JPH06211692A - Immunological enhancer - Google Patents

Abstract

PURPOSE:To obtain an immunological enhancer capable of minimizing side effects and enhancing the immunological enhancing effects. CONSTITUTION:This immunological enhancer comprises a human lysozyme as an active ingredient. The human lysozyme is useful for enhancing the immunological action of human lymphocytes and preventing or treating the onset of cancer, AIDS, etc., by parenterally administering the human lysozyme. When the human lymphocytes are used with the human lysozyme in combination, effects thereof can further be enhanced.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunopotentiating agent which has reduced side effects as much as possible and has enhanced immunopotentiating effect. The immunopotentiator of the present invention is parenterally administered to enhance immune activity, and is used as an anticancer agent, a therapeutic agent for AIDS, a cancer metastasis inhibitor, an infection preventive agent, etc. by its action.

[0002]

BACKGROUND OF THE INVENTION Human lysozyme is a basic protein consisting of 130 amino acids, including tears, mucus, saliva, egg white,
It is an enzyme that is present in milk and the like. This enzyme mainly hydrolyzes glycoside bonds of peptidoglycan, digests the cell wall of bacteria, and dissolves the bacteria. In addition, as the action of this enzyme, edema suppression, purulent mucus decomposition, anti-heparin activity, tissue repair activity, etc. are known,
Chloride of lysozyme is marketed as a compounding agent for enzyme preparations such as Acdime and cold remedies (see "Latest Medical Dictionary" by Ikuyaku Shuppan Co., Ltd.). The present inventors succeeded in producing a large amount of recombinant human lysozyme using baker's yeast as a host by utilizing the technique of recombinant DNA technology (see Japanese Patent Laid-Open No. 2-234673). Then, after various studies on its application, it was found that human lysozyme has a growth promoting action on animal cells and can be used as a serum-free medium component.
(JP-A-4-218365). When I proceeded with this research,
It was found that when human lymphocytes collected from the human body were cultured in a medium containing human lysozyme together with cytokine, the obtained human lymphocytes enhance the cytotoxic activity against cancer cells (Japanese Patent Application No. 3-199959).

[0003]

The present inventors further investigated the activity of human lysozyme, and found that human lysozyme has the effect of suppressing the growth of various tumor cells and the like and enhancing human immunity. The present inventors have completed the present invention by finding that they can be used as a medicine. Further, it was found that this action is markedly enhanced when human lysozyme and human lymphocytes coexist. That is, an object of the present invention is to provide a parenteral immunity enhancer containing human lysozyme as an active ingredient. A further object of the present invention is to provide a parenteral immunity enhancer containing human lysozyme and human lymphocytes as active ingredients. Although the immunopotentiating effect of egg white lysozyme has been studied (Tohoku Medical Journal 93 32 (1980)), egg white lysozyme has almost no immunopotentiating effect, and human lysozyme has a remarkable immunopotentiating effect that can be used as a medicine. What was found to be effective is truly amazing.

[0004]

Specifically, the present invention relates to a parenteral immunity enhancer containing human lysozyme as an active ingredient. Furthermore, the present invention relates to a parenteral immunity enhancer containing human lysozyme and human lymphocytes as active ingredients. Human lysozyme in the present invention, human placenta, white blood cells, urine,
It is found in tissues and secretions such as breast milk and tears, and can be extracted and purified from these or can be obtained by the above-mentioned genetic engineering techniques. The human lysozyme thus obtained can be further purified by using a cation exchange resin, a molecular sieve or the like to increase the purity to 99.99% or more.

