CS276333B6 - Process for preparing activated killer cells - Google Patents

Abstract

The solution concerns the field of immunology, medicine (e.g., oncology, internal medicine, etc.), pharmacology and biotechnology. It addresses the preparation of activated forms of cytotoxic cells and the production of cytokines. The purpose is to prepare activated forms of killer cells for specialized therapeutic use in oncology, clinical and experimental immunology, which is achieved by adding ergot alkaloids or their semi-synthetic derivatives to the culture of lymphocytes in vitro and subsequent cultivation for 3 to 7 days. The cultivation results in activated killer cells and/or the production of cytokines that induce increased proliferation and cytotoxicity of lymphoid cells. The application of ergot alkaloids in vivo achieves identical results.

Description

The invention relates to a process for the preparation of activated killer cells, i. cells playing an important role in anti-tumor and anti-viral immunity.

The mechanisms of immune recognition are among the important areas of maintaining the integrity of the organism. In addition to specific forms of receptor recognition, the existence of which on B and T cells is relatively well known, there is non-specific recognition, which can be immune or non-immune in nature. So far, a very heterogeneous trace of spontaneous cytotoxicity (SC) cells is included in this non-specific immunity. Cells that are the original lymphoid representatives are called natural killers (UK, K cells) and do not carry the membrane marker CD3 (International Classification of Leukocyte Surface Structures).

The group includes other representatives who rearrange the specific T cell receptor, carry the CD3 marker, and their recognition is not dependent on the major histocompatibility system (MHO). Thus, the cells are of different phenotype and, except for CD3 ⁺ cells, which belong to T lymphocytes, of not completely known origin.

They do not belong to the hitherto known T, B cell lines, although they arise from a common stem cell and bear some features common to T cells and cells of the myelomonocytic lineage. Spontaneous cytotoxic cells are able to recognize a wide range of cellular targets without prior sensitization and are not subject to restriction in association with MHO I and II. class. · Target cells are lysed similarly to specific cytotoxic lymphocytes (CIL) constitutively produced by lytic factors involving colloidal osmosis and together with as yet undescribed mechanisms that also require recognition processes. The final effect of the lysis is to damage the target cell by creating pores in the membrane (perforin), or fragmenting the DNA. The receptor of spontaneous cytotoxicity cells (especially NK cells) is of an unknown nature and there are real data and assumptions that one of the possible recognition mechanisms for which more substantial evidence has accumulated is the lectin-saccharide interaction. Together with specific cytotoxic lymphocytes, which they recognize through T cell receptors (superimmunoglobulin family), SC cells form an important defense system in anti-infective and anti-tumor immunity and play an important role in transplant immunity. In addition, they participate in a number of regulatory processes in hematopoiesis and thus play the role of immune surveillance, which is considered a basic condition for the existence of the organism. '

Activation of spontaneous cytotoxicity cells is one of the most serious problems in the preparation of naturally active substances and cellular systems, not only in terms of gaining basic knowledge, but also in terms of therapy. Key activators include interferon-type and interleukin-like lymphokines, which, along with the cells they activate, are thought to be promising in tumor immunotherapy. Cells described as activated killers (AK), lymphokines activated killers (LAK cells), are prepared and examined for the ability to lyse not only sensitive but also resistant cellular targets. Today, they form the basis of new treatment procedures in cases of malignant diseases of various kinds.

The disadvantage of the current method of activation using lymphokines (especially ID-2 and interferons) is their high toxicity and the resulting difficulties in administration to patients. In addition, the therapy is based on the use of recombinant preparations (bacterial products) which simultaneously contain bacterial antigens which increase the antibody response of patients, which leads to a reduction in the effectiveness of the treatment and the undesired production of antibodies against the bacterial antigens.

