Application of LSD1 inhibitor in preparation of combined radiotherapy drugs

Abstract

The invention relates to the technical field of biological medicine, in particular to application of an LSD1 inhibitor in preparation of a combined radiotherapy medicine, and the LSD1 inhibitor is used for regulating T cell failure induced by radiotherapy. In-vivo and in-vitro result experiments show that the LSD1 inhibitor can effectively improve the depletion condition of effector T cells in tumor tissues after radiotherapy and enhance the treatment effect of anti-PD-1 drugs, has no obvious side effects, and can be used as an active component for preparing drugs to be combined with radiotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an LSD1 inhibitor in the preparation of a combined radiotherapy drug. Background Art

[0002] Malignant tumors are malignant diseases characterized by the development of abnormal cells. These abnormal cells differentiate and proliferate uncontrollably and have the ability to infiltrate and destroy normal human tissues. At present, the incidence and mortality of malignant tumors continue to rise. Patients with advanced tumors usually require long-term treatment. Conventional treatment has a high drug resistance rate and poor patient prognosis. Radiotherapy, as a non-invasive treatment method, plays an important role in the field of tumor treatment. Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. Tumor radiotherapy is a local treatment method for tumors using radiation. Radiotherapy, with its excellent effects of reshaping the tumor immune microenvironment and inducing immunity, provides an important basis for combined radioimmunotherapy of tumors. However, in actual clinical applications, immune drugs, such as anti-PD-1 drugs, often have poor efficacy in combined radioimmunotherapy due to their low efficiency and drug resistance, and the patient's condition is further aggravated.

[0003] LSD1 is a histone demethylase, belonging to the flavin adenine dinucleotide (FAD)-dependent amine oxidase. It was initially thought to regulate the occurrence and development of tumors by specifically removing the monomethylation and dimethylation of lysine 4 (H3K4) and lysine 9 (H3K9) of histone H3. Studies in recent years have found that the expression of LSD1 is also involved in the regulation of the tumor immune microenvironment, improving the state of immune cells and inhibiting tumor progression. However, there are no relevant research reports on whether LSD1 is involved in regulating T cell exhaustion induced by radiotherapy. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the present invention aims to provide a use of an LSD1 inhibitor in the preparation of a combined radiotherapy drug.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: Use of an LSD1 inhibitor in the preparation of a combined radiotherapy drug, wherein the LSD1 inhibitor is used to regulate T cell exhaustion induced by radiotherapy.

[0006] Furthermore, the radiotherapy is external beam radiotherapy.

[0007] Furthermore, the external radiation therapy includes one or more of intensity modulated radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy and image-guided radiation therapy.

[0008] Furthermore, the radiotherapy is applied to tumor patients.

[0009] Furthermore, the tumor includes lung cancer.

[0010] Furthermore, the LSD1 inhibitor is selected from one or more of GSK2879552, SP2509, ORY-1001, GSK-LSD1, OG-L002, LSD1-IN-34, LSD1-IN-16 and trihydrochloride of CBB1007.

[0011] Furthermore, the drug includes a single preparation or a compound preparation with the LSD1 inhibitor as the first active ingredient.

[0012] Furthermore, the compound preparation also includes a second active ingredient, which is a PD1 antibody, and the LSD1 inhibitor is used to enhance the anti-tumor effect of the PD1 antibody.

[0013] Furthermore, the drug also includes at least one of pharmaceutically acceptable adjuvants, excipients and carriers.

[0014] Furthermore, the drug is an oral drug preparation.

[0015] The above one or more technical solutions in the embodiments of the invention have at least one of the following technical effects: The present invention provides the use of LSD1 inhibitors in the preparation of combined radiotherapy drugs, and LSD1 inhibitors are used to regulate T cell exhaustion induced by radiotherapy. In vivo and in vitro experiments show that LSD1 inhibitors can effectively improve the exhaustion of effector T cells in tumor tissues after radiotherapy, while enhancing the therapeutic effect of anti-PD-1 drugs, without obvious side effects, and can be used as active ingredients to prepare drugs for combined use with radiotherapy.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the tumor inhibition effect of different treatment groups on mice provided in Example 1 of the present invention.

