RIP2 inhibitor and immune checkpoint inhibitor combined pharmaceutical composition and application thereof

Abstract

The invention discloses a pharmaceutical composition of a receptor-interacting protein 2 (RIP2) inhibitor combined with an immune checkpoint inhibitor, and an application of the pharmaceutical composition. In particular, the present invention provides a pharmaceutical composition comprising an RIP2 inhibitor and an immune checkpoint inhibitor (ICI), and a pharmaceutical composition comprising an RIP2 inhibitor, an ICI, and a pharmaceutical composition comprising an RIP2 inhibitor, an ICI, and a pharmaceutical composition comprising an RIP2 inhibitor, an ICI, and a pharmaceutical composition comprising an RIP2 inhibitor and an ICI. The invention particularly relates to an application of an RIP2 inhibitor combined with ICI in preparation of drugs for treating and / or preventing tumors. The knockout RIP2 or RIP2 inhibitor has a novel application of sensitizing ICI antitumor, namely has an effect of enhancing the antitumor activity of the ICI drug. It is found for the first time that the anti-tumor activity of ICI drugs can be enhanced by knocking out RIP2 or using RIP2 inhibitors. After the RIP2 inhibitor and the ICI are jointly used, the proportion of CD3 + cells, CD8 + T cells, gamma delta TCR + T cells, effector memory T cells, CD8 effector T cells (IFN gamma + CD8 + T cells, GranzymeB + CD8 + T cells, TNF alpha + CD8 + T cells and CD107a + CD8 + T cells) in a tumor microenvironment is increased, and the treatment effect of the ICI medicine on tumors is enhanced.

Description

Pharmaceutical composition of RIP2 inhibitor combined with immune checkpoint inhibitor and application thereof

[0001] This application claims priority to Chinese patent application No. 202211400189.4, filed on November 9, 2022, and cites the full text of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the technical field of tumor treatment and relates to a pharmaceutical composition of a receptor-interacting protein 2 (RIP2) inhibitor combined with an immune checkpoint inhibitor (ICI) and its application; specifically, it relates to the application of a RIP2 inhibitor in enhancing the efficacy of anti-tumor ICI drugs. Background Art

[0003] Cancer is one of the major threats to human health, and the global cancer mortality rate is increasing year by year. Traditional cancer treatments include surgery, radiotherapy, chemotherapy, and targeted drug therapies. In recent years, with the deepening understanding and research of the tumor immune system, tumor immunotherapy has been widely used in clinical practice and has continuously achieved breakthroughs in efficacy. Tumor immunotherapy aims to control and eliminate tumors by reactivating suppressed specific immune responses and restoring normal anti-tumor immune activity. Immune checkpoint therapy, a key component of tumor immunotherapy, regulates T cell activity through a series of pathways, including co-inhibitory or co-stimulatory signals, to kill tumor cells. Currently, the most intensively studied immune checkpoint molecules include PD-1, PD-L1, CTLA-4, BTLA, TIM-3, TIGIT, VISTA, LAG-3, and IDO1. Immune checkpoint inhibitors (ICIs) that have been successfully used in the treatment of various tumors with good efficacy include PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors. However, clinical applications of ICIs have revealed significant shortcomings, such as low efficacy (PD-1 inhibitors have an efficacy of only 5%-30% and are effective in only 5% of colorectal cancer patients), strong cytotoxicity, treatment resistance, and a variety of immune-related adverse reactions. The low efficacy severely limits the benefits that cancer patients can gain from ICI therapy. Therefore, exploring new treatment modalities, improving the sensitivity of ICIs, and increasing their efficacy have long been research hotspots.

[0004] Summary of the Invention

[0005] The technical problem to be solved by the present invention is that ICI has poor effectiveness, cytotoxicity, treatment resistance and other shortcomings. The present invention provides a pharmaceutical composition of a receptor interacting protein 2 (RIP2) inhibitor combined with ICI and its application. The object of the present invention is to provide a new application of knocking out RIP2 or using a RIP2 inhibitor to significantly sensitize ICI drugs to fight tumors. In other words, the composition of the present invention effectively enhances the therapeutic effect of ICI drugs on tumors, has high effectiveness, and has a wide range of application prospects. The present invention first discovered that knocking out RIP2 or using a RIP2 inhibitor can enhance the anti-tumor activity of ICI drugs. After combining RIP2 inhibitors and ICI, the ratio of CD3+ cells, CD8+T cells, γδTCR+T cells, effector memory T cells, CD8 effector T cells (IFNγ+CD8+T cells, GranzymeB+CD8+T cells, TNFα+CD8+T cells, CD107a+CD8+T cells) in the tumor microenvironment is increased, thereby enhancing the therapeutic effect of ICI drugs on tumors.

[0006] The present invention provides a pharmaceutical composition of a RIP2 inhibitor combined with an ICI, comprising a RIP2 inhibitor and an ICI.

[0007] The pharmaceutical composition may further include pharmaceutical excipients. The amount of the pharmaceutical excipients used may be conventional in the art, and is generally selected by those skilled in the art based on the desired dosage form, properties, size, and compatibility with the active ingredient.

[0008] The RIP2 inhibitor is preferably one or more of GSK583 (CAS No. 1346547-00-9), WEHI-345, SB203580, OD36, OD38 and derivatives thereof, more preferably GSK583 and / or derivatives thereof.

[0009] The ICI is preferably one or more of a programmed cell death receptor 1 (PD-1) inhibitor, a programmed cell death receptor 1 ligand (PD-L1) inhibitor, a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor, a B lymphocyte or T lymphocyte attenuating factor (BTLA) inhibitor, a T cell immunoglobulin mucin 3 (TIM-3) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, a V-domain Ig inhibitor of T cell activation (VISTA) inhibitor, a lymphocyte activation gene 3 (LAG-3) inhibitor, and an indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor, and further preferably includes a PD-1 inhibitor and / or a PD-L1 inhibitor.

