavity or intravenously, intraperitoneally or subcutaneously.

Besides direct implantation of the cells into the animal body hybridoma cell lines may be multiplied while utilizing the nutrient body fluids supplied from the non-human warm-blooded animal through the use of a conventional diffusion chamber of any of various shapes and sizes. The diffusion chamber may be imbedded with a porous membrane filter, ultra-filter, or hollow fiber with pore sizes of about 10.sup.-7 to 10.sup.-5 m in diameter, which prevents contamination of the hybridoma cells with host cells within the diffusion chamber, yet allows the animal to supply the cells with its nutrient body fluid.

The diffusion chamber may be imbedded, for example, intraperitoneally, in the animal body, or it can be designed to be placed on the exterior of the host animal while being connected to the blood stream of the animal so as to permit the body fluid of the animal to circulate into the chamber. Placing the diffusion chamber on the exterior of the animal allows the advantages of enabling observation of the cell suspension in the chamber through a transparent side window equipped on the chamber wall, and also enables replacement and exchange with a fresh chamber. Cell multiplication thereby increases to a further higher level over the period of the animal's life, and the cell production per animal is further augmented without any sacrifice of the host animal. The multiplied human cells may be harvested easily without contamination by the animal's cells when using the diffusion chamber embodiment. Furthermore, the same improvement of productivity per cell of the cell line is achieved when using the diffusion chamber embodiment as when using the direct implantation embodiment.

Feeding of the host animal which has been implanted with the human cells, or which carries the diffusion chamber, may be carried out easily by conventional methods, and no special care is required. Maximum cell multiplication is obtained within 1-5 weeks after the cell implantation or the attachment of the diffusion chamber. The number of human cells obtained per host ranges from about 10.sup.7 to 10.sup.12 or more. In other words, the number of human cells transplanted in the animal body increases about 10.sup.2 to 10.sup.7 -fold or more, or about 10.sup.6 -fold more than that obtained by in vitro tissue culture methods.

After the hybridoma cells are multiplied in or on the non-human warm-blooded animal, they are collected and cultivated in vitro by an appropriate nutrient medium under conditions appropriate to accumulate a substantial amount of the peptide hormone in a manner well known per se. For example, the human.times.human hybridoma lymphoblastoid cells obtained after removal from the diffusion chamber or from suspension in acites, or after extracting and disaggregating the massive tumor formed subcutaneously, are su pended to give a cell concentration of about 10.sup.4 to 10.sup.8 cells per milliliter in a nutrient medium, pre-warmed at a temperature of about 20.degree. to 40.degree. C., and then incubated at this temperature for 1-50 hours to produce the human peptide hormone.

In some cases, it is necessary to subject the cells being so incubated in vitro to an inducer in order to cause the hormone to be released or to an agent for enhancing the hormone production. For example, the production of hGH, human insulin, human prolactin and hACTH usually must be induced by an appropriate inducing agent In the case of hGH the cells being incubated at the temperature of 20.degree. to 40.degree. C. are subjected to a growth hormone inducer at this temperature for about 1-20 hours to induce hGH. Preferable growth hormone inducers are amino acids, such as glycine, arginine, tryptophan, leucine, casamino acid and L-DOPA, amino acid metabolites, such as seratonin, and peptides.

In the case of insulin, the cells being incubated at 20.degree. to 40.degree. C. may be subjected to an insulin inducer at this temperature for about 1 to 20 hours to induce human insulin. Preferable insulin inducers are saccharides, such as glucose, mannose, fructose, ribose and xylitol; amino acids such as arginine, lyscine and leucine; peptide hormones, such as glucagon and adrenocorticotropic hormone (ACTH); and metal cations, such as K.sup.+ and Ca.sup.++.

Prolactin productive cells may be subjected to a prolactin inducer while incubating at a temperature of about 20.degree. to 40.degree. C. for about 1 to 50 hours to induce human prolactin. Preferable prolactin inducers are organic compounds such as reserpin, phenothiazine or chlorpromazine; hormones such as thyrotropin-releasing hormone and estrogen; amino acids such as lysine, arginine and cysteine; and inorganic salts such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate.

