WO2023217109A1

WO2023217109A1 - Combination of m6a rna methylase inhibitor and immune checkpoint inhibitor for treating tumors

Info

Publication number: WO2023217109A1
Application number: PCT/CN2023/092911
Authority: WO
Inventors: 陈捷凯; 孔祥谦; 吴凯昕; 吴红玲; 汤同柯
Original assignee: 中国科学院广州生物医药与健康研究院
Priority date: 2022-05-13
Filing date: 2023-05-09
Publication date: 2023-11-16
Also published as: CN117045801A

Abstract

A combination of a m6A RNA methylase inhibitor and an immune checkpoint inhibitor for treating tumors. The combination therapy is that the combination of a tumor immunotherapeutic agent and STM2457 or a derivative thereof is used for treating tumors comprising melanomas and ovarian cancer. The new combination therapy of anti-tumor drugs has great significance in temrs of drug development and clinical treatment of melanomas or ovarian cancer.

Description

m6A RNA methylase inhibitor combined with immune checkpoint inhibitor to treat tumors

Technical field

This disclosure belongs to the field of biomedicine and relates to the combined treatment of tumors with an m6A RNA methylase inhibitor and an immune checkpoint inhibitor.

Background technique

To date, more than 170 post-transcriptional RNA modifications have been discovered in humans. Among them, N6 methyladenosine (m6A) is the most common modification in eukaryotic messenger RNA (mRNA). m6A modified proteins include METTL3, METTL14, YTHDC1, YTHDF2, etc. m6A modification plays a key role in regulating RNA processing, splicing, nucleation, translation and stability, which is critical for the development of various human diseases such as cancer. m6A modifications are abnormal in various types of cancer and are associated with patient prognosis. Abnormalities in m6A modification are closely related to the occurrence, development and drug resistance of malignant tumors. As oncogenic factors, m6A-modified proteins promote tumor progression by upregulating oncogenes or downregulating tumor suppressor genes.

Programmed cell death protein 1 (PD-1) plays an important role in suppressing immune responses and promoting self-tolerance, including regulating the activity of T cells, activating antigen-specific T cell apoptosis programs and inhibiting the apoptosis of conventional T cells . PD-L1 is a transmembrane protein that is considered a common inhibitory factor of the immune response. It can combine with PD-1 to reduce the proliferation of PD-1-positive cells, inhibit their cytokine secretion, and induce cell apoptosis. PD-L1 also plays an important role in the progression of various malignant tumors. It inhibits the immune system's recognition and killing of tumor cells and promotes tumor immune escape. Blocking the PD-1/PD-L1 interaction through monoclonal antibodies has had a huge impact on cancer treatment.

Melanoma is a malignant tumor that originates from melanocytes. As an aggressive skin cancer, the incidence of melanoma is increasing worldwide. Many studies have explored the relationship between tumor cells and the tumor microenvironment. However, more than half of melanoma patients do not respond to immune checkpoint inhibitor treatment, which is closely related to the immunosuppressive microenvironment in which tumor cells live. By combining with other drugs, it is important to increase the level of tumor-infiltrating CD8+T cells and the CD8/Treg ratio in the tumor microenvironment, turn immune "cold" tumors into "hot" tumors, and improve the effectiveness of tumor immunotherapy. clinical significance.

Contents of the invention

In some embodiments, the present disclosure provides a composition comprising: an anti-tumor immunotherapeutic agent and STM2457 or a derivative thereof.

In some embodiments, the anti-tumor immunotherapeutic agent includes immune cells or one or more immune checkpoint inhibitors.

In some embodiments, the immune cells are selected from T cells, macrophages and/or monocytes or T cells containing artificial T cell receptors.

In some embodiments, the T cells are selected from one or more of natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and regulatory T cells.

In some embodiments, the T cells are selected from T lymphocytes.

In some embodiments, the T cells are selected from umbilical cord blood-derived T lymphocytes.