[0010] In the pharmaceutical composition, the programmed cell death receptor 1 (PD-1) inhibitor may be pembrolizumab.

[0011] In the pharmaceutical composition, the programmed cell death receptor 1 ligand (PD-L1) inhibitor may be atezolizumab.

[0012] In the pharmaceutical composition, the type of RIP2 inhibitor derivative can be a common type or an uncommon type of molecule in the art, and the derivative can achieve a RIP2 inhibitory effect through chemical and / or physical reactions in the in vivo environment, including but not limited to prodrugs of RIP2 inhibitors, such as ester derivatives, salt types, etc.

[0013] In the pharmaceutical composition, the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor may be ipilimumab or tremelimumab.

[0014] In the pharmaceutical composition, the B lymphocyte and T lymphocyte attenuating factor (BTLA) inhibitor can be Icatolimab.

[0015] In the pharmaceutical composition, the T cell immunoglobulin mucin 3 (TIM-3) inhibitor may be Cobolimab (TSR-022) or Sabatolimab (MBG453).

[0016] In the pharmaceutical composition, the T cell immunoreceptor (TIGIT) inhibitor having Ig and ITIM domains may be Tiragolumab.

[0017] In the pharmaceutical composition, the V-domain Ig inhibitor of T cell activation (VISTA) inhibitor may be CA-170 or JNJ-610588.

[0018] In the pharmaceutical composition, the lymphocyte activation gene 3 (LAG-3) inhibitor may be Relatlimab or Tebotelimab.

[0019] In the pharmaceutical composition, the indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor may be epacadostat or navoximod.

[0020] In the pharmaceutical composition, the mass ratio of the RIP2 inhibitor to the ICI may be 1:(1-60), for example, 1:6, 1:10, 1:15, 1:20, 1:30 or 1:60.

[0021] In the pharmaceutical composition, the mass ratio of the RIP2 inhibitor to the ICI may be 1:(6-10), for example, 1:6 or 1:10.

[0022] In a certain embodiment, the pharmaceutical composition consists of the RIP2 inhibitor and ICI.

[0023] In one embodiment, in the pharmaceutical composition, the RIP2 inhibitor is a derivative of GSK583, and the derivative of GSK583 is preferably GSK2983559.

[0024] In one embodiment, in the pharmaceutical composition, the ICI is a PD-1 inhibitor (e.g., pembrolizumab) or a PD-L1 inhibitor (e.g., atezolizumab).

[0025] In one embodiment, the pharmaceutical composition is GSK583 and a PD-L1 inhibitor.

[0026] In one embodiment, the pharmaceutical composition comprises GSK583 and a PD-L1 inhibitor, and the mass ratio of the GSK583 to the PD-L1 inhibitor is 1:6 to 1:10.

[0027] In one embodiment, the pharmaceutical composition comprises a RIP2 inhibitor and atezolizumab.

[0028] In one embodiment, the pharmaceutical composition is GSK583 and atezolizumab.

[0029] In one embodiment, the pharmaceutical composition is GSK2983559 and atezolizumab.

[0030] In one embodiment, the pharmaceutical composition is GSK583 and a PD-1 inhibitor.

[0031] In one embodiment, the pharmaceutical composition comprises GSK583 and a PD-1 inhibitor, and the mass ratio of the GSK583 to the PD-1 inhibitor is 1:6 to 1:10.

[0032] In one embodiment, the pharmaceutical composition is a RIP2 inhibitor and pembrolizumab.

[0033] In one embodiment, the pharmaceutical composition is GSK583 and pembrolizumab.

[0034] In one embodiment, the pharmaceutical composition is GSK2983559 and pembrolizumab.

[0035] In the pharmaceutical composition, the RIP2 inhibitor and ICI can be administered simultaneously or separately.

[0036] In the pharmaceutical composition, the RIP2 inhibitor and ICI can be administered isofrequently or unequally.

[0037] The “simultaneous administration” may be, for example, that the RIP2 inhibitor and the ICI are contained in a single pharmaceutical composition and administered simultaneously; or, alternatively, a “separate pharmaceutical composition comprising the RIP2 inhibitor” and a “separate pharmaceutical composition comprising the ICI” are administered simultaneously.

[0038] The “separate administration” method may be, for example, that the “separate pharmaceutical composition comprising a RIP2 inhibitor” and the “separate pharmaceutical composition comprising an ICI” are administered separately at different times, for example: one of the “separate pharmaceutical composition comprising a RIP2 inhibitor” and the “separate pharmaceutical composition comprising an ICI” is administered first, and the other is administered subsequently; for example, the “separate pharmaceutical composition comprising a RIP2 inhibitor” is administered first and then the “separate pharmaceutical composition comprising an ICI”; or the “separate pharmaceutical composition comprising an ICI” is administered first and then the “separate pharmaceutical composition comprising a RIP2 inhibitor”.

[0039] The "single pharmaceutical composition comprising a RIP2 inhibitor" may be a pharmaceutical composition comprising the RIP2 inhibitor and a pharmaceutical excipient. The "single pharmaceutical composition comprising an ICI" may be a pharmaceutical composition comprising the ICI and a pharmaceutical excipient.

[0040] The separate administrations may be close in time or remote in time. In the separate administrations, each individual pharmaceutical composition may be administered with equal or unequal frequencies, for example, once every 4 days or once every 7 days.

[0041] In the pharmaceutical composition, the RIP2 inhibitor (e.g., GSK583) is administered at different doses depending on the tumor conditions (tumor size, number of treatments, interval time, etc.), and can also be administered according to the patient's weight. The general dosage is 0.1 mg / kg-5 mg / kg (single dose), preferably 1 mg / kg (single dose).

