RU2402338C1 - Method for preparing activated mononuclear leukocytes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns microbiology and biotechnology. There is offered a method for prepared activated mononuclear leukocytes involving the use of interleukin-2 in combination with cyclodextrin for mononuclear cell activation, a lymphocyte and macrophage concentrate recovered from donor or patient blood is activated in vitro with a complex containing a pharmacopeia preparation of recombinant interleukin-2 within the range of suboptimal doses 50-100 MU in 1 ml of the culture medium and β-cyclodextrin in concentration 1×10^-5-1×10^-3 M in the culture medium at the molar ratio of components either 1:1, or 1:3, or 1:10, the lymphocyte and macrophage concentrate of donor or patient blood is processed in vitro with a complex containing a pharmacopeia preparation of recombinant interleukin-2 within the range of suboptimal doses 50-100 MU in 1 ml of the culture medium and β-cyclodextrin in concentration 1×10^-5-1×10^-3 M in the culture medium the molar ratio of components either 1:1, or 1:3, or 1:10.

EFFECT: invention provides the preparation of IL-2/CD-MNL with high cytotoxic activity to tumour cell lines; 10-fold reduction of IL-2 doses with preserved clinical effectiveness of the activated cells allows reducing the intensity of systemic by-effects of the preparation, and also the use of immunotherapy in debilitated oncological patients, including in the patients with a hyperreactive response to the IL-2 introduction.

2 ex, 1 tbl, 10 dwg

Description

The present invention relates to medicine, in particular to methods for producing activated mononuclear leukocytes (MNL), and can be used for immunotherapy of cancer.

Currently, for the adoptive immunotherapy of malignant neoplasms, a method of introducing activated blood cells into the body is used. The positive results of immunotherapy with interleukin-2 (IL-2) and lymphokinactivated killer cells (LAC) activated by this drug were achieved. IL-2 / LAC immunotherapy is used as an antitumor agent, especially in the treatment of tumor serositis (pleurisy, ascites and pericarditis), as well as chemoresistant neoplasms [Davydov MI, Normantovich VA, Kiselevsky MV, Volkov CM. Adaptive immunotherapy for tumor pleurisy: a clinical and laboratory study. Russian Oncology Journal. 2000. - No. 6 - p. 14-17; Kiselevsky M.V., Blumenberg A.G. Adaptive immunotherapy of ovarian cancer. - A collection of articles dedicated to the European School of Oncology. 2001, p. 164-176; Astul P., Viallat J.R., Laurent J.C., Bradely M., Boutin C. Intrapleural recombinant IL-2 in passive immunotherapy for malignant affusion. Chest. 1993. - Vol. 103 - No. 1 - p. 209-213; Borden E.C., Sondel P.M. Lymphokines and cytokines as cancer treatment: immunotherapy realized. Cancer 1990. - Vol. 65 - p. 800-814; Ebihara T., Koyama S. Lymphokine-activated suppressor (LAK) cells in patients with gastric carcinoma. Cancer Immunology Immunotherapy. 1989. - Vol. 28 - p. 218-224; Liu X., Li D., Zhang C, Ba D. Treatment of 121 patients with malignant effusion due to advanced lung cancer by intrapleural transfer of autologous or allogenic LAK cells combined with rIL-2. Medical Science Journal. 1993. - Vol.8 - p.186-189].

A known method of producing a LAC for the treatment of malignant neoplasms by in vitro incubation of MNL donated from the blood of a donor with preparations of recombinant interleukin-2 (IL-2) “Roncoleukin” (Biotech, RF) or “Prolekin” (Chiron, Holland) at a concentration of 500-3000 ME in 1 ml of culture medium for 48-72 hours [Biological methods of treating cancer. Ed. V.T. DeVita, S. Helman. - M .: Medicine, 2002]. Recommended for the treatment of lung cancer, the LAC administration route obtained by this method provides for 1-2-fold intrapleural administration of 10-100 million cells per week in combination with 4-5-fold daily injections of IL-2 intrapleurally at 500,000-1000000 ME [Dobritsa VP, Boterashvili N. M. et al. Modern immunomodulators for clinical use. A guide for doctors. - S.-P.: Polytechnic. 2001].