On the other hand, human lymphocytes are produced by stem cells derived from bone marrow. Therefore, it can be obtained by culturing such human lymphopoietic stem cells. There are B lymphocytes and T lymphocytes,
Any of these can be used, and further, K cells not belonging to these; NK cells and the like can be used. The amount of human lysozyme used varies depending on symptoms, sex, age, etc., but is usually 0.1-1,000 mg (25,000-25
0,000,000 units, measurement method Anal.Biochem., 12
8.77 (1988)), preferably 1-100 mg (250,000-
25,000,000 units) in 1 to several divided doses. The administration method is parenterally. Specifically, it is administered by subcutaneous injection, intramuscular injection, intravenous injection, arterial injection, intraperitoneal or intrathoracic injection, or the like. When it is administered orally, it is not desirable because its absorption efficiency from the intestine is low, its activity is weakened, and there is no immediate action. Therefore, it is administered parenterally.

[0006] The preparation is 0.1 g of human lysozyme (25,000,0
(00 units) as the hydrochloride salt, saline, phosphate buffer (PB
S) or Hanks' buffer, dissolved in 10 ml of distilled water, filled in an ampoule, and cold sterilized or heat sterilized to the extent that the activity is not lost. In addition, human lysozyme and these lysates may be dissolved before use.

In the present invention, human lymphocytes may be added thereto as an active ingredient. The human lymphocytes stored may be used, or the human lymphocytes may be collected from the body of a person to be treated and cultured in advance and administered together with human lysozyme. In this case, human lysozyme and human lymphocytes may be separately or mixed so that they can be administered in the form of a single agent, or after administration of human lymphocytes for a certain period of time, for example, 1 to 20 days later, human lysozyme is administered. Alternatively, after administration of human lysozyme, human lymphocytes may be administered for a certain period of time, for example, 1 to 30 days later. The immunopotentiator of the present invention includes
This includes not only such a dosage form in which human lysozyme and human lymphocytes can be administered simultaneously, but also dosage forms in which both are prepared individually and can be administered sequentially. An appropriate dose of human lymphocytes is about 10 ⁷ to 10 ⁸ per mg of human lysozyme, which is used together with the dose of human lysozyme as described above.

Next, the present invention will be specifically described with reference to examples.

Example 1 A secretory expression plasmid containing a synthetic human lysozyme gene was prepared by the method described in Japanese Patent Laid-Open No. 25527/1992, and this plasmid was introduced into yeast. Cultured in the source medium,
Human lysozyme was secreted into the culture medium. The resulting culture supernatant is filtered through a mesh 0.45 μm filter to remove the transformed yeast, and the filtrate is ultrafiltered with a molecular weight cut-off of 5,000 to 10,000 and concentrated approximately 20 times to desalt and human lysozyme. Was concentrated. 1,000 ml of this concentrated solution was used as a cation exchange column chromatography (cation exchange resin SP-Sepharose
HP, 700 cc), human lysozyme was adsorbed on the cation exchange resin, human lysozyme was desorbed and separated from the resin using the desorption solution NaCl, and this separation solution was further subjected to gel filtration column chromatography (packing resin Superdex 75PG, packing amount 5,000 A basic impurity similar to human lysozyme was separated and removed by applying 10 ml of an eluent of 10 mM NaCl. On the other hand, the separated solution containing human lysozyme was ultrafiltered to remove pyrogen-generating substances, and further filtered through a 0.22 μm mesh filter to remove bacteria, and the filtrate was lyophilized to obtain purified human lysozyme.

[0009]

[Example 2] Lymphocytes of a gastric cancer patient were collected and used as a serum or serum-free medium containing interleukin 2 (IL-2).
(The medium is NOC 905 medium developed by the inventors (JP-A-4-21
No. 8365 gazette)) and culture conditions 5% CO ₂ conditions 37
The cells were cultured at 0 ° C. to activate lymphocyte effect cells (E). This was contacted with ⁵¹ Cr-labeled cancer cell target cells (T) such as K-562, KATO-III, Daudi, and autologous tumor cells at each E / T (specifically, target cells ( the T) 1 × 10 ^4/10
00μl and effector cells (E) to ^{10 × 10 4 / 100μl (E} / T10), (T)
The 1 × 10 ⁴ / 100μl and the ^{(E) 20 × 10 4 /} 100μl (E / T 20)
, (T) a 1 × 10 ⁴ / 100μl and ^{E 40 × 10 4 / 100μl (} E / T
40) was added), the purified human lysozyme (HLY) obtained in Example 1 was added to the so-called effector phase, and the mixture was contacted at 37 ° C. for 4 hours to examine the ability to enhance the cytotoxic activity against cancer cells. . The results are shown in Tables 1 to 4 below. As shown in the results of this table, it was found that the damaging activity of activated lymphocytes was remarkably enhanced against any of the desired cells.