These disadvantages are eliminated by the killer cell activation process according to the invention. It consists in the induction of cytotoxic cells (NK, K, CIL) by various types of ergot alkaloids, both natural and semisynthetic, such as: peptide ergot alkaloids of the ergotamine group, ergergine aergostin, simple lysergic acid derivatives - ergometrine and ergin, clavic alkaloids - elymoclavine, agroclavine, chanoclavine and elymoclavin mono and difructoside, semisynthetic ergot alkaloids - dihydrolysergol, 10-alpha-methoxy-dihydrolysergol, 1-methyl-10-alpha-methoxy-dihydrolysergol and their nicotinoyl and 5-bromo-nicotinoyl esters, terguride, methylergometrine, [4a (S), 10b (S)] - 7-amino-3,4,4a, 5,6,10b-hexahydro-2-hydroxymethyl-4-methyl-6-oxobenzo-f) quinoline ( Ox-Elymoclavine), (4a (S)., 10b (S)) - 7-amino-3,4,4a, 5,6,10b-hexahydro-2,4-dimethyl-6-oxobenzo- (f) - quinoline (Ox-Agroclavine) and ox-dihydrolysergol and its 1-methyl, 10-alpha-methoxy and 1-methyl-10-alpha-methoxy

CS 276 333 B6 2 derivatives.

9-10 dihydroderivatives of natural ergot alkaloids and their 2 halogen derivatives can also be used.

The basic structures are shown in Figures 1 to 3.

The following immunological methods were used to test the preparation of activated killer (AK) cells and the preparation of cells with activated cytokine synthesis:

1. Testing of proliferative activity by incorporation of 1H-thymidine in vitro of human PBMC peripheral mononuclear cells.

2. Effect of supernatants obtained from 5-day cultures stimulated with ergot alkaloids on unstimulated PEMC cells.

3. Cytotoxic activity of cells obtained from 5-day cultures with ergot alkaloids stimulated. PBMC.

4. Effect of ergot alkaloids administered in vivo to Balb / c and nu / nu mice and in vitro monitoring of proliferative activity (spontaneous and PHA-induced) and spontaneous cytotoxicity of spleen lymphocytes divided on Ficoll-Verografin.

The first method of testing the method of preparation of activated killer cells consists in that the effect of ergot alkaloids is monitored continuously from the 1st to the 7th day of cultivation. Microscopically, the culture shows increased blast transformation and cell proliferation after only 3 days of culture. This effect deepens until day 7 of cultivation. Quantification of this phenomenon, ie. activation of the killer cells is performed by measuring the incorporation of 4 H-thymidine according to a commonly used methodology.

Another method that was used to activate the killer cells was to use supernatants from cultures stimulated with ergot alkaloids for 5 days. These supernatants are no longer substantially free of ergot alkaloids; on the one hand, they are bound to the receptor sites of the cell and, on the other hand, metabolized.

The addition of supernatants leads to the same phenomenon as after the addition of the alkaloids themselves, ie. that activation occurs by the action of other substances - cytokines (interleukins, interferons), which are released by activated cells. '

Another effect of ergot alkaloids on peripheral blood cell cultures is to increase their cytotoxic activity even against NK-resistant lines, which is manifested by the fact that after 5 days of culturing lymphocytes with ergot alkaloids, the killing activity of these cells increases. This effect is quantified by measuring the concentration of the released ^ ¹ Cr from target cells according to standard immunological methods.

The same effect also occurs after the addition of supernatants from cultures stimulated for 5 days in the presence of ergot alkaloids.

Balb / c (normal immune status) and nu / nu (thymus deficient) mice were used to test the activity of ergot alkaloids in vivo on the basis of in vitro experiments which showed an effect mainly on T lymphocytes. Following intraperitoneal administration of ergot alkaloid solutions, both proliferative and cytotoxic activity are increased. The results of experiments on nu / nu mice indicate that ergot alkaloids are able to modulate defective immune responses in the T cell region.

The use of the method for the preparation of activated killer cells according to the invention is envisaged mainly in the field of experimental focusing on finding the pathway of cell activation and the possibility of more detailed study of molecular aspects of activation by ergot alkaloids (it can be assumed This procedure would provide the knowledge necessary for the use of various forms of alkaloids in vivo and in therapeutic practice.

