CN107189989A - Oncolytic virus that a kind of immune effector cell is loaded and its preparation method and application - Google Patents

Abstract

The invention discloses an oncolytic virus loaded with immune effector cells, the immune effector cells are CIK cells, and the oncolytic virus is reovirus type 3 Dearing strain. It also discloses its preparation method and application. The oncolytic virus loaded with immune effector cells provided by the invention has better targeting and anti-tumor properties. After loaded with CIK cells, Reovirus mainly remained on the cell surface and significantly enhanced the cytotoxicity of CIK cells. CIK cells could deliver reovirus to tumor cells in the presence of neutralizing antibodies in vitro. The oncolytic virus loaded on the obtained immune effector cells can inhibit tumor growth and promote targeted anti-tumor effects. The preparation method of the invention is simple and easy, and the operating conditions are mild and easy to control. The obtained oncolytic virus loaded by immune effector cells can be used in the preparation of targeted antitumor drugs.

Description

Oncolytic virus loaded with immune effector cells and its preparation method and application

technical field

The invention relates to an oncolytic virus loaded with immune effector cells and a preparation method and application thereof, specifically belonging to the technical field of viruses.

Background technique

Oncolytic virus (OV) is a type of virus that is natural or artificially modified and can specifically replicate in large quantities in tumor cells and eventually destroy tumor cells, but has no killing effect on normal tissue cells. OVs that have been used in clinical research mainly include adenovirus, Newcastle disease virus, herpes simplex virus-1, reovirus and vaccinia virus. At present, there are more than 100 oncolytic virus clinical trials registered and carried out worldwide, involving most common tumor types. A series of clinical trials have been completed, and the safety and effectiveness of oncolytic virus therapy have been confirmed. Lots of encouraging progress. reovirus is a wild-type oncolytic virus, which widely exists in the human respiratory tract and digestive tract. The vast majority of people are asymptomatic after infection with the virus, and a small number of people can cause gastrointestinal and upper respiratory tract diseases after infection. It was found that almost all adults Antibodies to the virus are present in the human body. Canadian company Oncolytics Biotech has developed wild-type reovirus (Type 3dearing) as a patented product and carried out more than 30 clinical trials, among which the treatment of squamous cell carcinoma of the head and neck has entered phase III clinical trials. Good safety and tolerability have been observed in phase I/II clinical studies of intratumoral injections, and phase I clinical studies of intravenous reovirus alone have also confirmed the safety of the application, but the curative effect is limited, and the main reasons are explored is the appearance of neutralizing antibodies. Therefore, in order to overcome the shortcomings of traditional oncolytic virus drug delivery methods, people have gradually developed a new drug delivery method using cells as delivery vehicles, that is, the virus is adsorbed on cells with tumor recognition ability, and the tropism of cells to tumor tissue is used to Help the virus cross the complex blood environment and bring the virus to the tumor site.

Since autologous immune cells can escape the body's immune surveillance and infiltrate into tumor tissues, they may be effective carriers for transporting oncolytic viruses. Studies have reported that dendritic cells (DC) are used as the cell carrier of reovirus, and it is found that DC can internalize reovirus to protect it from neutralizing antibodies, and virus infection can also promote DC maturation, NK cells and T cells to kill tumors effect. Although in vitro experiments have shown the possibility of DC as reovirus carrier cells, there are still many shortcomings that will limit its clinical application, such as the difficulty in the large-scale expansion of DC in vitro and the inability to directly kill tumors. Therefore, in order to solve the above problems, it is particularly necessary to seek a new oncolytic virus loaded with an effective cell carrier.

Contents of the invention

In order to solve the deficiencies in the prior art, the object of the present invention is to provide an oncolytic virus loaded with immune effector cells and its preparation method and application. Preparation of targeted antitumor drugs.

In order to achieve the above object, the present invention adopts the following technical solutions:

An oncolytic virus loaded on immune effector cells, the immune effector cells are CIK cells (cytokine-induced killer cells), and the oncolytic virus is reovirus type 3 Dearing strain (reovirus type 3 Dearing strain).

Among the oncolytic viruses loaded on the aforementioned immune effector cells, the CIK cells loaded with reovirus have a density of 1×10 ⁶ -2×10 ⁶ cells/mL.