In some embodiments, the immune checkpoint inhibitor includes a polypeptide, a monoclonal antibody, or a chemically synthesized small molecule inhibitor.

In some embodiments, the immune checkpoint inhibitor includes a PD-L1 inhibitor, a PD-1 inhibitor, and/or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody or antigen-binding fragment thereof.

In some embodiments, the PD-1 inhibitor is a PD-1 antibody.

In some embodiments, the anti-tumor immunotherapeutic agent includes an antibody or antibody fragment that targets a tumor-specific antigen.

In some embodiments, the structural formula of STM2457 is as shown in formula (I):

In some embodiments, the composition further includes a pharmaceutically acceptable carrier, excipient, or stabilizer.

In some embodiments, the composition is an anti-tumor composition.

In some embodiments, the tumors include lung cancer (including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer), gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, Ovarian cancer, liver cancer, hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, myeloma, multiple myeloma, salivary gland cancer, kidney cancer, renal cell carcinoma, nephroblastoma, Basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or human mononuclear leukemia cells. In some embodiments, the inventors have found that PD-1 inhibitors (or PD-1 blockers) or T cells combined with STM2457 have very significant effects in the treatment of melanoma and ovarian cancer, and the effect of the combination group is far better than Single drug group.

In some embodiments, the combination even produces synergistic therapeutic effects that exceed the expectations of those skilled in the art.

Combining anti-CTLA-4 and anti-PD-1 therapies offers the potential for superior efficacy, which may be attributed to each agent acting in a complementary or even synergistic manner. But combination therapy may lead to greater toxicity. In patients with previously untreated melanoma or recurrent small cell lung cancer, drug-related grade 3-4 adverse events occurred in 54% to 55% of patients and in 24% to 27% of patients with concurrent blockade compared with 24% to 27% alone. The incidence was 24% to 27% with ipilimumab and 15% to 16% with nivolumab alone. Therefore, this disclosure finds new anti-tumor drug combination therapy, which is of great significance for drug development and clinical treatment of melanoma. In some embodiments, the present disclosure provides a kit comprising a first container, a second container and a package insert; the first container contains an anti-tumor immunotherapeutic agent, the second container contains STM2457 or its A derivative of a drug, and the package insert contains instructions for using the drug to treat cancer in an individual.

In some embodiments, the T cells are selected from T lymphocytes.

In some embodiments, the immune checkpoint inhibitors include polypeptides, monoclonal antibodies, or chemically synthesized small molecule inhibitors.

In some embodiments, the PD-1 inhibitor is a PD-1 antibody.

In some embodiments, the anti-tumor immunotherapeutic agent includes an antibody or antibody fragment targeting a tumor-specific antigen.

In some embodiments, the present disclosure provides the use of any of the compositions in the preparation of a medicament for the treatment of cancer.

In some embodiments, the cancer includes lung cancer (including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer), gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, Ovarian cancer, liver cancer, hepatoma, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, myeloma, multiple myeloma, salivary gland cancer, kidney cancer, renal cell carcinoma, nephroblastoma, Basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, esophageal cancer, or human mononuclear leukemia cells.

In some embodiments, the present disclosure provides a composition as described above for treating cancer.

In some embodiments, the present disclosure provides a method of treating cancer, comprising administering to a patient or subject any of the compositions described above.

In some embodiments, the present disclosure provides the use of STM2457 or a derivative thereof in the preparation of a medicament for the treatment of melanoma or ovarian cancer.

In some embodiments, the present disclosure provides a method of treating melanoma or ovarian cancer, comprising administering STM2457 or a derivative thereof to a patient or subject.

In some embodiments, the present disclosure provides STM2457 or derivatives thereof for the treatment of melanoma or ovarian cancer.

