CN113736732B - Method for in vitro activation of immune cells - Google Patents

Abstract

The invention relates to a method for activating immune cells in vitro, which cultures peripheral blood mononuclear cells by using a culture medium containing Muparfostat, effectively inhibits natural killer cells and T cells from expressing LAG-3 on the cell surface, and enhances the activities of the natural killer cells and the T cells by blocking the transmission of cell inhibitory signals.

Description

Method for in vitro activation of immune cells

Technical Field

The present invention relates to a method for in vitro activating immune cells.

Background

Immune cell therapy is a therapeutic method in which immune cells of a patient are proliferated and activated in vitro and then returned to the body. Currently, immune cell therapy has been gradually applied to cancer therapy, and the key to success is to understand the characteristics and functions of various immune cells and select the most appropriate immune cell types, such as natural killer cells (NK cells), T cells, etc., according to the condition and genetic characteristics of cancer patients.

Immune cells are present in the peripheral blood. In peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) isolated from blood, there are several types of cells, including blood cells, lymphocytes and platelets. Wherein the lymphocytes comprise immune cells such as NK cells, T cells, NKT cells, B cells and the like.

Since NK cell and T cell activity is regulated by activating or inhibitory receptors. Among them, LAG-3 expressed on NK cells and T cell surfaces belongs to inhibitory receptors, also called immune checkpoints. When LAG-3 binds to its ligand, it will transmit inhibitory messages to NK cells or T cells, thereby inhibiting NK cell or T cell activity. The challenge of cell therapy with NK cells and T cells is therefore whether the cells can be activated continuously without being inhibited by the inhibitory receptor messages. In recent years, it has been found that the use of anti-LAG-3 antibodies can block the inhibitory messages conducted by LAG-3, promoting NK cell or T cell activation. However, the use of anti-LAG-3 antibodies to culture cells only provides temporary blocking effects, not only at high cost but also in experimental stages, there is still a doubt about the injection of LAG-3 antibodies into humans, and thus there is still a great need to develop a convenient, efficient and safe method for activating immunocompetent cells in vitro.

Muparfastat, also known as PI-88, is sulfonated mannooligophosphate obtained by sulfonating natural yeast fermentation products, and is known as heparinase inhibitor (hepatapastoris) and has the effects of inhibiting angiogenesis and inhibiting cancer cell metastasis. The application of Muparfastat in vitro immune cell culture method and effect is not studied at present.

Disclosure of Invention

In one aspect of the invention, a method of activating immune cells in vitro is provided, comprising the steps of: separating peripheral blood mononuclear cells from a blood sample; suspending mononuclear cells from the peripheral blood in a culture medium containing Muparfastat and placing the culture medium in a cell culture dish; muparfastat was added daily to the medium and incubated for 4 to 18 days.

In a specific embodiment, the amount of Muparfastat added to the medium per day is 300 μg/ml; in a preferred embodiment, the medium further comprises a basal medium and a cytokine. In another preferred embodiment, the cytokine is interleukin 2; wherein the concentration of interleukin 2 in the culture medium is between 500 and 1000IU/ml.

In one embodiment, the immune cells comprise natural killer cells and T cells.

In another aspect, the invention provides a method of inhibiting the expression of LAG-3 by immune cells in vitro comprising the steps of: separating peripheral blood mononuclear cells from a blood sample; suspending mononuclear cells from the peripheral blood in a culture medium containing Muparfastat and placing the culture medium in a cell culture dish; muparfastat was added daily to the medium and incubated for 4 to 18 days.

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, the drawings show some, but not all, alternative embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. The accompanying drawings, which are incorporated in and constitute a part of this specification, help explain the principles of the invention.

Drawings

FIG. 1 is a flow chart of the method for in vitro activation of immune cells of the present invention.

FIG. 2 is a quantitative analysis of natural killer cells expressing LAG-3 after culturing PBMC according to the present invention.

FIG. 3 shows the analysis of the expression level of LAG3 protein in cells after culturing PBMC according to the present invention.

FIG. 4 is a quantitative analysis of LAG-3 expressing T cells after culturing PBMC and co-culturing them with a cancer cell line in accordance with the present invention. Wherein, fig. 4A is an analysis result of the co-culture of PBMCs and a549 cancer cell line; FIG. 4B shows the results of an assay for PBMC co-cultured with a HepG2 cancer cell line.