A method for preparing an oncolytic virus loaded with immune effector cells, comprising the following steps: collecting CIK cells activated and expanded in vitro, washing the cells with PBS, adjusting the density of the CIK cells through GT-T551 serum-free medium, and then taking the reo The isolated virus was incubated with CIK cells at 4°C, the unbound virus was washed away with PBS, and the virus was resuspended in GT-T551 serum-free medium.

Further, the preparation method of the above-mentioned oncolytic virus loaded with immune effector cells includes the following steps: collecting CIK cells activated and expanded in vitro until the 14th to 16th day, washing the cells with PBS for 2 to 4 times, passing through GT-T551 without Serum medium was used to adjust the density of CIK cells to 1×10 ⁶ to 5×10 ⁶ cells/mL, and then the reovirus was taken and incubated with CIK cells at 4°C for 2 to 4 hours at a titer of 1 to 10 pfu/cell. Wash away unbound virus with PBS, and resuspend in GT-T551 serum-free medium. By adopting the specific cell density and titer in the method of the present invention, the reovirus can be carried by the CIK cells in a maximum amount.

Preferably, the preparation method of the aforementioned oncolytic virus loaded with immune effector cells includes the following steps: collecting CIK cells that have been activated and expanded in vitro until the 14th to 16th day, washed the cells three times with PBS, and cultured by GT-T551 serum-free Adjust the density of CIK cells to 3×10 ⁶ cells/mL, then take reovirus, incubate with CIK cells at a titer of 5 pfu/cell at 4°C for 3 hours, wash off unbound virus with PBS, GT-T551 Serum-free medium after resuspended, that is.

Application of an oncolytic virus loaded with immune effector cells in the preparation of targeted antitumor drugs.

In the prior art, the treatment of tumors with reovirus has entered phase III clinical trial research, but in the early clinical trials, it faced difficulties such as the effect of neutralizing antibodies and poor targeting. CIK cells are a group of heterogeneous cell populations induced by PBMC in vitro with various cytokines and CD3 monoclonal antibodies, including CD8 ⁺ T, CD4 ⁺ T, NK and NKT cells, among which CD8 ⁺ T and NKT cells are considered It is the main effector cell. As one of the anti-tumor adoptive cell immunotherapy schemes, CIK has been widely used in clinical tumor treatment, but CIK cells cannot suppress tumor cells for a long time, so the curative effect is limited. In the present invention, CIK cells are selected as oncolytic virus-loaded cells, which have the advantages of strong in vitro expansion ability, high safety, broad-spectrum tumor killing ability, and strong blood vessel and tissue penetration ability. Loading oncolytic virus on CIK cells enhances its targeting, and oncolytic virus infection can promote the expression of NKG2D on CIK, so oncolytic virus infection can also enhance the activity of CIK. Therefore, the use of CIK cells to carry oncolytic virus for anti-tumor therapy will just take advantage of the advantages of both to make up for their respective shortcomings, and work together to achieve better anti-tumor effects.

In order to ensure that the technical solution of the present invention is scientific, reasonable and effective, the inventor has carried out a series of experiments.

1. Oncolytic virus loaded on immune effector cells

1. Materials

Reovirus type 3 Dearing strain was purchased from American type culture collection (ATCC), proliferated by L929 cells, aliquoted, and stored at -80°C for future use. Reovirus virus titer detection was performed using a modified methylcellulose virus plaque technique. The GT-T551 serum-free medium used for CIK cell culture was the product of Takara Company, recombinant human interleukin-2 (Interleukin-2, IL-2) was purchased from Beijing Sihuan Biological Company, and the anti-CD3 monoclonal antibody was purchased from R&D Company, interferon-γ (Interferon-γ, IFN-γ), recombinant human interleukin-1α (Interleukin-1α, IL-1α) were purchased from PeproTech. The human lymphocyte separation medium was produced by Tianjin Haoyang Biological Co., Ltd., and the methyl cellulose (M0152) was produced by Sigma.

