KR102195135B1 - A screening method of immunomodulators - Google Patents

Abstract

The present invention relates to a method for screening an immunomodulatory agent, wherein the screening method of the present invention can very effectively simulate the interaction between tumor cells and T cells in an in vitro environment, for example. Immunomodulators that can inhibit the action can be screened effectively.

Description

Screening method of immunomodulators {A screening method of immunomodulators}

The present invention relates to a method for screening an immunomodulatory agent.

The immune system works so that an organized response involving multiple cell types can be modulated, recognizing the foreign antigen and killing the foreign antigen, or pathogen or cell on which the foreign antigen is expressed. The immune system is very important not only for protection from invading microorganisms, including bacteria, viruses and parasites, but also for monitoring abnormal or mutated cells such as cancer cells and for the elimination of such cells.

On the other hand, T cell activation may be regulated by a balance between a positive signal and a negative signal provided by a co-stimulatory receptor, along with recognition of a specific antigen through a T cell receptor (TCR). In view of this, an immunomodulatory agent capable of blocking a functional antibody against a co-stimulatory activating molecule and a negative co-stimulatory molecule can enhance T cell stimulation, thereby promoting the lysis of cancer cells.

However, in the case of cancer patients, there is a disadvantage that the effect of anti-tumor immunotherapy is generally low or that the effect is not exhibited at all due to the regulation through the interaction between various signals. In particular, in the case of cancer cells, it is possible to self-modify the phenotype of the cell itself so that the expression of an inhibitory signal that can evade the immune system is up-regulated. For this reason, in the case of anti-tumor immunotherapy, there is a limitation in that it is difficult to lower the immune suppression in the region where cancer cells are present, and that it is difficult to restore a normal immune responsive network.

'T cell exhaustion', a major anti-tumor immune destruction mechanism, is a dysfunctional condition of T cells, which can be caused by prolonged exposure to antigens, and specifically binds to a modified phenotype of cancer cells. It can be caused by up-regulation of an inhibitory receptor that can be.

The pathway of PD-1 (Programmed cell death protein-1), which has been identified as a marker in T cells in T cell depletion state in chronic infection state, is an important immunosuppressive modulator of T cell depletion. Blocking of such a pathway can lead to activation, expansion, and enhanced functional functions of T cells. As such, the PD-1 allows the T cell response to be negatively regulated. When the interaction between PD-1 and Programmed cell death protein-Ligand 1 (PD-L1) or Programmed cell death protein-Ligand 2 (PD-L2) is blocked, the function of T cells can be partially restored.

Therefore, there is a need for an effective immunomodulatory agent that can overcome the limitations of immunotherapy due to T cell depletion, etc. However, research on a method for screening such an immunomodulatory agent is insufficient.

An object of the present invention is to provide a method for screening an immunomodulatory agent by simulating the interaction between tumor cells and T cells in vitro .

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned may be clearly understood by those of ordinary skill in the art from the following description.

The present inventors use a mixed lymphocyte reaction (MLR) that can induce an allogeneic or heterogeneous immune response to induce an immune response to peripheral blood mononuclear cells (PBMC), tumor cells and It was found that it can effectively simulate an in vitro environment such as interaction between T cells. In such an environment, immunomodulators are screened very effectively by treating an immunomodulatory agent that can inhibit the interaction between proteins expressed in various cells including T cells present in the peripheral blood mononuclear cells, and confirming the degree of division of T cells. The present invention has been completed by discovering that it can.

In the present invention, "immunomodulator" refers to a substance capable of controlling the differentiation of the first cell by controlling the interaction occurring in the first cell and the second cell or the third cell of the present invention, and the immunomodulatory agent May be at least one selected from the group consisting of antibodies, proteins, oligopeptides, organic molecules, polysaccharides, and polynucleotides.

