GB2170818A - Lymphokine containing compositions - Google Patents

Abstract

Somatic hybrids of fibroblasts and leukemia cells produce multiple lymphokines for indefinite periods. It has been found that effective concentrations of lymphokines and other growth factors are found to accumulate in the culture medium in which the leukemia x fibroblast cells are cultured. It is also found that a B-cell growth factor is produced by the antibiotic-resistant organism Mycoplasma orale when it is cultured with the hybrid cells.

Description

SPECIFICATION Lymphokine-containing compositions This invention relates to the biosynthesis of a useful composition of biologically-active molecules by a culture of novel hybrid, single-celled, living organisms produced by mixing and fusing ASL-1 mouse leukemia cells and L-M(TK-) mouse fibrolast cells, as described in my paper with W.L. Liang, "A somatic hybrid of TL(+) and TL(-) cells forms TL antigens, but fails to undergo modulation." Proc.

Nat Acad. Sci. 72:1873,1975.

i have discovered that colonies of the resulting hybrid cells (for identification, "UIC1LF") may be separated from both parents, and I have maintained a culture of these cells by asexual reproduction for more than a year. I have also created a second hybrid colony (for identification, "UIC2LF") by crossing a different strain (BW5147) of leukemia cells with L-M(TK-) cells; recently I have created yet another hybrid cell line designated "UIC3LF" by fusion of the ASL-1 leukemia cells and a different line of fibroblast cells designated NIH/3T3. All of these hybrid colonies have been assayed for the colony-stimulating factors. It is possible, and likely, that countless other such hybrids of cells may be created. Moreover, the creation of hybrids of cells from disparate species is well known. On the other hand, even within the same species, the formation of hybrid cells is the exception.Most mixtures of cells do not result in hybrids.

I have discovered that in the process of growth and replication of the leukemia x fibroblast hybrid cells a number of biologically active, and therefore valuable, molecules including colony-stimulating factors are secreted.l use the designation leukemia x fibroblast as an abbreviated way to designate the cells which result from the mixture of cells of the two indicated types, leukemia and fibroblast, with chemicals which break or weaken the cell membranes so that fusion occurs and "hybrid" cell types are created which enclose within one cell membrane some chromosomes of each of the "parent" cell lines.In the case of a mule or of other recognized hybrid plants and animals, all of the cells of the animal have the same chromosomes; but in the case of the hybrid cell lines, the initial composition of hybrid cells contains a large proportion of cells which have most or all of the chromosomes of each parental strain; but there are also cells with many less chromosomes, I believe that in the millions of cells that are present in such a hybrid culture, representatives might be found of countless, possible "karyotypes" representing the myriad of possible combinations which might be found from the available chromosomes.

Some of these types may not be capable of replication; others may reproduce only slowly, and some may contain "aging clocks" which may limit the number of successive divisions. By whatever mechanism, some of the resulting new karyotypes are apparently "immortal" in that they will reproduce their kind indefinitely, and some of these may multiply more rapidly than others with the result that in the period of more than three years that we have maintained one of these cultures, the "cell line" has approached a condition of apparent equilibrium wherein the average number of chromosomes which may be counted no longer diminishes significantly; but there remains large number of different karyotypes in the culture. For purposes of this application it is such a mixed colony that is referred to as "leukemia x fibroblast hybrid cells".The presence of and relative proportions of their products has been established by proven biological assays including immunochemical techniques. Some of these assays are reported below.

Lymphokines are a family of molecules that transmit growth and differentiation signals between various types of lymphocytes and are major effectors of immune regulation.

I believe that all species of mammals produce lymphokines that function in analogous ways; and I also believe that a systematic search will disclose other fibroblasts of other species, and leukemia cells of the same or similar species that will likewise form hybrids that to a greater or lesser extent will express some of these lymphokines. Moreover, I also believe that solid tissue cells other than fibroblasts will be found which form hybrids with immortal cells to produce some of the same or similar growth factors.