LAK cells used to treat cancer must be combined with interleukin-2 alone, which carries a high risk of side effects (nausea, fever, swelling, etc.). Although in the last period there is a shift to longer-term cultivation

CS 276 333 B6 with IL-2 and administration of smaller doses of IL-2 to patients, toxic side effects cannot be completely ruled out.

Preparation of activated killer cells using ergot alkaloids with fewer side effects is desirable not only because of the cost but also because of the necessary supportive effect of interleukins. '

The results obtained so far in vivo point to the possible treatment of many types of cancer and also indicate the direction that will be further developed using the natural mechanisms of immunity. Research into cells with killer capacity for their negative role is also important in transplant immunology.

The process for the preparation of ergot alkaloids activated killer cells is illustrated by the following non-limiting examples.

Example 1.

Human peripheral blood is separated into a Ficoll - Verografin mixture (8% Ficoll - Pharmacia in tissue with 35% Vergografin - Spofa 2.4: 1) after a 1: 1 dilution in RPMI 1,640 medium, pH 7 to 7.2 for 40 min at 40 min. 400 g. Interphase mononuclear lymphocytes are washed three times in RPMI 1,640, pH 7 to 7.2 (10 min, 250 g) and then diluted in RPMI culture medium L-glutamine, PNG / STM and 5% fetal calf serum to a concentration of 1.10 $ cells / ml.

The assay is performed in a 96-well tissue culture plate (NOTO). To 200 [mu] l of cell suspension (in triplets) is added 50 [mu] l of a solution of ergot alkaloids in RPMI pH 7.2 to a final concentration of 1 [mu] g / ml and 10 [mu] g / ml of cell suspension and after 5 days of culturing in a CO ₂ incubator ( at 37 ° C, 5% CO ₂ humidity 100®) was added 50 .mu.l of a solution of ^ H-thymidine (2.5 uCi _of). After 12 hours of incubation, the samples are harvested on a harvester and measured by the amount of thymidine incorporated by the lymphocytes, which expresses the degree of cell proliferative activity expressed in cpm. The degree of increase over the control is expressed by the stimulation index SI (SI = cpm of the sample / cpm of the control). The test results are shown in Table 1.

Table 1.

Increase of lymphocyte proliferative activity by the action of ergot alkaloids

alkaloid SI 10 / Ug / ml SI 1 / Ug / ml SI 0.5 μg / ml Elymoclavine 2.5 to 5.0 1.0 to 1.5 1.05 to 1.2 Blymoclavine fructoside 2.0 to 3.0 1, 0 to 1, 2 0.96 to 1.1 Elymoclavine difructoside 3.0 to 8.5 1.0 to 1.2 1.1 to 1.2 Ox-Blymoclavine 4.0 to 6.0 1.5 to 2.0 0.9 to 1.2. Dihydrolysergol. 1.5 to 2.0 2.0 to 5.0 1.2 to 1.4 10-alpha-methoxy-dihydrolysergol 1.5 to 2.0 1.5 to 3, o 1.3 to 1.5 Agroclavine 1.5 to 2.0 2.5 to 3.0 2.4 to 3.6 Ox-Agroclavine 3.0 to 4.0 1.5 to 2.5 3.1 to 5.6 Chanoklavin 1.0 to 1.5 1.5 to 2.5 3.1 to 5.6 -Χ-10-alpha-methoxy-dihydrolysergol 6.5 to 3.6 1.4 to 1.2 1-Methyl-10-alpha-methoxy-dihydrolysergol 1.0 to 0.8 1.1 to 0.96 1.2 to 1.02 Ergokristin 0.5 to 0.6 0.8 to 1.7 1.0 to 1.02 Alpha-ergocryptine 0.3 to 0.6 0.4 to 0.9 0.5 to 1.06 Nicergoline 0.4 to 0.7 0.7 to 0.8 1.0 to 1.2 Ergocorn 0.2 to 0.35 0.7 to 0.8 1.5 to 1.7 Ergometrine 0.5 to 0.8 0.6 to 0.9 1.4 to 1.6 Beta-ergocryptine 0.2 to 0.6 0.4 to 1.2 1.1 to 1.4 Ergotamine 0.7 to 0.85 0.5 to 1.0 1, 29 to 1.6

Example 2

Isolated human peripheral blood mononuclear cells were resuspended in the culture medium of Example 1 and cultured for 3 to 7 days in the presence of ergot alkaloids in CO _2.