The cells productive of hACTH are subjected to an adrenocorticotropic hormone inducer while being incubated at an temperature of about 20.degree. to 40.degree. C. for about 1 to 50 hours to induce hACTH. Preferable adrenocorticotropic inducers usable in the present invention are vasopressin, atropine methyl nitrate, insulin, glucagon, bacterial endotoxin, seratonin and propranolol.

Even if the use of inducer is not necessary in order to cause the cells to produce the peptide hormone, oftentimes the production can be enhanced by the use of certain known agents for this purpose. For example, production of hPL may be enhanced by carrying out the incubation in the presence of one or more amino acids such as leucine, lysine, arginine and cysteine; inorganic salts, such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate; or hormones, such as luteinizing hormone releasing hormone.

Human calcitonin producion enhancing agents are known to include amino acids, such as glycine, leucine, lysine, arginine and cysteine; inorganic salts, such as sodium chloride, postassium chloride, calcium chloride and magnesium sulfate; and hormones, such as dibutylcyclic AMP and prostaglandin E.

In the case of hPTH, the production may be enhanced if the incubation is carried out in the presence of amino acids, such as glycine, leucine, lysine, arginine, or cysteine; inorganic salts, such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate; or hormones, such as dopamine, isoproterenol, epinephrine and norepinephrine.

Enhancement of hTSH production may be obtained by carrying out the incubation in the presence of amino acids, such as glycine, leucine, lysine, arginine, or cysteine; inorganic salts, such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate; or a hormone, such as thyrotropin-releasing hormone.

It must be understood that the use of the particular inducing or enhancing agent is not a part of the present invention per se but is well known to the art. Those of ordinary skill in the art will be well aware of appropriate inducing or enhancing agents. Any such agents which are operable with the parent human peptide hormone-producing cells, when incubated in vitro, may also be used with the hybridoma cells multiplied in a non-human warm-blooded animal in accordance with the present invention.

The human peptide hormone obtained during the in vitro cultivation may be easily collected using conventional purification and separation techniques, such as salting out, dialysis, filtration, centrifugation, concentration and lyopholization. If a further purified human peptide hormone preparation is desirable, a hormone preparation of the highest purity can be obtained by the above mentioned techniques in combination with more sensitive conventional purification and separation techniques, such as adsorption and desorption with ion exchange resin, gel filtration, affinity chromatography, isoelectric point fractionation, electrophoresis, etc.

Occasionally, more than one hormone will be simultaneously produced when the cells obtained and multiplied in accordance with the present invention are cultivated in vitro for hormone production. For example, when the present process is being used for the production of human prolactin, human growth hormone may be produced simultaneously. Similarly, when hACTH is being produced, a large amount of human melanocyte-stimulating hormone (hMSH) and human lipotropin (hLPH) may be produced simultaneously. During hPL production, human chorionic gonadotropin (hCG) can be produced simultaneously. During hTSH production, other human hormones, such as human chorionic gonadotropin and human follicle-stimulating hormone (hFSH) can also be obtained. In all of these cases, the desired hormone can be separated and purified by the known techniques, such as those set forth above. Furthermore, the simultaneously produced hormones may also be separated and purified by similar techniques. The titer of these additional hormones will also be much higher than obtainable by means of the prior art.

The human peptide hormone preparation thus obtained is is advantageously usable alone or in combination with one or more agents for injection, or external, internal, or diagnostic administration in the prevention or treatment of human diseases.

There follow a number of examples of processes in accordance with the present invention for producing human peptide hormones. These examples are intended to be exemplary only and no limitations are intended thereby. In these examples, and elsewhere in the specification, the amount of hormone production is determined by the following procedures. The production of hGH in the culture medium was determined by the radio-immunoassay method as described in S. M. Glick et al, Nature, 199, 784 (1963), and expressed by weight in terms of hGH standard preparation distributed by the National Institutes of Health (U.S.A.).

The human insulin in culture medium was determined by the enzyme-immunoassay method as described in K. Kato et al, J. Biochem., 78, 235-237 (1975), and expressed in International Human Insulin Units (IU) wherein 1 IU of human insulin is defined as the amount of insulin that decreases the rabbit blood sugar level to 64 mg/dl within 1 hour, or to 45 mg/dl 2 hours after subcutaneous injection of the insulin preparation.

