Application of MEK1 / 2 inhibitor U0126 in treating CRS caused by CAR T cells

Abstract

The present invention relates to the field of biomedicine, and particularly to the application of the MEK1 / 2 inhibitor U0126 in the treatment of cytokine release syndrome (CRS) caused by chimeric antigen receptor T (CAR-T) cells. The technical solutions provided by the present invention include: the effects of U0126 at different concentration gradients on mTHP1, Raji, Nalm6, and CAR-T cells; the effect of U0126 on the CRS reaction caused by CAR-T; and the effect of U0126 on the anti-tumor activity of CAR-T cells targeting CD19. U0126 can significantly reduce the release of a large amount of inflammatory factors by macrophages mTHP1 stimulated by the co-culture supernatant of CAR-T cells and lymphoma cells such as Raji / Nalm6, can significantly inhibit CRS, and is expected to become a new target for the treatment of CRS. The new application of U0126 in CRS can expand its research in the field of medical technology and provide a new theoretical direction for clinical application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of the MEK1 / 2 inhibitor U0126 in the treatment of CRS caused by CAR T cells. Background Art

[0002] Chimeric Antigen Receptor T-Cell Immunotherapy (CAR T) is to edit T cells using genetic engineering technology for specific modification, so that they are loaded with receptors and co-stimulatory molecules that can recognize tumor antigens, and are amplified in large numbers in vitro and then infused back into the patient's body to recognize and kill tumor cells. At present, CAR T cell therapy has achieved good curative effects in the clinical treatment of hematological tumors and is a promising and highly efficient immunotherapy. Undoubtedly, CAR T therapy brings new hope for cancer treatment. However, this therapy also has serious adverse reactions, mainly cytokine release syndrome (also called cytokine release storm, CRS) and neurotoxicity. If not treated in time, it may endanger life. Therefore, it is necessary to closely monitor after infusion of CAR-T cells.

[0003] Cytokine release syndrome (CRS) is a severe systemic inflammatory response syndrome caused by the activation of immune cells and the release of a large amount of cytokines, which is characterized by fever, hypotension, and multiple organ dysfunction, etc. In severe cases, there will be dyspnea, renal failure, coagulation dysfunction, and even endanger life. CRS is mainly caused by the recognition of antigen-positive target cells (tumor cells or normal cells) after CART is infused into the body, resulting in a large number of activations and proliferations of T cells, and a large number of inflammatory factors are produced by immune cells such as monocytes / macrophages, thus presenting a series of clinical symptoms. As the main means currently used to control severe CRS, interleukin-6 receptor antagonists and hormones have greatly reduced the risk of CAR T cell therapy failure caused by severe CRS. However, there are still some severe CRS that are ineffective against this treatment. Even with the use of high-dose hormones, the clinical symptom progression of severe CRS cannot be controlled. The use of high-dose steroid hormones can rapidly reverse the clinical symptoms of CRS. However, steroid drugs can also inhibit the in vivo proliferation of CAR T, making patients treated with steroid drugs for CRS have a relatively high recurrence rate, affecting the curative effect of CAR T treatment. At present, although the IL-6R blocker tocilzumab, the IL-1 blocker Anakinra, and glucocorticoids have been clinically used to treat CRS, there are still many problems, such as poor curative effects in some patients and the influence of glucocorticoids on the amplification and antitumor activity of CAR-T cells. Therefore, it is crucial to find a new treatment method.

[0004] Currently, it is believed that CRS is an effect produced by the cooperation and amplification of multiple cells to cause inflammation. In particular, macrophages and the IL-6 and IL-1 released by them play a key role in this process. Some studies have shown that the occurrence of inflammation is closely related to the MEK pathway, and MEK and ERK inhibitors can significantly reduce the production of inflammatory factors such as IL-1β, TNF-α, and IL-6. The role of MEK inhibitors in CRS is not yet clear. Summary of the Invention

[0005] In view of this, the present invention provides the use of the MEK1 / 2 inhibitor U0126 in the treatment of CRS caused by CAR T cells.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0007] The present invention provides the use of the MEK-ERK pathway as a target in the preparation of a medicament for treating cytokine release syndrome.

[0008] The present invention also provides the use of MEK / ERK inhibitors in the preparation of a medicament for treating cytokine release syndrome.

[0009] In some specific embodiments of the present invention, the MEK / ERK inhibitor includes U0126.