In the present invention, the term "antibody" refers to a protein molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited, and as long as it has a polyclonal antibody, a monoclonal antibody, or an antigen-binding property, it may be included even if it is a part of the antibody, and all kinds of immunoglobulin antibodies may be included. In addition, special antibodies such as humanized antibodies may be included, and the antibody includes not only a complete form having two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules. The functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and may be Fab, F(ab'), F(ab') 2, Fv, etc., but is not limited thereto.

In the present invention, the term "organic molecule" is a compound having a carbon atom as a basic skeleton of a structure, and may be a synthesized material or extracted from a natural product.

In the present invention, "polynucleotide" refers to a polymer of nucleotides in which a nucleotide unit is formed in a long chain by covalent bonds, and refers to a DNA or RNA strand having a predetermined length or longer.

In the present invention, "oligopeptide" refers to a nucleic acid or nucleotide sequence including both RNA and DNA, and includes all of cDNA, genomic DNA, and synthetic DNA. In addition, the RNA includes all of mRNA, rRNA, and synthetic RNA. In addition, oligonucleotides, nucleic acids, and bases are used interchangeably in this specification.

In the present invention, "protein" refers to a polypeptide or protein in which amino acids are linked through an amino bond.

In the present invention, "polysaccharide" refers to a saccharide in which two or more monosaccharides form one molecule through a glycosidic bond, and its molecular weight varies from thousands to millions, and hypotonic polysaccharides such as starch or glycogen, and hypotonic polysaccharides such as cellulose or chitin. And structural polysaccharides.

In the present invention, the "major histocompatibility complex (MHC)" is a protein present on the surface of cells, and is composed of a major histocompatibility complex including amino acid sequences different from each other for each individual. Accordingly, when the main histocompatibility complex is the same in the immune system, it may be recognized as not an external antigen, and when the main histocompatibility complex is different, it may be recognized as an external antigen. The major histocompatibility complex is present in almost all cells present in an individual, type I, which presents proteins to cytotoxic T cells (CD8 ⁺ T cells), and type II, which is present only in immune cells such as macrophages or B cells. It can be classified as

In the present invention, the term "lymphocyte mixed culture" means that when mononuclear cells derived from a homogeneous individual are mixed at the same ratio, allogeneic immune rejection is induced, and the division of T cells contained in the mononuclear cells is increased. do. The allogeneic immune rejection reaction refers to a phenomenon of identifying and attacking when a foreign antigen derived from the same species enters an individual having an immune system.

In the present invention, the term "immune checkpoint" refers to an inhibitory pathway of the immune system, which is important for maintaining self-tolerance in order to minimize side tissue damage and regulating the duration and amplitude of the physiological immune response of peripheral tissues.

In the present invention, "PD-1 (Programmed cell death protein-1)" is a protein present on the surface of activated T cells, and is also referred to as CD279. Further, in the present invention, "PD-L1 (Programmed cell death protein-ligand 1)" is a protein on the surface of cancer cells or hematopoietic cells, and is also referred to as CD274. When the PD-1/PD-L1 of the present invention is bound, T cells cannot attack cancer cells by such binding.

One embodiment of the present invention provides a method for screening an immunomodulatory agent.

The screening method of the present invention comprises the steps of stimulating the first cells to express an immune checkpoint receptor in the first cells by mixing first cells and second cells having different major histocompatibility complexes; Mixing the stimulated first cells with third cells having different major tissue compatibility complexes with the first cells; Treating the mixed first and third cells with an immunomodulatory agent; And measuring the degree of division of the first cell.

Hereinafter, each step of the screening method will be described in detail.

Stimulating stage

The screening method of the present invention comprises the step of stimulating the first cell to express an immune checkpoint receptor in the first cell by mixing first and second cells having different major histocompatibility complexes. do.

The screening method of the present invention is based on a mixed lymphocyte culture reaction in which a first cell and a second cell or a third cell are mixed and cultured. When performing such a process, the homogeneous or heterogeneous immune response in the first cell is very effective. It can be irritating. The lymphocyte mixed culture reaction was developed to confirm in advance whether or not an allogeneic immune rejection reaction was performed in organ transplantation, but in the present invention, it is used to facilitate inducing T cell receptor (TCR) stimulation.