Human prolactin production is determined by the radioimmunoassay method as described in P. Hang, Proc. Nat. Acad. Sci. USA, 68, 1902-1906 (1971), and expressed by weight/ml cell suspension in terms of the NIH standard human prolactin preparation.

The hACTH production was determined by the radioimmunoassay method as described in S. Matsukura et al, J. Lab. Clin. Med., 77, 490-500 (1971), and expressed by weight per ml cell suspension. The hMSH and hLPH productions were determined by the radio-immunoassay method as described in K. Abe et al, J. Clin. Invest., 46, 1906-1916 (1967). First, the total amount of the hormones was determined with human .beta.-MSH standard, then the weight ratio of the hormones was calculated with the amounts of human .beta.-MSH and .beta.-LPH isolated by gel filtration.

The hPL production was determined by the radio-immunoassay method as described in P. Beck et al, J. Clin. Endocrinol., 25, 1457-1462 (1965), and expressed by weight in terms of the standard hPL preparation available from the National Institutes of Health (U.S.A.).

The hCG production was determined by the radio-immunoassay method as described in A. R. Midgley Jr., Endocrinology, 79, 10-18 (1966), and expressed in International Units (IU).

The hCT production in the culture medium was determined according to the radio-immunoassay method described in N. A. Samaan et al, J. Lab. Clin. Med., 81, 671-681 (1973), and expressed by weight which is determined with reference to dose-responsive curves obtained with an international reference preparation of calcitonin and available from the World Health Organization.

The hPTH production was determined by the bio-assay method as described in J. A. Parsons et al, Endocrinology, 92, 454-462 (1973), and expressed by weight in terms of the standard hPTH preparation, assigned 1300 USP units per mg, available from the National Institutes of Health (U.S.A.).

The hTSH production is determined by the radio-receptorassay method as described in S. W. Manley et al, J. Endocrinol. 61, 419-436 (1974), and expressed in International Units (IU) in the terms of the standard hTSH preparation available from the National Institute of Medical Research (England). The hFSH production was determined by radioimmunoassay method as described in C. Faiman et al, J. Clin. Endocrinol. Metab., 27, 444-447 (1967) and expressed in International Units (IU).

EXAMPLE 1

EXAMPLE 7

Human Insulin Production

A human.times.human hybridoma lymphoblastoid cell line capable of producing human insulin was obtained in a manner identical to that described for Example 1, except that human insuloma cells, obtained by extraction from an insuloma patient, was substituted for the human acidophile adenoma cells used in example 1. A human insuloma.times.Namalva hybridoma line capable of producing human insulin was thus produced.

The hybridoma cells so obtained were implanted intraperitoneally into adult nude mice which were then fed in the usual way for five weeks. The resulting massive tumors, about 15 g each, were disaggregated by extracting, mincing and suspending in a physiological saline solution containing collagenase. After washing the cells with Earle's 199 medium (pH 7.2), supplemented with 10 v/v % fetal bovine serum, the cells were resuspended, to give a cell concentration of about 10.sup.5 cells per ml, in a fresh preparation of the same medium which contained 30 mM L-arginine as the insulin inducer, and then incubated at 37.degree. C. for six hours to induce human insulin. Thereafter, the cells were ultra-sonicated, and the human insulin content of the supernatant was determined. The human insulin production was about 3,200 .mu.IU per cell.

EXAMPLE 7A

Human Insulin Control)

To compare the human insulin production of the hybridoma cells multiplied in vivo as in Example 7 with the human insulin production of the human insulin-producing parent cells incubated in vitro, the following experiment was conducted. The disaggregated human insuloma cells obtained by extracting from a patient suffering from insuloma, and then mincing, were directly cultivated in vitro in Earle's 199 medium (pH 7.2.), supplemented with 10 v/v % fetal bovine serum, and incubated at 37.degree. C. in the presence of 20 mM D-glucose as insulin inducer for four hours to induce human insulin. Thereafter, the cells were ultra-sonicated and the human insulin content of the supernatant was determined. The human insulin production was only about 200 .mu.IU per cell.