2. Preparation process

CIK cells were cultured by conventional methods, and the CIK cells that were activated and expanded in vitro to the 14th to 16th day were collected, washed with PBS for 2 to 4 times, and the density of CIK cells was adjusted to 1×10 ⁶ ～ by using GT-T551 serum-free medium. 5×10 ⁶ cells/mL density, then take reovirus, incubate with CIK cells at a titer of 1-10 pfu/cell at 4°C for 2-4 hours, wash off unbound virus with PBS, GT-T551 serum-free After the medium is resuspended, it is ready. The density of CIK cells loaded with reovirus was 1×10 ⁶ -2×10 ⁶ cells/ml, and stored at 4°C for use.

CIK cells can be cultured by conventional methods, and can be used in "The in vitro killing effect of CIK cells combined with paclitaxel on ovarian cancer SKOV-3 cells" (Xu Mei et al., Tumor, 2014, 07:591-595) or "Autologous CIK cell therapy on malignant The influence of immune function and quality of life in cancer patients" (Tong Gangling et al., Chinese Journal of Cancer Biotherapy, 2015, 04:504-508).

2. Validation of immune effector cells loaded with oncolytic virus

1. Laser confocal microscopy to observe the localization of reovirus virus in cells

Experiment description: Anti-reovirus-σ3 monoclonal antibody was used as the primary antibody, FITC-goat anti-mouse IgG antibody was used as the secondary antibody, DAPI was used for nuclear staining, and images were collected by Olympus FV1000 confocal microscope to observe the localization of reovirus in the cells. The result is shown in Figure 1. It can be seen from Figure 1 that the yellow-green fluorescence can be seen on the CIK cell membrane by laser confocal microscope observation. It shows that CIK cells are successfully loaded with reovirus.

2. Detection of reovirus loading into CIK cells by flow cytometry

CIK cells are a group of heterogeneous cell populations induced by PBMC in vitro with various cytokines and CD3 monoclonal antibodies, including CD8 ⁺ T, CD4 ⁺ T, NK and NKT cells, among which CD8 ⁺ T (CTL) and NKT Cells are considered to be primary effector cells. Block the cell sample with Fc blocking antibody anti-CD16/CD32 for 30 minutes, add flow cytometry antibody mixture or isotype control antibody (mouse anti-human APC-CD3, mouse anti-human PE-CD56/mouse anti-human PE-CD8, anti-reovirus -σ3 monoclonal antibody), incubated at 4°C for 30 minutes, washed the cells twice with PBS, added FITC-goat anti-mouse IgG antibody, and incubated at 4°C for 30 minutes. The percentage of cells bound to reovirus on the surface of CD8 ⁺ T cells and NKT cells was detected by flow cytometry. The result is shown in Figure 2. It can be seen from the figure that the persistence of reovirus can be detected on the surface of CD8 ⁺ T (CTL) cells and NKT cells. It shows that CIK cells are successfully loaded with reovirus.

3. Effects of loaded reovirus on CIK cells

1. Transmission electron microscope observation of ultrastructural changes of CIK cells loaded with reovirus virus

Ultrathin sections of 60-80nm were taken, double-stained with uranium and lead, dried overnight at room temperature, and then observed with FEI Tecnai G220TWIN electron microscope for ultrastructural changes and virus localization of CIK cells after virus loading. As shown in Figure 3. Under the electron microscope, a large number of virus particles were arranged in piles or clusters in the cytoplasm of tumor cells, and the cytoplasmic vacuoles increased. No virus particles were found in the cytoplasm of CIK cells loaded with reovirus. It showed that reovirus did not remain in CIK cells after being loaded into CIK cells, nor did it cause changes in the ultrastructure of CIK cells.

2. Flow cytometry to detect the changes of CIK cell subsets before and after loading reovirus

CIK cell samples before and after loading reovirus were blocked with Fc-blocking antibody anti-CD16/CD32 for 30 minutes, and then added flow cytometry antibody mixture or isotype control antibody (mouse anti-human APC-CD3, mouse anti-human PE-CD56/mouse anti- Human PE-CD8), combined at 4°C for 30 minutes, washed cells twice with FACS buffer, detected by Beckman FC500 flow cytometer, and analyzed the experimental results with Flowjo 10.0 software. As shown in Figure 4. The proportion of main effector cells in CIK cell subsets before and after reovirus loading was detected by flow cytometry, and no changes were found in the proportions of CD8 ⁺ T and CD3 ⁺ CD56 ⁺ NKT cell subsets after reovirus loading. It shows that the proportion of CIK cell subsets did not change after loading CIK cells with reovirus.