The disadvantages of this method are:

1) the need to use high concentrations of expensive drugs to activate lymphocytes;

2) the introduction of LAC obtained by this method, and IL-2 in recommended doses and regimens for cancer patients can cause negative side effects in the form of hyperthermia and chills;

3) IL-2 leads to the activation and proliferation of the predominantly lymphoid pool, without affecting the monocytic-macrophage link.

To increase the bioavailability, effectiveness and reduce the severity of side effects of cytokine therapy, which involves the introduction of blood cells activated by exogenous cytokines, new methods for their preparation are being developed. In particular, liposomal and pegylated forms of cytokine preparations are created for this purpose. A promising area in this area is the use of cyclodextrins (CDs), polysaccharides of bacterial and synthetic origin, as nanoplatforms for biologically active substances [Pharmaceutical Manufacturing Handbook: Production and Processes, ed. by Shayne Cox Gad. - John Wiley & Sons Inc., 2008; Lurri B., Cerchiara T., Bigucci F., Caponio D., Zecchi V. Bovine serum albumin nanospheres carring progesterone inclusion complexes. - Drug Delivery. 2005 .-- Vol. 12 - p. 281-287; Maestrelli F., Garsia-Fuentes M., Mura P., Alonso M.J. A new drug nanocarrier consisting of chitosan and hydroxypropylcyclodextrin. European Journal of Biopharmaceutical. 2006. - Vol.69 - No. 2 - p. 79-86].

Closest to the proposed method for producing activated MNL is the method described by Y. Kuroki et al. [Kuroki Y., Ochiai N., Kurokawa M., Niwayama S., Kishimoto C, Tazawa K., Fujimaki M. Augmentation of murine lymphokine-activated killer cell cytotoxicity by beta-cyclodextrin-benzaldehyde. Journal Cancer Research Clinical Oncology. 1991 - Vol.117 - p.109-114]. This method is adopted as a prototype. The prototype involves obtaining LAC from mononuclear leukocytes of the spleen of mice (MNL SM) of the C3H / He line after incubation with a combination of exogenous IL-2 and β-cyclodextrin-benzaldehyde (β-CD-BA). To release macrophages, spleen cells were incubated for 45 minutes at t = 37 ° C in RPMI-1640 medium (Sigma, USA) containing 10% quartz suspension (KAC-2; JIMRO, Takasaki, Japan) and 10% fresh blood serum C3H / He is a mice. Then, the cell suspension was layered on ficoll-pack (Pharmacia Inc., Piscataway, NJ), centrifuged at 3000 rpm for 30 min, cells were collected at interphase of ficoll-pack, and after washing twice with RPMI-1640 medium, they were placed in complete nutrient medium (PPS) - RPMI-1640 medium containing 10% fetal bovine serum inactivated at t = 56 ° C for 30 min (Filtron, Victoria, Australia), 2 mM L-glutamine, 100 IU / ml penicillin G, 100 μg / ml streptomycin , 20 μg / ml gentamicin, 2 μg / ml fungizone and 0.05 mm 2-mercaptoethanol. To MNL SM at a concentration of 5 × 10 ⁵ -6.5 × 10 ⁵ cells / ml, cultivated in PPS at t = 37 ° C in 5% CO ₂ for 7 days, β-CD-BA was added once on the 3rd day of incubation. at a dose of 200-400 μg per 1 ml of PPS and twice on the 3rd and 5th day of incubation of IL-2 at a dose of 500-700 ME per 1 ml of PPS. The described method provided an increase in the antitumor cytotoxic activity of MNL of the spleen of healthy animals in comparison with incubation of cells with IL-2 by 10-12%, in comparison with incubation of cells with β-CD-BA by 5-7%. The cytotoxic activity of MNL of the spleen of mice inoculated with sarcoma activated with a combination of IL-2 and β-CD-BA was 15-22% higher than the corresponding activity of cells cultured with IL-2 and β-CD-BA in mono-modes.