[0018] Figure 2It is a flow cytometric graph of the effects of different groups of enhanced CD8+ T cell function provided in Example 1 of the present invention, wherein both PD-1 and Tim-3 are immunosuppressive receptors.

[0019] Figure 3 is a statistical chart of the improvement of CD8+ T cell function exhaustion in different groups provided in Example 1 of the present invention, wherein PD-1 + Tim-3 - stands for precursor exhausted cells, PD-1 + Tim-3 + Represents terminal exhausted cells, among which PD-1 - Tim-3 + Represents cells with impaired effector function.

[0020] Figure 4 is a statistical diagram of the enhancement of CD8+ T cell function by different groups provided in Example 1 of the present invention, wherein GzmB + Represents granulamycin B positive, GzmB - Represents Granulocycin B negative, IFN-γ + Represents interferon gamma positive, IFN-γ - Stands for interferon gamma negative.

[0021] Figure 5 The in vitro tumor cells and CD8 + Schematic diagram of the T cell co-culture process.

[0022] Figure 6 The PD-1 selected according to Example 2 of the present invention + Tetramer + CD8 + T cells and sorted PD-1 - Tetramer + CD8 + Flow cytometry and statistical graphs of LSD1 expression in T cells, among which PD-1 - PD-1 - Tetramer + CD8 + Abbreviation for T cells, PD-1 + PD-1 + Tetramer + CD8 + Abbreviation for T cell.

[0023] Figure 7 The different groups provided in Example 2 of the present invention have different effects on CD8 + Flow cytometric plots of the effects on T cell function.

[0024] Figure 8 The different groups provided in Example 2 of the present invention have different effects on PD-1 + Tetramer + CD8 + Statistical graph of the results of the impact on T cell function.

[0025] Fig. 9 The different groups provided in Example 2 of the present invention have different effects on PD-1 + Tetramer + CD8 + Statistical graph of the effects of interferon-γ and granzyme B production on T cells.

[0026] Fig.10 This is a curve chart showing the inhibition of mouse tumor growth in different groups provided in Example 3 of the present invention.

[0027] Fig.11 These are photos of tumors of mice in different groups collected at the end of treatment provided in Example 3 of the present invention.

[0028] Fig.12 This is a statistical chart of the tumor inhibition status of mice in different groups at the end of treatment provided in Example 3 of the present invention.

[0029] Fig.13 The infiltrating CD3 in the tumors of different groups at the end of treatment provided in Example 3 of the present invention + CD45 + T cell percentage statistics.

[0030] Fig.14 The infiltrating CD3 in the tumors of different groups at the end of treatment provided in Example 3 of the present invention + CD8 + T cell percentage statistics.

[0031] Fig.15 is a statistical chart of the improvement of CD8+ T cell function exhaustion in mice in different groups at the end of treatment provided in Example 3 of the present invention, wherein PD-1 + Tim-3 - stands for precursor exhausted cells, PD-1 + Tim-3 + Represents terminal exhausted cells, among which PD-1 - Tim-3 + Represents cells with impaired effector function.

[0032] Fig.16 The different groups of CD3 provided in Example 3 of the present invention + CD8 + Statistical graph of the effects of interferon-γ and granzyme B production on T cells.

[0033] In the figure, Control is the PBS blank control group, RT is the radiotherapy group, iLSD1 is the LSD1 inhibitor GSK2879552 group, RT+iLSD1 is the radiotherapy combined with LSD1 inhibitor GSK2879552 group, RT+αPD-1 is the RT combined with anti-PD-1 treatment group, iLSD1+αPD-1 is the LSD1 inhibitor GSK2879552 combined with anti-PD-1 treatment group, Triple is the RT combined with anti-PD-1 and LSD1 inhibitor GSK2879552 group, and cells are cells. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0035] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0036] Example 1 In vivo radiation therapy (RT) combined with LSD1 inhibitor was used to treat lung cancer in mice.