[0010] In some specific embodiments of the present invention, the MEK / ERK inhibitor reduces the release of inflammatory factors by macrophages stimulated by the co-culture supernatant of CAR T cells and lymphoma cells. Among them,

[0011] the lymphoma cells include Raji cells and / or Nalm6 cells;

[0012] the macrophages include mTHP1 cells;

[0013] the inflammatory factors include IL-6, IL-1β, and / or TNF-α.

[0014] The occurrence of CRS activates many inflammation-related pathway proteins. In some specific embodiments of the present invention, the MEK / ERK inhibitor inhibits the ERK pathway activated by cytokine release syndrome.

[0015] In some specific embodiments of the present invention, the MEK / ERK inhibitor inhibits the expression of Phospho-ERK1 / 2.

[0016] As an ideal drug, it should not only have a good therapeutic effect on CRS, but also not inhibit the anti-tumor activity of CAR T cells. In some specific embodiments of the present invention, the MEK / ERK inhibitor has no obvious effect on the anti-tumor activity of CAR T cells.

[0017] The present invention provides the application of U0126 in the treatment of CRS. U0126 can significantly reduce a large amount of inflammatory factors released by macrophages mTHP1 stimulated by the co-culture supernatant of CAR T cells and lymphoma cells such as Raji / Nalm6. It is found that U0126 can significantly inhibit CRS and is expected to become a new target for the treatment of CRS. The new application of U0126 in CRS can expand its research in the field of pharmaceutical technology and provide a new theoretical direction for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0019] Figure 1 Showing the effects of different concentrations of U0126 on the apoptosis of mTHP1 cells;

[0020] Figure 2 Showing the effects of different concentrations of U0126 on the proliferation of Raji and Nalm6 cells; among them, A shows the flow cytometry analysis diagram of the effects of different concentrations of U0126 on the proliferation of Nalm6 cells; B shows the statistical chart of A; C shows the flow cytometry analysis diagram of the effects of different concentrations of U0126 on the proliferation of Raji cells; D shows the statistical chart of C;

[0021] Figure 3 Showing the effects of different concentrations of U0126 on the apoptosis of CAR T cells;

[0022] Figure 4 Showing that U0126 can effectively inhibit the ERK pathway activated by CRS;

[0023] Figure 5ELISA detection shows that U0126 can effectively inhibit the CRS reaction and reduce the levels of inflammatory factors such as IL-6, IL-1β, and TNF-α. Among them, A shows the inhibitory effect of U0126 on IL-6 produced by macrophages stimulated with the supernatant of co-culture of CAR T and Nalm6; B shows the inhibitory effect of U0126 on IL-1β produced by macrophages stimulated with the supernatant of co-culture of CAR T and Nalm6; C shows the inhibitory effect of U0126 on TNF-α produced by macrophages stimulated with the supernatant of co-culture of CART and Nalm6; D shows the inhibitory effect of U0126 on IL-6 produced by macrophages stimulated with the supernatant of co-culture of CAR T and Raji; E shows the inhibitory effect of U0126 on IL-1β produced by macrophages stimulated with the supernatant of co-culture of CAR T and Raji; F shows the inhibitory effect of U0126 on TNF-α produced by macrophages stimulated with the supernatant of co-culture of CAR T and Raji;

[0024] Figure 6 WB detection shows that U0126 can effectively inhibit the CRS reaction and reduce the levels of inflammatory factors such as IL-6 and IL-1β;

[0025] Figure 7(A) shows the statistical chart of the effect of U0126 on the killing of Raji cells by CAR T with different effector-target ratios;

[0026] Figure 7(B) shows the statistical chart of the effect of U0126 on the killing of Nalm6 cells by CAR T with different effector-target ratios;

[0027] Figure 7(C) shows the flow cytometry analysis chart of the effect of U0126 on the killing of Raji cells by CAR T with different effector-target ratios;

[0028] Figure 7(D) shows the flow cytometry analysis chart of the effect of U0126 on the killing of Nalm6 cells by CAR T with different effector-target ratios. Detailed implementation manners

[0029] The present invention discloses the application of the MEK1 / 2 inhibitor U0126 in the treatment of CRS caused by CAR T cells. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0030] The overall experimental scheme and results of the present invention are as follows:

[0031] Flow cytometry was used to detect the effect of different concentrations of U0126 on the apoptosis of mTHP1 cells. The results showed that when the concentration of U0126 was less than or equal to 2 μmol / L, the effect on the apoptosis of mTHP1 was not significant, but with the increase of the drug treatment time, the apoptosis rate increased. Flow cytometry was used to detect the effect of different concentrations of U0126 on the proliferation of Raji and Nalm6 cells after CFSE staining. The results showed that with the increase of the U0126 concentration, the inhibition of cell proliferation became more obvious. Finally, flow cytometry was used to detect the effect of different concentrations of U0126 on the apoptosis of CAR-T cells after 24 hours of treatment. The results showed that when the concentration of U0126 was greater than 2 μmol / L, the apoptosis of CAR T cells increased. In summary, we believe that a U0126 concentration of 2 μmol / L is more suitable as the drug concentration for the next experiment.

[0032] The occurrence of CRS activates many inflammation-related pathway proteins. Compared with the control group (NT + DMSO group), Phospho-ERK1 / 2 in macrophages in the CRS model group (CAR T + DMSO group) was significantly increased. At the same time, after using the MEK / ERK inhibitor U0126, Phospho-ERK1 / 2 returned to normal levels. The results showed that the occurrence of CRS is related to the MEK-ERK pathway, and U0126 has a certain therapeutic effect on CRS. To verify this conjecture, we successively used ELISA and Western blot to detect the anti-inflammatory effect of U0126 and found that U0126 could significantly inhibit CRS and reduce the levels of inflammatory factors such as IL-6, IL-1β, and TNF-α. The results showed that U0126 has an obvious inhibitory effect on CRS caused by CAR T cells.

[0033] As an ideal drug, it should not only have a good therapeutic effect on CRS but also not inhibit the anti-tumor activity of CAR T cells. Therefore, exploring the effect of U0126 on the anti-tumor activity of CAR T is also an indispensable research work. Flow cytometry was used to detect the effect of U0126 on the killing of Raji and Nalm6 by CAR T cells at different effector-to-target ratios. The results showed that U0126 had no obvious effect on the anti-tumor activity of CAR T cells.

[0034] In this example, the model construction and grouping are as follows:

[0035] THP1 cells in good growth state, every 1*10 6One cell was added with 1 μL of PMA (100 ng / μL) and cultured in the dark for 24 h to induce it into adherent-growing macrophages mTHP1. Then, it was changed to fresh pre-warmed RPMI-1640 medium (containing 10% fetal bovine serum, 100 kU / L penicillin, and 0.1 g / L streptomycin) and continued to be cultured for 4 - 24 h, and U0126 or the same dose of DMSO was added. After 24 h, the medium was changed to the co-culture supernatant of CAR T (NT cells as the control) and Raji / Nalm6 (1:

[0036] 1), and at the same time, U0126 or DMSO was continuously added and cultured for 24 h. The experiment was divided into four groups: control group (NT + DMSO), drug treatment group (NT + U0126), CRS model group (CAR T + DMSO), and model + drug group (CAR T + U0126).

[0037] In the application of the MEK1 / 2 inhibitor U0126 provided by the present invention in the treatment of CRS caused by CAR T cells, the raw materials and reagents used can all be purchased from the market.

[0038] The following further elaborates the present invention in combination with embodiments:

[0039] Example 1 Detection of apoptosis by Annexin V FITC-PI

[0040] Well-grown mTHP1 or CAR T cells were evenly seeded in 24-well plates at 5×10 6 cells / well, and the cells were treated with different concentrations of U0126 (1, 2, 4, 8, 16 μmol / L) or the same volume of DMSO according to the experimental design. After 24 h or 48 h, the cells were washed twice, then 100 μL of buffer, 3 μL of Annexin V, and 1 μL of 1×PI staining solution were added to each well, incubated at room temperature for 15 min, then 100 μL of buffer was added, and finally apoptosis analysis was performed by flow cytometry (the results are shown in Table 1 and Table 2).

[0041] The effect of different concentrations of U0126 on the apoptosis of mTHP1 cells was detected by flow cytometry. The results showed that when the concentration of U0126 was less than or equal to 2 μmol / L, the apoptosis of mTHP1 was not significantly affected, but with the increase of the drug treatment time, the apoptosis rate increased (as Figure 1 shown). The effect of different concentrations of U0126 on the apoptosis of CAR-T cells after acting for 24 h was detected by flow cytometry. The results showed that when the concentration of U0126 was greater than 2 μmol / L, the apoptosis of CAR-T cells increased (as Figure 3 shown). In summary, we believe that 2 μmol / L of U0126 is more suitable as the drug concentration for the next experiment.