[0042] In the pharmaceutical composition, the dosage of the ICI can be the conventional dosage in the art, and the general dosage is 2 mg / kg-10 mg / kg (single dose), for example, 6 mg / kg (single dose). For example, the dosage of the PD-L1 inhibitor (e.g., atezolizumab) is 6 mg / kg (single dose), and the dosage of the PD-1 inhibitor (e.g., pembrolizumab) is 6 mg / kg (single dose).

[0043] Preferably, the RIP2 inhibitor is administered at an equal frequency during the treatment, and the ICI is administered at an equal frequency or at an unequal frequency during the treatment.

[0044] Preferably, the RIP2 inhibitor is administered once every 4 days.

[0045] Preferably, the ICI is administered at an unequal frequency, once every 4 days or once every 7 days.

[0046] Preferably, the RIP2 inhibitor is administered once every 4 days, for a total of 4 to 7 times, for example, on the 0th, 4th, 8th and 12th days.

[0047] Preferably, the ICI is administered at an unequal frequency, once every 4 days or every 7 days, for a total of 3, 4 or 5 times, for example, on days 0, 4, 11 and 18.

[0048] Whether administered simultaneously or separately, the administration regimens of the RIP2 inhibitor and ICI (including administration route, administration frequency, administration dosage, administration interval, etc.) may be the same or different, and may be adjusted as needed by those skilled in the art to provide the optimal therapeutic effect.

[0049] In one embodiment, the RIP2 inhibitor is administered by injection (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular) or orally.

[0050] In one embodiment, the ICI is administered by injection (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular).

[0051] In one embodiment, the RIP2 inhibitor and the ICI are both administered by injection, such as intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection.

[0052] The present invention provides an application of the pharmaceutical composition in preparing a drug for treating and / or preventing tumors.

[0053] In the application, the tumor may be a tumor associated with the RIP2 signaling pathway, preferably intestinal cancer, melanoma, lung cancer, breast cancer, liver cancer, gastric cancer or pancreatic cancer.

[0054] The present invention provides a method for treating and / or preventing tumors, which comprises administering a therapeutically effective amount of the pharmaceutical composition to a patient in need.

[0055] In the method for treating and / or preventing tumors, the tumor may be a tumor associated with the RIP2 signaling pathway, preferably intestinal cancer, melanoma, lung cancer, breast cancer, liver cancer, gastric cancer or pancreatic cancer.

[0056] In the pharmaceutical composition, the RIP2 inhibitor and ICI can be administered isofrequently or unequally.

[0057] In the method for treating and / or preventing tumors, the RIP2 inhibitor and ICI can be administered simultaneously or separately.

[0058] The “simultaneous administration” may be, for example, that the RIP2 inhibitor and the ICI are contained in a single pharmaceutical composition and administered simultaneously; or, alternatively, a “separate pharmaceutical composition comprising a RIP2 inhibitor” and a “separate pharmaceutical composition comprising an ICI” are administered simultaneously.

[0059] The "separate administration" approach can include, for example, administering a "separate pharmaceutical composition comprising a RIP2 inhibitor" and a "separate pharmaceutical composition comprising an ICI" separately at different times, for example, administering one of the "separate pharmaceutical composition comprising a RIP2 inhibitor" and the "separate pharmaceutical composition comprising an ICI" first, and the other subsequently. For another example, administering the "separate pharmaceutical composition comprising a RIP2 inhibitor" first and then the "separate pharmaceutical composition comprising an ICI"; or administering the "separate pharmaceutical composition comprising an ICI" first and then the "separate pharmaceutical composition comprising a RIP2 inhibitor."

[0060] The "single pharmaceutical composition comprising a RIP2 inhibitor" may be a pharmaceutical composition comprising the RIP2 inhibitor and a pharmaceutical excipient. The "single pharmaceutical composition comprising an ICI" may be a pharmaceutical composition comprising the ICI and a pharmaceutical excipient.

[0061] The separate administrations may be close in time or remote in time. In the separate administrations, each individual pharmaceutical composition may be administered with equal or unequal frequencies, for example, once every 4 days or once every 7 days.

[0062] Preferably, the RIP2 inhibitor is administered isofrequently during the treatment, and the ICI is administered isofrequently or unequally during the treatment.

[0063] Preferably, the RIP2 inhibitor is administered once every 4 days, for a total of 4 to 7 times, for example, on the 0th, 4th, 8th and 12th days.

[0064] Preferably, the ICI is administered at an unequal frequency, once every 4 days or every 7 days, for a total of 3, 4 or 5 times, for example, on days 0, 4, 11 and 18.

[0065] In the method for treating and / or preventing tumors, the RIP2 inhibitor can be administered at different doses according to different tumor conditions (tumor size, number of treatments, interval time, etc.), or according to the patient's weight. The general dosage is 0.1 mg / kg-5 mg / kg (single dose), for example, 1 mg / kg (single dose). For example, the dosage of GSK583 is 1 mg / kg (single dose).

[0066] In the method for treating and / or preventing tumors, the ICI can be administered in different doses according to different tumor conditions (tumor size, number of treatments, interval time, etc.), and can also be administered according to the patient's weight. The general dosage is 2 mg / kg-10 mg / kg (single dose), for example, 6 mg / kg (single dose). For example, the dosage of PD-L1 inhibitors (e.g., atezolizumab) is 6 mg / kg (single dose), and the dosage of PD-1 inhibitors (e.g., pembrolizumab) is 6 mg / kg (single dose).

[0067] Whether administered simultaneously or separately, the administration regimens of the RIP2 inhibitor and ICI (including administration route, administration dose, administration interval, etc.) can be the same or different, and can be adjusted by those skilled in the art as needed to provide the optimal therapeutic effect.

