New uses of phenothiazines or compounds with similar structure in pharmaceuticals.

Abstract

To provide a pharmaceutical composition and a kit for cancer treatment, and to provide a novel use of phenothiazine compound in drug preparation.SOLUTION: Provided is a pharmaceutical composition for cancer treatment, containing: (a) an immune cell suitable for adoptive cell therapy as a first active ingredient; and (b) as a second active ingredient, a free phenothiazine compound selected from a compound of formula (I), a hydrate, a solvate or a reduced form thereof. The immune cells are selected from the group consisting of tumor-infiltrating lymphocytes, chimeric antigen receptor T cells, chimeric antigen receptor NK cells, and T cell receptor chimeric T cells, and the phenothiazine compounds are useful as PD-1 signal transduction inhibitors as it can inhibit PD-1 function and block PD-1 signal transduction.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of pharmaceutical technology, in particular to a new use of phenothiazines or compounds with similar structures, their hydrates, solvates or reduced forms in pharmaceuticals. Another aspect of the present invention relates to the combination of phenothiazine compounds with adoptive cell therapy. Another aspect of the present invention relates to the combination of phenothiazine compounds with tumor immunochemotherapeutic agents, in particular PD-1 antibodies. [Background technology]

[0002] Adoptive cell therapy is a therapeutic method that involves harvesting one or more different types of immune cells from a mammal, culturing and / or manipulating the harvested immune cells ex vivo, and returning the cultured and / or manipulated immune cells to the mammal. The ex vivo manipulation of the harvested immune cells includes introducing a recombinant nucleic acid into the immune cells. Adoptive cell therapy includes, but is not limited to, tumor infiltrating lymphocytes (TILs), lymphokine activated killer cells (LAKs), cytokine induced killer cells (CIKs), dendritic cells (DCs), natural killer cells (NKs), T cell receptor TCR-modified T cells (TCR-Ts), chimeric antigen receptor NK cells (CAR-NKs), and chimeric antigen receptor T cells (CAR-Ts).

[0003] The human immune system can combat and eliminate pathogenic microorganisms and mutated cells that invade from the outside, and immune cells are important cells that perform the human immune function. Among them, T cells are important immune cells in the human body, and can recognize antigens presented by major histocompatibility complexes (MHC) on target cells through antigen receptors (TCRs) expressed on their cell surface, which is called the first signal. In synergy with an appropriate second signal (e.g., B7 molecules) from the target cell, T cells are activated and exert their functions. For example, cytotoxic T cells (abbreviated as CTLs), a subclass of T cells, can eliminate virus-infected cells and tumor cells by directly killing target cells after activation.

[0004] During the process of T cell activation, T cells express negative feedback to initiate several molecules that inhibit their immune function, and PD-1 (i.e., programmed cell death 1) is one of the best-known molecules among them. When the PD-1 protein on the surface of CTL cells is stimulated by its ligand (usually PD-L1 on target cells (e.g., tumor cells)), PD-1 initiates its signal transduction, inhibiting the function of CTLs and causing apoptosis or anergy of T cells. Such signal transduction causes CTLs to lose their ability to kill target cells. Taking human tumor cells as an example, these tumor cells usually express PD-L1 on their cell surface, inhibiting their killing by CTLs, thereby allowing them to avoid immune monitoring. On the other hand, blocking the interaction between PD-L1 and PD-1 with drugs inhibits the signal transduction of PD-1 and restores the killing ability of CTLs against tumor cells. In addition to T cells, other immune cells can also express PD-1. For example, expression of PD-1 has been reported in macrophages, B cells, etc. Inhibiting the function of PD-1 with an appropriate drug can restore the function of these immune cells to some extent. For example, inhibiting the function of PD-1 changes macrophages from M2 (i.e., a type that inhibits the function of CTLs) to M1 (i.e., a type that promotes the function of CTLs) and eliminates tumor cells in the body. Currently, biologics such as antibody-based drugs have been approved by the FDA for clinical treatment. However, all drugs against PD-1 that have been used clinically so far are biologic antibodies, and no small molecule PD-1 signaling inhibitors have been reported.

[0005] https: / / baike.baidu.com / item / %E6%B0%AF%E5%8C%96%E7%89%A9https: / / baike.baidu.com / item / %E6%9F%93%E6%96%99Methylene blue (3,7-bis(dimethylamino)phenothiazine-5-ium chloride) is a phenothiazine salt, which is widely applied in chemical indicators, dyes, biological stains, etc. Recently, some studies have reported its use in medicine, for example, China Patent Publication No. CN 104027338A discloses a new use of methylene blue for anti-acute cerebral ischemia, and China Patent Publication No. CN 103417546B discloses a new use of methylene blue for promoting post-anesthetic awakening. In addition, some literature has disclosed that methylene blue can be used as a photosensitizer to prepare photosensitizers to perform photodynamic therapy. Researchers have found that when traditional photodynamic therapy is used for internal treatment, the intensity of the excitation light is attenuated due to the absorption and scattering of light by biological tissues, and the oxygen-deficient state of malignant tumor tissue reduces the production of singlet oxygen; therefore, when a simple photosensitizer is used as a photosensitizing drug, it does not have a full-fledged tumor treatment effect (Chinese Patent Publication No. CN 106668859A).

[0006] Therefore, although the prior art has disclosed some uses of methylene blue in medicine, there are no reports of the application of methylene blue to PD-1 signaling inhibition and its application in tumor prevention and treatment. At present, in clinical practice, there is still a lack of sufficiently effective drugs to treat cancer, so providing effective drugs for tumors has important clinical significance. Summary of the Invention

[0007] The present invention provides a pharmaceutical composition comprising (a) immune cells suitable for adoptive cell therapy, and (b) a phenothiazine compound selected from the compound of formula (I), a hydrate, solvate or reduced form thereof. [ka] (where Z is S+ , O + , C or N; Y is N or N + and Z is S + Or O + When Z is selected from the group consisting of C and N, then Y is N. + and X - is Z + Or N + To achieve electrical neutrality, one or more anions capable of forming a salt with R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted arylalkoxy, thioalkoxy, amine, nitro, amino, and halogen.

[0008] In some embodiments, the X is selected from an inorganic acid radical anion and an organic acid radical anion. Further, the inorganic acid radical anion is preferably Cl. - , Br - , I - The organic acid radical anion is preferably a methanesulfonate radical anion, an ethanesulfonate radical anion, a p-toluenesulfonate radical anion, a benzenesulfonate radical anion, an ethanedisulfonate radical anion, a propanedisulfonate radical anion, and a naphthalenedisulfonate radical anion.

[0009] In some embodiments, R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10are each independently selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted C5-C10 aryl, substituted or unsubstituted 5-10 membered heteroaryl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted arylalkoxy, thioalkoxy, amine, nitro, amino, and halogen. In some embodiments, the phenothiazine-based compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

[0010] In some embodiments, the phenothiazine-based compound is selected from 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of Formula II, a hydrate, solvate, or reduced form thereof. [ka] (Here, X is one or more anions, thereby achieving electrical neutrality, and has the same meaning as above.)