3. CCK-8 method to detect the survival rate of tumor cells and CIK cells after reovirus loading

Tumor cell lines (DLD-1, PC-3, NCI-H460) and CIK cells were mixed with different titers (0pfu/cell, 0.001pfu/cell, 0.01pfu/cell, 0.1pfu/cell, 1pfu/cell) Incubate with reovirus for 2 hours, wash cells with PBS for 3 times, remove unbound virus, add fresh cell culture medium to continue culturing for 24, 48 and 72 hours, collect cells at each time point, use CCK-8 kit to detect cell survival rate, and measure with microplate reader The absorbance value at 450nm was used to calculate the cell death rate according to the following formula.

Cell death rate=1-[(As-Ab]/(Ac-Ab)]×100%

Among them, As-the absorbance value of the experimental well, L/(g cm); Ac-the absorbance value of the control well (reovirus titer is 0 pfu/cell), L/(g cm); Ab-the blank well absorbance value, L /(g cm).

The results showed that the death rates of the three tested tumor cell strains all increased with the increase of the culture time after reovirus loading and the amount of loaded virus, while the cell death rate of CIK cells was lower than 5% within 72 hours of detection, suggesting that CIK cells No obvious cell death occurred within 72h after reovirus loading, the results are shown in Figure 5. It was shown that reovirus loading only caused the significant death of tumor cells, but did not affect the survival rate of CIK cells.

4. Performance of oncolytic virus loaded on immune effector cells

1. CCK-8 method was used to detect the oncolytic activity of reovirus delivered to tumor cells by CIK cells

CIK cells alone, CIK cells loaded with reovirus, and CIK cells loaded with UV-inactivated reovirus were used as effector cells respectively, and 8×10 ⁴ DLD-1 target cells were used according to the effector-target ratio of 10:1. Co-incubate for 2 hours under the condition of 7.5% human AB serum, wash away the CIK cells with serum-free medium, continue to culture the adherent tumor cells in the cell culture box for 24, 48 and 72 hours, and use CCK- 8 method to detect tumor cell death rate. The result is shown in Figure 6.

It can be seen from Figure 6 that at 72 hours, 13.53±12.02% and 18.63±8.06% of tumor cells died in the CIK cell alone group and the CIK cell loading UV inactivated reovirus group, respectively, while the CIK cell loading reovirus group had 77.20±0.98% % of tumor cells died. It shows that CIK cells loaded with reovirus can deliver reovirus to tumor cells in the presence of neutralizing antibodies.

2. Inhibitory effect of CIK cells loaded with reovirus on tumor growth in tumor-bearing nude mice

DLD-1 cells in the logarithmic growth phase were collected, the cell concentration was adjusted to 2×10 ⁷ /mL, 0.1 mL of cell suspension was subcutaneously injected into the left back skin of nude mice, and the treatment was started when the tumor volume reached 50 mm ³ . Tumor-bearing nude mice were randomly divided into three experimental groups, reovirus alone group (10 ⁸ PFU/mouse), CIK cell alone group (10 ⁷ cells/mouse), and reovirus-loaded CIK cell group (reovirus 10 ⁸ PFU +CIK10 ⁷ cells/mouse). Nude mice were intravenously injected with 100 μL of reovirus alone, CIK cells alone, and CIK cells loaded with reovirus, once a week for 3 consecutive weeks. The tumor size was measured with a caliper every other day, and the tumor volume was calculated according to length × width × height. The result is shown in Figure 7. The tumor volume was detected on the 15th day after treatment, and the tumor volume of the tumor-bearing nude mice in the CIK cell treatment group loaded with reovirus was significantly lower than that in the CIK cell alone group and the reovirus alone group. The CIK cell group loaded with reovirus treated tumor-bearing nude mice, delayed the growth of tumors, and prolonged the survival period of tumor-bearing mice. It shows that CIK cells loaded with reovirus have better anti-tumor activity in vivo.