Description of the drawings

Figure 1 shows the volume and mass measurements of mouse tumors in the four groups of regimens, showing that the combination of STM2457 and PD-1 antibodies can effectively inhibit the growth of melanoma in mice. (Figure 1A) Among the four-group regimens, the STM2457 and PD-1 antibody groups significantly reduced tumor weight compared to the NC control group. (Figure 1B) Among the four groups of regimens, the STM2457 and PD-1 antibody groups significantly reduced tumor volume compared with the NC control group.

Figure 2 shows the results of single-cell CD45 flow cytometry analysis of mouse tumors in the four groups of regimens. The combined use of STM2457 and PD-1 antibodies can effectively improve the tumor microenvironment, that is, increase the proportion of hematopoietic lineage cells in B16F0 tumors.

Figure 3 Analysis of tumor single cell sequencing data. The results show that the combined use of STM2457 and PD-1 antibodies can effectively promote the mitosis of T cell populations and the expression of genes related to the maintenance of stem cell populations in B16F0 tumors.

Figure 4 is a flow chart for the combined administration of STM2457 and PD-1 antibodies.

Figure 5 shows the combination of STM2457 and T cells in the treatment of ovarian cancer. (Figure 5A) Fluorescence photography of A1847 cells (marked with red fluorescence), T cells, and STM2457 treatment. (Figure 5B) Tumor cell death rate (%) calculated based on the number of A1847 cells detected by flow cytometry, STM2457 group vs NC+T group ***P<0.001.

Figure 6 shows the comparison of the inhibitory effects of m6A methylase inhibitor STM2457 alone on different tumor cells.

Figure 7 shows the combination of STM2457 and T cells to treat B16 melanoma. It is the tumor cell death rate (%) calculated based on the number of B16 cells detected by flow cytometry, STM2457 group vs NC+T group ***P<0.001.

Detailed ways

The technical solutions of the present disclosure are further described below through specific examples, which do not limit the scope of protection of the present disclosure. Some non-essential modifications and adjustments made by others based on the concepts of this disclosure still fall within the protection scope of this disclosure.

The β-cyclodextrin solution used in the following examples was purchased from Aladdin-e Company (https://www.aladdin-e.com/), and the product number is H108813. The PD-1 antibody used was purchased from BioXcell (https://bxcell.com) with the product number BE0146. The PBS buffer used was purchased from HyClone Company, product number SH30028.02.

"Comprises" or "includes" is intended to mean that the compositions (eg, media) and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" is meant to exclude other elements that are of any importance in combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. “Consisting of” means trace elements and substantial method steps excluding other components. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

As used herein, the components of a "composition" may be present in a mixed form or may be packaged separately. The separately packaged components may also contain their respective adjuvants. The adjuvant refers to a means in medicine that can assist the efficacy of a drug. Where the components of a composition are packaged separately, the separately packaged components may be administered simultaneously or in any sequential order, wherein the patient is first treated with one drug and then the other drug. . The patient refers to a mammalian subject, especially a human.

"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 immunotherapy agents" refer to drugs used for "tumor immunotherapy", including monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy and small molecule inhibitors, etc.

In this article, STM refers to "STM2457".

Example 1

1. Drug configuration

(1) Preparation method of m6A methylase inhibitor working solution:

1) Dissolve β-cyclodextrin in sterilized ultrapure water to obtain a β-cyclodextrin solution with a mass fraction of 20% (w/v), and filter it with a 0.22 μm filter membrane in a biological safety cabinet. Ensure its sterility;

2) Dissolve the STM2457 drug in the β-cyclodextrin solution described in step (1), shake and mix thoroughly at 42 degrees Celsius to form a homogeneous STM2457-β-cyclodextrin solution. The final concentration of STM2457 is 6.25mg/mL.

(2) Immune checkpoint drug configuration method:

The PD-1 antibody was diluted to 2.5mg/mL using PBS buffer.

