KR20230018665A - Immunocyte-tumor cell co-culture and method for screening antitumor and immune-modulating compounds - Google Patents

Abstract

The present invention relates to an immunocyte-tumor cell co-culture and a method for screening anticancer and immunomodulatory substances using the same. The immunocyte-tumor cell co-culture, which is an aspect of the present invention, is excellent in sensitivity to anticancer and immunomodulatory substances and has little variation in measurement results, and thus useful for discovery and development of effective substances.

Description

Immune cell-tumor cell co-culture and method for screening anti-cancer and immunomodulatory substances using the same

The present invention relates to an immune cell-tumor cell co-culture and a method for screening anti-cancer and immunomodulatory substances using the same.

Currently, most of the in vitro tests performed in the development of anticancer substances are performed using systems using cells cultured in a two-dimensional (2D) environment. However, the system utilizing cells cultured in 2D is different from the 3D bioenvironment, and due to problems such as lack of cell-cell and cell-substrate interactions formed in multiple layers on a solid surface, the drug effect can be accurately measured. There was a problem with unpredictability. Accordingly, in order to solve the problems of the 2D culture system, a 3D culture system has recently been attracting attention. The 3D culture system means culturing various cells in 3D in a 3D space composed of an extracellular matrix (ECM). The 3D culture system provides an environment similar to a living body in which nutrients, oxygen, drugs, etc. are supplied to cells through a diffusion gradient and permeation. In particular, interactions due to 3D cell-to-cell contact, interactions due to 3D cell-extracellular matrix contact, and paracrine signaling due to diffusion of cell secretions are different from 2D cell culture systems. It is a distinctive feature. However, the 3D culture system has various variables such as the type and shape of the extracellular matrix for mimicking the living environment, cell culture factors, and the shape of the cell culture container, so there are limitations in that the system is unstable and there is a large variation among individual spheroids. do.

KR 10-2021-0080228 A

In one aspect, an object of the present invention is to effectively screen anti-cancer substances and immunomodulatory substances.

In one aspect, an object of the present invention is to improve the accuracy and reliability of anti-cancer and immunomodulatory substance screening.

In one aspect, an object of the present invention is to provide a co-culture system that is easy to measure and analyze when evaluating the effectiveness of anti-cancer and immunomodulatory substances.

In order to achieve the above object, in one aspect, the present invention provides an immune cell-tumor cell co-culture, wherein the co-culture includes: immune cells; tumor cells; and an immune cell activator, wherein the immune cell activator includes a CD3 antibody and interleukin.

An immune cell-tumor cell co-culture, which is an aspect of the present invention, has excellent sensitivity to anticancer and immunomodulatory substances and has little variation in measurement results, and is therefore useful for discovering and developing effective substances.

1 is a photograph showing the immune cell-tumor cell co-culture of the present invention.
Figure 2 is the result of confirming the effect of the co-culture of the present invention.
Figure 3a shows the presence and composition of immune cell activators (CD3 mAb treatment concentration is 100 ng/ml, IL-2 treatment concentration is 10 ng/ml), and the ratio of immune cells and tumor cells constituting the co-culture. It is a diagram showing the change in Lloyd volume.
Figure 3b shows the change in spheroid volume according to the ratio of immune cells and tumor cells constituting the co-culture when treated with an immune cell activator containing 2.5ng/ml CD3 mAb and 0.25ng/ml IL-2. It is also
In the figure, CD3 represents CD3 mAb and IL2 represents IL-2.

Hereinafter, each configuration will be described in more detail, but this is only one example, and the scope of the present invention is not limited by the following content.

Terms or words used in the specification and claims of the present invention are not limited to the usual or dictionary meaning, and the inventor can properly define the concept of the term in order to explain his/her invention in the best way. Based on the principle, it should be interpreted as a meaning and concept consistent with the technical idea of the present invention.

In one aspect, the present invention is an immune cell-tumor cell co-culture, wherein the co-culture includes: immune cells; tumor cells; and an immune cell activator, wherein the immune cell activator is an immune cell-tumor cell co-culture containing a CD3 antibody and interleukin.

In the present specification, "co-culture" refers to the result of co-cultivation of homogeneous or heterogeneous cells, and collectively includes the co-cultured cells, extracellular materials used in co-culture, and materials produced during co-culture.

The tumor cells are not limited, for example, breast cancer cells, lung cancer cells, fibrosarcoma cells, gastric cancer cells, oral cancer cells, prostate cancer cells, liver cancer cells, ovarian cancer cells, thyroid cancer cells, cervical cancer cells, glioblastoma cells, melanoma cells , pancreatic cancer cells, renal cancer cells, bone cancer cells, lymphoma cells, brain tumor cells, etc., but are not limited thereto.

In one aspect, the immune cell-tumor cell co-culture is obtained by seeding a mixture of immune cells and an immune cell activator on a plate in which tumor cells are formed in a single spheroid state, followed by centrifugation to separate tumor cell single spheroids. It may include those in which the immune cells and immune cell activators are distributed around.