The benefit of the present invention lies in that the oncolytic virus loaded with immune effector cells provided by the present invention has better targeting and anti-tumor properties. In the present invention, CIK cells are selected as immune effector cells, and reovirus type 3 Dearing strain is used as oncolytic virus. As oncolytic virus-loaded cells, CIK cells have the advantages of strong in vitro expansion ability, high safety, broad-spectrum tumor killing ability, and strong blood vessel and tissue penetration ability. Enhancing targeting by loading CIK cells with oncolytic virus. The CIK cells loaded with oncolytic virus did not change significantly in terms of cell survival rate, ultrastructure and cell subpopulation ratio, which ensured that the loading of oncolytic virus would not affect the performance of CIK cells themselves. After loading CIK cells, reovirus mainly remained on the cell surface and significantly enhanced the cytotoxicity of CIK cells. CIK cells could deliver reovirus to tumor cells in the presence of neutralizing antibodies in vitro. The oncolytic virus loaded on the obtained immune effector cells can inhibit tumor growth and promote targeted anti-tumor effects. The preparation method of the invention is simple and easy, and the operating conditions are mild and easy to control. The obtained oncolytic virus loaded by immune effector cells can be used in the preparation of targeted antitumor drugs.

Description of drawings

Figure 1 is a laser confocal microscope observation of CIK cells loaded with reovirus (400×);

Figure 2 is the binding diagram of reovirus on the surface of CD8 ⁺ T and NKT cells;

Figure 3 is a map of cell ultrastructural changes and virus localization after reovirus loading;

Figure 4 is a graph showing the changes in the ratio of CD8 ⁺ T and NKT cells in CIK cells before and after reovirus loading;

Figure 5 is a graph of the cell survival rate after loading tumor cell lines and CIK cells in reovirus;

Figure 6 is a graph of tumor cell death rate after CIK cells deliver reovirus to tumor cells in the presence of neutralizing antibodies;

Figure 7 is a tumor volume diagram of tumor-bearing nude mice on the 15th day after treatment;

The meanings of reference signs in the figure: Figure 1: A-cells loaded with reovirus, B-cells not loaded with reovirus; Figure 2: (1) NKT, (2) CTL, A-cells not loaded with reovirus, B-isotype control, C - cells loaded with reovirus; Figure 3 and Figure 5: (1) DLD-1, (2) PC-3, (3) NCI-H460, (4) CIK; Figure 4: 1 - before loading, 2 - after loading; Figure 6: 1-CIK cells alone group, 2-CIK cells loaded with UV inactivated reovirus group, 3-CIK cells loaded with reovirus group, **P<0.05, ****P<0.0001; Figure 7: 1-loaded with reovirus CIK cell group, 2-reovirus alone group, 3-CIK cell alone group.

detailed description

The present invention will be further introduced below in conjunction with specific embodiments.

Example 1

An oncolytic virus loaded on immune effector cells, the immune effector cells are CIK cells (cytokine-induced killer cells), and the oncolytic virus is reovirus type 3 Dearing strain (reovirus type 3 Dearing strain). The density of CIK cells loaded with reovirus was 1×10 ⁶ cells/mL.

Example 2

An oncolytic virus loaded on immune effector cells, the immune effector cells are CIK cells (cytokine-induced killer cells), and the oncolytic virus is reovirus type 3 Dearing strain (reovirus type 3 Dearing strain). The density of CIK cells loaded with reovirus was 2×10 ⁶ cells/mL.

Example 3

An oncolytic virus loaded on immune effector cells, the immune effector cells are CIK cells (cytokine-induced killer cells), and the oncolytic virus is reovirus type 3 Dearing strain (reovirus type 3 Dearing strain). The density of CIK cells loaded with reovirus was 1.5×10 ⁶ cells/mL.

Example 4

An oncolytic virus loaded on immune effector cells, the immune effector cells are CIK cells (cytokine-induced killer cells), and the oncolytic virus is reovirus type 3 Dearing strain (reovirus type 3 Dearing strain). The density of CIK cells loaded with reovirus was 1.2×10 ⁶ cells/mL.

Example 5

An oncolytic virus loaded on immune effector cells, the immune effector cells are CIK cells (cytokine-induced killer cells), and the oncolytic virus is reovirus type 3 Dearing strain (reovirus type 3 Dearing strain). The density of CIK cells loaded with reovirus was 1.8×10 ⁶ cells/mL.