CS 276 333 B6 4 incubators. The stimulated cell suspension was then centrifuged for 10 min / 250 g and the supernatant was used in the proliferaee assay, see Example 1, with unstimulated human peripheral blood lymphocytes. To 200 [mu] l of unstimulated cell suspension was added 50 [mu] l of undiluted supernatant from stimulated lymphocytes. After 3 to 5 days of culture, the incorporation of 1 H-thymidine was measured. Supernatants used for proliferation testing were also tested in a spontaneous cytotoxicity assay against an NK-resistant tumor line (RÁJI) in a 96-well tissue culture plate (NOTO) in triplets.

Experimental scheme: To 100 μl of tumor cell suspension (concentration - 1x10 6 / ml) labeled for 90 min in a CO ₂ incubator (Amersham) - 100 μCi / 10 2 cells was added 100 μl of lymphocyte suspension prepared according to Example 1 at concentration 2 x 10 .mu.l / ml and 50 .mu.l of supernatant from stimulated cells. After a 4-hour incubation in a CO 2 incubator, the amount of Cr released in 100 .mu.l of culture medium was measured and the% cytotoxicity was calculated from the results obtained according to the formula:

% cytotoxicity = £ 22_ £ Si ^ _22S-2E2 ^ · _{x 1O} o cpm max. - cpm spont.

cpm exp. - the amount of released ^ 'cr from the mixture of lymphocytes and tumor cells cpm spont. - the amount released by Cr alone by the R1 tumor cells in cpm max. - maximal release of ^J Cr by tumor cells after use of detergents (2% Triton x 100)

To assess the efficacy of supernatants from stimulated lymphocytes to activate spontaneous cytotoxicity, a% increase in cytotoxicity calculated using the formula was used:

% increase ctx = x 100,% ctx K where

V - cytotoxic effect of the test supernatant, K - cytotoxic effect of the control culture.

The results are summarized in Table 2. Table 2

Effect of supernatants from lymphocytes stimulated with ergot alkaloids on proliferative and cytotoxic activity

alkaloid SI % increase in cytotoxicity Elymoclavine 4 to 8 80 to 100 Elymoclavine difructoside 2 to 4 40 to 50 Dihydrolysergol 5 to 9 80 to 200. 10-alpha-methoxy-dihydrolysergol 4 to 8 50 to 100

It can be seen from Table 2 that supernatants from lymphocytes stimulated with ergot alkaloids increase cell proliferation with spontaneous cytotoxicity. It follows that the effect is mediated by one or more cytokines produced by the stimulated cells.

Example 3

Lymphocytes prepared according to Example 1 were stimulated with ergot alkaloid solutions for 5 to 7 days at a concentration of 0.1 to 20 [mu] l / ml of cell suspension. The cells were washed twice with RPMI 1640 medium (10 min / 250 g) and then used in a cytotoxic assay, see Example 2, as effector cells against NK-resistant target lines (RAJI, BAUDI - they have the receptor for interferon alpha 2).

After 4 hours of incubation in a CO ₂ incubator, the amount of ^γ Cr released was measured.