EXAMPLE 7B

Human Insulin (Control)

In order to show the unexpected improvement in human insulin production when the hybridoma cells are multiplied in vivo in accordance with the present invention, as compared with the production obtained after in vivo multiplication of the non-hybridoma parent cells, the following control experiment was conducted. Disaggregated human insuloma cells, obtained by extracting from an insuloma patient and mincing, were implanted subcutaneously in adult nude mice which were then fed in the usual way for three weeks. The resulting subcutaneously formed massive tumors, about 10 g each, were extracted and treated similarly as in Example 7 to induce and determine the production of human insulin, except that 20 mM D-glucose was substituted for the 30 mM of L-arginine as used in Example 7. The human insulin production was about 1,000 .mu.IU per cell.

EXAMPLE 7C

Human Insulin (Control)

This control experiment was conducted in order to compare the amount of human insulin produced in accordance with Example 7 with the production obtained from the same hybridoma cells when incubated in vitro without the prior multiplication in vivo. The human insuloma.times.Namalva hybridoma Cells obtained in Example 7 were cultivated in vitro in a manner similar to that described in Example 7A using 30 mM L-arginine as the insulin inducer. The human insulin production was only about 100 .mu.IU per cell.

EXAMPLE 7D

The hybridoma cells so obtained were implanted intraperitoneally into adult nude mice, which were then fed in the usual way for five weeks. The resulting massive tumors, about 15g each, were disaggregated by extracting, mincing and suspending in a physiological saline solution containing trypsin. After washing the cells with Earle's 199 medium (pH 7.2), supplemented with 10 v/v % fetal bovine serum, the cells were resuspended in a fresh preparation of the same medium which contained 10 ng thyrotropin-releasing hormone as the growth hormone inducer and then incubated at 35.degree. C. for six hours to induce human prolactin. Thereafter, the cells were ultrasonicated, and the human prolactin content of the supernatant was determined. The human prolactin production was about 3,300 ng per ml cell suspension.

EXAMPLE 13A

Human Prolactin (Control)

To compare the human prolactin production of the hybridoma cells multiplied in vivo as in Example 13 with the human prolactin production of the human prolactin-producing parent cells incubated in vitro, the following experiment was conducted. The disaggregated human acidophile adenoma cells obtained by extracting from a patient suffering from acidophile adenoma of the pituitary gland and mincing, were directly cultivated in vitro in Earle's 199 medium (pH 7.2.), supplemented with 10 v/v % fetal bovine serum, and incubated at 37.degree. C. for six hours in the presence of 30 mM L-arginine as prolactin inducer to induce human prolactin. Thereafter, the cells were ultra-sonicated and the human prolactin content of the supernatant was determined. The human prolactin production was only about 100 ng per ml of suspension.

EXAMPLE 13B

Human Prolactin (Control)

In order to show the unexpected improvement in human prolactin production when the hybridoma cells are multiplied in vivo in accordance with the present invention, as compared with the production obtained after in vivo multiplication of the non-hybridoma parent cells, the following control experiment was conducted. Disaggregated human acidophile adenoma cells, obtained by extracting from a patient suffering from acidophile adenoma of the pituitary gland and mincing, were implanted subcutaneously in adult nude mice which were then fed in the usual way for three weeks. The resulting subcutaneously formed massive tumors, about 10 g each, were extracted and treated similarly as in Example 13A to induce and determine the production of human prolactin. The human prolactin production was about 600 ng per ml of cell suspension. There was simultaneously produced hGH in an amount of about 500 ng per ml cell suspension.

EXAMPLE 13C

Human Prolactin (Control)

This control experiment was conducted in order to compare the amount of human prolactin produced in accordance with Example 13 with the production obtained from the same hybridoma cells when incubated in vitro without the prior multiplication in vivo. The hybridoma cells obtained in Example 13 were cultivated in vitro in a manner similar to that described in Example 13A, using 10 ng thyrotropin-releasing hormone as the prolactin inducer. The human prolactin production was only about 200 ng per ml of cell suspension.