The oncolytic virus loaded on immune effector cells in Examples 1-5 was prepared by the method in Examples 6-8.

Example 6

A method for preparing an oncolytic virus loaded with immune effector cells, comprising the following steps: collecting CIK cells activated, expanded and cultured in vitro to the 14th to 16th day, washing the cells twice with PBS, and adjusting the virus with GT-T551 serum-free medium The density of CIK cells was 1×10 ⁶ cells/mL, and then the reovirus was taken, and the titer of 1 pfu/cell was incubated with CIK cells at 4°C for 2 hours, unbound virus was washed away with PBS, and GT-T551 was serum-free After the medium is resuspended, it is ready.

Example 7

A method for preparing an oncolytic virus loaded with immune effector cells, comprising the following steps: collecting CIK cells activated and expanded in vitro until the 14th to 16th day, washing the cells with PBS for 4 times, and adjusting the virus with GT-T551 serum-free medium The density of CIK cells was 5×10 ⁶ cells/mL, then the reovirus was taken, and the titer of 10pfu/cell was incubated with CIK cells at 4°C for 4 hours, unbound virus was washed away with PBS, GT-T551 was serum-free After the medium is resuspended, it is ready.

Example 8

A method for preparing an oncolytic virus loaded with immune effector cells, comprising the following steps: collecting CIK cells activated and expanded in vitro until the 14th to 16th day, washing the cells with PBS for 3 times, and adjusting the virus with GT-T551 serum-free medium The density of CIK cells was 3×10 ⁶ cells/mL, then the reovirus was taken, and the titer of 5 pfu/cell was incubated with CIK cells at 4°C for 3 hours, unbound virus was washed away with PBS, GT-T551 was serum-free After the medium is resuspended, it is ready.

The oncolytic virus carried by immune effector cells in Examples 1-5 is used in the preparation of targeted antitumor drugs.

Claims (6)

1. the oncolytic virus that a kind of immune effector cell is loaded, it is characterised in that：The immune effector cell is CIK cell, institute Oncolytic virus is stated for Dearing plants of SV 59 virus.

2. the oncolytic virus that immune effector cell according to claim 1 is loaded, it is characterised in that：It is loaded with and exhales the lonely disease of intestines The CIK cell density of poison is 1 × 10⁶~2 × 10⁶Cell/ml density.

3. the preparation method for the oncolytic virus that immune effector cell as claimed in claim 1 or 2 is loaded, it is characterised in that：Bag Include following steps：The CIK cell of Activated in Vitro amplification cultivation is collected, cell is washed with PBS, passes through GT-T551 serum free mediums CIK cell density is adjusted, then takes reovirus to be incubated with CIK cell at 4 DEG C, PBS washes away uncombined virus, GT- After T551 serum free mediums are resuspended, produce.

4. the preparation method for the oncolytic virus that immune effector cell according to claim 3 is loaded, it is characterised in that：Including Following steps：The CIK cell of Activated in Vitro amplification cultivation to the 14th~16 day is collected, cell is washed with PBS 2~4 times, passes through GT- T551 serum free mediums adjustment CIK cell density is 1 × 10⁶~5 × 10⁶Cell/mL density, then takes reovirus, with 1~10pfu/cell titre is incubated 2~4h with CIK cell at 4 DEG C, and PBS washes away uncombined virus, and GT-T551 is without blood After clear culture medium is resuspended, produce.

5. the preparation method for the oncolytic virus that immune effector cell according to claim 4 is loaded, it is characterised in that：Including Following steps：The CIK cell of Activated in Vitro amplification cultivation to the 14th~16 day is collected, cell is washed with PBS 3 times, passes through GT-T551 Serum free medium adjustment CIK cell density is 3 × 10⁶Cell/mL density, then takes reovirus, with 5pfu/cell's Titre is incubated 3h with CIK cell at 4 DEG C, and PBS washes away uncombined virus, after GT-T551 serum free mediums are resuspended, i.e., .

6. the oncolytic virus that immune effector cell as claimed in claim 1 or 2 is loaded is in anti-tumor drugs targeting is prepared Using.