[0037] The construction and treatment process of tumor-bearing mice are as follows: (a) 5- to 6-week-old female C57BL / 6 mice were used to construct tumor-bearing models; (b) LLC-OVA cells were counted after digestion at a rate of 1×10 6 The cells / 100 μL were inoculated subcutaneously into mice, where LLC-OVA cells are mouse lung cancer cells that overexpress ovalbumin; (c) The size of the transplanted tumor grows to 50-100 mm 3 Group treatment was performed on the left and right sides; (d) The mice were divided into four groups, with 4 mice in each group. The four groups were control group, LSD1 inhibitor group, RT group (8 Gy × 1f) and combined treatment group. Among them, the drug selected for the LSD1 inhibitor group was GSK2879552, 15 mg / kg, once a day, orally administered, the combined treatment group refers to the combined treatment of LSD1 inhibitor and RT, and the control group was treated with PBS buffer solution, the usage and dosage were the same as GSK2879552; (e) The mice inoculated with LLC-OVA cells were cultured for 14 days, and the weight of tumor (TW) and tumor volume (TV) of the mice were recorded from the 1st to the 11th day. The mice in different groups were treated accordingly, and the tumor tissues of the mice were collected on the 11th day. The results are as follows Figure 1 As shown, Figure 1 A in the figure is a curve chart showing the inhibition of tumor growth in different groups. Figure 1 B in the figure shows the tumor photos of mice in different groups collected at the end of treatment. Figure 1 C in the figure is a statistical chart of tumor inhibition in different groups. The results showed that the tumor volume and tumor weight of mice in the radiotherapy (8Gy×1f) combined with LSD1 inhibitor group were significantly reduced, *p value <0.05, **p value <0.01, ***p value <0.001, ****p value <0.0001.

[0038] The present invention treats CD8 infiltrating in tumors + The function of T cells was further analyzed. Figure 2 , Figure 3 , Figure 4 As shown in Figure 2, the use of LSD1 inhibitors can help reduce the increase in the proportion of terminally exhausted T cells induced by radiotherapy, while significantly promoting IFN-γ + G JZ + CD8 + The proportion of T cells increased, indicating that LSD1 inhibitors can effectively improve CD8 + Depletion of T cells enhances CD8 + T cell function, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001.

[0039] Example 2 Obtaining PD-1 in mouse tumor tissue after radiotherapy + and PD-1 - CD8 + In vitro co-culture experiments with tumor-infiltrating T cells.

[0040] like Figure 5As shown, on day 0, mice with successfully transplanted tumors were given a single dose of 8 Gy radiotherapy. On the third day after radiotherapy, the tumor tissues of the mice were collected to prepare single cell suspensions for flow cytometry sorting, and the sorted cells were co-cultured with tumor cells in vitro. After 2 days of co-culture, i.e., on the fifth day after radiotherapy, CD8 + Tetramer + PD-1 + T cells and CD8 + Tetramer + PD-1 - T cells were analyzed by flow cytometry. The specific steps are as follows: 1. The process of constructing the mouse transplant tumor model is as follows: (a) Tumor-bearing model was established using 5-6 week-old female C57BL / 6 mice; (b) LLC-OVA cells were digested and counted at 1×10 6 pcs / 100 μL were inoculated subcutaneously into mice; (c) The size of the transplanted tumor grows to 50-100 mm 3 Stereotactic radiotherapy (8 Gy × 1f) was performed on the left and right sides; 2. CD8 infiltrating in mouse tumors + The preparation process of T cell suspension is as follows: (a) Mice were euthanized 3 days after radiation treatment. (b) Soak the euthanized mice in alcohol for 5 minutes; (c) Using scissors and forceps to remove mouse tumor tissue; (d) The dissected tumor tissue was placed in a 6-well plate containing DMEM medium; (e) Use scissors and forceps to cut the peeled tissue into 1 mm pieces. 3 left and right size; (f) DNA hydrolase (500 μg / mL) and collagenase (1 mg / mL) were added to the 6-well plate. (g) Place the 6-well plate on a 37°C constant temperature horizontal shaker (200 g, 30 min); (h) After 30 min, the 6-well plate was removed, and the culture medium containing tumor tissue was aspirated through a 45 μm filter and collected in a 50 mL centrifuge tube; (i) Centrifuge at 4°C, 1500 rpm for 5 min, discard the supernatant, add 5 times the volume of red blood cell lysis buffer, and lyse for 5 min; (j) After lysis, add enough PBS to stop the lysis reaction, centrifuge at 1500 rpm at 4°C for 5 min, and discard the supernatant; (k) Resuspend the cells in 100 μL PBS, add a dead cell removal kit, pass through a column to remove dead cells, and obtain a single cell suspension in a live mouse tumor; (l) The single cell suspension in the mouse tumor was resuspended at 1 million / 100 ul, anti-PD-1 antibody and OVA-specific tetramer antibody were added, and CD8 + T cells; 3. In vitro CD8 + T cells are co-cultured with tumor cells.