EXAMPLE 13D

EXAMPLE 19

hACTH Production

A human.times.human hybridoma lymphoblastoid cell line capable of producing hACTH was obtained as follows. First, human lung carcinoma cells were extracted from a patient suffering from lung carcinoma and minced. These disaggregated human lung carcinoma cells were suspended together with the human Namalva leukemic lymphoblastoid line in a vessel, along with a salt solution containing 140 mM NaCl, 54 mM KCl, 1 mM NaH.sub.2 PO.sub.4 and 2 mM CaCl.sub.2. The cells were added in sufficient quantity to give respective cell concentrations of about 10.sup.4 cells per ml for each type of cell. The ice chilled cell suspension was mixed with a preparation of the same salt solution containing UV-irradiation pre-inactivated Sendai virus, transferred into a 37.degree. C. incubator about five minutes after their mixing and stirred therein for about 30 minutes to effect cell fusion. Obtained hybridoma cells which were found to produce hACTH were cloned according to conventional methods to obtain a human lung carcinoma.times.Namalva hybridoma line capable of producing hACTH which is usable in accordance with the present invention.

The hybridoma cells so obtained were implanted in adult nude mice, which were then fed in the usual way for five weeks. The resulting subcutaneously formed massive tumors, about 15g each, were extracted and disaggregated by mincing and suspended in a physiological saline solution containing trypsin. After washing the cells with Earle's 199 medium (pH 7.2), supplemented with 10 v/v % fetal calf serum, the cells were resuspended in a fresh preparation of the same medium which contained 0.1 .mu.g glucagon per ml as hACTH inducer to give a cell concentration of about 10.sup.6 cells per ml, and then incubated at 35.degree. C. for twenty hours to induce hormones. Thereafter, the cells were ultrasonicated, and the hACTH, hMSH and hLPH content in the supernatant was determined. The hACTH production was 250 .mu.g per ml cell suspension; hMSH 580 .mu.g per ml; and hLPH, 40 .mu.g per ml.

EXAMPLE 19A

hACTH (Control)

In order to show the unexpected improvement in hACTH production when the hybridoma cells are multiplied in vivo in accordance with the present invention, as compared with the production obtained after in vivo multiplication of the nonhybridoma parent cells, the following control experiment was conducted. The disaggregated human lung carcinoma cells, obtained by extracting from a patient suffering from lung carcinoma and mincing, were implanted in adult nude mice which were then fed in the usual way for five weeks. The resulting subcutaneously formed massive tumors, about 5 g each, were extracted and treated similarly as in Example 19 to induce and determine the production of hormones. The hACTH production was only 600 ng per ml cell suspension; hMSH, 1.2 .mu.g per ml; and hLPH, 350 ng per ml.

EXAMPLE 19B

EXAMPLE 25

hPL Production

A human.times.human hybridoma lymphoblastoid cell line capable of producing hPL was obtained as follows. First, human chorionic epithelioma cells were extracted from a patient suffering from chorionic epithelioma and minced. These disaggregated human chorionic epithelioma cells were suspended together with the human Namalva leukemic lymphoblastoid line in a vessel, along with a salt solution containing 140 mM NaCl, 54 mM KCl, 1 mM NaH.sub.2 PO.sub.4 and 2 mM CaCl.sub.2. The cells were added in sufficient quantity to give respective cell concentrations of about 10.sup.4 cells per ml for the two types of cells. The ice chilled cell suspension was mixed with a preparation of the same salt solution containing UV-irradiation preinactivated Sendai virus, transferred into a 37.degree. C. incubator about five minutes after their mixing and stirred therein for about 30 minutes to effect cell fusion. Obtained hybridoma cells which were found to produce hPL were cloned according to conventional methods to obtain a human chorionic epithelioma.times.Namalva hybridoma line Capable of producing hPL which is usable in accordance with the present invention.

The hybridoma cells so obtained were implanted intraperitoneally into adult nude mice, which were then fed in the usual way for five weeks. The resulting subcutaneously formed massive tumors, about 15g each, were disaggregated by extracting, mincing and suspending in a physiological saline solution containin trypsin. After washing the cells with Earle's 199 medium pH 7.2), supplemented with 10 v/v % fetal calf serum, the cells were resuspended to give a cell concentration of about 10.sup.6 cells per ml in a fresh preparation of the same medium which contained 30 mM L-arginine as a hormone production enhancing agent, and then incubated at 35.degree. C. for twenty hours to produce hPL. Thereafter, the cells were ultrasonicated, and the hPL content of the resulting supernatant was determined. The hPL production was about 340 .mu.g per ml cell suspension. The simultaneocs hCG production was about 230 IU per ml cell suspension.