[0010]

[Table 1] Cancer cell K-562 cytotoxicity ─────────────────────────────────── HLY concentration ( μg / ml) Cancer cytotoxicity (% Cx) E / T 10 E / T 20 E / T 40 ─────────────────────────── ───────── 0 36 41 40 1 59 60 57 10 68 71 74 50 73 79 80 100 75 80 82 ────────────────────── ────────────── The maximum release was the radioactivity when the target cells were treated with 1N-HCl. Cancer cells (T) were labeled with ⁵¹ Cr and their dose was measured by radioactivity.

[0011]

[Table 2] Toxicity of cancer cells KATO-III ─────────────────────────────────── HLY concentration ( μg / ml) Cancer cytotoxicity (% Cx) E / T 10 E / T 20 E / T 40 ─────────────────────────── ───────── 0 18 22 24 1 29 34 33 10 39 43 46 50 46 54 60 100 51 55 58 ────────────────────── ──────────────

[0012]

[Table 3] Damage of cancer cell Daudi ────────────────────────────────────HLY concentration (μg / ml) Cancer cytotoxicity (% Cx) E / T 10 E / T 20 E / T 40 ───────────────────────────── ─────── 0 45 52 52 1 58 59 60 10 70 73 78 50 80 89 90 100 81 87 90 ─────────────────────── ─────────────

[0013]

[Table 4] Cancer cell autologous tumor cell cytotoxicity ─────────────────────────────────── HLY concentration ( μg / ml) Cancer cytotoxicity (% Cx) E / T 10 E / T 20 E / T 40 ─────────────────────────── ───────── 0 31 38 42 1 50 52 51 10 63 71 77 50 71 82 88 100 72 85 90 ────────────────────── ──────────────

[0014]

Example 3 Human lymphocytes collected from a gastric cancer patient were cultured in a serum-free medium containing interleukin-2 and 50 μg / ml of human lysozyme in the medium for 2 weeks.
The phenotype of cultured lymphocytes was measured. The results are shown in Table 5.
Shown in. As shown in Table 5, human lysozyme is a CD
It was found that the ratio of 4-positive cells (helper T cells) was remarkably increased to have an immunopotentiating effect. In addition, collecting human lymphocytes from a patient suffering from lung cancer or gallbladder cancer,
When the same test as described above was performed, similar results were obtained.

[0015]

[Table 5] Phenotypes of cultured lymphocytes (average% of positive rate) ─────────────────────────────────── ── Number of cells CD3 CD4 CD8 CD16 CD25 ──────────────────────────────────── Without LAK HLY 6 31 10 32 23 19 Add LAK HLY 6 78 42 59 17 31 Add no TIL HLY 5 99 35 67 1 26 Add TIL HLY 5 99 68 75 1 33 ───────────────── ───────────────────── LAK is Lymphokine Activated Killer, TIL is T
umor Infiltrating Lymphocytes are shown. CD3 is T cell, CD4 is helper T cell, CD8 is killer T cell, CD16
Shows the surface antigen of each lymphocyte in the natural killer cell and CD25 in the activated T cell having the expression function of IL-2 receptor α chain. The positive rate is Fl
It was measured by ow cytometry method (FACS can). As shown in this table, when HLY was added, it was involved in the enhancement of the specific damaging action of cancer cells such as CD8, lymphocytes or CD8, which have an immunopotentiating action on CD3, CD4, CD25, etc. It has been found that the percentage of lymphocytes that are active is increased and that lymphocytes, such as CD16, that are not closely related to immunopotentiating activity are not increased or are decreased.