[0068] In one embodiment, the RIP2 inhibitor is administered by injection (eg, intravenous, subcutaneous, peritoneal, or intramuscular) or orally.

[0069] In one embodiment, the ICI is administered by injection (eg, intravenous, subcutaneous, peritoneal, or intramuscular).

[0070] In one embodiment, the RIP2 inhibitor and the ICI are both administered by injection, such as intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection.

[0071] In the method for treating and / or preventing tumors, the administration regimens (including administration route, administration dosage, administration interval, etc.) of the RIP2 inhibitor and the ICI may be the same or different, and may be adjusted by those skilled in the art as needed to provide the optimal therapeutic effect.

[0072] The present invention provides an application of an ICI in treating and / or preventing tumor diseases in mammals with RIP2 gene defects.

[0073] In the application, the tumor may be a tumor associated with the RIP2 signaling pathway, preferably intestinal cancer, melanoma, lung cancer, breast cancer, liver cancer, gastric cancer or pancreatic cancer.

[0074] The present invention provides the use of ICI in preparing a drug for treating and / or preventing tumor diseases in mammals with RIP2 gene defects.

[0075] In the application, the tumor may be a tumor associated with the RIP2 signaling pathway, preferably intestinal cancer, melanoma, lung cancer, breast cancer, liver cancer, gastric cancer or pancreatic cancer.

[0076] In the application, the ICI may be one or more of a programmed cell death receptor 1 (PD-1) inhibitor, a programmed cell death receptor 1 ligand (PD-L1) inhibitor, a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor, a B lymphocyte or T lymphocyte attenuating factor (BTLA) inhibitor, a T cell immunoglobulin mucin 3 (TIM-3) inhibitor, a T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitor, a V-domain Ig inhibitor of T cell activation (VISTA) inhibitor, a lymphocyte activation gene 3 (LAG-3) inhibitor, and an indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor, preferably including a PD-1 inhibitor and a PD-L1 inhibitor.

[0077] In the application, the programmed cell death receptor 1 (PD-1) inhibitor may be pembrolizumab.

[0078] In the application, the programmed cell death receptor 1 ligand (PD-L1) inhibitor may be atezolizumab.

[0079] In the application, the dosage of the ICI can be the conventional dosage in the art, and the general dosage is 2 mg / kg-10 mg / kg (single dose), for example, 6 mg / kg. For example, the dosage of the PD-L1 inhibitor (e.g., atezolizumab) is 6 mg / kg (single dose).

[0080] Preferably, the ICI is administered at an isofrequency or unequal frequency during the treatment.

[0081] In one embodiment, the ICI is administered unequally frequently, such as once every 4 days or once every 7 days.

[0082] In one embodiment, the ICI is administered at an unequal frequency, once every 4 days or every 7 days, for a total of 4 times, for example, on days 0, 4, 11, and 18.

[0083] In one embodiment, the ICI is administered by injection (eg, intravenous, subcutaneous, peritoneal, or intramuscular).

[0084] Explanation of terms:

[0085] Unless otherwise specified, the following terms used in this invention have the following meanings:

[0086] As used herein, the term "treat" refers to therapeutic treatment. With respect to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that leads to or causes the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0087] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical composition that, when administered to a patient, is sufficient to effectively treat a disease or condition described herein. The amount of the pharmaceutical composition that constitutes a "therapeutically effective amount" will vary depending on the pharmaceutical composition, the condition and its severity, and the age of the patient to be treated, but can be adjusted as needed by one skilled in the art.

[0088] The term "pharmaceutical composition" as used herein refers to a composition containing specified active ingredients that can be prepared into a single dosage form.

[0089] As used herein, the term "patient" refers to any animal, preferably a mammal, and most preferably a human, that is about to receive or has received a compound or composition according to embodiments of the present invention. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being the most preferred.

[0090] As used herein, the term "pharmaceutical excipients" refers to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These excipients are all substances contained in pharmaceutical preparations, other than the active ingredient. For a complete list, see the Pharmacopoeia of the People's Republic of China (2020 Edition), Part IV, or the Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009 Sixth Edition).

[0091] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0092] The reagents and raw materials used in the present invention are commercially available.

[0093] The positive progressive effect of the present invention is that the pharmaceutical composition of the present invention can enhance the anti-tumor activity of ICI drugs, and enhance the therapeutic effect of ICI drugs on tumors by upregulating the proportion of CD3+ cells, CD8+T cells, γδTCR+T cells, effector memory T cells, and CD8 effector T cells (IFNγ+CD8+T cells, GranzymeB+CD8+T cells, TNFα+CD8+T cells, CD107a+CD8+T cells) in the tumor microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 shows the wild type (WT) and RIP2 knockout (RIP2 - / - Figure 1A shows the construction scheme of the mouse colorectal cancer model. Figure 1B shows the treatment of C57BL / 6 WT and RIP2 with atezolizumab. - / - Dosage regimen for the mouse colorectal cancer tumor-bearing model.

[0095] Figure 2 shows that knocking out the RIP2 gene significantly enhances the inhibitory effect of the PD-L1 inhibitor (atezolizumab) on colorectal cancer growth in mice. Figure 2A is a graph showing the changes in tumor volume growth in different dosing groups. Figure 2B is a graph showing the tumor tissue weight results in different dosing groups. Figure 2C is a graph showing the tumor weight inhibition rate results in different dosing groups.

[0096] Figure 3 shows the construction of a C57BL / 6 mouse colorectal cancer (MC38) tumor model and the dosing schedule for the RIP2 inhibitor GSK583 combined with the PD-L1 inhibitor (atezolizumab). Figure 3A shows the construction schedule for the mouse colorectal cancer tumor model. Figure 3B shows the dosing schedule for GSK583 combined with atezolizumab in the mouse colorectal cancer tumor model.