EXAMPLE 25A

hPL (Control)

This control experiment was conducted in order to compare the amount of hPL produced in accordance with Example 25 with the amount produced after similar in vivo multiplication of the non-hybridoma parent cells. The disaggregated human chorionic epithelioma cells, obtained in the manner described in Example 25, were implanted subcutaneously in adult nude mice which were then fed in the usual way for five weeks. The resulting subcutaneously formed massive tumors, about 5 g each, were extracted and disaggregated, and then treated similarly as in Example 25 to induce hPL. The hPL production was only 3 .mu.g per ml cell suspension.

EXAMPLE 25B

EXAMPLE 31

EXAMPLE 37

hPTH Production

A human.times.human hybridoma lymphoblastoid cell line capable of producing hPTH was obtained as follows. First, human parathyroid tumor cells were extracted from a parathyroid tumor patient and minced. These disaggregated parathyroid tumor cells were suspended together with the human Namalva leukemic lymphoblastoid cell line in a vessel, along with a salt solution containing 140 mM NaCl, 54 mM KCl, 1 mM NaH.sub.2 PO.sub.4 and 2 mM CaCl.sub.2, to give respective cell concentrations of about 10.sup.4 cells per ml for both types of cells. The ice chilled cell suspension was mixed with a preparation of the same salt solution containing UV-irradiation pre-inactivated Sendai virus, transferred into a 37.degree. C. incubator about five minutes after their mixing and stirred therein for about 30 minutes to effect cell fusion. Obtained hybridoma cells which were found to produce hPTH were cloned according to conventional methods to obtain a human parathyroid tumor.times.Namalva hybridoma line capable of producing hPTH which is usable in accordance with the present invention.

The hybridoma cells so obtained were implanted intraperitoneally into adult nude mice, which were then fed in the usual way for five weeks. The resulting massive tumors, about 15 g each, were disaggregated by extracting, mincing and suspending in a physiological saline solution containing trypsin. After washing the cells with Earle's 199 medium (pH 7.2), supplemented with 10 v/v % fetal calf serum, the cells were resuspended to give a cell concentration of about 10.sup.5 cells per ml in a fresh preparation of the same medium which contained 20 mM CaCl.sub.2 and 30 mM L-arginine as hPTH enhancing agents, and then incubated at 37.degree. C. for forty hours to produce hPTH. Thereafter, the cells were ultrasonicated, and the hPTH content of the resulting supernatant was determined. The hPTH production was about 830 ng per ml cell suspension.

EXAMPLE 37A

hPTH (Control)

To compare the hPTH production of the hybridoma cells multiplied in vivo as in Example 37 with the hPTH production of the hPTH-producing parent cells incubated in vitro, the following experiment was conducted. The disaggregated human parathyroid tumor cells were directly cultivated in vitro in Earle's 199 medium (pH 7.2), supplemented with 10 v/v % fetal calf serum. These cells were treated similarly as in Example 37 to produce hPTH. The hPTH production was only 4 ng per ml cell suspension.

EXAMPLE 37B

EXAMPLE 43

EXAMPLE 46B

It will be obvious to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is described in the specification.

Claims

What is claimed is:

1. In the process for producing human peptide hormone, excluding human erythropoietin, which comprises culturing human cells capable of producing a human peptide hormone on an in vitro nutrient medium under conditions appropriate to accumulate a substantial amount of the human peptide hormone, and recovering the accumulated human peptide hormone from the culture, the improvement, by which the amount of human peptide hormone produced during said in vitro culturing is substantially increased, comprising using as said human cells capable of producing a human peptide hormone, cells obtained by a process comprising: implanting a human.times.human hybridoma lymphoblastoid cell line capable of producing said human peptide hormone in a non-human warm-blooded animal; feeding the animal to allow said human hybridoma cell line to utilize the nutrient body fluid of the animal for its multiplication; and extracting and disaggregating the resultant tumor, formed in the animal, for use in the in vitro production of said human peptide hormone.

