T-cell decrease inhibitor used for inhibiting t-cell decrease caused by bendamustine

Abstract

One embodiment of the present disclosure addresses the problem of providing an agent for inhibiting a T-cell decrease caused by bendamustine or a pharmaceutically acceptable salt thereof.　This T-cell decrease inhibitor, which is used to inhibit a T-cell decrease caused by bendamustine or a pharmaceutically acceptable salt thereof, contains a substance that impedes at least one signal transduction transmitted by a TNFR superfamily member expressed in the T cell.

Description

T-cell reduction inhibitors used to suppress the reduction of T cells caused by bendamustine

[0001] One embodiment of the present disclosure relates to an agent for suppressing T cell decline used to suppress T cell decline caused by bendamustine or a pharmaceutically acceptable salt thereof. Another embodiment of the present disclosure relates to a method for screening such an agent for suppressing T cell decline.

[0002] Bendamustine was developed in the former East Germany in the 1960s, but in the 21st century, multiple international clinical trials have demonstrated its high effectiveness against a wide range of B-cell lymphomas and chronic lymphocytic leukemia, and it is now one of the most frequently used anticancer drugs for these diseases.

[0003] However, as a side effect of bendamustine, T lymphocytes (especially CD4 + It is known that bendamustine treatment results in a decrease in T lymphocytes, and that T lymphocyte recovery persists for six months even after six months of treatment (Non-Patent Document 1). Therefore, there is a high risk of opportunistic infections for approximately one year after the start of bendamustine treatment (Non-Patent Document 2). In recent years, it has become a concern that coronavirus infections after bendamustine treatment are more likely to become severe. Furthermore, with the introduction of immunotherapies such as CAR-T cell therapy and bispecific antibodies into routine clinical practice for some B-cell tumors, there are concerns that the decrease in T cells caused by bendamustine may weaken the effectiveness of these immunotherapies. Consequently, the use of bendamustine has been discouraged worldwide in recent years.

[0004] It has previously been reported that bendamustine has the effect of strongly inhibiting LUBAC, which is expressed in B-cell tumors (Non-patent Document 3), but the mechanism by which bendamustine causes a decrease in T lymphocytes has not been elucidated.

[0005] Saito H. et al., Blood Cancer Journal, 2015; 5(10):e362. doi: 10.1038 / bcj.2015.86. Marcus R, et al., The New England Journal of Medicine, 2017; Oct 377:1331-1344. doi: 10.1056 / NEJMoa1614598De Cezare et al., Cell Chemical Biology, 2018; 25: 1117-1127. doi: 10.1016 / j.chembiol.2018.06.004

[0006] An object of one embodiment of the present disclosure is to provide an agent for suppressing T cell reduction caused by bendamustine or a pharmaceutically acceptable salt thereof.An object of another embodiment of the present disclosure is to provide a method for screening an agent for suppressing T cell reduction caused by bendamustine or a pharmaceutically acceptable salt thereof.

[0007] The present inventors have conducted extensive research to solve the above problems and have found that (1) OX40 expressed in T cells is a molecule involved in the survival and proliferation of T cells, and that bendamustine converts the OX40-mediated signal into a cell death signal, resulting in a decrease in the number of T cells; (2) in the treatment of B-cell tumors using bendamustine, blocking the binding between OX40 and OX40L expressed in T cells and inhibiting OX40-mediated signal transduction does not impede the anti-tumor effect of bendamustine; and (3) blocking the binding between OX40 and OX40L expressed in T cells and inhibiting OX40-mediated signal transduction suppresses the decrease in T cells caused by bendamustine.

[0008] Based on the above findings, the present inventors have found that by inhibiting signal transduction mediated by at least one TNFR superfamily member expressed in T cells, it is possible to suppress bendamustine-induced T cell depletion without interfering with the therapeutic effects of bendamustine. Furthermore, based on the above findings, the present inventors have also found that by incubating T cells in the presence of at least one ligand of a TNFR superfamily member expressed in T cells, bendamustine or a pharmaceutically acceptable salt thereof, and a test substance, it is possible to screen for candidate substances that can suppress bendamustine-induced T cell depletion using the viability of T cells after incubation as an indicator. The present disclosure has been completed through further investigation based on these findings.