2. Evaluation of the combined use of STM2457 and PD-1 antibody on the growth inhibition of mouse melanoma B16F0

Mouse melanoma B16F0 (or "B16") was inoculated into 6-8 week old C57BL/6J mice, and each mouse was inoculated with 2*10^5 cells. After the tumor mass can be touched, start injecting different drug combinations (a total of four groups, namely: NC, STM2457, PD-1 antibody, STM2457+PD-1 antibody), and regularly detect changes in the mouse's weight and tumor volume. Variety. The results showed that the STM2457+PD-1 antibody group had the most significant effect in inhibiting tumor growth (Figure 1).

3. Evaluation of the combined use of STM2457 and PD-1 antibodies on changes in the microenvironment of melanoma

Take the tumor obtained after the dissection of the above mouse, clean it with PBS, discard the necrotic part of the tissue, take part of the tumor into a 1.5mL centrifuge tube, cut it into pieces of about ^1mm3 , and then add 1.5mL preheated DMEM Medium (containing final concentration of 2mg/mL collagenase P, 20ug/mL DNase I enzyme). Digest for 15 minutes at 37°C, shake and pipet, filter with a 70um filter membrane, and centrifuge to remove the supernatant. Add 700ul red blood cell lysis solution to the EP tube, lyse it at 4 degrees for 10 minutes, add an equal volume of serum to terminate, and then obtain a single cell sample, then stain it with FITC-CD45 antibody, and then conduct flow cytometry analysis. The results show that the three groups of regimens (STM2457, PD-1 antibody, STM2457+PD-1 antibody) can increase the proportion of hematopoietic lineage cells compared with the NC control group, proving that the combined use of STM2457 and PD-1 antibody can effectively improve the microbiology of melanoma. environment (Figure 2).

4. Evaluation of the impact of combined use of STM2457 and PD-1 antibodies on global gene expression in melanoma

Take the cell sample obtained in step 3 above and perform single-cell transcriptome (scRNA-seq) analysis. Data analysis shows that compared to the PD-1 antibody alone group, the STM2457+PD-1 antibody group can effectively promote T cell mitosis and the expression of stem cell population maintenance-related genes in the immune microenvironment of B16FO tumors, indicating that STM2457 can enhance T cell activity. Reduce T cell exhaustion (Figure 3).

Note: The four groups in Figures 1-3 are:

(1)NC group (or NC+NC):

(2)NC+PD1 group (or PD-1 antibody group):

(3)STM+NC group (STM2457 group):

(4)STM+PD1 group (STM2457&PD-1 antibody group):

The STM injection dose is 50mg/kg, approximately 1.25mg/mouse, once a day; the PD-1 antibody injection dose is 250ug/mouse, twice a week. A 2-week dosing period started from the time when mice were subcutaneously inoculated with B16F0 tumors and had obvious touch sensation (about 5-7 days later). The flow chart is shown in Figure 4.

Example 2 Evaluation of the combined use of STM2457 and T cells to inhibit the growth of ovarian cancer cells

At the cellular level, human ovarian cancer cell A1847 was treated with 1 μM m6A methylase inhibitor STM2457 and then co-incubated with cord blood-derived T cells.

The specific steps are: seed 1,000 A1847 cells in a 96-well plate, treat and culture them at a concentration of 1uM STM2457 for 3 days, then add umbilical cord blood-derived T cells in different combinations (the number of T cells added is based on the ratio of A1847 to T cells). 1:1), a total of four groups (respectively: NC; STM2457; NC+T; STM2457+T). After incubation for 24 hours, the cells were collected. The number of A1847 cells was detected by flow cytometry, and the killing of tumor cells was calculated.