[0042] Table 1 Effects of U0126 on apoptosis of mTHP1 cells

[0043]

[0044] Note: * indicates P < 0.05 compared with 0 mol / L, 48 h; ** indicates P < 0.01 compared with 0 mol / L, 48 h; *** indicates P < 0.001 compared with 0 mol / L, 48 h.

[0045] Table 2 Effects of U0126 on apoptosis of CAR T cells

[0046]

[0047] Note: * indicates P < 0.05 compared with 0 mol / L; ** indicates P < 0.01 compared with 0 mol / L.

[0048] Example 2 Detection of cell proliferation by CFSE

[0049] Centrifuge Raji / Nalm6 cells with good growth status, resuspend them with pre-warmed 1*PBS at 37 °C, add 1 μL of CFSE (5 μmol / L) to every 5*10 6 cells, mix gently, and place them in an incubator (37 °C, 5% CO2) for 15 minutes for staining. Then add 5 volumes of medium to the incubator for 5 minutes to terminate the staining, centrifuge and resuspend, and inoculate 1*10 5 cells / well into a 24-well plate and place it in the incubator for culture. After 3 days, centrifuge, resuspend with pre-cooled 1*PBS, and detect cell proliferation by flow cytometry (the results are shown in Tables 3 and 4).

[0050] Use flow cytometry to detect the effects of different concentrations of U0126 (1, 2, 4, 8, 16 μmol / L) on the proliferation of Raji and Nalm6 cells stained with CFSE and inoculated in 6-well plates (5*10 5 cells / well) for 3 days. The results show that as the concentration of U0126 increases, the inhibition of cell proliferation becomes more obvious (as Figure 2 shown).

[0051] Table 3 Effects of U0126 on the proliferation of Nalm6 cells

[0052]

[0053] Note: ** indicates P < 0.01 compared with 0 mol / L; **** indicates P < 0.0001 compared with 0 mol / L.

[0054] Table 4 Effects of U0126 on the proliferation of Raji cells

[0055]

[0056] Note: *** indicates P < 0.001 compared with 0 mol / L; **** indicates P < 0.0001 compared with 0 mol / L.

[0057] Example 3 Detection of CAR T killing function by CFSE-PI co-staining

[0058] Raji / Nalm6 cells were stained with CFSE and seeded at 2.5 * 10 5 cells / well in a 24-well plate. At the same time, Raji / Nalm6 blank wells (i.e., cells without treatment) and Raji / Nalm6 wells single-stained with CFSE were seeded. Then, CAR T cells were seeded at different effector-to-target ratios (0.5:1, 1:

[0059] 1, 2:1, 5:1). The experiment was divided into two groups. One group was treated with U0126 (2 μmol / L), and the other group was treated with the same dose of DMSO as the control group. After 24 hours of treatment, the cells were collected by centrifugation, resuspended in 100 μL of pre-cooled 1*PBS, and then the experimental group and control group cells were stained with PI staining solution in the dark at room temperature for 15 minutes. 100 μL of pre-cooled 1*PBS was added and gently mixed. Flow cytometry was used to detect the killing of Raji / Nalm6 cells (the results are shown in Tables 5 and 6).

[0060] Flow cytometry was used to detect the effect of U0126 (2 μmol / L) on the killing of Raji and Nalm6 by CAR-T cells at different effector-to-target ratios. The results showed that 2 μmol / L of U0126 had no significant effect on the efficacy of CAR-T cells (as shown in Figure 7).

[0061] Table 5 Effect of U0126 on the killing of Nalm6 cells by CAR T cells

[0062]

[0063]

[0064] Note: There was no statistical significance in each group.

[0065] Table 6 Effect of U0126 on the killing of Raji cells by CAR T cells

[0066]

[0067] Note: There was no statistical significance in each group.