The disadvantages of the described method include:

1) removal of macrophages and dendritic cells, target cells for CD polysaccharide, from the cell suspension, which excludes the possibility of enhancing the activity of antitumor immunity effectors due to the co-stimulating effect of macrophages and dendritic cells activated by cyclodextrin;

2) a slight increase in the effect when using a combination of IL-2 and β-CD-BA to stimulate healthy mouse cells relative to the effect of exposure to these substances in mono mode;

3) to achieve the effectiveness of the combination of IL-2 / β-CD-BA, relatively high concentrations of its components were used;

4) the non-pharmacopeia drug IL-2 was used in the work;

5) the method involves the use of a relatively expensive preparation of β-cyclodextrin-benzaldehyde;

6) the testing of this method was carried out only on cells of mice of the line C3H / He.

An object of the present invention is to provide a more efficient method for producing activated MNL from the blood of a donor or patient.

The proposed method involves the following stages:

a) the allocation of MNL from the blood of a patient or donor by separating them from blood serum, red blood cells and neutrophils;

b) incubation of the isolated MNL with the IL-2 / CD complex containing IL-2 at a concentration of 50-100 IU / ml and β-CD at a concentration of 1 × 10 ^-5 -1 × 10 ^-3 M in 1 ml, and obtaining of activated cells - IL-2 / CD-MNL.

The method is as follows: MNL is isolated from heparin-stabilized at a dose of 25 units / ml peripheral blood on a single-stage ficoll gradient (PanEco, RF or Sigma, USA) with a density of 1.077 g / cm ^{3 by} centrifugation at 400 g for 20-30 minutes. Lymphoid cells forming an interphase ring are pipetted and washed three times in RPMI-1640 medium (Sigma, USA or PanEco, RF). After each washing in a 10-fold volume of medium, the cells are pelleted by centrifugation at 200 g. Then, MNL is precipitated by centrifugation, counted using a 5% trypan blue solution (PanEco, RF) and resuspended in PPS medium RPMI-1640 (Sigma, USA or PanEco, RF) containing 10% fetal bovine serum inactivated at t = 56 ° C for 30 minutes (HyClone Laboratories, Logan, UK), 2 mM L-glutamine, 100 μg / ml penicillin and 100 μg / ml streptomycin sulfate (Sigma, USA or PanEco, RF). The concentration of cells in suspension should correspond to 5 × 10 ⁵ -6 × 10 ⁵ cells / ml. To obtain IL-2 / CD-MNL, 1 ml of a solution of the IL-2 / CD complex obtained by incubation on a ST-3L shaker (Elmi, Latvia) at 800-1200 rpm for 45-60 minutes was added to 10 ml of MNL PPS of the drug “Roncoleukin” (Biotech, RF) or “Prolekin” (Chiron, Holland) at a concentration of 50-100 IU / ml and β-CD at a concentration of 1 × 10 ^-5 -1 × 10 ^-3 M in a molar ratio of 1: 1, or 1: 3, or 1:10. As a control, take MNL activated by the IL-2 preparation at the concentration used in the prototype, corresponding to 500 ME in 1 ml of culture medium. Cells are incubated in costar 100-250 ml plastic bottles or similar bottles of other companies in a CO ₂ incubator (Guan, France) for 18-48 hours at t = 37 ° C in 5% CO ₂ .