[0097] Figure 4 shows that GSK583 significantly enhances the inhibitory effect of the PD-L1 inhibitor (atezolizumab) on the growth of colorectal cancer in mice. Figure 4A is a graph showing the changes in tumor volume growth in different dosing groups. Figure 4B is a graph showing the tumor tissue weight results in different dosing groups. Figure 4C is a graph showing the tumor weight inhibition rate results in different dosing groups.

[0098] Figure 5 shows that GSK583 combined with the PD-L1 inhibitor (atezolizumab) can increase the proportion of different types of immune cells in tumor tissues of colorectal cancer bearing models. Figure 5A shows the proportion of CD3+ cells in immune cells in tumor tissues of different dosing groups. Figure 5B shows the proportion of CD8+ cells in T cells in tumor tissues of different dosing groups. Figure 5C shows the proportion of CD4+ cells in T cells in tumor tissues of different dosing groups. Figure 5D shows the proportion of γδTCR+ T cells in T cells in tumor tissues of different dosing groups. Figure 5E shows the proportion of effector memory T cells in immune cells.

[0099] Figure 6 shows that GSK583 combined with the PD-L1 inhibitor (atezolizumab) can increase the proportion of CD8 effector T cells in tumor tissues of colorectal cancer bearing models. Figure 6A shows the proportion of GranzymeB+CD8+T cells in T cells in tumor tissues of different dosing groups. Figure 6B shows the proportion of IFNγ+CD8+T cells in T cells in tumor tissues of different dosing groups. Figure 6C shows the proportion of TNFα+CD8+T cells in T cells in tumor tissues of different dosing groups. Figure 6D shows the proportion of CD107a+CD8+T cells in T cells in tumor tissues of different dosing groups.

[0100] Figure 7 shows the construction of a C57BL / 6 mouse colorectal cancer (MC38) tumor model and the dosing regimen for the RIP2 inhibitor GSK583 combined with the PD-1 inhibitor (pembrolizumab). Figure 7A shows the construction scheme for the mouse colorectal cancer tumor model. Figure 7B shows the dosing regimen for GSK583 combined with pembrolizumab in the mouse colorectal cancer tumor model.

[0101] Figure 8 shows that GSK583 significantly enhances the inhibitory effect of the PD-1 inhibitor (pembrolizumab) on the growth of colorectal cancer in mice. Figure 8A is a graph showing the changes in tumor volume growth in different dosing groups. Figure 8B is a graph showing the tumor tissue weight results in different dosing groups. DETAILED DESCRIPTION

[0102] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not specify the manufacturer are conventional products available on the market.

[0103] Unless otherwise specified, “*” in the drawings of this application indicates significant differences, where “*” indicates p<0.05, “**” indicates p<0.01, “***” indicates p<0.001, “****” indicates p<0.0001, and “ns” indicates p>0.05, indicating no significant difference.

[0104] Example 1: Knockout of the RIP2 gene significantly enhances the inhibition of PD-L1 inhibitor (atezolizumab) on the growth of colorectal cancer (MC38) in C57BL / 6 mice

[0105] The test substance, atezolizumab (Roch) and the IgG control antibody (inVivoMAb human IgG1 isotype control; BioX cell, BE0297) were prepared in normal saline. Experimental animals and tumor lines: SPF-grade male wild-type (WT) C57BL / 6 mice (20-25 g), RIP2 gene knockout (RIP2 - / - Mice, 6-8 weeks old (20-25 g), were purchased from Southern Model Organisms Co., Ltd. Gene knockout was performed using CRISPR-Cas9 technology and bred at the Tsinghua University Animal Experimental Center. Cells used in the experiment were mouse colorectal cancer cells (MC38), purchased from the China Union Medical College Cell Bank.

[0106] According to the method shown in (Figure 1A and Figure 1B), the MC38 cell seeding concentration was 5×10 6 / ml, 0.1ml / mouse, subcutaneous inoculation, and wait until the tumor grows to a volume of 100mm 3 In the range of WT mice and RIP2 - / - Mice were randomly divided into two groups according to tumor volume, with 6-8 mice in each group, and the drug administration was started. The drug administration schedule was as follows: ①WT-blank antibody control group (WT-IgG control, 150μg / mouse); ②WT-atezolizumab group (150μg / mouse); ③RIP2 - / - Blank antibody control group (RIP2 - / - -IgG control, 150 μg / mouse); ④RIP2 - / - - Atezolizumab group (150 μg / animal). IgG control antibody and atezolizumab were administered by intraperitoneal injection on Days 0, 4, 11, and 18, for a total of four doses. The day of group administration was designated Day 0. During the administration process, the animals were weighed every 2-3 days, and the long and short diameters of the tumors were measured with a vernier caliper. The formula was: (1 / 2) × long diameter × (short diameter) 2 Tumor size was calculated. The experiment ended on day 20, mice were euthanized, tumors were removed and weighed, and the tumor inhibition rate was calculated using the formula: (tumor weight of the treated group - average tumor weight of the control group) / average tumor weight of the control group * 100%).

[0107] Test results (as shown in Figure 2):

[0108] In WT and RIP2 - / -In the C57BL / 6 mouse colorectal cancer (MC38) tumor-bearing model, compared with the corresponding IgG control group, atezolizumab can significantly inhibit tumor growth, and the growth rate of tumor volume is slower than that of the corresponding control group, and RIP2 - / - -The inhibitory effect of atezolizumab group was more significant; compared with WT mice, atezolizumab had a significant inhibitory effect on RIP2 - / - The effect of inhibiting tumor growth in mice was also significantly improved (Figure 2A). After administration, the tumor weight of atezolizumab was significantly increased in WT and RIP2 mice compared with the tumor weight of the corresponding control group. - / - The results showed that atezolizumab significantly reduced the expression of RIP2 in the RIP2 - / - The tumor inhibition effect in WT mice was significantly stronger than that in WT mice (Figure 2B, Figure 2C). This shows that knocking out RIP2 significantly enhances the inhibitory effect of PD-L1 inhibitors on the growth of colorectal cancer in mice.

