JPH0465052B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an antitumor agent containing a novel lymphotoxin having an amino acid sequence clearly distinguishable from others and a sugar chain-linked human interferon-γ. More specifically, it contains a novel lymphotoxin with antitumor activity and carbohydrate-linked human interferon-γ having the same effect as active ingredients, and the coexistence of both lymphokines has a synergistic effect on antitumor activity. The present invention relates to an antitumor agent having the following characteristics.

(Prior art) Lymphotoxins are induced to be produced inside and outside cells by applying antigens to sensitized lymph cells or by applying mitogens such as phytohemaagglutinin or concanavalin A to cells.
Alternatively, it is a proteinaceous substance produced from cell lines that proliferate in tissue cultures derived from certain lymphocytes, and is a type of lymphokine defined as a substance that exhibits cytotoxicity.

It has been reported that lymphotoxin in biological species is not a homogeneous protein in vitro, and subtypes differ in molecular weight, charge, and stability. For example, in humans, more than five types of lymphotoxin have been identified, and the representative ones are αLT (molecular weight 70,000 to 90,000), βLT (molecular weight 30,000 to 50,000), and γ-LT.
(molecular weight 12,000 to 20,000), α-heavy LT (molecular weight 140,000 to 160,000), and LT complexes (molecular weight 200,000 or more) [JJ Devlin et al. Lymphokine 7
Volume 313 (1984) Academic Press Publishing]. Lymphotoxins are involved in the regulation of the immune system [Evans, CH Cancer Immunology and Immunotherapy.
and immunotherapeutics) Volume 12, Page 181 (1982
)] or both in vivo and in vitro [Papermaster et al.
Pharmacology (Res.Comm.in Chem.Patho.
and Pharmacology) vol. 8, p. 413 (1974),
Papermaster Clinical Immunology and
Immunopathology (Clinical immuno.and
immunopathology) Volume 5, Page 31 (1976),
Papermaster, Annual New York Academy of Science (Ann.NWAcad.
Sci.) Vol. 332, p. 451 (1979), Khan et al., Hematology and Oncology
and oncology), vol. 11, p. 128A (1981), Gately
vol. 27, p. B2 (1976), Rosenberg et al., Journal of Immunology, vol. 110, p. 1623 (1973).
), Sawada et al., Japanese Journal
Of Medicine (Japanese.of Med.) Volume 46,
263 (1976)], it suppresses the growth of tumor cells or acts cytolytically. Further in
Under vitro conditions, this effect is more pronounced in tumor cells than in normal cells for cells obtained from the same species.
It is reported to be more powerful [William
et al., Cell Immunology, vol. 6, p. 171 (1973
), Evans et al.
Res., Vol. 35, p. 1035, (1975)], and because of these functions, it has been a substance that has been expected to be a therapeutic agent for malignant tumors since its discovery.

Interferon is an antiviral active substance discovered by Isaacs and Lindemann in 1975, and due to differences in its producing cells, induction methods, physicochemical and immunological properties, etc.
Three types are known: β and γ [Science of Interferon by Shigeyasu Kobayashi, Kodansha Scientific Publishing]. Further, as a result of subsequent research, it has been found that various interferons have antitumor activity in addition to antiviral activity, and in particular, γ-type interferon (hereinafter referred to as interferon-γ) has higher antitumor activity than α- and β-types. Because of its stronger tumor activity, it is expected to be used as an antitumor agent.

Previous research has shown that both lymphotoxin and interferon-γ are expected to be used as antitumor agents, but the only way to produce them is to induce them from human lymphocytes or human spleen cells, making it difficult to obtain large quantities of human cells. Because the amount of lymphokine induced was small, and it was difficult to purify due to its physicochemical instability,
Its exact biochemical properties remained unknown.

However, recently, a method for easily mass-producing it using recombinant DNA technology has been developed, and it is being confirmed that it is useful as a therapeutic agent for malignant tumors.

The present inventors also previously used recombinant DNA technology,
Method for producing natural interferon-γ with sugar chains using animal cells (Patent application 119648/1982)
In addition, we developed an amino acid sequence that is clearly distinguishable from the amino acid sequence of lymphotoxin derived from lymphoblastoid cells (RPMI 1788 strain) reported by Gray et al. [Gray et al., Nature, Vol. 312, p. 721 (1984)]. We have discovered a method for producing a new type of lymphotoxin (hereinafter referred to as lymphotoxin-K) in animal cells and microbial cells, and have been conducting intensive research to see if it is useful as a therapeutic agent for malignant tumors. As a result, it has been confirmed that this new lymphotoxin-K has significantly higher antitumor activity in vivo and in vitro than other previously reported lymphotoxin.

By the way, in 1983, Williams et al. revealed that interferon-α exhibits a synergistic effect with lymphotoxin in an in vitro cell growth inhibition experiment, suggesting a synergistic effect between other β- and γ-type interferons and lymphotoxin. [Willam et al., Journal of Immunology,
Volume 130, page 518 (1983)]. This discovery has drawn attention to the possibility that combination preparations of interferons and lymphokines may act more strongly as antitumor agents than either alone, and a synergistic effect with interferon-γ has also been confirmed. [Williamson et al., Proceedings of the National Academy of Sciences (Pro.
NAS) vol. 80, p. 5397, (1983), JP-A-60-
89434].