[0016]

Example 4 Limit by TIL collected from gastric cancer patient
CD4 ⁺ clone by ing dilution method (100% CD4 positive cells)
Of CD4 ⁺ clones that have an immunopotentiating effect (N
OC 905 medium with IL-2 200 U / ml added as the basic medium
The effect of human lysozyme on (culturing at 37 ° C for 4 days) was examined (only at conference presentation). To determine the amount of proliferation,
DNA synthesizing ability, that is, ³ H-thymidine uptake (37
It was carried out by measuring the uptake reaction) at 4 ° C. for 4 hours. The results are shown in Table 6. As shown in Table 6,
Human lysozyme was found to be effective in enhancing the proliferative capacity of CD4 ⁺ clones.

[0017]

[Table 6] ────────────────────────────────── HLY (μg / ml) ³ H-thymidine incorporation Amount cpm (4 days of culture, uptake reaction 4 hr) ────────────────────────────────── 0 32,000 1 56,000 10 79,000 50 96,000 100 105,000 ──────────────────────────────────

[0018]

Example 5 Formulation Example (1) 1 g of the purified human lysozyme obtained in Example 1 was dissolved in 100 ml of physiological saline, sterilized with a filter, and dispensed into ampoules in 10 ml portions. Was prepared. (2) 1 g of the purified human lysozyme obtained in Example 1
Active lymphocytes separately packaged in 10 mg and obtained in Example 2
10 ⁸ to 10 ⁷ pieces were prepared so that both could be used together when used. In this method, active lymphocytes are administered by intravenous injection, and then purified human lysozyme is administered by intravenous injection within 1 to 30 days.

[0019]

INDUSTRIAL APPLICABILITY When the immunopotentiator of the present invention is administered parenterally, human lysozyme enhances the immune action, kills cancer cells, and prevents or treats the development of cancer. It is considered that this action is caused by enhancing the ability of human lysozyme to bind to human lymphocytes administered from inside or outside the body to cancer cells, or to enhance the ability of human lymphocytes to release cancer cytotoxic factor. Furthermore, it is also possible to directly act on the tumor cells themselves to increase the sensitivity of the tumor cells to lymphocytes. Therefore, when human lymphocytes cultured outside the body of a cancer patient by adoptive immunotherapy are returned to the cancer patient and human lysozyme is administered together, the effect of killing the cancer cells can be further enhanced.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Hiroshi Tanimura 7-27 Wakayama City, Wakayama Wakayama Medical University Gastroenterological Surgery Department (72) Inventor Yuuki Yamagami 7-27 Wakayama City, Wakayama Prefecture Wakayama Prefectural Government Inside the Department of Gastroenterological Surgery, Medical University (72) Takuya Kakuda 7-27 Wakayama, Wakayama Prefecture Wakayama Prefectural Inside the Department of Gastroenterological Surgery, Medical University (72) Inventor Shizuma Mizobata 7-27 Wakayama, Wakayama Prefectural Inside the Department of Gastroenterological Surgery, Medical University (72) Kohei Noguchi, 7-27, Wakayama City, Wakayama Prefecture Wakayama Prefectural Office Inside the Department of Gastroenterological Surgery, Medical University (72) Makoto Iwahashi, 7-27 Wakayama City, Wakayama Prefecture Inside the Department of Gastroenterological Surgery, Medical University (72) Mikiko Tamai 7-27, Wakayama City, Wakayama Prefecture Wakayama Medical University Gastroenterological Surgery Room (72) Inventor Tsukasa Hotta 7-27, Wakayama City, Wakayama Prefecture, Department of Gastroenterological Surgery, Wakayama Medical University (72) Inventor, Kazuo Arii 7-27, Wakayama City, Wakayama Prefecture Gastroenterological Surgery, Wakayama Medical University In the classroom

Claims (2)

[Claims] 1. A parenteral immunity enhancer comprising human lysozyme as an active ingredient. 2. A parenteral immunity enhancer comprising human lysozyme and human lymphocytes as active ingredients.