CS 276 333 B6

Table 3

Effect of ergot alkaloid stimulation on cytotoxic cell activity

alkaloid % increase in cytotoxicity C. b. PARADISE 10 / Ug / ml 1 / Ug / ml ( ), 5 / Ug / ml Elymoclavine. 40 to 100 20 to 30 Elymoclavine difructoside 40 to 60 20 to 30 Ox-elymoclavin ' 20 to 25 30 to 40 Bysergol 9 to 10 1 1 to 20 Dihydrolysergol 80 to 90 10 to 20 10-alpha-methoxy-dihydrolysergol 40 to 90 30 to 55 Ox-10-alpha-methoxy-dihydrolysergol 50 to 95 10 to 20 Agroclavine 0 to 5 10 to 20 Ox-agroclavine 60 to 100 50 to 60 Chanoklavin 35 to 80, 20 to 40 30 to 50 Ergokristin 0 20 to 30 20 to 30 Ergocorn. 10 to 20 20 to 30 50 to 60 Nicergoline 20 to 30 30 to 40 50 to 60 Ergotamin ' 30 to 40 60 to 70 60 to 70 Ergometrine 15 to 20 35 to 45 20 to 30 Beta-ergocryptine 0 to 5 30 to 40 30 to 40 Alpha-ergocryptine 0 10 to 20 5 to 10

The table shows that all ergot alkaloids tested increase the ability of lymphocytes to kill NK-resistant tumor cells RAJI and 10-alpha-methoxy-dihydrolysergol in addition to DAUDI cells.

Example 4

Balb / c (normal immune response) and nu / nu (defective team development) mice were given a single intraperitoneal solution of ergot alkaloids at a dose of 0.1 to 20 mg / kg body weight. A control group of mice from both strains received the same volume of medium in which the individual substances were dissolved. After 7 days, the mice were sacrificed by cervical dislocation and the spleen cells were divided into Ficoll-Verografin after homogenization. Spleen lymphocytes were tested for proliferative activity, after 3 days according to Example 1 and spontaneous cytotoxicity according to Example 2.

Table 4

Effect of in vivo application of solution, ergot alkaloids on proliferative activity (SI) and spontaneous cytotoxicity (% ctx) of splenic lymphocytes of Balb / c mice

alkaloid 10 .ug / ml 1st dose 2nd dose % ctx CB-YAC1 1, ug / mi 1st dose 2nd dose % ctx CB-YAC1 Elymoclavine 6.5 2.47 24.0 4.75 2.27 37.0 Elymoclavine difructoside Ox-elymoclavine 4.81 2.28 18.25 5.37 1.43 14.5 Bysergol · 1.53 2.28 25.14 4.42 4.96 17.34 Dihydrolysergol. 4.45 6.85 28.16 2.25 - 1 O-alpha-methoxy-dihydrolysergol 2.8 1.67 2.10 20.86 Ox-10-alpha-methoxy-dihydrolysergol 5.50 5.70 21, 62 Agroclavine 4.08 1, 08 3.96 0.68 14.33 Ox-agroclavine 8.45 11.05 ⁺ ) 0.95 2.63 ⁺⁾ 40.90 24.19 ⁺⁾ 1.85 2.65 22.06 Chanoklavin 3.59 0.92 30.63 Beta-ergocryptine 7.15 5.49 22.74

CS 276 333 B6 6 Ergotamine 1.33 2.09 25.23 Alpha-ergocryptine 4.33 2.79 21, 42 Ergometrine 3.29 2.75 25.76 Ergocorn 6.31 ¹ , 93 11.68 Ergokristin 2.15 1.36 24.83 Control 1.00 1.00 4.28 1, 00 1, 00 4.28

⁺ ) 0.5 .mu.g / ml

Table 5 '

Effect of ergot alkaloids on proliferative activity and spontaneous cytotoxicity of spleen lymphocytes in nu / nu mice

alkaloid and dose spontaneous proliferation SI proliferation in the presence of PHA SI % increase in cytotoxicity Elymoclavine 10 mg / kg 2.0 to 2.5 3.4 to 4.5 90 to 100 Dihydrolysergol 1 mg / kg 2.5 to 3.0 6.0 to 7.0 80 to 100 10-alpha-methoxy-dihydrolysergol 1 ug / kg 2.4 to 2.8 4.0 to 5.0 90 to 110 Agroclavine 1 mg / kg 2.7 to 3.0 1.5 to 2.0 40 to 60

From the results shown in Tables 4 and 5, it is clear that the proliferative activity of the lymphocytes increases after administration of ergot alkaloid solutions to mice in vivo. In addition, Table 5 shows that ergot alkaloids act mainly on the T cell population (as can be seen from the results of proliferation in the presence of PHA) and are able to largely correct the defective immune response, in the area of T lymphocytes.