The results are shown in Figures 5A-5B. It can be seen that the combination of STM2457 and T cells can effectively inhibit the growth of ovarian cancer cells. Among them, the steps of the method for isolating T cells derived from umbilical cord blood include:

Umbilical cord blood is separated from mononuclear cells by density gradient centrifugation. The specific steps are: (1) dilute the umbilical cord blood 2 times with PBS and centrifuge it in a clean sterile collection tube at 25°C and 750g for 30 minutes; (2) transfer the white middle layer of MNC into a new 50mL centrifuge tube; (3) Add 30mL PBS to each tube, centrifuge at 350g for 10min at 4℃; (4) Discard the supernatant, add 5mL of red blood cell lysis solution, incubate at 25℃ for 10min, fill with PBS to dilute the red blood cells for lysis solution, centrifuge at 300g for 10 minutes at 4°C; (5) Resuspend the cell pellet in 25 mL of PBS, centrifuge at 300g for 10 minutes at 4°C, and repeat once; (6) Resuspend the cells in PBS, count, and the cells obtained are mononuclear cells. (7) Add biotin-conjugated antibodies (CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, CD235a) to the resuspended mononuclear cells and incubate for 5 minutes, followed by incubation with anti-biotin magnetic beads for 10 minutes. The magnetically labeled cells are removed through a magnetic column to isolate high-purity T lymphocytes.

Example 3 Inhibitory effects of m6A methylase inhibitor STM2457 alone on different tumors

At the cellular level, 5 μM m6A methylase inhibitor STM2457 was used to treat mouse melanoma cells B16; human breast cancer cells MDA-MB-231 and MCF7; human monocytic leukemia cells Thp1; mouse lung cancer cells LLC; and human Melanoma cells A375; human lung cancer cells H1299, H23, H460, and H522 were processed.

Specifically: seed 1,000 cells in a 96-well plate, treat with 5 μM STM2457 for 4 days, add 10ul CCK8 (Beyotime) to each well, incubate for 1 hour in an incubator, and use a microplate reader to detect the absorbance value at 450 nm.

The results are shown in Figure 6. It can be seen that THP-1 and MDA-MB-231 cells are sensitive to STM2457, MCF7, LLC, and H460 have about 20% inhibitory effect, and the other cancer cells are not very sensitive.

Example 4 Evaluation of the combined use of STM2457 and T cells to inhibit the growth of B16 melanoma cells

At the cellular level, murine melanoma B16 cells were treated with 1 μM m6A methylase inhibitor STM2457 and then co-incubated with mouse spleen-derived T cells.

The specific steps are: seed 1,000 B16 cells in a 96-well plate, treat and culture them at a concentration of 1 μM STM2457 for 3 days, then add mouse spleen-derived T cells in different combinations (the number of T cells added is based on the number of B16 and T cells). Ratio 1:2), a total of four groups (respectively: NC; STM2457; NC+T; STM2457+T). After incubation for 24 hours, the cells were collected. The number of B16 cells was detected by flow cytometry, and the killing of tumor cells was calculated.

The results are shown in Figure 7. It can be seen that the combination of STM2457 and T cells can effectively inhibit the growth of melanoma cells.

Among them, the steps of the method for isolating T cells derived from mouse spleen include:

Mononuclear cells were isolated from the spleen obtained by dissection of mice. The specific procedures are as follows: (1) Soak the spleen of the dissected mouse in PBS, use a 1mL syringe to back-grind the spleen to 1mL, and filter with a filter. Centrifuge the filtrate collection tube at 750g for 5 minutes at 25°C; (2) Discard the supernatant, add 5mL of red blood cell lysate, incubate at 25°C for 10 minutes, fill with PBS diluted red blood cell lysate, and centrifuge at 300g for 10 minutes at 4°C; (5) Resuspend with 25mL of PBS Suspend the cells to pellet, centrifuge at 300g for 10 minutes at 4°C, and repeat once; (6) Resuspend the cells in PBS and count. The cells obtained are mononuclear cells. (7) Add biotin-conjugated antibodies (CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, MHC class II and Ter-119) to the resuspended mononuclear cells and incubate for 5 minutes, followed by anti- The biotin magnetic beads are incubated for 10 minutes, and the magnetically labeled cells are removed through the magnetic column to isolate high-purity T lymphocytes.