[0009] That is, the present disclosure relates to a technology for suppressing T cell depletion caused by bendamustine or a pharmaceutically acceptable salt thereof, and provides the following inventions. Item 1-1. A T cell depletion inhibitor used to suppress T cell depletion caused by bendamustine or a pharmaceutically acceptable salt thereof, the T cell depletion inhibitor comprising a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells. Item 1-2. The T cell depletion inhibitor according to Item 1-1, used in patients with B cell tumors to be administered bendamustine or a pharmaceutically acceptable salt thereof. Item 1-3. The T cell depletion inhibitor according to Item 1-1 or 1-2, wherein the substance inhibits the interaction between at least one TNFR superfamily member and its ligand. Item 1-4. The T cell decline inhibitor according to Item 1-3, wherein the substance is at least one selected from the group consisting of an antibody that binds to a TNFR superfamily member or its ligand, an antibody fragment thereof, an antagonist of a TNFR superfamily member, an siRNA against a TNFR superfamily member or its ligand, an antisense nucleic acid against a TNFR superfamily member or its ligand, and a ribozyme against a TNFR superfamily member or its ligand. Item 1-5. The T cell decline inhibitor according to any of Items 1-1 to 1-4, wherein the substance inhibits signal transduction mediated by OX40. Item 1-6. The T cell decline inhibitor according to Item 1-5, wherein the substance inhibits the interaction between OX40 and OX40L. Item 1-7. The agent for suppressing T cell decline according to Item 1-6, wherein the substance is at least one selected from the group consisting of an anti-OX40L antibody, an anti-OX40 antibody, an antibody fragment thereof, an OX40 antagonist, an siRNA against OX40 or OX40L, an antisense nucleic acid against OX40 or OX40L, and a ribozyme against OX40 or OX40L. Item 1-8. A therapeutic drug set comprising a first pharmaceutical composition containing bendamustine or a pharmaceutically acceptable salt thereof, and a second pharmaceutical composition containing a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells.Item 1-9. The therapeutic drug set according to Item 1-8, which is used to treat B-cell tumors. Item 1-10. The therapeutic drug set according to Item 1-8 or 1-9, wherein the substance inhibits the interaction between at least one TNFR superfamily member and its ligand. Item 1-11. The therapeutic drug set according to Item 1-10, wherein the substance is at least one selected from the group consisting of an antibody that binds to a TNFR superfamily member or its ligand, an antibody fragment thereof, an antagonist of a TNFR superfamily member, an siRNA against a TNFR superfamily member or its ligand, an antisense nucleic acid against a TNFR superfamily member or its ligand, and a ribozyme against a TNFR superfamily member or its ligand. Item 1-12. The therapeutic drug set according to any of Item 1-8 to 1-11, wherein the substance inhibits signal transduction mediated by OX40. Item 1-13. The therapeutic drug set according to Item 1-12, wherein the substance inhibits the interaction between OX40 and OX40L. Item 1-14. The therapeutic drug set according to Item 1-13, wherein the substance is at least one selected from the group consisting of an anti-OX40L antibody, an anti-OX40 antibody, an antibody fragment thereof, an OX40 antagonist, an siRNA against OX40 or OX40L, an antisense nucleic acid against OX40 or OX40L, and a ribozyme against OX40 or OX40L. Item 1-15. Use of a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells, for the manufacture of a T cell decline inhibitor used to inhibit T cell decline caused by bendamustine or a pharmaceutically acceptable salt thereof. Item 1-16. A substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells, for use in treatment to inhibit T cell decline caused by bendamustine or a pharmaceutically acceptable salt thereof.Item 1-17. A method for suppressing a decrease in T cells caused by bendamustine or a pharmaceutically acceptable salt thereof, comprising administering to a patient requiring treatment with bendamustine or a pharmaceutically acceptable salt thereof a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells, and bendamustine or a pharmaceutically acceptable salt thereof. Item 1-18. A method for treating a disease for which bendamustine or a pharmaceutically acceptable salt thereof is administered, comprising administering to a patient requiring treatment with bendamustine or a pharmaceutically acceptable salt thereof a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells, and bendamustine or a pharmaceutically acceptable salt thereof.