FIG. 5 is a quantitative analysis of natural killer cells (NK cells) expressing LAG-3 after culturing PBMC and co-culturing it with a cancer cell line according to the present invention. Wherein, FIG. 5A is the analysis result of the co-culture of PBMC and A549 cancer cell line; FIG. 5B shows the results of an assay for PBMC co-cultured with a HepG2 cancer cell line.

Detailed Description

In view of the above-mentioned problems to be solved, the present invention provides a method for activating immune cells in vitro, which comprises culturing peripheral blood mononuclear cells in a culture medium containing Muparfastat, effectively inhibiting the expression of LAG-3 on the cell surface by natural killer cells and T cells, and enhancing the activity of natural killer cells and T cells by blocking the transmission of cytostatic signals.

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document and its definitions will control.

As used herein, the terms "about," "approximately," or "approximately" shall generally refer to within 20%, preferably within 10%, and more preferably within 5% of a particular value or range. As used herein, the numerical values are approximations, by use of the antecedent "about," "about," or "approximately" unless expressly stated otherwise.

In the present invention, "natural killer cells" (natural killer cell) or "NK cells" refer to cells whose surface antigen presents CD3 ^- CD56 ⁺ Is a cell of (a) a cell of (b).

In the present invention, "T cell" refers to a cell surface antigen presenting CD3 ⁺ Is a cell of (a) a cell of (b).

In the present invention, "immune cells" refer to lymphocytes in peripheral blood mononuclear cells.

In the present invention, immune cells are "activated" as determined by: when NK cells or T cells do not express LAG-3, the NK cells or the T cells have better activity.

Materials and methods

The peripheral blood sample is prepared by collecting whole blood from the arm of a subject according to the plan passed by the ethical committee, placing the whole blood in a sterile blood collection tube, and storing the whole blood at room temperature for subsequent treatment.

The basal medium used in the invention can be selected from the following materials: commercially available basal media such as CellGro SCGM (CellGenix), KBM 501 (Kohjin Bio Inc.), AIM-V (Thermo Fisher Inc.), X-VIV015 (Lonza Inc.), DMEM or RPM 1-1640.

The medium of the present invention may contain appropriate components such as proteins, cytokines, antibodies, serum, and compounds. The cytokines are sometimes interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), or interleukin-21 (IL-21).

Methods for preparing muparfastat (PI-88) and the phosphomannose sulfide compositions contained therein have been disclosed in U.S. Pat. No. 20140135282 and P.N. Handley et al (2017). The general chemical structure of the phosphomannose sulfide component of PI-88 is disclosed in "new structural insight into the oligosaccharide phosphate fraction of Saccharomyces Pichia (Hansenula) holstii NRRL Y2448 phosphomannose" on pages 68 to 75 of saccharide research 446-447, N.Handley et al, supra:

method for separating peripheral blood mononuclear cells from blood sample

7.5-8ml of blood is drawn into a blood collection tube containing anticoagulant heparin and Ficoll-Hypaque reagent, and a polyester gel barrier is added to separate the two liquids. The blood collection tube was centrifuged at 1800g for 20 minutes at room temperature. After centrifugation is complete, the plasma fraction is collected for subsequent cell culture, leaving a5 to 10mm plasma layer on the interface, without disturbing the cell layer during operation. The peripheral blood mononuclear cell layer of the interface was then collected into a 15ml conical tube with a pipette, the PBMC was washed with 10ml phosphate buffered saline (Phosphate buffer saline; PBS for short) and the conical tube was inverted 5 times, and centrifuged at 400g for 5 minutes. After repeating the aforementioned washing step twice, the cells were resuspended in 5ml of PBS. Counting the number of cells, typically 1ml whole blood can isolate 1.3x10 ⁶ Is a PBMC of (C).

Method for in vitro activation of immune cells

PBMCs were used at 1x10 per well ⁶ The density of cells was placed in a six-well cell culture dish and medium was added at 37℃and 5% CO ₂ Is cultured in the environment of (2). The culture medium is based on AIM-V culture medium (Thermo Fisher Scientific company) and is supplemented with interleukin-2 (Novartis company) with a concentration ranging from 500 to 1000IU/ml, and Muparfostat with a concentration of 300 μg/ml. Thereafter, the growth of cells was observed every two to three days, and fresh NK cell medium was changed according to the growth of cells until day 18. Interleukin-2 (Novartis company) in a concentration range of 500-1000 IU/ml was added every two to three days during the culture; muparfastat was added daily at a concentration of 300. Mu.g/ml. And finally, transferring the mixed solution of the cells in the culture dish and the culture medium to a centrifuge tube, collecting the cells in the mixed solution in a centrifugal way, washing the cells with PBS, and uniformly scattering the cells with the PBS after repeating the steps for three times, thereby obtaining the activated immune cells.