MNL activated by the IL-2 / CD complex exhibit higher cytotoxic activity than LAC (Fig. 1). In our experiments, as target cells were used cells of human erythromyeloid leukemia line K-562 (figure 2). In contrast to LACs stimulated by optimal (500 IU / ml) and suboptimal (50 IU / ml) doses of IL-2 and MNL, cultivated in the presence of β-CD, a large number of large adherents appear in the IL-2 / CD-MNL culture dendritic cells (FIGS. 3-6). This is confirmed by the results of flow cytometry (Fig.7-10). This method made it possible to establish the features of the IL-2 / CD-MNL phenotype represented by CD25 ^high , CD58 ^high , CD3 / CD16 ^high ; CD56 ^high ; CD16 / CD56 ^high ; HLA-DR ^high , in comparison with LAC and MNL (tab.).

Example 1. Evaluation of the antitumor effect of IL-2 / CD-MNL on cells of the line K-562

The cytotoxic activity of LAK and IL-2 / CD-MNL was determined relative to tumor cells of the K-562 line. Tumor cells (1 × 10 ⁴ in 1 ml) were incubated in PPS with IL-2 / CD-MNL and LAC in a ratio of 1: 5 in flat-bottomed 96-well plates (Costar, USA) for 18 hours at t = 37 ° C in 5% CO ₂ . Cells were scanned and photographed using an Axiovert invertoscope (Zess, Germany). Then, MTT vital dye was added to the wells, and the cytotoxic activity index (ICA) was calculated from the value of optical density measured on a plate photometer (Multiscan MS, Sweden) [Shpakova AP, Pavlova KS, Bulycheva T.I. MTT is a colorimetric method for determining the cytotoxic activity of natural killer cells. Clinical laboratory diagnostics. 2000. - No. 2 - p.20-23]. As shown in figure 1, the cytotoxic activity of IL-2 / CD-MNL is 20% higher compared to the LAC obtained by incubating MNL with IL-2 at an optimal dose of 500 ME in 1 ml of culture medium.

Example 2. Determination of the features of the phenotype of IL-2 / CD-MNL by flow cytometry (FACS analysis).

The expression of surface markers was determined using monoclonal antibodies against the corresponding antigens (Caltag Laboratories, USA), the results were taken into account by flow cytometry using a FACScan flow pitometer (Becton Dickinson, USA). At MNL, the expression levels of the differentiating antigens CD3, CD4, CD16 were examined; activation antigen CD25; adhesion molecules CD56, CD58; antigen-presenting molecules CD83, HLA-DR; LPS receptor CD14, as well as costimulatory molecules CD80, CD11a. Gate cell populations were established based on a combination of direct and side light scattering and cell size. When taking into account the results, 5000 events in the gate were counted. Statistical processing of the results was carried out using the WINMDI 2.8 software package.

As follows from the table, IL-2 in combination with CD, in contrast to the mono-mode of IL-2 at a suboptimal concentration, leads to an increase in the expression of NK antigens (CD 16), adhesion molecules (CD58; CD56) and antigenic presentation (HLA-DR), as well as an activation marker, a receptor for IL-2 (CD25).

The invention is illustrated by the following figures.

Figure 1 - Increasing the intensity of the index of cytotoxic activity (ICA) IL-2 / CD-MNL in comparison with MNL, LAC and MNL activated β-CD in mono mode (CD-MNL).

Figure 2 - Photograph of intact cells of the line K-562. Increase 100. Added MTT.

Figure 3 - Photograph of LAC activated by IL-2 at an optimal concentration of 500 ME in 1 ml of medium, after 18 hours of incubation with K-562 cells. Increase 100. Added MTT.

Figure 4 - Photograph of LAC activated by IL-2 at a suboptimal concentration of 50 ME in 1 ml of medium, after 18 hours of incubation with K-562 cells. Increase 100. Added MTT.

Figure 5 - Photograph of MNL activated by β-CD in mono mode, after 18 hours of incubation with K-562 cells. Increase 100. Added MTT.

6 is a photograph of IL-2 / CD-MNL after 18 hours of incubation with K-562 cells. Increase 100. Added MTT.