[0011] In some embodiments, the reduced form of the phenothiazine-based compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, a hydrate or solvate thereof. [ka] (Here, X is one or more anions, thereby achieving electrical neutrality, and has the same meaning as above.)

[0012] In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium Dimesylate It is.

[0013] In some embodiments, the immune cells are selected from tumor infiltrating lymphocytes (TILs), chimeric antigen receptor T cells (CAR-Ts), chimeric antigen receptor NK cells (CAR-NKs), and T cell receptor (TCR) chimeric T cells (TCR-Ts).

[0014] In some embodiments, the immune cells are T cell receptor (TCR) chimeric T cells.

[0015] In some embodiments, the TCR is capable of binding to the SIINFEKL peptide.

[0016] Yet another aspect of the invention provides a kit comprising: a) any of the immune cells described above formulated in a first dosage form; and b) any of the compounds described above formulated in a second dosage form.

[0017] Yet another aspect of the present invention provides the use of any of the above compounds in the manufacture of a medicament for the medicament for treating cancer.

[0018] In some embodiments, the cancer is melanoma, breast tumor, lung cancer, prostate cancer, breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, lymphoma, esophageal cancer, bladder cancer, urethral cancer, non-Hodgkin's lymphoma, renal cancer, or brain cancer.

[0019] Another aspect of the invention also provides the use of any of the above phenothiazine compounds in the manufacture of a medicament for enhancing immune cell function.

[0020] Another aspect of the invention provides the use of any of the above phenothiazine compounds in the manufacture of a medicament for inhibiting PD-1 signaling.Another aspect of the invention provides the use of any of the above phenothiazine compounds in the manufacture of a medicament for blocking PD-1 downstream signaling pathways.Another aspect of the invention provides the use of any of the above phenothiazine compounds in the manufacture of a medicament for blocking SHP2 protein recruitment by PD-1.

[0021] Another aspect of the invention provides a method of inhibiting PD-1 signaling comprising blocking a PD-1 downstream signaling pathway. In some embodiments, the blocking is achieved by blocking PD-1-induced recruitment of SHP2 protein. In some embodiments, the blocking of PD-1-induced recruitment of SHP2 comprises contacting a cell with an effective amount of any of the phenothiazine compounds described herein.

[0022] Another aspect of the invention provides a method of treating cancer in a subject, the method comprising inhibiting PD-1 signaling in the subject. In some embodiments, inhibiting PD-1 signaling in the subject comprises blocking a PD-1 downstream signaling pathway. In some embodiments, blocking the PD-1 downstream signaling pathway comprises blocking SHP2 recruitment by PD-1. In some embodiments, the step of blocking SHP2 recruitment by PD-1 comprises administering to the subject an effective amount of any of the phenothiazine compounds described herein.

[0023] Another aspect of the invention provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of any of the phenothiazine compounds described herein and administering a second therapy to the subject. In some embodiments, the second therapy is selected from chemotherapy, radiation therapy, and surgery. In some embodiments, the chemotherapy is tumor immunotherapy. In some embodiments, the tumor immunotherapy comprises administering to the subject a PD-1 antibody or a PD-L1 antibody or a functional fragment thereof. In some embodiments, the method according to this aspect exerts a synergistic anti-tumor effect.

[0024] Another aspect of the present invention provides a pharmaceutical composition for treating cancer, the pharmaceutical composition comprising any of the phenothiazine compounds described in the present invention and a second chemotherapeutic agent. In some embodiments, the second chemotherapeutic agent is a tumor immunotherapeutic agent. In some embodiments, the tumor immunotherapeutic agent is a PD-1 antibody or a PD-L1 antibody or a functional fragment thereof.

[0025] Another aspect of the invention provides a method of enhancing the therapeutic effect of a PD-1 or PD-L1 antibody, or a functional fragment thereof, in a subject, comprising administering to the subject an effective amount of any of the phenothiazine compounds described in the invention prior to, simultaneously with, or following administration of the PD-1 or PD-L1 antibody, in some embodiments exerting a synergistic anti-tumor effect.

[0026] Through a great deal of creative work, the inventors of the present invention have discovered that the combination of the phenothiazines of the present invention and compounds having a similar structure with immune cells (especially genetically modified immune cells) can significantly improve the killing action of the immune cells against target cells and achieve a synergistic effect. The phenothiazines of the present invention and compounds having a similar structure can inhibit the function of PD-1 and block PD-1 signaling, and are useful as PD-1 signaling inhibitors.

[0027] In vitro experiments have found that such compounds can restore the functions of immune cells inhibited by PD-1, improve the cytokine secretion and target cell killing functions of immune cells such as CTLs, and improve the immune function of the living body. In animal models, the phenothiazines of the present invention and compounds having similar structures can shrink transplanted tumors or in situ lung cancer in mice, thereby achieving the purpose of treating cancer. Furthermore, compared to large molecules such as PD-1 antibodies, small molecule PD-1 signaling inhibitors have the advantages of low cost, simple manufacturing technology (e.g., by chemical synthesis), multiple administration routes, high patient compliance, safety and high reliability. In addition, experiments of the present invention have also demonstrated that the phenothiazine compounds of the present invention have tumor inhibitory effects equal to or greater than those of PD-1 antibodies. [Brief description of the drawings]

[0028] [Figure 1] 1 shows the experimental results of Example 1. A shows the expression of PD-1 protein on the surface of untransfected and transfected Jurkat cells, B shows the expression of PD-L1 protein on the surface of untransfected and transfected Raji cells, C shows the result of transfected Raji cells inducing phosphorylation of PD-1 protein Y248 in transfected Jurkat cells, D shows the result of MTC promoting IL-2 secretion in Jurkat-PD-1-NFAT-luc cells, and E shows that MTC or LMT (N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium dimesylate) promotes the increase of luciferase activity in Jurkat-PD-1-NFAT-luc cells. [Diagram 2] 1 shows the promotion of OT-1 cell division by MTC in Example 2. FIG. 1A shows the result of expressing PD-1 protein on the surface of OT1-CTL cells, and FIG. 1B shows the result of MTC promoting CTL division. [Diagram 3]In Example 2, promotion of effector molecule secretion from OT-1 cells by MTC is shown, and MTC promotes secretion of IL-2, IFNγ, perforin and granzyme B from CTL cells. [Figure 4] (A) shows that MTC restores the ability of OT-1 T cells to kill target cells, (B) shows that MTC promotes the killing of EL4-OVA (PD-L1) by CTLs, (C) shows the induction of PD-L1 protein expression in B16-F10 by IFNγ, (D) shows that MTC or LMT synergistically increase the killing of B16-OVA cells by CTLs, and (E) shows that MTC or LMT have no killing effect on B16-WT cells and no killing effect on cells even when combined with CTL cells. [Diagram 5] Figure 1 shows the results of MTC promoting the elimination of tumors formed by target cells by cytotoxic T cells (CTLs). A is a schematic diagram of the transplanted tumors, B, C, and D show that MTC inhibits the growth of transplanted tumors in Rag1- / - mice, and E shows that MTC inhibits the growth of transplanted tumors in C57BL / 6J mice. [Figure 6] Figure 1 shows the results of primary tumor treatment with MTC. A is a schematic diagram of an experiment to treat a tumor in situ, and B shows the results of MTC eliminating the tumor through CD8+ T cells. [Figure 7] FIG. 1 shows the results of MTC blocking PD-1-induced SHP2 recruitment. A demonstrates that MTC reduces the binding of PD1 to SHP2, B shows that MTC inhibits PD1-induced SHP2 recruitment, and C shows that MTC inhibits PD1-induced SHP2 binding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The term "adoptive cell therapy" involves the transfer of immune cells with anti-tumor activity into a cancer patient. In some embodiments, ACT is a treatment method that involves using lymphocytes with anti-tumor activity, expanding these cells in vitro to large numbers, and injecting these cells into a host suffering from cancer.