Lymphokines have potential value in the treatment of malignant diseases. They act as adjuvants and enhance the immune response and enable the cloning of differentiated cells involved in the immune response. The cloning of growth-factor-dependent hybridomas for treatment of malignancy and other immune therapies is enhanced in the presence of this growth-factor-containing hybridcell medium. Pharmaceutical chemical and research laboratories involved in related work will make use of these materials in these and other ways.

Because of the presence of lymphoid-cell growth factors, doctors using marrow transplantation for therapy may use the hybrid-cell-containing medium to promote in-vitro culture of white blood cells, which are then transfused into the recipient.

Products including growth factors and other biologically-active materials are produced and marketed commercially in the United States by companies such as Collaborative Research, Inc. 128 Spring Street, Lexington, MA 02173.

Lymphokines are biologically-active hormonallike intercellular chemical messengers. They are integral components of the mammalian immune system converting antigen-stimulated lymphocytes to form antibodies and to engage in cell-mediated immune reactions. Examples are: (a) B-cell growth factors which are specialized types of lymphokines that are required for the proliferation and differentiation of antigen-stimulated "resting" B-cells into cells which form specific antibodies, (b) colony stimulating factors which convert undifferentiated stem cells into phagocytic cells, into (c) interleukins regulating the differentiation and growth of T-cells.

It is known that lymphokines are produced by lec tin-stimulation of malignant mouse-leukemia cell lines; but the yield is meager. Moreover, extensive subsequent separation and partial-purification steps are required. The formation of lymphokines by lectin-stimulated cells is unreliable; and in any case new cultures are required usually within one week.

I have found that the activities expressed by these mouse hybrids affect, to some extent, rat, rabbit, and mouse cells of various H-2 types. This suggests that between these species the analogous lymphokines are chemically similar, but not the same.

Accordingly, in one aspect, this invention is a new and useful composition of matter, the mixture of growth factors expressed or exuded from the new hybrid cell strains and the as-yet-not isolated active molecules in the mixture, particularly lymphokines. (I prefer to say "expressed" rather than "secreted" or "exuded" because the more familiar terms have in some contexts become words of art.) In another aspect, the invention is a new method of producing lymphokines.

Yet another aspect of the invention resides in the new and useful hybrid cells. Although I have published, Cohen and Liang, supra, my method of producing hybrid strains and have indicated the parental cell types, without the hereditary material publications alone might not have enabled others heretofore to replicate the UIC1LF, UIC2LF and UIC3LF cells. I have also discovered that in addition to these hybrid-cell lines, one of the abovementioned original UIC1LF cultures had become infected with antibiotic-resistant Mycoplasma orale and that in the conditioned co-culture of the hybrid cells and this organism, a B-cell growth factor is found which is not produced when this organism is excluded. The method of producing this B-cell growth factor is thus, a further feature of the invention.

For several reasons, not presently understood, these L-M(TK-) fibroblast cells, which in their natural state have been known to generate traces of several classes of lymphokines, when so hybridized with leukemia cells are deregulated by something passed to the hybrid from the leukemia parental cells for uninhibited lymphokine production; so that the hybrid cells produce the active molecules in relatively very large quantities.

This process is to be distinguished from two other general methods for the biosynthesis of complex molecules. Human insulin, for example, is now produced by bacteria, the genes of which have been altered to carry the insulin-producing trait of human cells. It is also known that antibodies are produced in enhanced quantities by socalled "hybridomas"--hybrids of cancer cells and somatic cells. Such a hybrid, with the cells of a particular individual generates the antibodies that individual would generate, but in enhanced quantities. The present discovery is an outgrowth of my work on hybridomas. The new method combines features of these two methods. It produces physiological macromolecules the tedium of genetic engineering.

Manuel and Defendi originally described (Manuel, J. and Defendi, V. "Regulation of DNA synthesis in mouse macrophages. I. Sources, action and purification of macrophage growing factor (MFG)." Exp. Cell Research, 65, 33, 1971) the existence of Macrophage-growth factor. There are no reports of which I am aware of the stable continuous production of such growth factors by leukemia x fibroblast cells. I have found that cultures of murine leukemia x fibroblast, hybrid cells express two of the known colony stimulating factors in relatively large quantities continuously as the cells are cultured. No special conditions of cell culture, hybridcell induction, or cell stimulation are required.