In the mixed lymphocyte culture of the present invention, when mononuclear cells derived from homogeneous individuals are mixed at the same ratio, allogeneic immune rejection is induced, and the division of T cells contained in the mononuclear cells is increased. The allogeneic immune rejection reaction refers to a phenomenon of identifying and attacking when a foreign antigen derived from the same species enters an individual having an immune system.

The first to third cells of the present invention are derived from the bone marrow or blood of another individual of the same kind, and may be included without limitation as long as they include lymphocytes, lymphocyte progenitor cells, reactive lymphocytes, antigen presenting cells, and the like. Preferably, the first to third cells may be peripheral blood mononuclear cells or bone marrow mononuclear cells (BMMC), more preferably peripheral blood mononuclear cells. However, it is not limited thereto.

The first cell of the present invention may be a T cell, and the second or third cell may be an antigen presenting cell, but is not limited thereto.

The T cells of the present invention are lymphocytes responsible for antigen-specific adaptive immunity, and are composed of CD8+ T cells, CD4+ T cells, γδ T cells, MAIT (Mucosal associated invariant T) cells, and natural killer T (Natural killer T) cells. It may be at least one selected from the group, but is not limited thereto.

The antigen-presenting cell (APC) of the present invention is a generic term for a cell capable of presenting an antigen after transfecting a short protein fragment into a cell, pulverizing it for a shorter time in the cell, and B cells, It may be at least one selected from the group consisting of macrophages and dendritic cells, but is not limited thereto.

The first cell of the present invention is a cell stimulated by a cell different from the first cell, and may be a cell having a major histocompatibility complex different from the second cell or the third cell.

The second cell or the third cell of the present invention is a cell capable of presenting a foreign antigen to the first cell, and has a major histocompatibility complex different from the first cell, and the second cell or the third cell It may have the same or different major histocompatibility complexes.

The second or third cells of the present invention may have inhibited cell division. As for the method of inhibiting cell division, any physical or chemical method may be used as long as the cells are inhibited from further cell division, and preferably, radiation is applied to the second or third cells to inhibit cell division. It may be, but is not limited thereto.

The major histocompatibility complex of the present invention is a protein present on the surface of a cell, and is composed of a major histocompatibility complex comprising amino acid sequences different from each other for each individual. Accordingly, when the main histocompatibility complex is the same in the immune system, it may be recognized as not an external antigen, and when the main histocompatibility complex is different, it may be recognized as an external antigen. The major histocompatibility complex is present in almost all cells present in an individual, type I, which presents proteins to cytotoxic T cells (CD8 ⁺ T cells), and type II, which is present only in immune cells such as macrophages or B cells. It can be classified as

The stimulated first cell of the present invention may be stimulated through a process of mixing with the second cell, thereby expressing the immune checkpoint receptor in the cell.

The immune checkpoint of the present invention is an inhibitory pathway of the immune system that is important in maintaining self-tolerance to minimize side tissue damage and regulating the duration and amplitude of the physiological immune response of peripheral tissues. These immune checkpoints are a major mechanism that causes immune resistance by tumor cells, etc., through the process of binding the immune checkpoint receptor expressed on the surface of T cells stimulated by antigen-presenting cells and ligands present in the antigen-presenting cells. Can be induced. For the purposes of the present invention, the immune checkpoint receptor may be any receptor involved in the immune checkpoint pathway for T cells specific for a tumor antigen, for example, Programmed cell death protein-1 (PD-1), Tim-3 (T cell immunoglobulin and mucin domain 3), Lag-3 (Lymphocyte-activation gene 3), VISTA (V-domain Immunoglobulin Suppressor of T cell Activation), TIGIT (T cell immunoreceptor with Ig and ITIM domains), CTLA -4 (cytotoxic T lymphocyte antigen 4), BTLA (B and T lymphocyte attenuator), B7-H3, B7-H4, KIR (Killer Cell Immunoglobulin-Like Receptor), IDO (Indoleamine-2,3-dioxygenase), A2AR ( adenosine A2a receptor), GITR, ICOS (Inducible T-cell COStimulator), CD200R, CD112R, CD96, CD137, 2B4, OX40, CD27, CD40L, CD226, CD122, CD28, but may be at least one selected from the group consisting of, It is not limited thereto.