[0010] That is, the present disclosure provides the following inventions related to technology for screening drugs for suppressing T cell reduction caused by bendamustine or a pharmaceutically acceptable salt thereof. Item 2-1. A method for screening test substances for candidate substances effective in suppressing T cell reduction caused by bendamustine or a pharmaceutically acceptable salt thereof, the screening method comprising: a first step of incubating T cells in the presence of at least one ligand of a TNFR superfamily member expressed in T cells, bendamustine or a pharmaceutically acceptable salt thereof, and the test substance, and determining the T cell viability; and a second step of selecting, as the candidate substance, a test substance that results in a high T cell viability determined in the first step. Item 2-2. The screening method according to Item 2-1, wherein the ligand is OX40L.

[0011] According to one embodiment of the present disclosure, it is possible to suppress the decrease in T cells caused by treatment with bendamustine or a pharmaceutically acceptable salt thereof. Therefore, according to the present disclosure, it is possible to suppress the occurrence of, or the progression of, infectious diseases such as opportunistic infections and coronavirus infections in patients receiving treatment with bendamustine or a pharmaceutically acceptable salt thereof, and to prevent such infections from becoming severe. Furthermore, immunotherapies such as CAR-T cell therapy and bispecific antibodies have traditionally been used for some B-cell tumors, and there have been concerns that the decrease in T cells caused by bendamustine or a pharmaceutically acceptable salt thereof may weaken the efficacy of these immunotherapies. However, according to the present disclosure, it is possible to suppress the decrease in T cells even after treatment with bendamustine or a pharmaceutically acceptable salt thereof, thereby also suppressing adverse effects on subsequent immunotherapies.

[0012] Furthermore, according to one embodiment of the present disclosure, a drug that is effective in suppressing the decrease in T cells caused by bendamustine or a pharmaceutically acceptable salt thereof can be screened using a simple method.

[0013] This figure shows the results of determining the ratios of dead cells (PI-positive) and apoptotic cells (PI-negative and caspase-positive T cells) in T cells after co-culture of T cells with OX40L-expressing L cells or mock L cells in the presence of 1 to 75 μM bendamustine. This figure shows the results of administering anti-OX40L antibody or control antibody (day 1 and day 5) and bendamustine (day 1 and day 5) to a mouse model of B cell lymphoma, and measuring tumor volume over time. This figure shows the results of administering anti-OX40L antibody or control antibody (day 1 and day 5) and bendamustine (day 1 and day 5) to wild-type C57BL / 6 female mice, and measuring the number of T cells and B cells in the peripheral blood on days 0, 17, and 31. A shows the number of CD4 in the peripheral blood. + T cell count, B is CD8 in peripheral blood +A is a box plot showing the number of T cells in the control antibody group, and B is a box plot showing the number of B cells in the peripheral blood. In A to C, "control" refers to the control antibody-administered group, and "anti-OX40L antibody" refers to the anti-OX40L antibody-administered group. In A to C, ns, *, **, and *** indicate no significant difference, p value < 0.05, p value < 0.01, and p value < 0.001, respectively, by Student's t-test.