Unlike other methods, selection of positive (versus nonproducing) hybrid cell clones is unnecessary. The activities of the hybrid cell clones is unnecessary. The activities of the lymphokines so produced exhibit biological and molecular characteristics indistinguishable from those fomed naturally. One such hybrid cell cultures have been tested to be stable for lymphokine production for periods in excess of 36 months.

Malignant leukemia x nonmalignant-fibroblast hybrid cells have some of the specialized characteristics of each parental source. Normal fibroblast cells form lymphokines. According to Stanley et al (Stanley, E.R.; Cifoni, M.; Heard, P.M.; Defendi, V.

"Factors Regulating Macrophage Growth Factor." J. Expt Med. 143 631-647 (1976)) monocyte/macrophage colony-stimulating factor was detected in their conditioned medium of cultured mouse fibroblast cells. This might have suggested that fibroblast cells fused with immortal cells would similarly produce growth factors; but it has been found that these hybrid cells produce the lymphokines at rates that are much greater than the modest production by normal cells. Moreover, I do not known whether all of the cells in the culture produce each growth factor, or any of the growth factors, whether the several cells are specialists in producing one or the other of the factors; and I do not know if some of the cells in the culture are just free riders and do not participate at all in the observed yield.It is possible that there is a symbiotic relationship between specialist karyotypes in which the factors produced by some of the types support the growth and productivity of other types in the cuiture; and that this symbiotic relationship tends to control the population and proportions of the equilibrium composition of the culture. I have found that unlike unfused cells in general and fibroblast cells in particular, hybrids of fibroblasts with leukemia cells form a stable culture resulting in a sustained cell line producing lymphokines in large quantities in marked contrast to their modest and regulated production by normal fibroblast cells. Evidence for the presence of the identified lymphokines is found in the following observations: 1. Hybrid-cell-conditioned medium stimulates the proliferation of spleen and bone marrow cells of nude (BALBIc) mice; the stimulus is concentration dependent, as shown by tritium-labelled thymi dine-incorporation assay.

2. Leukemia x fibroblast hybrid-cell-conditioned medium stimulates the proliferation of bone marrow and thymus cells of normal mice. In the case of bone marrow, the stimulatory effect is reduced more than 90% if the conditioned medium is treated with antibodies to monocyte/macrophage colony-stimulating factor beforehand as shown by incorporation of tritiated thymidine (3H-thymidine).

3. By microscopic analysis of colonies of bone marrow cells growing in leukemia x fibroblast hybrid-cell-conditioned medium, "mixed" cell types are present indicating that Granulocyte/Monocyte Colony-Stimulating Factor is present.

4. Leukemia x fibroblast conditioned medium supports the growth of normal mouse macrophages for periods in excess of 4 months, indicating that Macrophage Colony-stimulating factor (M CSF) is present.

5. Conditioned media from each of two types of leukemia x fibroblast hybrid cells, similarly conditioned, both stimulate the proliferation of normal mouse spleen and bone marrow cells, as shown by tritium-uptake assay.

6. Tests by radioimmunoassay for the presence in the conditioned medium of leukemia x fibroblast hybrid cells of the monocyte/macrophage colonystimulating factor (M-CSF) are positive.

7. Tests for the effect of hybrid-cell conditioned medium on the natural killer activity of normal spleen cells are positive.

8. The proliferative stimulus of murine leukemia x fibroblast hybrid-cell-conditioned medium is apparently not species-specific; it affects spleen cells from rat and rabbit as well as blood cells of humans, although to a lesser extent that it affects spleen cells from mice.

9. It is not H-2 restricted; the proliferative stimulus of the leukemia x fibroblast hybrid-cell-conditioned medium from cells which are (H-2a/k) on spleen cells from H-2 non-identical mice is equivalent to that for H-2 identical mice of all types.