Example 5

Isolated human peripheral blood mononuclear cells were resuspended in the culture medium of Example 1 and cultured for 5 days in the presence of ergot alkaloids and 20 to 100 IU / ml rIL-2 in a CO 2 incubator. Proliferation activity was determined as in Example 1. The results are summarized in Table 6.

Table 6

Proliferative activity of PEL and KK cells (99% purity) after simultaneous stimulation with rIL-2 and ergot alkaloids (cpm)

AND

alkaloid PBL. NK Elymoclavine 40 500 130 200 Elymoclavine difructoside 45 980 127 900 Chanoklavin 40 600 3,000 Ox-elymoclavine 54 400 120 100 Lysergol 59 200 148 850 Dihydrolysergol 38 200 142 520 10-alpha-methoxy-dihydrolysergol . 59 700 161 220 Ox-10-alpha-methoxy-DH-lysergol · 45 800 103 500 Agroclavine 47 700 121 700 Alpha-ergocryptine 51 300 166 400 Nicergoline 45 230 145 050 Control + rIL-2 50 000 120 000

It follows from the above Example 5 that ergot alkaloids generally act synergistically with rIL-2, in particular lysergol and its derivatives, as well as alpha-ergocryptine and cergoline, have a slightly stimulating effect. In the case of chanoclavine, there is a significant attenuation of proliferative activity

NK cells stimulated with rIL-2.

Claims (6)

P A T B E I 0 CLAIMS A process for the preparation of activated killer cells, the activation of which is achieved by increasing the proliferative activity, characterized in that cultures of human peripheral blood lymphocytes are treated with ergot alkaloids. 2. The process according to item 1, characterized in that peptide ergot alkaloids of the ergotamine, ergotoxin and ergoxin groups, simple lysergic acid derivatives - ergometrine and ergin, clavine alkaloids - elymoclavine, agroclavine, chanoclavin and mono and difructoside elymoclavin are used as natural ergot alkaloids. or mixtures thereof. 3- The process according to item 1, characterized in that dihydrolysergol, 10-alpha-methoxy-dihydrolysergol, 1-methyl-10-alpha-methoxy-dihydrolysergol and their nicotinoyl and 5-bromo-nicotinoyl esters are used as semisynthetic ergot alkaloids, Lisuride, Terguride, methylergometrine, [4a (S), 10b (S)] - 7-amino-3,4,4a, 5,6,10b-hexahydro-2-hydroxyethyl-4-methyl-6-oxobenzo-f) quinoline (Ox-Elymoclavine (, / 4a (S), 10b (S)) - 7-amino-3,4,4a, 5,6,1Ob-hexahydro-2,4-dimethyl-6-oxobenzo- (f) -quinoline (Ox-Agroclavine), further ox-dihydrolysergol and its 1-methyl, 10-alpha-methoxy and 1-methyl-10-alpha-methoxy derivatives and further 9-10 dihydroderivatives of natural ergot alkaloids and their 2 halogen derivatives or their mixtures . 4. The method of claim 1, wherein the ergot alkaloids are added to the culture in the form of a solution in a standard medium of pH 7 to 7.2 at concentrations of 0.1 to 20 .mu.g / ml of culture. 5. A process for the preparation of activated killer cells, characterized in that the activation is achieved by adding a supernatant from 3 to 7 day cultures of lymphocytes stimulated with ergot alkaloids, preferably elymoclavine, dihydrolysergol, 10-alpha-methoxy-dihydrolysergol and elymoclavine difructoside. 6. A process for the preparation of activated killer cells, wherein the activation is achieved by increasing the cytotoxic activity against NK-resistant target cells, characterized in that the culture is performed in the presence of ergot alkaloids, preferably elymoclavine, elymoclavine difructoside, dihydrolysergol and 10-alpha- methoxy-dihydrolysergol.