[0109] Example 2: RIP2 inhibitor GSK583 significantly enhances the inhibition of PD-L1 inhibitor (atezolizumab) on the growth of colorectal cancer (MC38) in C57BL / 6 mice in vivo

[0110] The test substance, atezolizumab (Roch) and the IgG control antibody (inVivoMAb human IgG1 isotype control; BioX cell, BE0297), were prepared in normal saline. GSK583 was prepared as a 20× DMSO stock solution and diluted to the dosing concentration immediately before use using normal saline containing 5% Cremophor EL.

[0111] Experimental Animals and Tumor Lines: SPF-grade male wild-type C57BL / 6 mice, 6-8 weeks old (20-25 g), were purchased from the Animal Experiment Center of Tsinghua University. Cells used in the experiment were mouse colorectal cancer cells (MC38), purchased from the China Union Medical College Cell Bank.

[0112] According to the method shown in (Figure 3A and Figure 3B), the MC38 cell seeding concentration was 5×10 6 / ml, 0.1ml / mouse, subcutaneous inoculation, and wait until the tumor grows to a volume of 100mm 3Within the range, mice were randomly divided into 4 groups according to tumor volume, with 6-8 mice in each group, and drug administration began. The dosing regimen was: ① blank antibody control group (IgG control, 150μg / mouse); ② GSK583 (1mg / kg) group; ③ atezolizumab group (150μg / mouse); ④ atezolizumab (150μg / mouse) + GSK583 (1mg / kg). GSK583 was administered intravenously once every 4 days, for a total of 4 times; IgG control antibody and atezolizumab were administered by intraperitoneal injection on DAY 0, 4, and 11, for a total of 3 times. The group administration day was set as DAY 0. During the administration process, the animals were weighed every 2-3 days, and the long and short diameters of the tumor were measured with a vernier caliper. The formula: (1 / 2) × long diameter × (short diameter) 2 The tumor size was calculated. The experiment ended on DAY 15, and the mice were euthanized. The tumors were removed and weighed, and the tumor inhibition rate was calculated.

[0113] Test results (as shown in Figure 4):

[0114] In a C57BL / 6 mouse colorectal cancer (MC38) tumor-bearing model, both the atezolizumab group and the atezolizumab + GSK583 combination group significantly inhibited tumor growth compared to the IgG control group, with tumor volume growth rates slower than in the control group (Figure 4A). After administration, tumor weights in both groups were significantly and statistically significantly reduced compared to the control group (Figures 4B and 4C). The atezolizumab + GSK583 group exhibited a significantly stronger tumor inhibitory effect than the atezolizumab alone group (Figures 4B and 4C). GSK583 alone had no significant inhibitory effect on tumor growth (Figures 4A, 4B, and 4C). This suggests that the RIP2 inhibitor GSK583 significantly enhances the inhibitory effect of PD-L1 inhibitors on the growth of colorectal cancer in mice in vivo.

[0115] Example 3: Flow cytometry detection of the proportion of immune cells in tumor tissues of colorectal cancer-bearing mice after treatment with GSK583 combined with PD-L1 inhibitor (atezolizumab)

[0116] Preparation of enzyme lysis buffer: Take 1g collagenase IV (Collagenase; Sigma, Cat: C5138), 100mg hyaluronidase (Hyaluronidase; Sigma, Cat: H6254) and 20,000 units of DNase, type IV (Sigma Cat: D5025) and add to 80ml balanced salt solution (Hanks Balanced Salt Solution, HBSS; EallBio Life Science, Cat: 03.15009C), mix well and add HBSS solution to the volume of 100ml to obtain 100× tissue lysis buffer. After filtering with a 0.22μm filter membrane, store at -20℃ and dilute to 1× with HBSS before use.

[0117] Tumor tissue dissociation: Tumor tissue obtained from each group in Example 2 was removed and weighed (0.04-1 g). The tissue was then cut into 2×2 mm pieces using a scalpel. 2.5 ml of lysis mixture was added and the pieces were transferred to a gentleMACS C Tube (Miltenyl Biotec; Cat: 130-096-334). The C Tube was inverted on a gentleMACS Dissociator (Miltenyl Biotec; Cat: 130-093-235) and the program "gentleMACS Program m_impTumor_02" was run. The C Tube was removed from the gentleMACS Dissociator and incubated at 37°C for 40 min. The C Tube was inverted again on the gentleMACS Dissociator and the program "gentleMACS Program m_impTumor_03" was run.

[0118] Filter using a 70 μm filter membrane (BD biosciences, Cat: 352235), centrifuge at 300 × g for 7 min, and discard the supernatant; add 5 ml of red blood cell lysis buffer (Biolegend, Cat: 420301) and lyse at room temperature for 5 min, add 10 ml of phosphate-buffered saline (PBS; EallBio Life Science, Cat: 03.15018C) to terminate the reaction; centrifuge at 1000 rpm at 4°C for 5 min, discard the supernatant; add Cell Staining Buffer (Biolegend, Cat: 420201) and mix thoroughly.