The second cell or the third cell of the present invention may be one in which a ligand capable of specifically binding to the immune checkpoint receptor expressed in the first cell is expressed, preferably PD-L1 (Programmed cell death protein-ligand 1) or PD-L2 (Programmed cell death protein-ligand 2), and more preferably PD-L1, but is not limited thereto.

PD-1 expressed in the stimulated first cells of the present invention can be expressed in T cells exposed to foreign antigens, and interacts with PD-L1 or PD-L2 mainly expressed in inflammatory or tumor cells. I can. Such interactions can occur in the effector stage of the T cell response in peripheral tissues, and by this phenomenon, the process of recognizing abnormal cells and dissolving them is inhibited, thereby increasing the effectiveness of cancer treatment through immunomodulators. It can be hindered.

Mixing;

The screening method of the present invention may perform the step of mixing the first cells stimulated in the stimulating step with third cells having different major histocompatibility complexes.

The immunomodulator can be effectively screened in a state in which both the stimulated first cell, the second cell, or the third cell of the present invention expresses both the protein that inhibits the binding of the immunomodulator.

Referring to FIG. 1 of the present invention, an immunomodulator should be treated in a state in which both PD- expressed in the first cell and PD-L1 or PD-L2 expressed in the second or third cell of the present invention are present. However, even if the immunomodulator is treated at the time when TCR (T cell receptor) signal is increased (refer to section 1 in Fig. 1(A)), the first cell (T cell) is immune due to low PD-1 expression. Even if there is no target to which the modulator can be bound, and even if the immunomodulator is treated at the time when PD-1 is expressed at its maximum (see section 2 in Fig. 1(A)), the second cell is killed and PD- It is useless because neither L1 nor PD-L2 exists.

The step of mixing the third cells of the present invention is to solve the problem caused by the difference in the parallax in which PD-1 and PD-L1 or PD-L2 can be expressed maximally, as described above, On the sixth day from the step , the third cells capable of stimulating the first cells may be mixed again in the same manner as in the'stimulating step ' to apply additional stimulation to the first cells. Under these conditions, the first cells with increased expression of PD-1 and the newly administered third cells have increased expression of PD-L1 or PD-L2 due to allogeneic immune rejection (Fig. 1 See (B) of), it is possible to create an optimal environment in which the first cell and the third cell can interact.

Treating an immunomodulatory agent;

The screening method of the present invention may perform the step of treating the mixed first and third cells with an immunomodulatory agent after the mixing step.

Since the step of treating the immunomodulatory agent of the present invention is performed in an optimal environment in which the first cell and the third cell can interact, whether or not such an interaction can be effectively suppressed by the treated immunomodulatory agent. You can check about.

The immunomodulatory agent of the present invention may include any substance capable of controlling the differentiation of the first cell by controlling the interaction between the first cell and the second cell or the third cell of the present invention. For the purpose of the present invention, it may be a substance that regulates the differentiation of T cells by controlling the interaction of the immune checkpoint receptor and the ligand, and preferably, a group consisting of antibodies, proteins, oligopeptides, organic molecules, polysaccharides, and polynucleotides. It may be at least one selected from, more preferably an antibody, an organic molecule, or a combination thereof (antibody and an organic molecule), but is not limited thereto.

The antibody of the present invention may include any antibody capable of specifically binding to an immune checkpoint receptor, and preferably may be an antibody specific for PD-1, but is not limited thereto.

The organic molecule of the present invention may be included as long as it is a substance capable of modulating the interaction of an immune checkpoint receptor and a ligand, and may preferably be a compound represented by the following Formulas 1 to 3, but is not limited thereto.