[0041] (a) LLC-OVA cells were cultured and expanded in vitro using DMEM medium (containing 10% FBS and 1% double antibody); (b) When the cell confluence reaches 70%, digestion and counting are performed, where cell confluence refers to the degree to which cells aggregate with each other in tissues or culture medium; (c) The CD8 + T cells and tumor cells were co-cultured at a ratio of 5:1, and PBS, GSK2879552 (1 μg / mL), or anti-PD-1 drugs (20 μg / mL) were added during the co-culture process; (d) After 2 days of co-culture, cells were collected and analyzed by flow cytometry; The results are as follows Figure 6 As shown in the figure, the sorted PD-1 + Tetramer + CD8 + T cells (including precursor and terminally exhausted T cells) compared to PD-1 - Tetramer + CD8 + The expression of LSD1 in T cells was significantly increased, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001. This result also suggests that the expression of LSD1 in the exhausted cell population is higher than that in the non-exhausted cell population. + Represents tetramer positivity, indicating tumor-infiltrating CD8 + Effector T cells.

[0042] like Figure 7 and Figure 8 As shown, in the co-culture system, PD-1 + Tetramer + CD8 + In the T cell group, the LSD1 inhibitor combined with anti-PD-1 treatment group significantly upregulated PD-1 compared with the anti-PD-1 monotherapy group. + Tim-3 -The proportion of precursor exhausted cells and PD-1 + Tim-3 + The proportion of terminally exhausted T cells decreased significantly. - Tetramer + CD8 + Compared with the PD-1 monotherapy group, the LSD1 inhibitor combined with anti-PD-1 therapy in the T cell group + Tim-3 - The proportion of precursor exhausted cells and PD-1 + Tim-3 + The proportion of terminal T cells did not change significantly. This result shows that LSD1 inhibitors can significantly improve PD-1 + Tetramer + CD8 + The degree of T cell exhaustion promotes an increase in the proportion of precursor exhausted cells and a decrease in the proportion of terminal exhausted cells, thereby enhancing tumor-infiltrating CD8 + T cell responses to anti-PD-1 therapy, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001.

[0043] like Fig. 9 As shown, in PD-1 + CD8 + Tetramer + Among the T cell groups, the combined treatment group had the highest production of IFN-γ and GzmB. - CD8 + Tetramer + The proportion of T cells in each group did not change significantly. This result suggests that LSD1 inhibitors will help enhance CD8 + The function of the exhausted T cell population and the response to anti-PD-1 treatment were significantly enhanced, whereas this phenomenon was not evident in the non-depleted cell population.

[0044] In conclusion, LSD1 inhibition helps improve CD8 + The degree of T cell exhaustion promotes the expansion of precursor exhausted T cells and enhances the production of effector factors in combined radioimmunotherapy, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001.

[0045] Example 3 In vivo RT combined with LSD1 inhibitor and anti-PD-1 for the treatment of lung cancer in mice.