[0119] Flow cytometry detection: After counting the isolated tumor tissue dissociation solution, adjust the cell concentration to 200 μl / well (5-10×10 5 cells / well) were added to a 96U plate; centrifuged at 350×g for 5 min, and the supernatant was discarded; blocking solution was added at 100 μl / well to CD16 / CD32 (Biolegend; Cat: 101302) in a cell staining buffer at a ratio of 1:200 and incubated at 4°C in the dark for 15-20 min; then centrifuged at 350×g for 5 min, and the supernatant was discarded; the prepared antibody mixture was added at 100 μl / well and incubated at 4°C in the dark for 15-20 min. Incubate with light for 30 minutes; centrifuge at 350×g for 5 minutes and discard the supernatant; then add 100 μl / well of cell staining buffer for washing, centrifuge at 350×g for 5 minutes, discard the supernatant, and repeat washing twice; after the final wash, resuspend the cells in 4% paraformaldehyde (Biyuntian, P0099) at 100 μl / well and incubate at 4°C overnight; centrifuge at 350×g for 5 minutes and discard the supernatant; resuspend the cells in 100 μl / well of cell staining buffer and then detect on the instrument.

[0120] Experimental results (as shown in Figure 5 or Figure 6):

[0121] Compared with the IgG control group, the GSK583 alone group, and the atezolizumab alone group, the proportions of CD3+ cells, CD8+ T cells, γδTCR+ T cells, and effector memory T cells in tumor tissues in the atezolizumab and GSK583 combined group were significantly increased, while the proportion of CD4+ T cells was not affected (Figures 5A-5E). Further detection of different types of CD8 effector T cells revealed that the proportions of IFNγ+CD8+T cells, GranzymeB+CD8+T cells, TNFα+CD8+T cells, and CD107a+CD8+T cells in CD8 effector T cells were significantly increased in the atezolizumab and GSK583 combined group (Figures 6A-6D). Therefore, it can be judged that the combined use of RIP2 inhibitors and ICIs upregulates the proportions of CD3+ cells, CD8+T cells, γδTCR+T cells, and effector memory T cells in the tumor microenvironment; and increases the proportions of IFNγ+CD8+T cells, GranzymeB+CD8+T cells, TNFα+CD8+T cells, and CD107a+CD8+T cells in CD8 effector T cells, thereby enhancing the effect of ICI drugs in inhibiting tumor growth.

[0122] Example 4: RIP2 inhibitor GSK583 significantly enhances the inhibition of PD-1 inhibitor (pembrolizumab) on the growth of colorectal cancer (MC38) in C57BL / 6 mice

[0123] The test substance, pembrolizumab (Merck, Inc.) and the IgG control antibody (inVivoMAb human IgG1 isotype control; BioX cell, BE0297), were prepared in normal saline. GSK583 was prepared as a 20× stock solution in DMSO and diluted to the dosing concentration with normal saline containing 5% Cremophor EL immediately before use.

[0124] Experimental Animals and Tumor Lines: SPF-grade male wild-type C57BL / 6 mice, 6-8 weeks old (20-25 g), were purchased from the Animal Experiment Center of Tsinghua University. Cells used in the experiment were mouse colorectal cancer cells (MC38), purchased from the China Union Medical College Cell Bank.

[0125] According to the method shown in (Figure 7A and Figure 7B), the MC38 cell seeding concentration was 5×10 6 / ml, 0.1ml / mouse, subcutaneous inoculation, and wait until the tumor grows to a volume of 100mm 3 Within the range, mice were randomly divided into 3 groups according to tumor volume, with 6-7 mice in each group, and drug administration began. The dosing regimen was: ① blank antibody control group (IgG control, 150μg / mouse); ② pembrolizumab group (150μg / mouse); ③ pembrolizumab (150μg / mouse) + GSK583 (1mg / kg) group. GSK583 was administered intravenously once every 4 days, for a total of 7 times; IgG control antibody was administered intraperitoneally on DAY0, 4, 11, and 18, for a total of 4 times; pembrolizumab was administered intraperitoneally on DAY0, 4, 11, 18, and 25, for a total of 5 times. The group administration day was set as DAY0. During the drug administration process, the animals were weighed every 2-3 days, and the long and short diameters of the tumor were measured with a vernier caliper. The formula: (1 / 2)×long diameter×(short diameter) 2 Calculate tumor size. The average tumor volume of mice in the IgG control group on DAY 20 was close to 2000 mm 3 The mice in this group were euthanized; the experiment was ended on DAY28, and the mice in the pembrolizumab group and the pembrolizumab (150 μg / mouse) + GSK583 (1 mg / kg) group were euthanized, and the tumors were removed and weighed after dissection.

[0126] Test results (as shown in Figure 8):

[0127] In a C57BL / 6 mouse colorectal cancer (MC38) tumor-bearing model, both pembrolizumab and pembrolizumab + GSK583 significantly inhibited tumor growth compared with the IgG control group on Day 20, with tumor volume growth rates slower than those in the control group (Figure 8A). After the end of dosing on Day 28, tumor weight in the pembrolizumab + GSK583 group was significantly and statistically reduced compared with the pembrolizumab group (Figure 8B). This suggests that the RIP2 inhibitor GSK583 significantly enhances the inhibitory effect of PD-1 inhibitors on the growth of colorectal cancer in mice in vivo.

[0128] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A pharmaceutical composition of a RIP2 inhibitor combined with an ICI, comprising a RIP2 inhibitor and an ICI.

