Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1995-01-18
2004-02-03
Carlson, Karen Cochrane (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S416000, C530S422000, C530S415000, C530S351000
Reexamination Certificate
active
06686455
ABSTRACT:
This application relates to lymphokines. In particular, it relates to cytotoxic factors secreted by lymph cells and methods for making same in recombinant cells.
Immune cells such as B cells, T Cells, natural killer cells and macrophages are known to elaborate substances that exert cytotoxic activity toward tumor cells but which are innocuous to normal cells. These substances have been variously named, for example, lymphotoxin, tumor necrosis factor, NK cell cytotoxic factor, hemorrhagic necrosis factor, macrophage cytotoxin or macrophage cytotoxic factor. At the present time the identities of the proteins associated with these names are unclear. The principal difficulties have been that the biological assays employed to detect the proteins do not discriminate among them, the proteins appear to be found in nature as aggregates or hydrolytic products, and the amounts heretofore obtained have been so small that the high degree of purification needed to fully characterize the proteins has not been reached.
Typically, such cytotoxic substances are found in the sera of intact animals, or in the culture supernatants of lymph cells or cell lines after the animals or cells had been treated with a substance known to stimulate the proliferation of immune cells (an “inducer”). Thereafter the serum or supernatant is recovered and assayed for cytotoxic activity towards a target tumor cell line. A standard target is L-929, a murine tumor cell line. This cell line and others used in bioassays of this type are nonspecific in their lytic response because a variety of apparently discrete lymph cell products are able to effect lysis. Similar nonspecific responses are observed in in vitro tumor necrosis assays. Thus, cytolytic assays which observe for the lysis of cell lines in vitro or tumor necrosis in vivo are inadequate to distinguish among the various cytotoxic lymph products.
Cytotoxic factors tentatively have been classified on the basis of the lymph cells from which they are induced. For example, lymphotoxin is a name commonly applied to the cytotoxic secretory products of B or T lymphocytes, or cell lines derived therefrom, while tumor necrosis factor often is used to describe cytotoxic products of macrophages or their derived cell lines. This classification system, however, has not been developed to the point where there is any assurance that only a single protein is being referred to, or that proteins referred to by different names are in fact different.
Attempts have been made to purify and characterize the cytotoxic factors secreted by each cell type. To the extent that reports vary as to a property of a cytotoxic factor, or are completely inconsistent as to a given property, it could be concluded either that the characterization was erroneous or that a plurality of discrete cytotoxic factors are secreted by each cell type. For example, the cytotoxic products derived from macrophages, monocytes or monocytic cell lines, while sometimes generally referred to as tumor necrosis factor, have been reported to have properties that appear inconsistent with a theory of a single cytotoxic product. See for example the following literature: R. MacFarlan et al., 1980, “AJEBAK” 58(pt 5): 489-500; D. Mannel et al., 1980, “Infection and Immunology” 30(2): 523-530; H. Ohnishi et al., UK patent application 2,106,117A; and J. Hammerstrom, 1982, “Scand J. Immunol.” 15: 311-318.
On the other hand C. Zacharchuk et al., 1983, “Proc. Nat. Acad. Sci. USA”, 80: 6341-6345 suggest that guinea pig lymphotoxin and a cytotoxic factor from guinea pig macrophages are immunochemically similar, if not identical. Similar conclusions are advanced in Ruff et al., 1981
, Lymphokines
Vol. 2 pp 235-272 at pp 241-242.