[0046] The construction and treatment process of tumor-bearing mice are as follows: (a) 5-6 week-old female C57BL / 6 mice were used to establish a mouse tumor-bearing model; (b) LLC-OVA cells were digested and counted at 1×10 6 pcs / 100 μL were inoculated subcutaneously into tumor-bearing mice; (c) The transplanted tumor grows to a size of 50-100 mm 3 Group treatment was performed on the left and right sides; (d) The mice were divided into 5 groups, with 5 mice in each group, namely PBS group (100 μL, once a day), RT (8 Gy × 1f) combined with LSD1 inhibitor group (GSK2879552, 15 mg / kg, once a day, oral), RT (8 Gy × 1f) combined with anti-PD-1 treatment (150 μg / mouse, twice a week) group, LSD1 inhibitor (GSK2879552, 15 mg / kg, once a day, oral) combined with anti-PD-1 treatment (150 μg / mouse, twice a week), RT (8 Gy × 1f) combined with anti-PD-1 (150 μg / mouse, twice a week) and LSD1 inhibitor (GSK2879552, 15 mg / kg, once a day, oral) group. The tumor volume and body weight of the mice were recorded from the 13th day after tumor inoculation, and the tumor-bearing mice were treated accordingly. The tumor tissues of the mice were collected from the 27th day after tumor inoculation. like Fig.10 , Fig.11 , Fig.12 As shown, the tumor volume and tumor weight of mice in the radiotherapy (8Gy×1f) combined with LSD1 inhibitor plus anti-PD-1 treatment group were significantly reduced.

[0047] like Fig.13 , Fig.14 , Fig.15 , Fig.16 As shown, the present invention further analyzed the function of CD8+ T cells infiltrating mouse tumors and found that LSD1 inhibitors combined with radiotherapy plus anti-PD-1 treatment significantly promoted the infiltration of T lymphocytes and increased the proportion of CD8+ T cells. In addition, the functional status of tumor-infiltrating CD8+ T was analyzed, and it was found that the three combined treatment groups significantly increased the proportion of precursor-exhausted T cells compared with the radiotherapy plus anti-PD-1 treatment group, and the proportion of CD8+IFN-γ+GzmB- T lymphocytes and CD8+IFN-γ+GzmB+ T lymphocytes was significantly increased. The above results once again confirmed that LSD1 inhibition helps to promote the increase in the proportion of CD8+ precursor-exhausted T cells in radiotherapy combined with anti-PD-1 treatment, and enhance the effect of radiotherapy and / or immunotherapy.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of LSD1 inhibitor in the preparation of combined radiotherapy drugs, characterized in that: The LSD1 inhibitor is used to regulate T cell exhaustion induced by radiation therapy.

2. The use of the LSD1 inhibitor according to claim 1 in the preparation of a combined radiotherapy drug, characterized in that: The radiation therapy is external beam radiation therapy.

3. The use of the LSD1 inhibitor according to claim 2 in the preparation of a combined radiotherapy drug, characterized in that: The external radiation therapy is selected from one or more of intensity modulated radiation therapy, stereotactic radiation therapy, three-dimensional conformal radiation therapy and image-guided radiation therapy.

4. Use of the LSD1 inhibitor according to any one of claims 1 to 3 in the preparation of a combined radiotherapy drug, characterized in that: The radiation therapy is applied to patients with tumors.

5. The use of the LSD1 inhibitor according to claim 4 in the preparation of a combined radiotherapy drug, characterized in that: The tumor includes lung cancer.

6. Use of the LSD1 inhibitor according to claim 1 in the preparation of a combined radiotherapy drug, characterized in that: The LSD1 inhibitor is selected from one or more of GSK2879552, SP2509, ORY-1001, GSK-LSD1, OG-L002, LSD1-IN-34, LSD1-IN-16 and trihydrochloride of CBB1007.

7. Use of the LSD1 inhibitor according to claim 1 in the preparation of a combined radiotherapy drug, characterized in that: The medicine includes a single preparation or a compound preparation with the LSD1 inhibitor as the first active ingredient.

8. Use of the LSD1 inhibitor according to claim 7 in the preparation of a combined radiotherapy drug, characterized in that: The compound preparation also includes a second active ingredient, which is a PD1 antibody, and the LSD1 inhibitor is used to enhance the anti-tumor effect of the PD1 antibody.

9. Use of the LSD1 inhibitor according to claim 1 in the preparation of a combined radiotherapy drug, characterized in that: The drug also includes at least one of pharmaceutically acceptable adjuvants, excipients and carriers.

10. Use of the LSD1 inhibitor according to claim 1 in the preparation of a combined radiotherapy drug, characterized in that: The medicine is an oral pharmaceutical preparation.

Images