[0030] The term "tumor infiltrating lymphocytes" or TILs refers to white blood cells that have left the bloodstream and migrated to a tumor. The term "CAR-T" is an abbreviation for "chimeric antigen receptor T cells," and the chimeric antigen receptor (CAR) is the core component of CAR-T, which confers the T cells with the ability to recognize antigens on target cells (e.g., tumors) in an HLA-independent manner, allowing CAR-modified T cells to recognize a broader range of targets compared to the native T cell surface receptor TCR.

[0031] The term "TCR-T (T cell receptor (TCR) chimeric-T cell)" refers to a T cell expressing an engineered TCR or an artificial TCR. The engineered T cell receptor or artificial T cell receptor is genetically modified to have a structure that targets an antigen of interest while retaining domains and / or helper molecules in the TCR signaling pathway. In some embodiments, the TCR-T retains all the helper molecules in the TCR signaling pathway, and therefore, with a small amount of antigen stimulation, it can become fully activated and cause a killing effect on the target cell. Compared to CAR-T, these TCR-T retain and apply all the helper molecules in the TCR signaling pathway, and therefore, the TCR-T has a higher recognition sensitivity to low-concentration, low-copy-number antigens than some CAR-Ts, and is expected to have therapeutic potential.

[0032] Tyrosine protein phosphatase non-receptor type 11 (PTPN11), also called protein tyrosine phosphatase 1D (PTP-1D), is an enzyme that contains domain phosphatase-2 (SHP-2) or protein tyrosine acid phosphatase 2C (PTP-2C) in Src homology region 2 and is encoded by the human PTPN11 gene. SHP-2 is widely expressed in various tissues and cell types and is involved in multiple signaling pathways, including growth factors such as PDGF, EGF, and IGF-1, cytokines such as IL-3, GM-CSF, and EPO, and insulin and interferon. SHP-2 has a compound signaling function. Various signaling processes, such as the Ras-Raf-MAP-ERK pathway, the Jak-Stat pathway, and the PI3K-Akt pathway, appear to be involved. It has also been shown that it can bind to various signal intermediates, including Grb2, FRS2, Jak2, p85 subunit of PI3 kinase, IRS-1, Gab1, and Gab2. SHP-2, a downstream molecule of the PD-1 receptor, is involved in the inhibitory signal transduction of T cells. Research has shown that SHP-2 is a downstream molecule of PD-1 signaling and not only inhibits T cell activation but also promotes T cell dysfunction. Research has also shown that knocking out SHP2 on T lymphocytes induces antitumor immunity and inhibits the development of murine colitis-associated cancer.

[0033] Methionine (MT) is a redox molecule and, depending on the environmental conditions (e.g., pH, oxygen, reducing agents), it can form either a reduced form, a 10H-phenothiazine-based compound (i.e., N,N,N',N'-tetramethyl-10H-phenothiazine-3,7-diammonium (LMT)), or an oxidized form (MT + ) exists in equilibrium with

[0034] The oxidized form of the salt MTX is shown in Formula II. [ka] When LMT exists in the form of a salt thereof, it is referred to as a LMTX salt, as shown in formula III. [ka] X - is selected from inorganic acid radical anions and organic acid radical anions, and examples of suitable organic acid radical anions include 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethylenedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxyacetic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethylsulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, lysine acid, glycerol ... The organic acid anions include, but are not limited to, organic acid anions derived from organic acids such as pentane, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, benzenesulfonic, propanedisulfonic, propionic, pyruvic, salicylic, stearic, succinic, p-aminobenzenesulfonic, tartaric, toluenesulfonic, p-toluenesulfonic, naphthalenedisulfonic, and valeric acids. Suitable polymeric organic anions include, but are not limited to, polymeric organic anions derived from polymeric acids such as tannic acid and carboxymethylcellulose. Furthermore, the inorganic acid root anion is preferably Cl-, Br-, or I-. The organic acid root anion is preferably a methanesulfonic acid root anion.

[0035] Methylene blue (MTC), also referred to herein as 3,7-bis(dimethylamino)phenothiazine-5-ium chloride, is an oxidized form of methionine (MT) (i.e., MT + ), a low molecular weight (319.86) water-soluble tricyclic organic compound having the following structural formula: [ka] Also, N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium Dimesylate The structure of is shown below. [ka]

[0036] The term "solvate" as used herein has its general meaning and refers to a complex of a solute (e.g., a compound, a salt of a compound) and a solvent. If the solvent is water, the solvate can be conveniently referred to as a hydrate, such as a monohydrate, a dihydrate, a trihydrate, etc. Unless otherwise specified, when a specific compound is referred to, the specific compound also includes its solvated form.

[0037] As used herein, "treatment" includes administering a compound of the present application or a composition of the present application to alleviate symptoms or complications of a disease or disorder, or to eliminate a disease or disorder. As used herein, the term "alleviation" describes a process in which the severity of a sign or symptom of a disorder is reduced. A symptom may be alleviated but not eliminated. In one embodiment, administering a composition of the present application eliminates the sign or symptom.

[0038] Uses and Treatments One aspect of the present invention relates to the use of a phenothiazine compound selected from the group consisting of a compound represented by formula (I) and its hydrates, solvates or reduced forms in the manufacture of a PD-1 signaling inhibitor. [ka] (where Z is S + , O + , C or N; Y is N or N + and Z is S + Or O + When Z is selected from, Y is selected from N; when Z is selected from C or N, Y is N + Selected from X - is Z+ Or N + To achieve electrical neutrality, one or more anions capable of forming a salt with R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted arylalkoxy, thioalkoxy, amine, nitro, amino, and halogen.

[0039] In some embodiments, the phenothiazine-based compound is selected from 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of formula II, a hydrate, solvate, or reduced form thereof. [ka] (Here, X is one or more anions to achieve electrical neutrality, and has the same meaning as above.)

[0040] In some embodiments, the reduced form of the phenothiazine-based compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, a hydrate or solvate thereof. [ka] (Here, X is one or more anions to achieve electrical neutrality, and has the same meaning as above.)

[0041] In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX), preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride.

[0042] Another aspect of the present invention relates to the use of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof in the manufacture of a cancer medicament. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate It is.