2. A process as set forth in claim 1, wherein said human peptide hormone is human insulin.

3. A process as set forth in claim 1, wherein said human peptide hormone is human growth hormone.

4. A process as set forth in claim 1, wherein said human peptide hormone is human prolactin.

5. A process as set forth in claim 1, wherein said human peptide hormone is human adrenocorticotropic hormone.

6. A process as set forth in claim 1, wherein said human peptide hormone is human placental lactogen.

7. A process as set forth in claim 1, wherein said human peptide hormone is human thyroid-stimulating hormone.

8. A process as set forth in claim 1, wherein said human peptide hormone is human parathyroid hormone.

9. A process as set forth in claim 1, wherein said human peptide hormone is human calcitonin.

10. A process as set forth in claim 1, wherein said non-human warm-blooded animal is a fowl or a mammal.

11. A process as set forth in claim 1, wherein said non-human warm-blooded animal is a chicken, pigeon, dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, nude rat, hamster, mouse or nude mouse.

12. A process as set forth in claim 14, wherein said fusing step comprises: suspending said parent human cells together with said human lymphoblastoid line in a salt solution containing an effective amount of a cell fusion inducing agent; allowing the resultant cell suspension to stand for a period sufficient to effect cell fusion; and selecting and cloning a hybridoma line capable of producing human peptide hormone.

13. A process as set forth in claim 12, wherein said cell fusion inducing agent is Sendai virus or polyethylene glycol.

14. A process as set forth in claim 1, wherein said human.times.human hybridoma lymphoblastoid cell line is obtained by fusing parent human cells inherently capable of producing a human peptide hormone with a human lymphoblastoid line.

15. A process as set forth in claim 14, wherein said parent human cells are normal or tumor human cells.

16. A process as set forth in claim 14, wherein said human peptide hormone is human insulin and said parent human cells are human insuloma cells.

17. A process as set forth in claim 14, wherein said human peptide hormine is human insulin and said parent human cells are human pancreas Langerhans island .beta.-cells.

18. A process as set forth in claim 14, wherein said human peptide hormone is human growth hormone and said parent human cells are human acidophile adenoma cells.

19. A process as set forth in claim 14, wherein said human peptide hormone is humanprolactin and said parent human cells are human acidophile adenoma cells.

20. A process as set forth in claim 14, wherein said human peptide hormone is human adrenocorticotropic hormone and said parent human cells are human chromophobe adenoma cells of the pituitary gland.

21. A process as set forth in claim 2, wherein said human peptide hormone is human placental lactogen and said parent human cells are human chorionic epithelioma cells.

22. A process as set forth in claim 14, wherein said human peptide hormone is humanplacental lactogen and said parent human cells are human syncytiotrophoblast cells from chorionic villi.

23. A process as set forth in claim 14, wherein said human peptide hormone is human thyroid-stimulating hormone and said parent human cells are human basophile adenoma cells.

24. A process as set forth in claim 14, wherein said human peptide hormone is human thyroid-stimulating hormone and said parent human cells are human chromophobe adenoma cells.

25. A process as set forth in claim 14, wherein said human peptide hormone is human parathyroid hormone and said parent human cells are human parathyroid tumor cells.

26. A process as set forth in claim 14, wherein said human peptide hormone is human parathyroid hormone and said parent human cells are human kidney carcinoma cells.

27. A process as set forth in claim 14, wherein said human peptide hormone is human parathyroid hormone and said parent human cells are human ovarian tumor cells.

28. A process as set forth in claim 14, wherein said human peptide hormone is human calcitonin and said parent human cells are human thyrophyma cells.

29. A process as set forth in claim 14, wherein said human peptide hormone is human calcitonin and said parent human cells are human carcinoid cells.

30. A process as set forth in claim 14, wherein said parent human cells are human lung carcinoma cells.

31. A process in accordance with claim 14, wherein said human lymphoblastoid line is of leukemic origin.

32. A process as set forth in claim 14, wherein said human lymphoblastoid line is a member selected from the group of cell lines consisting of Namalva, BALL-1, NALL-1, TALL-1 and JBL.

33. In the process for producing human peptide hormone, excluding human erythropoietin, which comprises culturing human cells capable of producing said peptide hormone on an in vitro nutrient medium under the conditions appropriate to accumulate a substantial amount of the human peptide hormone, and recovering the accumulated human peptide hormone from the culture, the improvement, by which the amount of human peptide hormone produced during said in vitro culturing is substantially increased, comprising using as said human cells capable of producing a human peptide hormone, cells obtained by a process comprising: suspending a human.times.human hybridoma lymphoblastoid cell line capable of producing said human peptide hormone in a device in which the nutrient body fluid of a non-human warm-blooded animal can be supplied to said human hybridoma cell line; embedding or placing said device in or on a non-human warm-blooded animal in a manner such that the nutrient body fluid of said animal is supplied to the human hybridoma cell line within said device; feeding the animal to allow said human hybridoma cell line to utilize the nutrient body fluid for its multiplication; and harvesting the multiplied human hybridoma cells from the device for use in the in vitro production of said human peptide hormone.

