Production of Tumor Angiogenesis Factor by Cell Culture

This invention relates to the in vitro production of human tumor angiogenesis factor from the human liver adenocarcinoma cell line SK-HEP-1.

It has been known for some time that unless a solid tumor is provided with blood vessels by its host it remains small and dormant. The substance that is released by tumors and provides vascularization has been named tumor angiogenesis factor (TAF) by Dr. Judah Folkman of the Harvard Medical School. J. Exptl. Med. 133, 275-88 (1971); Cancer Res. 34, 2109-13 (1974).

Ever since it was recognized that tumor induced neovascularization represents the breaching of an important barrier in the control of tumor growth, considerable effort has been spent in searching for ways to inhibit neovascularization. It was early suggested by Dr. Folkman that blockade of this factor (inhibition of angiogenesis) might arrest solid tumors at a tiny diameter of a few millimeters (avascular phase). New Engl. J. Med. 285, 1182 (1971); J. Exptl. Med. 133, 275-88 (1971). One suggested approach was the raising of antibodies against TAF extracts for the production of an anti-serum. Folkman, Ann Surg. 175, 409-16 (1972); Phillips et al., Int. J. Cancer, 17, 549-58 (1976). Another approach to inhibiting neovascularization which has received much attention recently consists in treatment of tumor cells with extracts of cartilage. Eisenstein et al, Am J. Pathol. 73, 765-74 (1973); Sorgente et al., Lab. Invest. 32, 217-22 (1975); Eisenstain et al., Amer. J. Pathol. 81, 337-48 (1975; "Medical News", JAMA 232 (1), 14-15 (1975); Brem and Folkman, J. Exptl. Med. 141, 427-39 (1975); and Kuettner et al., U.S. Pat. No. 4,042,457. These efforts are limited, of course, to the amount of TAF which is available and suitable for test purposes.

TAF finds further use in the development of tests such as an angiogenenic assay or a diagnostic screening test for neoplasia, Klagsbrun et al., Cancer Res. 36, 110-14 (1976); and Brem et al., Science 195, 880-81 (1977); Cancer 41, 239-44 (1978).

TAF also has been proposed as useful for wound healing. Rettusa et al, FASEB, Abstract No. 4309, 61st Ann. Meet., Apr. 1-8, 1977, Chicago, Ill.

Various human tumor cells have been reported heretofore as capable of elaborating TAF when measured by certain assays. Thus, extracts from human neuroblastoma, Wilms' tumor and human hepatoblastoma were found to contain TAF which was able to cause the formation of new blood vessels in the subcutaneous fascia of rats. Folkman et al., J. Exptl. Med. 133, 275-88 (1971).

So also, WI-38 embryonic lung, SV W126 (SV 40 virus transformed W126), glioblastoma, meningioma and HeLa cells (the latter only in suspension culture and not in monolayers) grown in T-75 tissue culture flasks were described as giving a positive TAF response as measured by bioassay on the chick chorioallantoic membrane (CAM). Folkman and Klagsbrun, Chapter 31 of "Fundamental Aspects of Neoplasia", at page 402, (Gottlieb et al, eds.), Springer-Verlag, New York, 1975.

Subsequently, extracts from hypernephroma, haemangioma and human kidney were similarly described as eliciting a TAF response which resulted in the growth of new capillaries in the subcutaneous fascia of rats. Phillips et al., Int. J. Cancer, 17, 549-58 (1976).

In still other experiments, Hubler and Wolf demonstrated TAF responses from human cutaneous melanoma implanted in transparent hamster-cheekpouch chambers. Cancer 38, 187-92 (1976).

All of the foregoing tumor cells are derived from fresh tumors or primary cultures except the WI-38, SV W126 and HeLa cells. Fresh tumors and primary cultures are not, however, generally suitable sources of TAF except for limited research purposes or small scale production. In order to provide a commercially significant source of TAF in terms of ready availability and adequate supply, production from a suitable established cell line is deemed necessary. As a practical matter, the cell line should have not only specific TAF activity, but it should also have good cell growth characteristics in terms of rapid growth, good suspension growth, adaptability to large-scale culture and economical nutrient requirements.

The terms "cell line" and "established cell line" are used herein in conformance with the definitions published by Federoff in the Tissue Culture Association Manual, Vol. 1. No. 1, pp. 53-57 (1975).

Applicants have investigated numerous human tumor cell lines for the production of TAF, but most of them have been eliminated as unsuitable candidates in view of their poor growth characteristics as above-defined.