[0043] Another aspect of the present invention relates to the use of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof in the manufacture of a medicament for improving immune cell function. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate It is.

[0044] Another aspect of the present invention relates to the use of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof in the manufacture of a medicament for preventing or treating the recurrence of cancer. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate It is.

[0045] Another aspect of the present invention relates to the use of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof in the manufacture of a medicament for blocking PD-1 downstream signaling pathway. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate It is.

[0046] Another aspect of the present invention relates to the use of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof, in the manufacture of a medicament for blocking recruitment of SHP2 protein by PD-1. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate It is.

[0047] Another aspect of the present invention provides a method for treating cancer, comprising administering to a subject suffering from cancer a therapeutically effective amount of a phenothiazine compound of formula I of the present invention, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate In some embodiments, the cancer is melanoma, thymic tumor, lung cancer, prostate cancer, breast cancer, ovarian cancer, colorectal cancer, pancreatic cancer, liver cancer, lymphoma, esophageal cancer, bladder cancer, urethral cancer, non-Hodgkin's lymphoma, renal cancer, or brain cancer. In some embodiments, the cancer is melanoma. In some embodiments, the subject is a mammal, preferably a human.

[0048] Another aspect of the invention provides a method of improving immune cell function comprising administering to a subject a therapeutically effective amount of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate In some embodiments, the improvement of immune cell function can achieve an antiviral effect. In some embodiments, the subject is a mammal, preferably a human.

[0049] Another aspect of the present invention provides a method for preventing or treating recurrence of cancer, comprising administering to a subject suffering from said cancer a therapeutically effective amount of a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium salt (MTX) of formula II, a hydrate, solvate or reduced form thereof. In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III. In this aspect, preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium. Dimesylate In some embodiments, the subject has undergone surgery, radiation therapy, or chemotherapy.

[0050] In some embodiments, the method may be combined with other cancer therapies. For example, the method may be combined with surgery, radiation therapy, or chemotherapy. Thus, in some embodiments, the cancer treatment method of the present invention further comprises administering a therapeutically effective amount of a second therapeutic agent to the subject suffering from the cancer.

[0051] In some embodiments, the second therapeutic agent is an immune cell suitable for adoptive cell therapy. In these embodiments, the second therapeutic agent is administered before, simultaneously with, or after administration of a phenothiazine compound of Formula I, a hydrate, solvate, or reduced form thereof.

[0052] In some embodiments, the second therapeutic agent is a chemotherapeutic agent, preferably, the chemotherapeutic agent is a tumor immunochemotherapeutic agent, more preferably, the chemotherapeutic agent is a PD-1 antibody, a PD-L1 antibody, or a fragment thereof. In some embodiments, the second therapeutic agent is administered before, simultaneously with, or after administration of a phenothiazine compound of Formula I, a hydrate, solvate, or reduced form thereof.

[0053] When the second therapeutic agent is not administered simultaneously with the phenothiazine compound of the present invention, its hydrate or solvate, the two are administered at an interval of about 0.1 hours to about 72 hours, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, or 72 hours.

[0054] When a second therapeutic agent is administered simultaneously with the phenothiazine compound of the present invention, its hydrate, solvate or reduced form, in some embodiments, the phenothiazine compound of the present invention, its hydrate, solvate or reduced form is provided in the form of a pharmaceutical composition separate from the second therapeutic agent. In some embodiments, the separate pharmaceutical compositions are provided in the same kit. When a second therapeutic agent is administered simultaneously with the phenothiazine compound of the present invention, its hydrate, solvate or reduced form, in other embodiments, the phenothiazine compound of the present invention, its hydrate, solvate or reduced form is provided in the form of a single pharmaceutical composition with the second therapeutic agent.

[0055] Pharmaceutical Compositions and Kits One aspect of the present invention provides a pharmaceutical composition comprising a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof for the treatment of cancer. [ka] (where Z is S + , O + , C or N; Y is N or N + and Z is S + Or O + When Z is selected from, Y is selected from N; when Z is selected from C or N, Y is N + Selected from X - is Z + Or N + To achieve electrical neutrality, one or more anions capable of forming a salt with R1, R2, R3, R4, R5, R6, R7, R8, R9 and R 10 are each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkoxy, substituted or unsubstituted arylalkoxy, thioalkoxy, amine, nitro, amino, and halogen.

[0056] In some embodiments, the X is selected from an inorganic acid radical anion and an organic acid radical anion. Further, the inorganic acid radical anion is preferably Cl. - , Br - , I - The organic acid radical anion is preferably a methanesulfonate radical anion, an ethanesulfonate radical anion, a p-toluenesulfonate radical anion, a benzenesulfonate radical anion, an ethanedisulfonate radical anion, a propanedisulfonate radical anion, or a naphthalenedisulfonate radical anion.

[0057] In some embodiments, the phenothiazine-based compound is selected from 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX) of formula II, a hydrate, solvate, or reduced form thereof. [ka] (Here, X is one or more anions to achieve electrical neutrality, and has the same meaning as above.)

[0058] In some embodiments, the reduced form of the phenothiazine-based compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, a hydrate or solvate thereof. [ka] (Here, X is one or more anions to achieve electrical neutrality, and has the same meaning as above.)

[0059] In some embodiments, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium salt (MTX), preferably, the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazin-5-ium chloride (MTC).

[0060] In some embodiments, the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium Dimesylate It is.

[0061] Another aspect of the present invention provides a pharmaceutical composition for the treatment of cancer comprising a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof, and immune cells suitable for adoptive cell therapy, wherein formula I and its substituents are as defined above.

[0062] In some embodiments, the immune cells are selected from tumor infiltrating lymphocytes (TILs), chimeric antigen receptor T cells (CAR-Ts), chimeric antigen receptor NK cells (CAR-NKs), and T cell receptor (TCR) chimeric T cells.

[0063] In some embodiments, the immune cells are T cell receptor (TCR) chimeric T cells.

[0064] In some embodiments, the TCR is capable of binding to the SIINFEKL peptide.

[0065] Another aspect of the present invention provides a pharmaceutical composition for treating cancer comprising a phenothiazine compound of formula I, a hydrate, solvate or reduced form thereof, and an immunotherapeutic agent for cancer chemotherapy, wherein formula I and its substituents are as defined above.

[0066] In some embodiments, the immunotherapeutic agent for cancer chemotherapy is a PD-1 antibody, a PD-L1 antibody, or a functional fragment thereof.

[0067] Thus, the pharmaceutical composition of the present invention may comprise an active ingredient (e.g., any compound of formula I) present in the composition in a form mixed with a pharma- ceutically acceptable carrier. The composition of the present invention may also comprise two active ingredients (e.g., any compound of formula I and adoptive therapy immune cells, or any compound of formula I and PD-1 antibody) simultaneously, which are included in the same pharmaceutical composition in an appropriate form, and upon administration of the pharmaceutical composition, the two active ingredients are administered to the subject simultaneously or sequentially. For example, the compound of formula I, immune cells suitable for adoptive cell therapy, and carriers are present in the pharmaceutical composition in the form of a mixture in a predetermined ratio. Alternatively, the compound of formula I and carriers constitute one part of the pharmaceutical composition in a predetermined ratio, and immune cells suitable for adoptive cell therapy and carriers constitute another part of the pharmaceutical composition in a predetermined ratio, and the two parts are combined, for example, combined in a core-shell structure to form the pharmaceutical composition. Other methods available in pharmaceuticals or pharmaceutical engineering may also be used to combine two active ingredients without affecting the exertion of the action of each active ingredient after administration.