The attempts at characterization of immune cytotoxic factors also have focused on using as starting material the sera or peritoneal fluid of animals that have been exposed to immunogenic antigens. These sources contain the entire cornucopia of the stressed immune system, in contrast to the product or products of a single cell type or line. The following should be consulted as examples of publications of this type: S. Green et al., 1982, “J. Nat. Cancer Inst.” 68(6): 997-1003 (“tumor necrosis-inducing factor”); M. Ruff et al., 1980, “J. Immunology” 125(4): 1671-1677 (“tumor necrosis factor”); H. Enomoto et al., European Patent Application 86475 (“antitumor substance”); H. Oettgen et al., 1980, “Recent Results Cancer Res.” 75: 207-212 (“tumor necrosis factor”); F. Kull et al., 1981, “J. Immunol.” 126(4): 1279-1283 (“Tumor Cell Cytotoxin:); D. Mannel et al., 1980, “Infection and Immunity” 28(1): 204-211 (“cytotoxic factor”); N. Matthews et al., 1980, “Br. J. Cancer: 42: 416-422 (“tumor necrosis factor”); S. Green et al., 1976, “Proc. Nat. Acad. Sci. USA:, 73(2): 381-385 (“serum factor”); N. Satomi et al., 1981, “Jpn J. Exp. Med.” 51(6): 317-322; N. Matthews, 1979, “Br. J. Cancer” 40: 534-539 (“tumor necrosis factor”); K. Haranaka et al., 1981, “Jpn. J. Exp. Med.” 51(3): 191-194 (“tumor necrosis factor”); and L. Old et al., European Patent Application 90892; T. Umeda et al., 1983, “Cellular and Molecular Biology” 29(5): 349-352; H. Enomoto et al., 1983, European Patent Application 86,475.
Further literature which should be consulted is J. Nissen-Meyer et al., 1982, “Infection and Immunity” 38(1): 67-73; J. Klostergaard et al., 1981, “Mol. Immunol.” 18(12): 1049-1054; N. Sloane, U.S. Pat. No. 4,359,415; and H. Hayashi et al., U.S. Pat. No. 4,447,355; K. Hanamaka et al., 1983, European Patent Application 90,892; and G. Granger et al., 1978, “Cellular Immunology” 38: 388-402.
European Patent Application Publn. No. 100641 describes a cytotoxic polypeptide which was purified substantially free of impurities from a human lymphoblastoid cell culture. This polypeptide was designated lymphotoxin, although its relationship to other reported cytotoxic polypeptides under the name lymphotoxin is conjectural. It was not known whether this was the sole cytotoxic polypeptide elaborated by immune cells, as suggested by Zacharchuk et al. (Id.), or whether it was one of a potential family of cytotoxic factors.
The polypeptide of the '641 Application has two amino termini, a larger variant ending with Leu Pro Gly Val Gly Leu Thr Pro Ser Ala Ala Gln Thr Ala Arg Gln His Pro Lys Met His Leu Ala His Ser Thr . . . and a smaller variant with the truncated amino terminus His Ser Thr Leu Lys Pro Ala Ala . . . The amino acid sequence of the lymphotoxin of the '641 Application is disclosed in copending U.S. Ser. No. 616,503, filed May 31, 1984, wherein the term “lymphotoxin” is defined.
According to the prior literature the interferons, which exhibit some tumor inhibitory activity, and a poorly characterized protein having an AlaAla amino terminus (U.K. Patent Application Publn. No. 2,117,385A), were candidates for non-lymphotoxin cytotoxic factors. As will be seen, the tumor necrosis factor of this invention is not an interferon, is not lymphotoxin and does not have an AlaAla amino terminus.
It is an object of this invention (a) to conclusively determine whether or not another tumor necrosis factor than lymphotoxin exists and, if so, to identify it in such a way as to clearly distinguish other such factors; (b) to produce such a factor by methods other than induced cell culture, which is expensive and yields product which is contaminated with the inducing agent, or by induction of peripheral blood lymphocytes, which is economically impractical, poorly reproducible, and produces product contaminated with homologous cellular and plasma proteins; (c) to obtain DNA encoding such tumor necrosis factor and to express the DNA in recombinant culture; (d) to synthesize such factor in recombinant culture in the mature form; (e) to modify the coding sequence or structure of such factor; (f) to formulate such factor into therapeutic dosage forms and to administer same to animals for the treatment of tumors; and (g) to produce diagnostic reagents relating to such factor.
SUMMARY
A cytotoxic factor has been purified to homogeneity, characterized
Carlson Karen Cochrane
Genentech Inc.
Marschang Diane L.
LandOfFree
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