The above-indicated procedures are representative of, but not exhaustive of the known assay methods for such physiologically active macromolecules. The foregoing properties of the conditioned medium have been demonstrated by these standard assay methods which clearly prove the presence of two of the colony-stimulating factors and other growth factors.

Preferred embodiment As described in my above paper with Liang, the hybrid cell line is produced as follows: 1. Mix together leukemia cells and fibroblast cells along with polyethylene glycol for about one minute to weaken the cell membranes.

2. Add about nine times the volume of a standard cell-growth medium such as Gibco Laboratories, Grand Island, N.Y. Medium 1640 14072 to which is added mammalian blood serum, preferably about 10% fetal-calf serum obtainable from Hyclone Labs, 1725 S. State Hwy 89-91, Logan, Utah 84321, and centrifuge to wash out the glycol.

3. Resuspend cells in the growth medium and spread this mixture to plastic petri plates, which are the preferred substrate on which to grow the adherent cells.

4. Twenty-four hours later the medium is renewed with a similar medium containing effective amounts of a selection medium comprising a combination of hypoxanthine, aminopterin, and thymidine (HAT). This kills the fibroblast cells which lack the enzyme thymidine kinase. The leukemia cells do not adhere to the plates. With the passage of time colonies of hybrid cells are observed to appear as spots on the plates, which are harvested, the leukemia cells being washed out.

The hybrid-cell conditioned medium is prepared as follows: 1. Ten million hybrid cells are added to ten milliliters of the standard growth medium and incubated for 24 hours at 37 degrees C.

2. The medium is decanted by centrifuging and filtered through a 0.45 micron nitrocellulose filter.

3. This "hybrid-cell-conditioned medium" may be stored at -20 degrees C. until used The hybrid-cells, treated with 10% dimethyl sulfoxide (DMSO) may be kept at liquid-nitrogen temperature indefinitely.

The time and proportions have been standardized so that the resulting lot of medium will have the same potency as other lots. It would be entirely feasible to grow a culture until it failed for lack of nutrition.

I have maintained cultures of the UIC1 LF leukemia x fibroblast cells for years. I have not measured any difference in the production of the active factors and resultant potency from successive tenmillion cell cultures; but I have observed the chromosomes of these cells under the microscope and note that the number of chomosomes in the hybrid cells varies substantially from one cell to the next, moreover that the average number of chromosomes per cell decreases with successive generations of the hybrid cell line. The assumption is that some genetic configurations (karyotypes) have greater stability in reproduction; and that the cell line would evolve into a few karyotypes with an average of 74 +/- 4 chromosomes which are propagated.

This tendency for spontaneous changes in the propagating hybrid-cell line leads me to suppose that cultures made from different individuals from the same standardized mouse strains will also express the valuable activity in substantially the same quantities and that the separate cell lines will tend to converge on the same karyotypes.

It has been the policy of the University of Illinois Medical School at Chicago to make available at cost any of the cell lines it maintains. The L-M (TK ) cell line was reported in Exp. Cell Res. 31:297312, 1963 and has been deposited with the American Type Culture Collection, Rockville, Maryland (ATCC No. CCL 1.3). The NIH/3T3 cell line is from a mouse bladdder fibroblast carcinoma and is deposited under ATCC No. CRL 1658. The ASL-1 mouse leukemia cell line has been maintained for many years by the Memorial Sloan Kettering Institute in New York City. Dr. Edward Boyse of that In stitute gave me the leukemic mice from which I have propagated the cell line about five years ago.

since that time the University of Illinois also has maintained both frozen cells and active cultures of the ASL-I line. Because it has been the policy of the Sloan Kettering to make its cell lines available upon request, the ASL-1 line is believed to be widely dispersed. The UICILF cell line has been deposited under ATCC No. CRL 8722. It is also available from the University of Illinois, as are the UIC2LF and UIC3LF lines. The BW5147 strain of leukemia cells is from a C57B1/6 mouse and has been widely used at the University of Illinois.