[0068] Another aspect of the present invention provides a kit comprising a first pharmaceutical composition comprising a therapeutically effective amount of immune cells suitable for adoptive cell therapy, and a second pharmaceutical composition comprising a therapeutically effective amount of a phenothiazine compound of formula I or a hydrate or solvate thereof, which are present independently. Thus, in some embodiments of the kit, the first pharmaceutical composition can be present in a single dosage form, and the second pharmaceutical composition can be present in another single dosage form, which may be the same or different. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition in the kit are each contained in a separate container.

[0069] Another aspect of the present invention provides a kit comprising a first pharmaceutical composition comprising a therapeutically effective amount of a phenothiazine compound of formula I or a hydrate or solvate thereof, and a second pharmaceutical composition comprising a therapeutically effective amount of a second chemotherapeutic agent (e.g., an immunotherapeutic agent such as a PD-1 antibody), which are present independently. Thus, in some embodiments of the kit, the first pharmaceutical composition can be present in a single dosage form, and the second pharmaceutical composition can be present in another single dosage form, which may be the same or different. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition in the kit are each contained in a separate container.

[0070] The therapeutically or prophylactically effective amount of the active ingredient to be administered can be determined by standard procedures, and factors to be taken into consideration include, for example, the IC 50 These may include biological half-life, age, size and weight of the subject, and disorders associated with the subject. The importance of these and other factors is well known to those of skill in the art. In general, dosages are between about 0.01 mg / kg and 50 mg / kg, preferably between 0.1 mg / kg and 20 mg / kg, of the subject being treated.

[0071] Carriers or excipients may be used to prepare pharmaceutical compositions. The carriers or excipients may be selected to facilitate administration of the compound. Examples of carriers include calcium carbonate, calcium phosphate, various sugars (e.g., lactose, glucose, and sucrose), or starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible solvents. Examples of physiologically compatible solvents include water for injection (WFI) sterile solution, saline solution, and glucose.

[0072] The appropriate dosage form depends in part on the route of administration, such as oral, transdermal, transmucosal, inhalation, or injection (parenteral). Such dosage form must allow the active ingredient to reach the target cells. The drug or drug composition of the present invention can be administered by different routes, including intravenous, intraperitoneal, subcutaneous, intramuscular, oral, transmucosal, rectal, transdermal, or inhalation. In some embodiments, oral administration is preferred. For oral administration, for example, the compound may be prepared as a conventional oral dosage form, such as capsules, tablets, and liquid preparations, such as syrups, elixirs, and concentrated drops.

[0073] Working Example The present invention will now be described in more detail with reference to specific examples. In the following examples, MTC refers to 3,7-bis(dimethylamino)phenothiazine-5-ium chloride of the following structural formula as a PD-1 signaling inhibitor. [ka] In the following examples, LMT is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium having the following structural formula: Dimesylate It is. [ka]

[0074] Example 1 Improvement of human T cell function by MTC 1. Experimental method: (1) A stably transfected cell line highly expressing PD-1 was constructed using Jurkat cells. The pCAGin-PD-1 plasmid was electrotransfected into Jurkat cells, then screened with 900 μg / mL G418 antibiotic, and a single clone was flow-sorted. A DNA fragment controlling the luciferase gene (denoted as NFAT-LUC) by the NFAT binding site was then electrotransfected into Jurkat-PD-1 cells. A single clone was then flow-sorted and named clone 5. Using this cell, the expression level of IL-2 can be reflected by measuring the activity of luciferase. A Raji cell stably expressing PD-L1 was constructed, and the pCDNA-PD-L1 plasmid was electrotransfected into Raji cells, then screened with 2 μg / mL puromycin resistance, and a single clone was then flow-sorted and named clone 1. (2) PD-1 molecules on Jurkat-PD-1 were stimulated using Raji cells stably expressing PD-L1. Raji cells are human-derived B lymphocytes expressing B7 molecules, which may provide a second signal required for T cell activation. Jurkat-PD-1-NFAT-LUC cells were stimulated with 2μg / mL CD3 antibody / 2μg / mL CD28 antibody, and then co-cultured with Raji-PD-L1 cells at a 1:1 ratio for 6 hours. Phosphorylation of PD-1 at Y248 position was detected by immunoblotting. (3) A 96-well plate was coated with 10 μg / mL CD3 antibody / 10 μg / mL CD28 antibody and left at 4℃ overnight. After that, excess antibody was washed off with PBS. Raji cells or Raji-PD-L1 cells were co-cultured with Jurkat-PD-1-NFAT-LUC cells at a 1:1 ratio, or Raji-PD-L1 cells and Jurkat-PD-1-NFAT-LUC cells mixed at a 1:1 ratio were co-cultured with 1 μM MTC or 10 μg / mL PD1 antibody. After co-culture for 6 hours, the supernatant was collected and IL-2 secretion was measured by ELISA. (4) In a 96-well plate, the plate was coated with 10 μg / mL CD3 antibody / 10 μg / mL CD28 antibody and left at 4℃ overnight, then excess antibody was washed off with PBS, and Raji cells or Raji-PD-L1 cells were co-cultured with Jurkat-PD-1-NFAT-LUC cells at a 1:1 ratio, or Raji-PD-L1 cells and Jurkat-PD-1-NFAT-LUC cells mixed at a 1:1 ratio were added with 4.86 μM MTC or LMT, and co-cultured for 6 hours, after which luciferase substrate was added and luciferase was read out using a microplate reader. II. Experimental results and analysis: (1) For Jurkat cells stably expressing PD-1, FACS showed that clone 5 correctly expressed PD-1 molecules (Figure 1A). For Raji cells stably expressing the constructed PD-L1, FACS showed that clone 1 correctly expressed PD-L1 (Figure 1B). (2) After co-culture of Raji-PD-L1 cells and Jurkat-PD-1 cells for 6 hours, phosphorylation of PD-1 at Y248 was confirmed by immunoblotting (Figure 1C), indicating that PD-1 and PD-L1 bind to each other when Jurkat cells expressing PD-1 are co-cultured with Raji cells stably expressing PD-L1. (3) The results of IL-2 secretion showed that Jurkat-PD-1-NFAT-LUC significantly stimulated IL-2 secretion after the addition of PD1 antibody (anti-PD1, Nivolumab, Bristol Myers Squibb) or Raji cells. In the experimental group with Raji PDL1, the amount of IL-2 secretion was reduced compared to the Jurkat-PD-1-NFAT-LUC group without any stimulation, but the amount of IL-2 secretion increased rapidly when MTC or PD1 antibody was added (Figure 1D). In addition, MTC significantly improved the function of Jurkat cells inhibited by PD1 compared to the PD1 antibody group. (4) Jurkat-PD-1-NFAT-LUC showed limited increase in luciferase activity upon stimulation with CD3 antibody / CD28 antibody / Raji cells, and cells treated with Raji-PD-L1 showed reduced luciferase activity. Addition of 4.86 μM MTC or LMT to cells treated with Raji-PD-L1 rapidly increased luciferase activity (Figure 1E), indicating that MTC or LMT could improve the function of Jurkat cells inhibited by PD-1.