[Formula 1]

[Formula 2]

[Formula 3]

Formula 1 is 4-(5,6-dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo(1,2-b)pyrazol-3-yl)-6-quinoli Necarboxamide (4-(5,6-Dihydro-2-(6-methyl-2-pyridinyl)-4H-pyrrolo(1,2-b)pyrazol-3-yl)-6-quinolinecarboxamide) and , Formula 2 is ( R )-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl) piperidine-2,6-dione (( R )- 3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione), wherein Chemical Formula 3 is an isomer of Chemical Formula 2 ( S ) -3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl) piperidine-2,6-dione (( S )-3-(4-Amino-1 -oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione), but is not limited thereto.

Measuring the degree of division of the first cell;

In the screening method of the present invention, after the step of treating the immunomodulatory agent, the step of measuring the degree of division of the first cell may be performed.

The step of measuring the degree of division of the first cell of the present invention is to determine the degree of division of the first cell, that is, the T cell, which is caused by suppressing the interaction between the first cell and the third cell by an immunomodulatory agent. , Preferably, a flow cytometer may be used, but is not limited thereto.

The screening method of the present invention is a fluorescent reagent such as CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester) before or after the stimulating step and the mixing step so that measurement can be facilitated using a flow cytometer. The step of staining the first to third cells through may be further included, but is not limited thereto.

The screening method of the present invention can very effectively simulate the interaction between tumor cells and T cells in an in vitro environment, for example, and effectively screen immunomodulators capable of inhibiting the interaction. I can.

Figure 1 (A) and (B) shows a schematic diagram of a case where the cells according to an embodiment of the present invention are treated with an antibody specific for PD-1.
2 is a schematic diagram of a method for screening an immunomodulatory agent according to an embodiment of the present invention.
3 shows the result of confirming the fraction of divided T cells according to the treatment of the antibody and/or Galunisertib using the method according to an embodiment of the present invention.
FIG. 4 shows the results of confirming the fraction of divided T cells according to treatment with an antibody and/or lenalidomide using a method according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

[Example 1] Induction phase

A first PBMC was prepared by separating peripheral blood mononuclear cells (PBMCs) of a healthy first subject group using a Ficoll concentration gradient separation method. In addition, using the same method, a second PBMC was prepared by separating PBMC from the peripheral blood of a second subject group different from the first subject group. The separated PBMC was put in a freezing medium and stored in liquid nitrogen until used in the following examples. In order to reduce deviations that may occur due to differences in individuals, PBMCs separated from at least 5 or more individuals included in the first or second subject group are mixed in the same ratio in each of the first and second PBMCs. Made it possible. Here, the first PBMC was set as a cell in which an immune rejection reaction occurred due to stimulation in the induction of the immune rejection reaction, and the second PBMC was set as a cell that applied stimulation to the reaction cell.

In the case of the stimulating cells, since cell division did not occur and only stimulation was applied to the reaction cells, gamma rays were irradiated to the second PBMC at an intensity of 3000 rad. Then, the 1 to 2 X 10 ⁶ cells/mL of the first and second PBMCs were suspended in CFSE (5(6)-Carboxyfluorescein diacetate N-succinimidyl ester) buffer (PBS containing 5% FBS) and , 1 μM of CellTrace red dye was added to the suspended first PBMC, and 5 μM of CFSE dye was added to the suspended second PBMC, followed by incubation at 37° C. for 20 minutes. Into each of the first and second PBMCs after the cultivation was completed, 5 times the amount of the initially added CFSE buffer was added, and incubated at room temperature for 5 minutes. Thereafter, pellets of each of the first and second PBMCs were obtained through centrifugation, and the pellet was resuspended in RPMI1640 medium containing 10% fetal calf serum and penicillin/streptomycin (Penicillin/streptomycin). The resuspended 1st PBMC and 2nd PBMC for each well of a 24-well plate were dispensed by the number of 3 x 10 ⁶ cells, respectively, and mixed so as to mix well with each other. Then, it was incubated for 3 to 4 days at 37°C in a CO ₂ incubator. Thereafter, half of the medium present in the well is removed, the other half is replaced with a new medium, and further cultured for 2 days to be included in the first PBMC in which a protein such as PD-1 is expressed maximally by a heterologous immune response. T cells (hereinafter referred to as'reaction cells') were obtained.