[0068] Example 4 Detection of the expression level of inflammatory factors by ELISA kit

[0069] CAR T and Raji / Nalm6 cells were co-cultured at an effector-to-target ratio of 1:1 for 24 h, and then centrifuged to obtain the supernatant for later use. THP1 cells in good growth condition were seeded in a 6-well plate at a density of 2*10 6 and induced with PMA for 24 h to become macrophages mTHP1. Then the prepared supernatant was added to the 6-well plate at 2 mL / well to culture mTHP1 cells, so as to stimulate the production of a large amount of inflammatory factors. Then the culture supernatant was taken, and the experimental operations were carried out according to the instructions of the ELISA kit (the ELISA kit was purchased from Proteintech Company, including the kit for detecting IL-6 with the product number KE00139, the kit for detecting IL-1β with the product number KE00021, and the kit for detecting TNF-α with the product number KE00154) to detect the expression levels of IL-6, IL-1β and TNF-α (the results are shown in Tables 7 and 8).

[0070] The anti-inflammatory effect of different concentrations of U0126 was detected by ELISA kit, and it was found that U0126 had a good anti-inflammatory effect at the protein level (as Figure 5 shown).

[0071] Table 7 Effects of U0126 on the expression of IL-6, IL-1β and TNF-α in the supernatant of mTHP1 cells stimulated by the co-culture supernatant of CAR T and Nalm6 cells

[0072]

[0073] Note: NT-DMSO group = control group, CART-DMSO group = model group, U0-2 mol / L group, U0-4 mol / L group and U0-8 mol / L group = model establishment plus U0126 group; * indicates P < 0.05 when the model group is compared with the control group; **** indicates P < 0.0001 when the model group is compared with the control group; # represents P < 0.05 when the model establishment plus U0126 group is compared with the model group; # represents P < 0.0001 when the model establishment plus U0126 group is compared with the model group.

[0074] Table 8 Effects of U0126 on the expression of IL-6, IL-1β and TNF-α in the supernatant of mTHP1 cells stimulated by the co-culture supernatant of CAR T and Raji cells

[0075]

[0076] Note: NT-DMSO group = control group, CART-DMSO group = model group, U0-2 mol / L group, U0-4 mol / L group, and U0-8 mol / L group = model + U0126 group; **** indicates P < 0.0001 when the model group is compared with the control group; # represents P < 0.0001 when the model + U0126 group is compared with the model group.

[0077] Example 5 Western blot experiment

[0078] Lyse mTHP1 cells (human myeloid leukemia mononuclear cells (THP-1) are from the Cell Bank of the Chinese Academy of Sciences in Shanghai and are induced into mTHP1 by PMA) with NP40 lysis buffer, extract proteins, and after quantifying the proteins with a BCA kit (the BCA protein concentration assay kit (enhanced) is purchased from Shanghai Beyotime Biotechnology Co., Ltd., and the product number is P0010), boil the proteins at 100 °C for 5 min. Prepare the stacking gel and separating gel, keep the protein sample loading amount consistent, and then perform electrophoresis under the conditions of 100 V for 120 min. The membrane transfer conditions are: 100 V for 70 min. After the membrane transfer is completed, block with 5% skim milk at room temperature for 2 h. After blocking, incubate with the primary antibody overnight at 4 °C, and the dilution ratio is uniformly 1:2000. The next day, wash the membrane with TBST buffer and then incubate with the secondary antibody at room temperature for 2 h, and the dilution ratio is uniformly 1:2000. Finally, after washing the membrane with TBST buffer, detect the protein expression with a chemiluminescence instrument.

[0079] As an MEK1 / 2 inhibitor, U0126, detect the changes in the expression levels of ERK1 / 2 and phosphorylated ERK1 / 2 under the action of U0126 (2 μmol / L) by western blot, and it is found that the expression level of phosphorylated ERK1 / 2 is indeed significantly inhibited (as Figure 4 shown). Finally, use the WB detection results to verify the high anti-inflammatory level of U0126 again. The results show that 2 μmol / L of U0126 has a good inhibitory effect on CRS caused by the supernatant of the co-culture of CAR-T cells and Raji / Nalm6 (1:1) stimulating mTHP1 (as Figure 6 shown).

[0080] Table 9 Information of each antibody in Western blot

[0081]

[0082] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a MEK / ERK inhibitor in the preparation of a medicament for treating cytokine release syndrome caused by CAR T therapy; the MEK / ERK inhibitor is U0126.

2. The application according to claim 1, characterized in that, The MEK / ERK inhibitor inhibits the ERK pathway activated by cytokine release syndrome.

3. The application according to claim 2, characterized in that The MEK / ERK inhibitor inhibits the expression of Phospho-ERK1 / 2.

4. The application according to any one of claims 1 to 3, characterized in that, The MEK / ERK inhibitor has no significant effect on the anti-tumor activity of CAR T cells.

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