2. The pharmaceutical composition according to claim 1, wherein The composition satisfies one or more of the following conditions: (1) The pharmaceutical composition further comprises a pharmaceutical excipient; (2) The ICI is one or more of a programmed cell death receptor 1 inhibitor, a programmed cell death receptor 1 ligand inhibitor, a cytotoxic T lymphocyte-associated protein 4 inhibitor, a B lymphocyte or T lymphocyte attenuating factor inhibitor, a T cell immunoglobulin mucin 3 inhibitor, a T cell immunoreceptor inhibitor with Ig and ITIM domains, a V-domain Ig inhibitor of T cell activation, a lymphocyte activation gene 3 inhibitor, and an indoleamine 2,3-dioxygenase 1 inhibitor; preferably, the ICI includes a PD-L1 inhibitor and / or a PD-1 inhibitor, the PD-L1 inhibitor may be atezolizumab, and the PD-1 inhibitor may be pembrolizumab; (3) The mass ratio of the RIP2 inhibitor to the ICI is 1:(1-60), for example, 1:6, 1:10, 1:15, 1:20, 1:30 or 1:60, preferably 1:(6-10); and (4) the RIP2 inhibitor is one or more of GSK583, WEHI-345, SB203580, OD36, OD38 and derivatives thereof, preferably GSK583 and / or derivatives thereof; The derivative of GSK583 is preferably GSK2983559.

3. The pharmaceutical composition according to claim 1 or 2, wherein The pharmaceutical composition consists of a RIP2 inhibitor and an ICI; Preferably, the pharmaceutical composition is GSK583 and a PD-1 inhibitor, such as GSK583 and pembrolizumab, and the mass ratio of the GSK583 to the PD-1 inhibitor is further preferably 1:6 to 1:10; alternatively, the pharmaceutical composition is GSK583 and a PD-L1 inhibitor, such as GSK583 and atezolizumab, and the mass ratio of the GSK583 to the PD-L1 inhibitor is further preferably 1:6 to 1:

10.

4. The pharmaceutical composition according to at least one of claims 1 to 3, characterized in that The pharmaceutical composition satisfies one or more of the following conditions: (1) The RIP2 inhibitor is administered at a dose of 0.1 mg / kg to 5 mg / kg, for example, 1 mg / kg; (2) The ICI is administered at a dose of 2-10 mg / kg, for example, 6 mg / kg; (3) the RIP2 inhibitor is administered once every 4 days; (4) the ICI is administered once every 4 days or every 7 days; and (5) the RIP2 inhibitor is administered at an equal frequency during the treatment, and the ICI is administered at an equal frequency or an unequal frequency during the treatment.

5. The pharmaceutical composition according to at least one of claims 1 to 4, characterized in that The pharmaceutical composition satisfies one or more of the following conditions: (1) The RIP2 inhibitor is administered once every 4 days, for a total of 4 to 7 times, for example, on days 0, 4, 8, and 12; (2) The ICI is administered at an unequal frequency, once every 4 days or every 7 days, for a total of 3, 4, or 5 times, for example, on days 0, 4, 11, and 18; and (3) the RIP2 inhibitor and the ICI are both administered by injection, such as intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection.

6. Use of the pharmaceutical composition according to at least one of claims 1 to 5 in the preparation of a medicament for treating and / or preventing tumors.

7. The use according to claim 6, characterized in that The tumor is a tumor related to the RIP2 signaling pathway, preferably colorectal cancer, melanoma, lung cancer, breast cancer, liver cancer, gastric cancer or pancreatic cancer.

8. A method for treating and / or preventing tumors, characterized in that: It comprises administering a therapeutically effective amount of at least one pharmaceutical composition according to claims 1 to 5 to a patient in need thereof.

9. The method according to claim 8, wherein The RIP2 inhibitor is administered at a dose of 0.1 mg / kg-5 mg / kg, for example, 1 mg / kg of GSK583; or the ICI is administered at a dose of 2 mg / kg-10 mg / kg, for example, 6 mg / kg of atezolizumab or pembrolizumab.

10. The method according to claim 8 or 9, characterized in that The method satisfies one or more of the following conditions: (1) The RIP2 inhibitor is administered at a dose of 1 mg / kg; (2) The ICI is administered at a dose of 6 mg / kg; (3) the RIP2 inhibitor is administered once every 4 days, for a total of 4 to 7 times, for example, on days 0, 4, 8, and 12; (4) The ICI is administered at an unequal frequency, once every 4 days or every 7 days, for a total of 3, 4, or 5 times, for example, on days 0, 4, 11, and 18; (5) The RIP2 inhibitor and the ICI are both administered by injection, such as intravenous injection, subcutaneous injection, intramuscular injection or intraperitoneal injection; and (6) the tumor is a RIP2-related tumor, preferably melanoma, lung cancer, breast cancer, intestinal cancer, gastric cancer or pancreatic cancer.

11. Use of ICI in the preparation of a drug for treating and / or preventing tumor diseases in mammals with RIP2 gene defects.

12. The use according to claim 11, characterized in that The application meets one or more of the following conditions: (1) The tumor is a tumor related to the RIP2 signaling pathway, preferably colorectal cancer, melanoma, lung cancer, breast cancer, liver cancer, gastric cancer or pancreatic cancer; (2) The ICI is one or more of a programmed cell death receptor 1 inhibitor, a programmed cell death receptor 1 ligand inhibitor, a cytotoxic T lymphocyte-associated protein 4 inhibitor, a B lymphocyte or T lymphocyte attenuating factor inhibitor, a T cell immunoglobulin mucin 3 inhibitor, a T cell immunoreceptor inhibitor with Ig and ITIM domains, a V-domain Ig inhibitor of T cell activation, a lymphocyte activation gene 3 inhibitor, and an indoleamine 2,3-dioxygenase 1 inhibitor; preferably, the ICI comprises a PD-L1 inhibitor and / or a PD-1 inhibitor, the PD-L1 inhibitor may be atezolizumab, and the PD-1 inhibitor may be pembrolizumab; (3) The ICI is administered at a dose of 2-10 mg / kg, for example, 6 mg / kg; (4) The ICI is administered at an unequal frequency, once every 4 days or once every 7 days; and (5) the ICI is administered by injection, such as intravenous injection, subcutaneous injection, peritoneal injection or intramuscular injection.

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