(Means for resolving the problem) During the development of a new lymphotoxin-K antitumor agent, the present researchers discovered that a combination agent with natural interferon-γ could be used to treat previously reported lymphoblastoid cells. We found that this combination exhibited significantly higher efficacy than the results of an in vitro cell growth inhibition test by Ajyarul et al. (Japanese Patent Application Laid-Open No. 60-89434), which combined lymphotoxin derived from the human body and interferon-γ. The present invention was completed after confirming that the drug was extremely suitable for clinical application.

The content of the present invention will be explained in more detail below with reference to Examples, but the present invention is not limited to these Examples. For example, as a specific example of lymphotoxin-K of the present invention, sugar chain-bound lymphotoxin-K produced in BHK cells or non-sugar chain-bound lymphotoxin-K produced in Escherichia coli may be used.
There is no problem even if the amino acids at the N-terminal portion of these proteins are in various analogue types. In addition, as an example of inhibiting the growth of tumor cells in the present invention,
Although HeLa cells derived from cervical cancer are used, the target disease for the new lymphotoxin-K and glycan-linked human interferon-γ complex is any tumor that is sensitive to this complex. It is also applicable to malignant tumors such as breast cancer, lung cancer, liver cancer, bladder cancer, stomach cancer, kidney cancer, leukemia, lymphoma, and brain tumor.

Lymphotoxin-K in the composite formulation of the present invention
The composition ratio of sugar chain-bound human interferon-γ is considered to be 100,000/1 to 1/100,000, preferably within the range of 20/1 to 1/20, but this is just a guideline. It will be easily understood that these are predictions based on the examples, and the present invention is not limited to these numerical ranges.
The combination preparation may be administered clinically in the form of an intravascular or intramuscular injection with a pharmaceutically acceptable carrier, or may be administered directly to the cancerous tissue, but is not limited to these. Oral and other administration methods are also possible.
In addition, the dosage will further depend on the route of administration, the type of tumor,
It will be determined by your doctor as appropriate depending on your symptoms, body weight, drug sensitivity, etc. Therefore, the dosage form of the composite preparation can be any appropriate dosage form depending on the administration method, and may be solid, semi-solid, or liquid, such as injections, tablets, pills, capsules, etc. Dosage forms such as powders, solutions, suspensions, etc. can be used.

(Example 1) Inhibition of cell proliferation by lymphotoxin-K and carbohydrate-linked human interferon-γ alone HeLa cells seeded at 1 x 10 ⁵ cells in a 12-well multiplate (manufactured by Flow Laboratory) (purchased from Dainippon Pharmaceutical), planted 24
After hours, 50-400 U of lymphotoxin-K per ml.
(produced in BHK cells), or 1 to 200 U/ml of carbohydrate-linked human interferon-γ (CHO-K1
Eagle containing 5% fetal bovine serum (produced in cells)
The medium was replaced with 1 ml of ME medium (manufactured by Nissui) and cultured for 3 days. After culturing, the cells were collected by treatment with a 0.02% EDTA-0.25% trypsin solution, and after staining with trypan blue, the number of viable cells was measured. Figure 1 shows the ratio of the number of viable cells in each lymphokine-containing plot, expressed as a percentage, when the number of viable cells in the control plot that does not contain lymphotoxin-K or carbohydrate-linked human interferon-γ is taken as 100%. It was hot. Lymphotoxin-K is approximately
No cell growth inhibition effect was observed up to a concentration of 50 U/ml, and the concentration that showed 50% cell growth inhibition was approximately
It was 300U/ml. Furthermore, with sugar chain-bound human interferon-γ, no effect was observed up to a concentration of 4 U/ml, and the concentration that showed 50% cell growth inhibition was 20 U/ml.
It was hot in ml.

(Example 2) Synergistic effect between lymphotoxin-K and carbohydrate-linked human interferon-γ One of the most standard and recognized methods for showing synergistic effect between two or more drugs is The desired result can be obtained with a combination that is one-quarter or less of the MIC (minimum inhibitory concentration) of each drug [Goodman.A. et al., The Pharmacological Basis of Therapeutics].
Pharmacological basis of therapeutics), published by Maximilian Publications. Therefore, cell growth is inhibited by simultaneous addition of a concentration of 10 to 20 U/ml, which is about one-fourth of the observed MIC of lymphotoxin-K, and a concentration of 1 to 4 U/ml of glycan-linked human interferon-γ. The results were examined in the same manner as in Example 1. As a result, as shown in Figure 2, even in the treated group with lymphotoxin-K10U/ml and carbohydrate-linked human interferon-γ1U/ml, in which both lymphokines had concentrations less than a quarter of their MICs, the concentration of both lymphokines was close to 25%. Inhibition of cell proliferation was observed, and an extremely strong synergistic effect on cell proliferation by lymphotoxin-K and sugar chain-bound human interferon γ was found.

[Brief explanation of the drawing]

Figure 1 shows the effect of lymphotoxin on cell proliferation.
2 is a graph showing the effects of K and sugar chain-bound human interferon-γ when used alone. Figure 2 shows
It is a graph showing the synergistic effect of lymphotoxin-K and sugar chain-bound human interferon-γ on cell proliferation.

Claims (1)

[Scope of Claims] 1. An antitumor agent containing human lymphotoxin-K and sugar chain-linked human interferon-γ represented by the general formula [Table] [Table]. 2 The blending ratio of human lymphotoxin-K and carbohydrate-linked human interferon-γ is
The antitumor agent according to claim 1, characterized in that the ratio is from 100,000:1 to 1:100,000. 3. Claims 1 or 2, characterized in that by containing human lymphotoxin-K and sugar chain-bound human interferon-γ, they act synergistically on cells rather than each alone.
The antitumor agent described in section.