Frozen cells from these cell lines are available upon request upon payment of handling and shipping costs by contacting Mr. Kenneth Johnson, Department of Microbiology and Immunology, University of Illinois, College of Medicine at Chicago, P.O. Box 6998, Chicago, Illinois 60680 (312) 996-9479.

Claims (40)

1. A method of producing a composition of matter comprising the steps of: a. treating with chemicals a mixture of leukemia cells propagated from a mammal and of connective-tissue cells from an animal to weaken the membranes of said cells to produce a mixture of hybrid cells of many karyotypes containing chromosomes of each parent cell type together with said parent cell types; b. washing said cells to remove said chemicals; c. suspending said mixture of cells in a suitable cell-growth medium and spreading the resulting suspension over a suitable culture substrate; d. incubating the resulting culture at substantially the normal body temperature of the species of said mammal for a period of time sufficient to establish colonies of cells adherent to said substrate;; e. renewing said nutrient medium with added doses of a selection medium effective to kill any colonies of said parent connective-tissue cells; f. growing said cells in said medium at substantially said temperature, thereby establishing visible colonies of said hybrid cells on said substrate; g. harvesting said cells and adding them to a quantity of said growth medium; h. incubating the harvested cells at said temperature for a time sufficient to express effective quantities of biologically-effective molecules; and i. clarifying the resulting conditioned medium to yield said composition of matter.

2. The method of Claims 1 or 10 wherein said animal is a mammal.

3. The method of Claim 2 where said animal is of the same species as said mammal.

4. The method of Claim 1 wherein said connective-tissue is of a fibroblast.

5. The method of Claim 4 wherein said animal and said mammal are mice.

6. The method of Claims 1, 2, 3, and 4 wherein said chemicals comprise polyethylene glycol.

7. The method of Claim 4 or Claim 10 or Claim 11 wherein: i. said chemicals comprise polyethylene glycol; ii. said cell-growth medium comprises mammalian blood serum; iii. said substrate is a plastic petri dish; iv. said selection medium comprises a combination of hypoxanthine, aminopterin, and thymidine; and wherein v. said animal and said mammal are mice.

8. A method as defined by Claim 5 wherein said composition of matter is characterized in that it comprises: i. Granulocyte/Monocyte Colony-Stimulating Factor, and ii. Macrophage Colony-Stimulating Factor.

9. The method of Claim 8 wherein said composition of matter is further characterized in that it: i. stimulates the proliferation of spleen and bone marrow cells of nude (BALB/c) mice; ii. stimulates the proliferation of bone marrow and thymus cells of normal mice; iii. stimulates the proliferation of normal mouse spleen and bone-marrow cells; and iv. stimulates the proliferation of rat and rabbit spleen cells.

10. A method for producing cell growth factors which comprises the steps of: a. treating with chemicals a mixture of leukemia cells propagated from a mammal and of adherent solid-tissue cells from an animal to weaken the membranes of said cells to produce a mixture of hybrid cells of many karyotypes containing chromosomes of each parent cell together with said parent cell types; b. washing said cells to remove said chemicals; c. suspending said mixture of cells in a suitable cell-growth medium and spreading the resulting suspension over a suitable culture substrate; d. incubating the resulting culture at substantially the normal body temperature of the species of said mammal for a period of time sufficient to establish colonies of cells adherent to said substrate;; e. renewing said nutrient medium with a dose of selection medium effective to kill any colonies of said parent adherent solid-tissue cells; f. growing said cells in said medium at substantially said temperature, thereby establishing visible colonies of said hybrid cells on said substrate; g. harvesting said cells and adding them to a quantity of said growth medium; h. incubating the harvested cells at said temperature for a time sufficient to express effective quantities of biologically-effective molecules; and i. clarifying the resulting conditioned medium to yield said growth factors in solution.