Claims

A composition, characterized in that the composition includes: an anti-tumor immunotherapeutic agent and STM2457 or a derivative thereof.
The composition of claim 1, wherein the anti-tumor immunotherapeutic agent includes immune cells or one or more immune checkpoint inhibitors.

Preferably, the immune cells are selected from T cells, macrophages and/or monocytes or T cells containing artificial T cell receptors;

Preferably, the T cells are selected from one or more of natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells;

Preferably, said T cells are selected from T lymphocytes.
The composition according to any one of claims 1-2, wherein the immune checkpoint inhibitor includes a polypeptide, a monoclonal antibody, or a chemically synthesized small molecule inhibitor;

Preferably, the immune checkpoint inhibitor includes a PD-L1 inhibitor, a PD-1 inhibitor and/or a CTLA-4 inhibitor;

Preferably, the PD-1 inhibitor is an antibody or an antigen-binding fragment thereof;

Preferably, the PD-1 inhibitor is a PD-1 antibody.
The composition of any one of claims 1-3, wherein the anti-tumor immunotherapeutic agent includes an antibody or antibody fragment targeting a tumor-specific antigen.
The composition according to any one of claims 1 to 4, wherein the structural formula of STM2457 is as shown in formula (I):
The composition according to any one of claims 1 to 5, further comprising a pharmaceutically acceptable carrier, excipient, or stabilizer;

Preferably, the composition is an anti-tumor composition;

Preferably, the tumor includes lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, hepatoma, bladder cancer, breast cancer, colon cancer, colon cancer Rectal cancer, endometrial cancer, myeloma, multiple myeloma, salivary gland cancer, kidney cancer, renal cell carcinoma, nephroblastoma, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, Esophageal cancer, or human mononuclear leukemia cells;

Preferably, the cancer includes ovarian cancer or melanoma.
A pharmaceutical kit, characterized by comprising a first container, a second container and a packaging insert; the first container contains an anti-tumor immunotherapeutic agent, the second container contains a drug of STM2457 or its derivatives, and The package insert contains instructions for using the drug to treat cancer in an individual.
The kit of claim 7, wherein the anti-tumor immunotherapeutic agent includes immune cells or one or more immune checkpoint inhibitors;

Preferably, the immune cells are selected from T cells, macrophages and/or monocytes or T cells containing artificial T cell receptors;

Preferably, the T cells are selected from one or more of natural killer (NK) cells, cytotoxic T lymphocytes (CTL) and regulatory T cells;

Preferably, the T cells are selected from T lymphocytes.

Preferably, the immune checkpoint inhibitor includes polypeptides, monoclonal antibodies, or chemically synthesized small molecule inhibitors;

Preferably, the immune checkpoint inhibitor includes a PD-L1 inhibitor, a PD-1 inhibitor and/or a CTLA-4 inhibitor;

Preferably, the PD-1 inhibitor is an antibody or an antigen-binding fragment thereof;

Preferably, the PD-1 inhibitor is a PD-1 antibody.

Preferably, the anti-tumor immunotherapeutic agent includes an antibody or antibody fragment targeting a tumor-specific antigen;

Preferably, the structural formula of the STM2457 is as shown in formula (I):
The use of the composition according to any one of claims 1 to 6 in the preparation of drugs for the treatment of cancer;

Preferably, the cancer includes lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, hepatoma, bladder cancer, breast cancer, colon cancer, colon cancer Rectal cancer, endometrial cancer, myeloma, multiple myeloma, salivary gland cancer, kidney cancer, renal cell carcinoma, nephroblastoma, basal cell carcinoma, melanoma, prostate cancer, vulvar cancer, thyroid cancer, testicular cancer, Esophageal cancer or human mononuclear leukemia cells;

Preferably, the cancer includes ovarian cancer or melanoma.
The use of STM2457 or its derivatives in the preparation of drugs for the treatment of melanoma or ovarian cancer;

Preferably, the structural formula of the STM2457 is as shown in formula (I):