[Example 2] Restoration phase

It was confirmed whether antibodies or immunomodulators could be screened by confirming the process of restoring cells in which immune rejection was induced in Example 1 above.

The reaction cells obtained in Example 1 and the second PBMC irradiated with gamma rays were dispensed into a 24-well plate, and stained with 5 μM of CellTrace red dye and 5 μM of CFSE dye in the same manner as in Example 1. Then, in the well, with or without an antibody specific for PD-1, 0, 0.01, 0.1 or 1 μM of Galunisertib or 0, 0.1, 1, 5 and 10 μM of Lenalidomide was treated. Then, the cells were cultured for 5 days in a CO ₂ incubator at 37° C., and the fraction of the divided CD8 ⁺ T cells was confirmed using a flow cytometer, and the results are shown in FIGS. 3 and 4.

As shown in FIGS. 3 and 4, it was confirmed that the fraction of CD8 ⁺ T cells was increased in the case where the antibody specific for PD-1 was treated compared to the case where the antibody specific for PD-1 was not treated. In addition, the fraction of CD8 ⁺ T cells was increased depending on the concentration when galunictive or lenalidomide was treated compared to the control group. In addition, the fraction of CD8 ⁺ T cells up to 20% or 40% or more when the antibody and immunomodulator were treated together compared to the case where the antibody or immunomodulator (galunic acid or lenalidomide) was treated, respectively. It was confirmed that it was increased.

As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (11)

Stimulating the first cells to express an immune checkpoint receptor in the first cells by mixing first cells and second cells having different major histocompatibility complexes (MHC);
Mixing the stimulated first cells with third cells having different major tissue compatibility complexes with the first cells;
Treating the mixed first and third cells with an immunomodulatory agent; And
Including the step of measuring the degree of division of the first cell,
The ligand capable of specifically binding to the immune checkpoint receptor expressed on the surface of the first cell is PD-L1 (Programmed cell death protein-ligand 1) or PD-L2 (Programmed cell death protein-ligand 2). Phosphorus, a method for screening immunomodulators. The method of claim 1,
The immune checkpoint receptor is PD-1 (Programmed cell death protein-1), the method for screening an immunomodulatory agent. The method of claim 1,
The second cell or the third cell is a method for screening an immunomodulatory agent that expresses a ligand capable of specifically binding to the immune checkpoint receptor expressed in the first cell. delete The method of claim 1,
The first to third cells are those derived from bone marrow or blood, a method for screening an immunomodulatory agent. The method of claim 5,
Wherein the first to third cells are peripheral blood mononuclear cells (Peripheral blood mononuclear cells; PBMC) or bone marrow-derived mononuclear cells (Bone marrow mononuclear cells; BMMC), the screening method of an immunomodulatory agent. The method of claim 5,
The first cell is a T cell,
The second or third cell is an antigen-presenting cell (APC), the method for screening an immunomodulatory agent. The method of claim 7,
The T cells are at least one selected from the group consisting of CD8+ T cells, CD4+ T cells, γδ T cells, MAIT (Mucosal associated invariant T) cells, and natural killer T (Natural killer T) cells. Screening for immunomodulatory agents Way. The method of claim 1,
The second cell or the third cell is that cell division is suppressed, the method for screening an immunomodulatory agent. The method of claim 1,
The immunomodulatory agent is at least one selected from the group consisting of antibodies, proteins, oligopeptides, organic molecules, polysaccharides, and polynucleotides. The method of claim 1,
The step of measuring the degree of division of the first cell is to be measured through a flow cytometer, an immunomodulatory screening method.

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