[0075] Example 2 Promotion of OT-1 cell division and effector molecule secretion by MTC CD8 in OT-1 transgenic mice + TCR expressed on T cells can recognize the SIINFEKL-H-2Kb complex of chicken ovalbumin. Therefore, when spleen cells from OT-1 mice were cultured in vitro, stimulation with the SIINFEKL peptide segment increased the number of CD8 + The T cells are activated and extensively expanded; they are called CTL cells. 1. Experimental method: (1) Preparation of CTL cells: The spleen of OT1 mouse was collected, crushed, and red blood cells were dissolved by adding red blood cell lysis solution to prepare a single cell suspension. The culture medium was adjusted to 1640 + 10% FBS + 50μM βME + 10nM IL-2, the cell density was adjusted to about 2-4 million / mL, 10nM OVA257-264 was added, and the cells were cultured in a 37℃, 5% CO2 cell incubator. From the day of CTL preparation, the cells were stained with FITC-labeled PD-1 antibody every day, and then the changes in PD-1 expression on the surface of the cells were measured by flow spectrometry. (2) The above CTL cells were labeled with 5 nM CFSE and stimulated with 10 nM SIINFEKL and 10 nM IL-2. At the same time, 10 μg / mL mouse PD-L1 protein was added. In the experimental group, 100 nM of the small molecule compound MTC was also added. After stimulation for 24, 48, and 72 hours, respectively, the effects of PD-L1 protein and MTC on CTL cell division were observed by flow cytometry. (3) CTL cells and target cells EG7 (EL4 lymphoma cells expressing chicken ovalbumin) were mixed and cultured at a concentration of 1:1, and 1 μM of the protein transport inhibitor GolgiPlug was added. TM After treatment with for 6 hours, the cells were fixed with 4% paraformaldehyde and perforated with 0.1% saponin. Then, the cells were stained with antibodies against each effector molecule, and the effect of MTC on the secretion of CTL effector molecules was detected by flow cytometry. II. Experimental results and analysis: (1) The amount of PD-1 on the cell surface of CTL cells gradually increased with increasing induction time throughout the entire induction process (Figure 2A). (2) When CTLs were stimulated with SIINFEKL and IL-2 and PD-L1 protein was added, the PD-L1 protein inhibited the division of CTL cells, whereas the addition of 100 nM MTC could reverse this inhibition (Figure 2B). (3) EG7 are EL4 lymphoma cells that express chicken ovalbumin, so that CTL cells can recognize and perform their killing function. When activated OT-1 mouse spleen cells and EG7 cells are mixed in a 1:1 ratio, the CTL cells start to secrete effector molecules such as IL-2, IFNγ, Perforin, and Granzyme B. Protein transport inhibitor GolgiPlug TM (PTI) was added to the EG7 cells and detected by FACS. As a result, we found that EG7 cells overexpressing PD-L1 on the surface (EG7-PD-L1 cell stable line) strongly inhibited the secretion of IL-2, IFNγ, perforin, and granzyme B by CTL cells. Meanwhile, after treatment with MTC, the secretion of IL-2, IFNγ, perforin, and granzyme B by CTL cells increased rapidly (Figure 3). This indicated that MTC improved the inhibition of PD-1 and promoted the secretion of IL-2, IFNγ, perforin, and granzyme B by CTL cells.

[0076] Example 3: Restoration of target cell killing by OT-1 T cells by MTC 1. Experimental method: (1) For preparation of CTL cells, see Example 2. (2) EG-7-PD-L1 cells were labeled with 5 nM CFSE, and CTL cells and EG-7-PD-L1 were mixed in a 5:1 ratio, then treated with 1 μM, 5 μM, or 10 μM MTC for 4 hours. Then, they were stained with 10 μg / mL PI, and the apoptosis of the target cells EG-7-PD-L1 was detected by flow cytometry to determine the killing ability of CTL cells against the target cells. (3) Melanoma cells B16-OVA (B16 cells expressing the OVA gene) were treated with 10 μg / mL IFN-γ for 24 hours, then mixed and cultured with CTL cells at a ratio of 1:5 for 4 hours and simultaneously treated with MTC (1 μM). B16-OVA cells not treated with IFN-γ but treated with DMSO and MTC alone served as a control. Apoptosis of B16-OVA was observed under a microscope to determine the killing ability of CTL cells against target cells. (4) Among melanoma cells B16-OVA (B16 cells expressing the OVA gene), the B16-OVA group was not treated with anything, the Water group had its medium replaced with water, and the remaining groups were treated with 10 μg / mL of IFNγ. After 24 hours, all groups except the B16-OVA group were mixed and cultured with CTL cells in a 1:5 ratio for 4 hours and simultaneously treated with MTC (1 μM), LMT (1 μM) or anti-PD1 (PD-1 antibody, 10 μg / mL, Nivolumab, BMS). The death of B16-OVA in each group was observed under a microscope to determine the killing ability of CTL cells against target cells. In addition, the non-genetically engineered melanoma B16-WT was also provided. The B16-WT group received no treatment, while the IFN-γ+MTC group and the IFN-γ+LMT group were treated with 10 μg / mL IFN-γ. After 24 hours, they were co-cultured with CTL cells in a 1:5 ratio for 4 hours and simultaneously treated with MTC (1 μM) or LMT (1 μM). On the other hand, the MTC and LMT groups were treated only with MTC (10 μM) and LMT (10 μM), and their killing ability against target cells was observed. II. Experimental results and analysis: (1) Activated OT-1 cells (CTL cells) express PD-1 on their surface and therefore have limited ability to kill EG-7-PD-L1 cells (OVA-modified mouse T lymphoma cells EG7 overexpress mouse PD-L1) (Figure 4A). However, in this system, MTC could significantly restore the killing ability of CTL cells against EG-7-PD-L1 (Figure 4A). (2) As shown in Figure 4E, MTC and LMT have no killing effect on B16-WT cells, and OT-1 cells cannot recognize B16-WT cells, so the combination of MTC / LMT and CTL cells has no killing effect on B16-WT. On the other hand, B16-OVA cells express low levels of MHC-I molecules, and the SIINFEKL peptide segment is not normally presented, so OT-1 cells cannot recognize and kill B16-OVA cells (Figures 4C and 4D). CTL cells have killing effect when they present the SIINFEKL peptide segment after treating B16-OVA cells with IFNγ (Figures 4C and 4D). However, after IFNγ treatment, PD-L1 expression of B16-OVA was induced (Figure 4B). Since the killing effect of CTL against B16-OVA is reduced by the interaction between PD-1 and PD-L1, the killing effect of CTL alone is not significant. In addition, the addition of MTC or LMT significantly improved the killing of B16-OVA cells by CTL cells under the above conditions (Figure 4C and Figure 4D). Furthermore, as shown in Figure 4D, the killing effect of the combination of MTC and CTL cells was equivalent to that of the combination of LMT and CTL, and was significantly stronger than that of the anti-PD-1 group. Therefore, the combination of MTC or LMT and CTL cells can synergistically enhance the killing effect against B16-OVA.