11. The method as defined by Claim 10 wherein said growth factors comprise colony stimulating growth factors.

12. In a method for producing a composition of matter which comprises colony-stimulating growth factors, the steps of: a. combining mammalian leukemia x solid-tissue adherent hybrid cells and a soluble cell-growth medium; b. incubating the resulting mixture at a temperature to promote cell-colony growth to produce a crop of cells; c. harvesting and discarding the cells; and d. clarifying the resulting medium to yield said composition of matter.

13. The method of Claim 12 wherein said composition of matter comprises lymphokines.

14. The method of Claim 12 wherein: i. said composition of matter comprises colonystimulating factors; and ii. said cells are hybrids of leukemia and fibroblast cells.

15. The method of Claim 13 wherein said cells are hybrids of leukemia cells and fibroblast cells.

16. The method of Claim14 wherein said fibroblast cells are of the L-M(TK-) cell line.

17. The method of Claim 16 wherein said leukemia cells are of the ASL-1 leukemia cell line. ASL-1 mouse.

18. The method of Claim 16 wherein said leukemia cells are of the BW5147 strain of mouse leukemia cells.

19. The method of Claim 2 wherein said connective-tissue cells are fibroblast cells.

20. The method of Claim 15 wherein said fibro blast cells are of the L-M(TK-) cell line.

21. The method of Claim 15 wherein said com position is characterized in that it stimulates the proliferation of spleen and bone marrow cells of nude (BALB/c) mice.

22. The method of Claim 15 wherein said com position is characterized in that it stimulates the proliferation of bone marrow and thymus cells of normal mice.

23. The method of Claim 15 wherein said com position is characterized in that it supports the growth of normal mouse macrophages for periods in excess of 4 months.

24. The method of Claim 15 wherein said com position is characterized in that it stimulates the proliferation of normal mouse spleen and bone marrow cells.

25. The method of Claim 15 wherein said com position is characterized in that it stimulates the proliferation of rat and rabbit spleen cells.

26. The method of Claim 13 wherein said hy brid cells are hybrids of leukemia and fibroblast cells.

27. The method of Claim 14 wherein said fibro blast cells are of the NIH/3T3 cell line.

28. The method of Claim 27 wherein said leuke mia cells are of the ASL-1 cell line.

29. The method of Claim 27 wherein said leuke mia cells are of the BW5147 strain of mouse cells.

30. A clear fluid composition which comprises cell growth factors, and is made by the process comprising the steps of: a. combining as a culture leukemia x fibroblast hybrid cells and a suitable cell-growth medium comprising about 10% mammalian blood serum, b. incubating the resulting mixture at a tempera ture to promote growth for a time sufficient to pro duce a crop of cells, c. harvesting and discarding said cells, and d. clarifying by filtration to yield said fluid composition.

31. The composition as defined by Claim 30 wherein parent cells of hybrid cells are of the L M(TK-) cell line.

32. The composition as defined by Claim 30 wherein parent cells of hybrid cells are of the ASL1 cell line.

33. The composition defined by Claim 30 wherein parent cells of said hybrid cells are of the BW5147 strain of mouse cells.

34. The composition defined by Claim 30 wherein said hybrid cells are of mouse leukemia cells and L-M(TK-) fibroblast cells.

35. The composition as defined by any of Claim 30-34 inclusive, wherein said factors comprise the Granulocyte/Monocyte Colony-Stimulating Factor.

36. The composition as defined by any of Claims 30-34 inclusive, wherein said factors comprise the Macrophage Colony-Stimulating Factor.

37. A composition as defined by any of Claims 30-34 inclusive, wherein said factors comprise a Bcell growth factor, and wherein said culture is modified by the presence and co-culture therein of the organism Mycoplasma orale.

38. A composition as defined by Claim 37 comprising a Granulocyte/Monocyte Colony Stimulating Factor.

39. A leukemia x fibroblast hybrid cell line comprising cells having substantially between 70 and 78 chromosomes and obtained by propagating the hybrid cells created by crossing murine leukemia cells with murine fibroblast cells.

40. A cell line as defined by Claim 37, further characterized in that it expresses Granulocyte/Monocyte Colony-stimulating Factor.