In one aspect, the centrifugation may include performing under RT conditions for about 1 to 3 minutes under a centrifugal force of about 150 to 350 xg.

In one aspect, the antibody may include a monoclonal antibody.

The CD3 antibody may react with the epsilon-subunit of the CD3 complex, but is not limited thereto.

Also, in one aspect, the interleukin is not limited, and interleukin 1, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, interleukin 12, and interleukin 13, interleukin 15, interleukin 17, etc., preferably interleukin 2.

Also, in one aspect, the immune cells and tumor cells may be included in a ratio of 3 to 10:1. Specifically, the immune cells and tumor cells may be included in a ratio of 3 to 7:1 or 4 to 6:1. When the immune cells and tumor cells are included in the above ratio, a co-culture with maximized sensitivity to anti-cancer substances can be obtained. For example, when the immune cells and tumor cells are included in the same ratio as above, a higher cancer cell growth inhibition rate may be exhibited when the same amount or a small amount of the anti-cancer substance is treated compared to the control group.

Also, in one aspect, the CD3 antibody and interleukin may be included in a concentration ratio of 5 to 20:1. Specifically, the CD3 antibody and interleukin have a concentration ratio of 5 to 15: 1, or a concentration ratio of 7 to 15: 1, or a concentration ratio of 7 to 13: 1, or a concentration ratio of 8 to 12: 1, or 9 It can be included in a concentration ratio of ~11:1.

In one aspect, the CD3 antibody may be included at a concentration of 1 to 5 ng/ml, or may be included at a concentration of 1.5 to 5 ng/ml, or may be included at a concentration of 1.5 to 4.5 ng/ml, or may be included at a concentration of 1.5 to 5 ng/ml. It may be included at a concentration of 4 ng/ml, or may be included at a concentration of 2 to 3 ng/ml.

Further, in one aspect, the interleukin may be included at a concentration of 0.1 to 0.5 ng/ml, or may be included at a concentration of 0.1 to 0.4 ng/ml, or may be included at a concentration of 0.15 to 0.4 ng/ml, Or it may be included in a concentration of 0.2 ~ 0.4ng / ml, or may be included in a concentration of 0.2 ~ 0.3ng / ml.

In one aspect, the immune cells may include peripheral blood mononuclear cells (PBMC).

Also, in one aspect, the tumor cells may be in the form of a single spheroid.

Specifically, in the immune cell-tumor cell co-culture, the tumor cells are located in the center of the co-culture, and the immune cells may be in a three-dimensional form surrounding the tumor cells.

In one aspect, the present invention is a method for screening anticancer substances or immunomodulatory substances comprising the step of administering a candidate substance to the immune cell-tumor cell co-culture.

In the present specification, "anti-cancer substance" is a generic term for substances capable of inhibiting and killing cancer cells.

In the treatment of the above drugs, the method performed to evaluate the efficacy of anticancer substances and / or immunomodulatory substances in vitro may be by a conventional method in the art, for example, a culture seeded with a co-culture It may be a type of culturing by adding an anticancer substance and/or an immunomodulatory substance to a medium for a certain period of time, but is not necessarily limited thereto.

In one aspect, the anti-cancer substance and/or immunomodulatory substance screening method may further include incubating the co-culture and the candidate substance.

The candidate substance may refer to a substance expected to prevent or treat tumor or cancer and/or a substance expected to specifically or non-specifically enhance or suppress an immune response. The form of the candidate material is not limited, and may be, for example, a single compound or a complex compound, and all materials existing in nature may be a target.

In addition, the anticancer substance screening method may further include measuring the expression level of a tumor marker in the cultured co-culture or determining whether or not the tumor cells are viable.

In addition, the immunomodulatory substance screening method may further include measuring the expression level of immune-related markers in the cultured co-culture.

In addition, in one aspect, the present invention is an immune cell-tumor cell co-culture composition. The immune cell-tumor cell co-culture composition may include immune cells and an immune cell activator. The immune cell activator may include a CD3 monoclonal antibody and IL-2. The CD3 monoclonal antibody and IL-2 may be included in a concentration ratio of about 5 to 20:1. In the composition for co-culture of immune cells-tumor cells, when the composition is administered to tumor cells, the tumor cells and immune cells are co-cultured at a ratio of 1:3 to 10, and the CD3 monoclonal antibody is about 1 to 5 ng/ml of concentration, IL-2 may be administered at a concentration of about 0.1-0.5 ng/ml.

Hereinafter, the present invention will be described in more detail with reference to the following preparation examples, examples and experimental examples. However, these preparation examples, examples, and experimental examples are provided only for illustrative purposes to aid understanding of the present invention, and the scope and scope of the present invention are not limited by the following examples.