[0077] Example 4 MTC assists cytotoxic T cells in eliminating tumors formed by target cells The experimental results of Examples 1 to 3 above confirmed that MTC strongly restores cytotoxic T cells that kill target cells overexpressing PD-L1. This Example further investigated the tumor treatment ability of MTC in vivo. 1. Experimental method: (1) Chicken ovalbumin (OVA) and mouse PD-L1-overexpressing lymphoma cells, EL4-OVA-mPDL1 cells, were resuspended in BD Matrigel (2 × 10 cells per side per mouse). 6 Resuspend cells in 100 μl BD Matrigel). (2) The mixture of the above cells and Matrigel was inoculated subcutaneously at the left and right lumbar region of Rag1− / − mice at 100 μl each. (3) After about 5 days of inoculation and tumor fixation, activated OT-1 T cells (i.e., 2 × 10 6 Mice were injected with 10 mg / kg / day CTL cells (100 mg / kg / day MTC) and divided into three groups: CTL cells alone, CTL cells + 10 mg / kg / 2 days PD-1 antibody, and CTL cells + 40 mg / kg / day MTC. (4) From the day of tail vein injection of CTL cells, the length and width of the transplanted tumor in each group of mice were measured every other day, and the length x width was calculated. 2 The tumor volume was calculated using the formula: / 2 (Figure 5A). II. Experimental results and analysis: (1) In mice that received a simultaneous intraperitoneal injection of PD-1 antibody, tumor growth was inhibited, as were increases in tumor volume and weight, compared to the control group that received tail vein injections of CTL cells alone. This demonstrated that tumor growth was inhibited in the PD-1 antibody group. (2) Tumor growth was also strongly inhibited by intragastric administration of MTC to mice bearing transplanted tumors (Fig. 5B, C, D). After the end of treatment, the group injected with only CTL cells showed rapid tumor growth in both tumor weight and tumor volume, indicating that administration of CTL alone was not effective in inhibiting tumor growth. On the other hand, both the CTL+PD-1 antibody treatment group and the CTL+MTC treatment group were able to significantly inhibit tumor growth, and the therapeutic effect of the CTL+MTC treatment group was superior to that of the CTL+PD-1 antibody treatment group. Similar results were obtained in C57BL / 6J mice using EG7-mPDL1 cells in the same manner as in this example (Figure 5E). Tumors grew faster in mice without CTL administration (CTL free), whereas administration of CTL alone inhibited tumor growth compared to CTL free, but the inhibitory effect was weaker than that of the CTL+PD-1 antibody administration group and the CTL+MTC combination group. This indicates that MTC can effectively assist cytotoxic T cells in eliminating subcutaneous tumors formed by target cells.

[0078] Example 5 Effective Treatment of Tumors in Situ with MTC The advantage of transplanted tumors is that the antigen epitopes recognized by T cells are very clear, and the system is single, clarifying the problem. The disadvantage is that this model cannot simulate the complex carcinogenesis of primary cancer, the process of tumor formation, and the interaction between tumors and interstitial cells. Therefore, mouse transplanted tumor models have low predictability for the effects of drugs in human patients. Therefore, in this example, a transgenic lung cancer mouse model was used to further investigate the tumor treatment effect of the MTC of the present invention. By feeding EGFR-L858R transgenic mice with tetracycline (DOX)-containing food, it was possible to induce the expression of human EGFR-L858R mutants (mutants commonly found in human lung cancer) in mouse lung epithelial cells, and after 1 to 2 months, mouse lung cancer could be detected and recorded by computer tomography (CT). 1. Experimental method: (1) EGFR-L858R mutant mice were fed DOX diet to induce in situ tumor models for approximately 40 days. (2) Tumor size and severity were recorded using computer tomography (CT). (3) Tumor-bearing mice were randomly assigned to groups and administered placebo (HKI solution) or MTC (40 mg / kg / day in HKI solution). After 2 weeks of treatment, tumor size was recorded again by CT. (4) MTC induces CD8 + To demonstrate that T cells can be used to achieve the objective, the following experiment was also carried out in this example. First, CD8 antibody was intraperitoneally injected (200 mg / mouse / 3 days) for one week to investigate the CD8 + The cells were removed, then treated with both MTC and CD8 antibody for 2 weeks, and tumor size was re-recorded by CT. II. Experimental results and analysis: The principle of this experiment is that when these mice are fed DOX-containing food, rtTA in the lung epithelial cells binds to DOX, changes its conformation, and binds to a sequence called TetO, initiating the expression of EGFR mutants controlled by TetO. These mice developed in situ lung adenocarcinomas 40 days after being fed tetracycline. These lung adenocarcinomas realistically simulated the entire clinical process in which lung epithelial cells become cancerous due to the action of EGFR mutants, the cancer progresses, and finally the organism dies from cancer. The experimental results of this example showed that the tumors in the placebo-treated group grew rapidly, while the tumors in the mice treated with MTC for 14 days were almost completely removed by imaging (Figure 6B), and CD8 + In T cell-depleted mice, tumor growth was not inhibited after MTC treatment (Fig. 6B), indicating that MTC can effectively treat in situ tumors caused by EGFR-L858R mutation by activating endogenous CTLs.