34. A process as set forth in claim 33, wherein said human peptide hormone is selected from the group consisting of human insulin, human growth hormone, human prolactin, human adrenocorticotropic hormone, human placental lactogen, human thyroid-stimulating hormone, human parathyroid hormone, and human calcitonin.

35. A process as set forth in claim 33, wherein said device is a diffusion chamber equipped with a membrane filter, ultra-filter or hollow fiber having a nominal pore size in the range of 10.sup.-7 -10.sup.-5 m.

36. A process as set forth in claim 33, wherein said non-human warm-blooded animal is a fowl or a mammal.

37. A process as set forth in claim 33, wherein said non-human warm-blooded animal is a chicken, pigeon, dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, nude rat, hamster, mouse or nude mouse.

38. A process as set forth in claim 33, wherein said fusing step comprises: suspending said parent human cells together with said human lymphoblastoid line in a salt solution containing an effective amount of a cell fusion inducing agent; allowing the resultant cell suspension to stand for a period sufficient to effect cell fusion; and selecting and cloning a hybridoma line capable of producing human peptide hormone.

39. A process as set forth in claim 38, wherein said cell fusion inducing agent is Sendai virus or polyethylene.

40. A process as set forth in claim 33, wherein said human.times.human hybridoma lymphoblastoid cell line is obtained by fusing parent human cells inherently capable of producing a human peptide hormone with a human lymphoblastoid line.

41. A process as set forth in claim 40, wherein said parent cells are normal or tumor human cells.

42. A process as set forth in claim 40, wherein said human peptide hormone is human insulin and said parent human cells are human insuloma cells.

43. A process as set forth in claim 40, wherein said human peptide hormine is human insulin and said parent human cells are human pancreas Langerhans island .beta.-cells.

44. A process as set forth in claim 40, wherein said human peptide hormone is human growth hormone and said parent human cells are human acidophile adenoma cells.

45. A process as set forth in claim 40, wherein said human peptide hormone is humanprolactin and said parent human cells are human acidophile adenoma cells.

46. A process as set forth in claim 40, wherein said human peptide hormone is human adrenocorticotropic hormone and said parent human cells are human chromophobe adenoma cells of the pituitary gland.

47. A process as set forth in claim 40, wherein said human peptide hormone is human placental lactogen and said parent human cells are human chorionic epithelioma cells.

48. A process as set forth in claim 40, wherein said human peptide hormone is humanplacental lactogen and said parent human cells are human syncytiotrophoblast cells from chorionic villi.

49. A process as set forth in claim 40, wherein said human peptide hormone is human thyroid-stimulating hormone and said parent human cells are human basophile adenoma cells.

50. A process as set forth in claim 40, wherein said human peptide hormone is human thyroid-stimulating hormone and said parent human cells are human chromophobe adenoma cells.

51. A process as set forth in claim 40, wherein said human peptide hormone is human parathyroid hormone and said parent human cells are human parathyroid tumor cells.

52. A process as set forth in claim 40, wherein said human peptide hormone is human parathyroid hormone and said parent human cells are human kidney carcinoma cells.

53. A process as set forth in claim 40, wherein said human peptide hormone is human parathyroid hormone and said parent human cells are human ovarian tumor cells.

54. A process as set forth in claim 40, wherein said human peptide hormone is human calcitonin and said parent human cells are human thyrophyma cells.

55. A process as set forth in claim 40, wherein said human peptide hormone is human calcitonin and said parent human cells are human carcinoid cells.

56. A process as set forth in claim 40, wherein said parent human cells are human lung carcinoma cells.

57. A process in accordance with claim 40, wherein said human lymphoblastoid line is of leukemic origin.

58. A process as set forth in claim 40, wherein said human lymphoblastoid line is a member selected from the group of cell lines consisting of Namalva, BALL-1, NALL-1, TALL-1 and JBL.

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Abstract

Description