One cell line that has now been found to have good growth characteristics in suspension culture and is able to elaborate the desired TAF in suitable quantities is the human liver adenocarcinoma cell line SK-HEP-1. The established cell line SK-HEP-1 was derived from a liver adenocarcinoma by G. Trempe of Sloan-Kettering Institute for Cancer Research in 1971, and cultures of the cell line are available from that institute. This cell line has been known to produce tumors in nude mice but it has not heretofore been known as a suitable source of TAF. Prior publications on the establishment and characterization of cell line SK-HEP-1 include the following:

Fogh and Trempe, "New Human Tumor Cell Lines" in Human Tumor Cells In Vitro (Fogh. ed.), Plenum Publ. Corp., New York, 1975, pp. 115-54;

Fogh et al., J. Natl. Cancer Inst. 58, 209-14 (1977; and

Fogh et al., J. Natl. Cancer Inst. 58, 209-14 (1977); Fogh et al., N. Natl. Cancer Inst. 59, 221-25 (1977).

Initially, the medium used by applicants for maintenance and growth of this cell line was Dulbecco's modification of Eagle's minimum essential medium (MEM) containing 4 mg/ml of glucose. The culture medium was supplemented with 15% by volume fetal bovine serum without addition of any antibiotics, although lower concentrations of fetal bovine serum, for example, about 10-15%, also can be used. The cells were first grown at 37.degree. C. in attached culture in 75 cm.sup.2 T-flasks (Falcon plastics) and were then converted to agitated, liquid suspension culture in the same medium in 4-liter and 100-liter vessels in accordance with the procedure described in U.S. Pat. No. 4,059,485. The cells were then harvested after times ranging from 5 to 11 days growth since inoculation, and TAF was extracted and assayed according to the general procedures described in U.S. Pat. No. 4,059,486.

Specifically, the cells after growth were washed successively three times in lactated Ringer's solution, resuspended in phosphate buffered saline (pH 7-7.4) and stirred for 4 hours at 4.degree. C. The supernatant after removal of the cells by centrifugation was retained as the cell extract. The cells pellet from the extract was resuspended in distilled water and stirred at 37.degree. C. for 20 minutes. The supernatant after removal of the cell debris by centrifugation was retained as the cell lysate.

In various runs of 4 liters and 100 liters the cell extracts and cell lysates were assayed to contain the following amounts of total protein (Lowry protein assay):

The cell extracts from the 4-liter runs gave strongly positive tests in the bioassay for TAF in the chorioallantoic membrane (CAM) of chick embryos, in accordance with the procedure described by Folkman, Cancer Res. 34, 2109-13; 36, 110-14 (1976).

The cell extracts from the 100-liter runs were subjected to further purification by CM-Sephadex.RTM. (Pharmacia) ion exchange chromatography and lyophilized. The purified material also gave a positive test in the CAM bioassay for TAF. The use of CM-Sephadex chromatography for obtaining a TAF active fraction from tumor cells is described by Tuan et al., Biochemistry 12 (17), 3159-65 (1973).

It will be appreciated that other nutrient culture media for culture of the SK-HEP-1 cells can be used in place of Dulbecco's MEM, for example, any of the well-known tissue culture media described by H. J. Morton, In Vitro 6, 89-108 (1970). These conventional culture media contain known essential amino acids, mineral salts, vitamins and carbohydrates. They are also frequently fortified with mammalian sera such as fetal bovine serum. Suitable growth of the cells can be carried out at about 35.degree.-38.degree. C. but cell proliferation is best at 37.degree. C. Growth under these cell culture conditions for about 4-8 days generally is sufficient to produce the desired TAF.

Other suitable equipment and procedures for growing cells in agitated, liquid suspension which can be adapted for culture of the cell line SK-HEP-1 will be readily apparent to the person skilled in the art by reference to well-known texts on cell culture such as, for example, Paul, "Cell and Tissue Culture," The Williams and Wilkins Company, Baltimore, 4th ed. 1970, at pages 277-291; Kruse and Patterson, "Tissue Culture Methods and Applications, " Academic Press, New York, 1973, at pages 333-377.

It should also be understood that various means can be used to separate and purify TAF-containing fractions from the cells and cell culture product other than the illustrative specific recovery procedures described above. These various recovery means include the known techniques for the separation and purification of proteinaceous substances in general such as, for example, dialysis, salt and solvent precipitation, adsorption with gels, cellulose ion exchange chromatography, Sephadex gel filtration, electrophoresis, and lyophilization. Thus, TAF can be obtained by extraction from the cells followed by subjecting the extract to dialysis against NaCl (0.15M), Sephadex G-100 chromatography, dialysis against water and lyophilization analagous to the methods described by Folkman et al., J. Exptl. Med. 133, 275-88 (1971) and Phillips et al., Int. J. Cancer 17, 549-58 (1976). According to Phillips et al, id, when the dialysate is subjected to chromatography on a column of Sephadex G-100 in 0.15 M NaCl, the fractions between 35,000 and 300,000 molecular weight contain the greatest TAF activity. According to Folkman, Cancer Res. 34, 2109-13 ( 1974), TAF is a proteinaceous substance having a molecular weight of approximately 100,000. It will be appreciated, however, that the aforesaid molecular weights are estimates based on partially purified TAF-containing products and the inventors are not bound by such estimates or by any particular purification procedure or TAF purity.

Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. All such further examples are included within the scope of the appended claims.