[Preparation Example] Preparation of immune cell-tumor cell co-culture

Tumor cells in a single spheroid state were prepared by culturing for 1 day in a 37°C, CO ₂ incubator and seeded in a 384-well plate. In addition, the medium (MS-9384UZ, PrimeSurface ^® 3D culture) was prepared by pre-warming. Pre-warmed medium and immune cells (PBMC) were transferred to a clean bench and prepared. After that, the immune cells were placed in the medium so that the ratio of tumor cells and immune cells was 1:5. Then, CD3 monoclonal antibody (CD3 mAb, Invitrogen, OKT3 (16-0037-81)) and IL-2 (Peprotech, 200-02-50UG), which are immune cell activators, were added at a final concentration of 2.5 ng/ ml and 0.25 ng/ml were seeded in the medium. Thereafter, centrifugation was performed at 250xg, 2 minutes, RT, so that the dispensed immune cells were gathered toward a single spheroid formed on the U-shaped bottom to prepare a co-culture.

[Experimental example]

[Experimental Example 1] Confirmation of therapeutic substance effect using co-culture

Patient-derived brain tumor cells ((PDXC0008T, PDXC0042T1, PDXC0046T, PDXC0050T, PDXC0017T, PDXC0093T, AMB-BT-0093T, AMB-BT-0022T), each 375 cells/30μl/well) and peripheral blood mononuclear cells collected from healthy adults A co-culture prepared in the same manner as in Preparation Example was prepared using. Each of the prepared co-cultures was administered with immunomodulatory drugs (Atezolizumab, At406, Birinapant, Enbrel) at 200nM, 10μM, 10μM, and 200nM, respectively, and the size change of the spheroids was observed after 7 days. As a result, when using the co-culture of the present invention, it was confirmed that the sensitivity to the therapeutic drug was greatly improved.

[Experimental Example 2] Confirmation of the effect of the ratio of tumor cells and immune cells constituting the co-culture, and the composition of the immune cell activator

The ratio of tumor cells and immune cells constituting the co-culture was adjusted (1:0, 1:2.5, 1:5, 1:10), and the composition of the immune cell activator (CD3 mAb and IL-2 were not included). Sensitivity and deviation to the treatment drug were measured according to the case (NT), the case where only one of the two was included (CD3 mAb, IL-2), and the case where both were included (CD3 mAb+IL-2)). At this time, the concentrations of CD3 mAb and IL-2 were treated at 100 ng/ml and 10 ng/ml, respectively. Except for the above conditions, the experiment was performed in the same manner as in Experimental Example 1.

As a result, it was confirmed that treatment with CD3 mAb and IL-2 was insignificant, whereas treatment with IL-2 at the same time showed the effect. too high (Fig. 3a).

Therefore, in order to lower the inhibition level appropriate for the assay, the CD3 mAb and IL-2 concentrations were treated at 2.5 ng/ml and 0.25 ng/ml, respectively.

As a result, when immune cells and tumor cells are included at a ratio of 1:5, and the immune cell activator contains both CD3 mAb and IL-2 (2.5ng/ml, IL-2 0.25ng/ml), it is different from other cases. By comparison, it was possible to confirm a significant spheroid volume change with little deviation and significant drug effect verification (Fig. 3b).

Claims (13)

As an immune cell-tumor cell co-culture,
The co-culture,
immune cells;
tumor cells; and
Contains an immune cell activator;
The immune cell-tumor cell co-culture, wherein the immune cell activator comprises a CD3 antibody and interleukin. According to claim 1,
The CD3 antibody is an immune cell-tumor cell co-culture comprising a CD3 monoclonal antibody. According to claim 1,
The CD3 antibody, comprising reacting with the epsilon-subunit of the CD3 complex (CD3 complex), immune cell-tumor cell co-culture. According to claim 1,
The interleukin is an immune cell-tumor cell co-culture comprising interleukin-2. According to claim 1,
The immune cells and tumor cells are included in a ratio of 3 to 10: 1, immune cell-tumor cell co-culture. According to claim 1,
The CD3 antibody and interleukin are contained in a concentration ratio of 5 to 20: 1, immune cell-tumor cell co-culture. According to claim 6,
The CD3 antibody is included at a concentration of 1 to 5 ng/ml,
The interleukin is contained in a concentration of 0.1 ~ 0.5ng / ml, immune cell-tumor cell co-culture. According to claim 1,
The immune cells, including peripheral blood mononuclear cells (PBMC), immune cell-tumor cell co-culture. According to claim 1,
The tumor cells are in the form of single spheroids, immune cell-tumor cell co-culture. According to claim 1,
The immune cell-tumor cell co-culture,
An immune cell-tumor cell co-culture, wherein the tumor cells are located at the center of the co-culture, and the immune cells are in a three-dimensional form surrounding the tumor cells. A method for screening anti-cancer substances comprising the step of administering a candidate substance to the immune cell-tumor cell co-culture of any one of claims 1 to 10. According to claim 11,
The method is for the screening of immune anti-cancer substances, anti-cancer substance screening method. A method for screening immunomodulatory substances comprising the step of administering a candidate substance to the immune cell-tumor cell co-culture of any one of claims 1 to 10.

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