7 is a Graph reflecting the expression level of surface markers of intact MNL (control). The horizontal axis of the graph is the signal intensity for this parameter. The vertical axis is the number of events.

Fig. 8 is a graph showing the expression level of surface markers of LAK activated by IL-2 at an optimal concentration of 500 ME in 1 ml of medium. The horizontal axis is the signal intensity for this parameter. The vertical axis is the number of events.

Fig. 9 is a graph showing the expression level of surface markers of LAK activated by IL-2 at a suboptimal concentration of 50 ME in 1 ml of medium. The horizontal axis is the signal intensity for this parameter. The vertical axis is the number of events.

Figure 10 is a Graph showing the expression level of surface markers of IL-2 / CD-MNL. The horizontal axis is the signal intensity for this parameter. The vertical axis is the number of events.

Table - Features of the IL-2 / CD-MNL phenotype in comparison with CD-MNL; intact cnl; MNL, activated IL-2 at a dose of 50 ME per 1 ml of medium and CD with sequential administration; LAC activated by IL-2 in optimal (500 IU / ml) and suboptimal (50 IU / ml) concentrations.

Technical result

Compared with the method of obtaining LAK, represented only by lymphocytes, the proposed method for producing IL-2 / CD-MNL provides activation and proliferation of not only lymphoid, but also antigen-presenting cells. CD also allows you to reduce the effective concentration of IL-2 by an order of magnitude, which may have clinical significance when conducting immunotherapy. Reducing the dose of IL-2 while maintaining the clinical effectiveness of activated cells can reduce the severity of systemic side effects of the drug and will allow the use of immunotherapy in weakened cancer patients, including patients with a hyperreactive response to the introduction of IL-2. The use of low doses of expensive drugs will expand the scope of IL-2.

The method of obtaining activated mononuclear leukocytes Expressed molecules Cnl control Sequential administration of IL-2 and CD LAC (dose IL-2-50 IU / ml) LAC (dose IL-2-500 IU / ml) CD-MNL IL-2 / TsD-MNL Cd3 21.73 41.65 33.59 45.59 23.6 49.19 * CD3 / CD16 0.86 7.73 11.82 16.56 6.55 22.3 * CD16 5.22 6.02 10.56 6.22 5.21 8.3 Cd4 6.46 1,6 12.16 17.64 15.71 0.67 CD4 / CD25 13.05 9.61 18.82 21.76 11.16 6.14 CD25 2,58 5.07 1.8 3.21 4.7 7.8 * CD58 4.18 10.89 7.56 9.32 10.64 14.45 * CD58 / HLA-DR 16.01 fourteen 16.8 20.31 16.34 12,99 HLA-DR 3.26 3.24 1.68 3.98 5.71 * 5.22 * CD80 0.57 0.25 0.47 0.78 0.14 0.26 CD80 / CD83 9.81 9.05 9.18 12,42 8.25 8 Cd83 2.89 3.76 3 3 4.43 2.59 CD14 0.08 0.53 0.43 0.56 0.12 0.29 CD14 / CD11c 31.7 14.58 10.96 13.95 9.91 13.57 Cd11c twenty 10.94 10.53 8.91 14.11 13.44 Cd56 0.5 0 1.3 1,57 1,5 6.3 * CD56 / CD16 7.82 2.2 5.1 9.1 0 12.3 * * - significant changes compared to control (p <0.05)

Claims (1)

A method of producing activated mononuclear leukocytes, characterized by using interleukin-2 in combination with cyclodextrin to activate mononuclear cells, characterized in that the activation in vitro of a concentrate of lymphocytes and macrophages isolated from the blood of a donor or patient is carried out by a complex comprising a pharmacopoeial preparation of recombinant interleukin-2 in the range of suboptimal doses of 50-100 ME in 1 ml of culture medium and β-cyclodextrin in a concentration of 1 · 10 ^-5 -1 · 10 ^-3 M in the culture medium with a molar ratio nii components 1: 1, or 1: 3, or 1:10.

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