Analysis of cell surface antigens by flow cytometry

At 2x10 ⁵ Cells/200. Mu.l activated cells were placed in 96 well plates and reacted at 4℃for 30 minutes with 3. Mu.l of fluorescent-labeled antibody, followed by washing 3 times with PBS, then 400. Mu.l of PBS was added to suspend the cells, and then the fluorescent labeling on the cell surface was analyzed by flow cytometry. The fluorescent-labeled antibody includes anti-CD3 antibody (clone UCTH 1), anti-CD56 antibody (clone HCD 56), and anti-LAG-3 antibody (clone 11C3C 65).

Test of Co-culture of PBMC and cancer cell lines

Will be 1x10 ⁶ A549 cancer cell line and 3x10 ⁶ The HepG2 cancer cell line of (C) is placed in a T25 cell culture flask, and is cultured for one day after adding a proper amount of culture medium. Every other day, cancer cell lines were washed with PBS and then mixed with freshly prepared 3X10 ⁶ Peripheral blood mononuclear cells of (C) were co-cultured at 37℃in an environment of 5% CO2 until the fourth day. Muparfastat was added at a concentration of 300. Mu.g/ml per day to the culture medium of the experimental group; the control group was not supplemented with Murpafostat during the incubation period. On the fourth day, the mixture of cells and culture medium was transferred to a centrifuge tube, the cells in the mixture were collected by centrifugation, and washed with PBS at 2X10 ⁵ Cells/200. Mu.l activated cells were placed in 96 well plates and reacted at 4℃for 30 minutes with 3. Mu.l of fluorescent-labeled antibody, followed by washing 3 times with PBS, then 400. Mu.l of PBS was added to suspend the cells, and then the fluorescent labeling on the cell surface was analyzed by flow cytometry. The above-mentioned fluorescent-labeled antibody contains anti-CD3 antibody (clone UCTH 1), anti-CD56 antibody (clone HCD 56) and anti-LAG-3 antibody (clone 11C3C 65).

Analysis of LAG-3 protein expression by Western blotting

After peripheral blood mononuclear cells were cultured to day 18 in the manner described above under the "method for activating immune cells in vitro", the cultured PBMC and CMV3-His-LAG-3 transfected 293ft cells (positive control group) were lysed with RIPA solution (Tris-HCl 50mM pH 7.5,NaCl 150mM,NP-40%, SDS 0.1%). Proteins from PBMC and 293ft cells were separated by 12% SDS-PAGE and transferred to PVDF membrane (BIO-RAD). The PVDF membrane was previously blocked with PBS containing 4% calf serum (BSA) for one hour at room temperature. Next, PVDF moiety was bound to a primary antibody comprising an anti-LAG-3 (17B 4) antibody (added at a ratio of 1:1000; novus Biologicals Co.) and an anti-GAPDH (GT 239) antibody (GeneTex Co.). PVDF membrane was washed three times with PBST for ten minutes, and then secondary antibody, peroxidase-AffiniPure Goat Anti-Mouse IgG+IgM (H+L) antibody (added in a ratio of 1:10000; jackson ImmunoResearch Inc Co.) was added thereto, and reacted at room temperature for one hour. Next, the PVDF membrane was washed three times with PBST, and then the signal was detected with Trident femto WesternHRP Substrate (GeneTex Co.). And finally, judging the relative protein quality by using an image J image analysis program.

Examples

EXAMPLE 1 culture results of Muparfastat addition to Medium

FIG. 2 is a quantitative analysis of natural killer cells expressing LAG-3 after culturing PBMC according to the present invention. As can be seen from FIG. 2, when Muparfostat was not added to the medium, on day 18, LAG-3 expressing natural killer cells (i.e., the surface antigen was LAG-3) ⁺ CD3 ^- CD56 ⁺ Is the cells of (2) in the PBMC) account for 83% of the total number of NK cells in the PBMC; in contrast, when Muparfastat was added to the medium at a concentration of 300. Mu.g/ml per day, LAG-3 was added on day 18 ⁺ CD3 ^- CD56 ⁺ The cell number was decreased, accounting for only 64% of the total number of NK cells. The result shows that adding Muparfastat into the culture medium effectively increases the number of NK cells which do not express LAG-3, and the NK cells have better activity.