[0079] Example 6 Blockade of SHP2 recruitment by PD-1 by MTC In this example, we preliminarily investigated the mechanism by which MTC inhibits PD-1 transduction signaling and investigated the ability of MTC to affect SHP2 recruitment by PD-1. By binding to its ligand, PD-1 can recruit SHP2 protein to the periphery of the T cell receptor, thereby inhibiting the activation of the proximal kinase of the T cell receptor and reducing Lck-mediated ZAP-70 protein phosphorylation and the initiation of downstream signaling pathways. Luciferase can be divided into two fragments, N-terminal and C-terminal, and after these two fragments are fused with other proteins, respectively, if the N-terminal and C-terminal luciferase fragments are close to each other for the interaction of these fusion proteins, the activity of luciferase can be detected, so that the strength of the interaction of the fused proteins can be indirectly reflected by detecting the activity of luciferase. 1. Experimental method: (1) First, we lipofected 293T cells with two fusion genes, PD-1-Cluc and Nluc-SHP2, to establish a stably transfected cell line. We then added different concentrations of MTC to the cells and cultured them for 6 hours to observe the effect of MTC on the luciferase readout. (2) Two fusion genes, PD-1-GFP and SHP2-mCherry, were lipofected into 293T cells to construct a stably transfected cell line. The cells were then cultured with different concentrations of MTC for 6 hours, after which they were treated with 1 μM PVD (a phosphatase inhibitor) for 5 minutes and fixed with 4% paraformaldehyde. The effect of MTC on the localization of GFP and mCherry, i.e., PD-1 and SHP2, was observed using Confocal. (3) 293T cells were co-transfected with PD-1-Flag plasmid and SHP2 plasmid, and then treated with different concentrations of MTC to detect the ability of PD-1 to recruit SHP2 by Co-IP. II. Experimental results and analysis: (1) When we added different concentrations of MTC to 293T cells stably transfected with PD-1-Cluc and Nluc-SHP2, we found that MTC reduced the luciferase readout, indicating that MTC could block PD-1-induced SHP2 recruitment (Figure 7A). (2) To further substantiate the above conclusions, we used 293T cells stably transfected with PD-1-GFP and SHP2-mCherry. Confocal results showed that GFP and mCherry colocalized well when treated with PVD, a phosphatase inhibitor, indicating that phosphorylated PD-1 recruited SHP2 (Figure 7B). (3) In the system described above in (2), addition of MTC caused mCherry to dissociate from the membrane and show cytoplasmic distribution (Figure 6B). (4) Co-IP results showed that MTC blocked the binding of PD-1 to SHP2 and unexpectedly blocked downstream signaling of PD-1, thereby improving immune cell function and enhancing tumor cell killing when combined with adoptive cell therapy or other tumor immunotherapies (Figure 7C).

[0080] The technical features of the embodiments described above may be combined in any manner. For convenience of explanation, all possible combinations of the technical features in the embodiments described above have not been described. However, as long as there is no contradiction in the combinations of these technical features, they should all be considered to be within the scope described in this specification.

[0081] The above examples only show some embodiments of the present invention, and the description is specific and detailed, but this should not be understood as a limitation on the scope of the invention patent. However, those skilled in the art can make some modifications and improvements without departing from the concept of the present invention, all of which are within the scope of protection of the present invention. Therefore, the scope of protection of the invention patent shall be subject to the scope of the attached claims.

Claims

1. (a) as a first active ingredient, immune cells suitable for adoptive cell therapy; (b) a pharmaceutical composition for treating cancer comprising, as a second active ingredient, a free phenothiazine compound selected from the compound of formula (I), its hydrate, solvate or reduced form, 【Chemistry 1】 (where Z is S ＋ , O ＋ , C or N; Y is N or N ＋ and Z is selected from S ＋ Or O ＋ When Z is selected from the group consisting of C and N, then Y is N. ＋ and X － Is Z ＋ Or N ＋ and achieving electrical neutrality by providing one or more anions capable of forming a salt with R １ , R ２ , R ５ , R ６ , R ９ and R １０ is hydrogen, R ３ , R ４ , R ７ and R ８ is methyl.) wherein the reduced form is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, a hydrate or a solvate thereof; 【Chemistry 2】 (Here, X － is made one or more anions to achieve electrical neutrality.) wherein the immune cells are selected from the group consisting of tumor infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor NK cells (CAR-NK cells), and T cell receptor (TCR) chimeric T cells (TCR-T cells).

2. The X － , Cl － , B － , I － , methanesulfonate radical anion, ethanesulfonate radical anion, p-toluenesulfonate radical anion, benzenesulfonate radical anion, ethanedisulfonate radical anion, propanedisulfonate radical anion, and naphthalenedisulfonate radical anion.

3. 2. The pharmaceutical composition of claim 1, wherein the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium salt (MTX) of formula II, a hydrate or a solvate thereof. 【Chemistry 3】 (Here, X － is made one or more anions to achieve electrical neutrality.)

4. 2. The pharmaceutical composition of claim 1, wherein the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride.

5. 2. The pharmaceutical composition according to claim 1, wherein the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium dimesylate.

6. The pharmaceutical composition of claim 1 , wherein the immune cell is a T cell receptor (TCR) chimeric T cell.

7. The pharmaceutical composition of claim 6 , wherein the TCR is capable of binding to the SIINFEKL peptide.

8. (a) as a first active ingredient, immune cells suitable for adoptive cell therapy formulated in a first formulation; (b) a kit for treating cancer, comprising, as a second active ingredient, a free phenothiazine compound selected from the compound represented by formula (I) or a hydrate, solvate or reduced form thereof, which is formulated in a second formulation, 【Chemistry 4】 (where Z is S ＋ , O ＋ , C or N; Y is N or N ＋ and Z is selected from S ＋ Or O ＋ When Z is selected from the group consisting of C and N, then Y is N. ＋ and X － Is Z ＋ Or N ＋ To achieve electrical neutrality, one or more anions capable of forming a salt with R １ , R ２ , R ５ , R ６ , R ９ and R １０ is hydrogen, R ３ , R ４ , R ７ and R ８ is methyl.) wherein the reduced form is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, a hydrate or a solvate thereof; 【Chemistry 5】 (Here, X － is made one or more anions to achieve electrical neutrality.) wherein the immune cells are selected from the group consisting of tumor infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T cells), chimeric antigen receptor NK cells (CAR-NK cells), and T cell receptor (TCR) chimeric T cells (TCR-T cells).

9. 1. Use of a phenothiazine compound in the manufacture of a cancer medicament, comprising: wherein the phenothiazine compound is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium dimesylate; wherein the cancer is lung cancer or lymphoma.

10. Use of a phenothiazine compound in the manufacture of a PD-1 signaling inhibitor, wherein the phenothiazine compound is selected from the group consisting of a compound represented by formula (I) and its hydrate, solvate or reduced form, 【Chemistry 6】 (where Z is S ＋ , O ＋ , C or N; Y is N or N ＋ and Z is selected from S ＋ Or O ＋ When Z is selected from the group consisting of C and N, then Y is N. ＋ and X － Is Z ＋ Or N ＋ To achieve electrical neutrality, one or more anions capable of forming a salt with R １ , R ２ , R ５ , R ６ , R ９ and R １０ is hydrogen, R ３ , R ４ , R ７ and R ８ is methyl.) wherein the reduced form is N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium salt (LMTX) of formula III, a hydrate or a solvate thereof. 【Chemistry 7】 (Here, X － is made one or more anions to achieve electrical neutrality.) ,use.

11. X － , Cl － , B － , I － 11. The use according to claim 10, wherein the anion is selected from the group consisting of methanesulfonate, ethanesulfonate, p-toluenesulfonate, benzenesulfonate, ethanedisulfonate, propanedisulfonate and naphthalenedisulfonate.

12. The use according to claim 10, wherein the phenothiazine compound is selected from 3,7-bis(dimethylamino)phenothiazine-5-ium salt (MTX) of formula II, a hydrate or a solvate thereof. 【Chemistry 8】 (Here, X － is made one or more anions to achieve electrical neutrality.)

13. The use according to claim 10, wherein the phenothiazine compound is 3,7-bis(dimethylamino)phenothiazine-5-ium chloride or N3,N3,N7,N7-tetramethyl-10H-phenothiazine-3,7-diammonium dimesylate.

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