To further explore the mechanism of action of muparfastat on activating immune cells, analysis was performed on the expression level of LAG3 protein in cells. FIG. 3 shows the analysis of the expression level of LAG3 protein in cells after culturing PBMC according to the present invention. The results showed that the expression level of LAG-3 protein was 58% of that of the control group, compared to the control group without Muparfastat, after the addition of Muparfastat. As can be seen from FIG. 3, muparfastat helps to inhibit the expression level of LAG-3 protein in immune cells, thereby blocking the inhibitory message transmitted by LAG-3 and achieving the effect of activating immune cells.

Example 2 test results of Co-culture with cancer cell lines

To simulate the scenario of immune cell interaction with cancer cells, PBMCs were co-cultured with cancer cell lines in this example. The cancer cell lines include an a549 cancer cell line (non-small cell lung cancer cell line) and a HepG2 cell line (liver cancer cell line). FIG. 4 is a quantitative analysis of LAG-3 expressing T cells after culturing PBMC and co-culturing with a cancer cell line in accordance with the present invention. Among them, FIG. 4A shows the results of analysis of the co-culture of PBMC with A549 cancer cell lines, showing that in the control group without Muparfastat, the amount of LAG-3-expressing T cells was 5.3% of the total number of PBMC, whereas in the experimental group with Muparfastat, the amount of LAG-3-expressing T cells was reduced, accounting for only 3.2% of the total number of PBMC. FIG. 4B is an analysis of the co-culture of PBMC with HepG2 cancer cell line, showing that LAG-3 expressing T cells account for 5.2% of the total PBMC in the control group without Muparfastat addition, whereas LAG-3 expressing T cells account for only 1% of the total PBMC in the experimental group with Muparfastat addition.

FIG. 5 is a quantitative analysis of natural killer cells (NK cells) expressing LAG-3 after culturing PBMC and co-culturing it with a cancer cell line according to the present invention. Among them, FIG. 5A is an analysis result of the co-culture of PBMC with the A549 cancer cell line, which shows that in the control group to which Muparfastat was not added, NK cells expressing LAG-3 accounted for 5% of the total number of PBMC, whereas in the experimental group to which Muparfastat was added, the number of NK cells expressing LAG-3 decreased, accounting for only 3.2% of the total number of PBMC. FIG. 5B is an analysis result of the co-culture of PBMC with HepG2 cancer cell line, which shows that the NK cells expressing LAG-3 account for 7.7% of the total number of PBMC in the control group without Muparfostat, whereas the NK cells expressing LAG-3 account for only 1.5% of the total number of PBMC in the experimental group with Muparfostat. In summary, adding Muparfastat to the culture medium effectively increases the number of NK cells and T cells which do not express LAG-3, and the NK cells and the T cells have better activity, so that the effect of activating immune cells can be achieved.

Claims (4)

1. A method of activating immune cells in vitro comprising the steps of:

(a) Separating peripheral blood mononuclear cells from a blood sample;

(b) Suspending the peripheral blood mononuclear cells in a culture medium containing Muparfastat and placing the culture medium in a cell culture dish for culture, wherein the culture medium comprises a basic culture medium, interleukin-2 with the concentration range of 500-1000 IU/ml is added, and Muparfastat with the concentration of 300 mug/ml is added; and

(c) Muparfastat was added to the medium daily for 4 to 18 days, wherein the amount of Muparfastat added to the medium daily was 300. Mu.g/ml.

2. The method of claim 1, wherein the immune cells comprise natural killer cells and T cells.

3. A method of inhibiting LAG-3 expression in immune cells in vitro comprising the steps of:

(a) Separating peripheral blood mononuclear cells from a blood sample;

(b) Suspending the peripheral blood mononuclear cells in a culture medium containing Muparfastat and placing the culture medium in a cell culture dish for culture, wherein the culture medium comprises a basic culture medium, interleukin-2 with the concentration range of 500-1000 IU/ml is added, and Muparfastat with the concentration of 300 mug/ml is added; and

(c) Muparfastat was added to the medium daily for 4 to 18 days, wherein the amount of Muparfastat added to the medium daily was 300. Mu.g/ml.

4. The method of claim 3, wherein the immune cells comprise natural killer cells and T cells.

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