[0014] 1. Definitions It should be understood that the terms used in this disclosure are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0015] In the present disclosure, "TNFR superfamily members expressed in T cells" refer to receptors that belong to the tumor necrosis factor (TNF) receptor superfamily and are present in T cells, and specifically include OX40 (also known as TNFRSF4, CD134, ACT35), TNFR1 (also known as TNFRSF1A, CD120a), TNFR2 (also known as TNFRSF1B, CD120b, p75), GITR (also known as TNFRSF18, CD357, AITR), CD27 (also known as TNFRSF7), 4-1BB (also known as TNFRSF9, CD137), DR3 (also known as TNFRSF25, TRAMP), HVEM (also known as TNFRSF14, CD270, ATAR), and Fas (also known as APO These include 10 members of the TNFR superfamily, including TNFR-1 (also known as DR4, APO-2), TRAILR1 (also known as DR4, APO-2), and TRAILR2 (also known as DR5, KILLER, TRICK2). Each member of the TNFR superfamily is expressed in T cells and exists as a transmembrane receptor in T cells.

[0016] In the present disclosure, "signal transduction mediated by a TNFR superfamily member" refers to signal transduction that occurs when a ligand of the TNFR superfamily member binds to its receptor. Specific examples of the ligands for each receptor of the TNFR superfamily member include OX40L for OX40, TNF and LTA for TNFR1, TNF and LTA for TNFR2, GITRL for GITR, CD70 for CD27, 4-1BBL for 4-1BB, TL1A for DR3, LIGHT for HVEM, FasL for Fas, TRAIL for TRAILR1, and TRAIL for TRAILR2.

[0017] In the present disclosure, "bendamustine" is 4-[5-[bis(2-chloroethyl)amino]-1-methyl-2-benzimidazolyl]butyric acid, a compound that possesses the structures of both an alkylating agent and an antimetabolite.

[0018] In this disclosure, "B cell neoplasm" refers to a malignant tumor derived from B cells.

[0019] In the present disclosure, a "test substance" is a substance that is the subject of a screening method for confirming the presence or absence of a desired effect.

[0020] 2. Agent for Inhibiting T Cell Decline One embodiment of the present disclosure provides an agent for inhibiting T cell decline caused by bendamustine or a pharmaceutically acceptable salt thereof, the agent comprising a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells. The agent for inhibiting T cell decline of the present disclosure is described in detail below.

[0021] [Active ingredient] The T cell decline inhibitor of the present disclosure contains, as an active ingredient, a substance (hereinafter sometimes referred to as an inhibitor) that inhibits at least one of the signal transduction mediated by TNFR superfamily members expressed in T cells.

[0022] The inhibitor used as an active ingredient may be any substance that inhibits at least one signal transduction pathway mediated by a TNFR superfamily member expressed in T cells, including, for example, a substance that inhibits the interaction between at least one TNFR superfamily member and its ligand. Specific examples include at least one of the following: a substance that inhibits the interaction between OX40 and OX40L; a substance that inhibits the interaction between TNFR1 and TNF and / or LTα; a substance that inhibits the interaction between TNFR2 and TNF and / or LTα; a substance that inhibits the interaction between GITR and GITRL; a substance that inhibits the interaction between CD27 and CD70; a substance that inhibits the interaction between 4-1BB and 4-1BBL; a substance that inhibits the interaction between DR3 and TL1A; a substance that inhibits the interaction between HVEM and LIGHT; a substance that inhibits the interaction between Fas and FasL; a substance that inhibits the interaction between TRAILR1 and TRAIL; and a substance that inhibits the interaction between TRAILR2 and TRAIL. Among these inhibitors, a preferred example is a substance that inhibits the interaction between OX40 and OX40L.

[0023] Specific examples of inhibitors used as active ingredients include antibodies or antibody fragments thereof that bind to TNFR superfamily members or their ligands, antagonists of TNFR superfamily members, siRNAs against TNFR superfamily members or their ligands, antisense nucleic acids against TNFR superfamily members or their ligands, ribozymes against TNFR superfamily members or their ligands, etc. Among these inhibitors, a preferred example is an antibody or antibody fragment thereof that binds to a TNFR superfamily member or its ligand.

[0024] The antibody that binds to a TNFR superfamily member or its ligand may be any antibody capable of inhibiting the binding of a TNFR superfamily member to its receptor, including, for example, an antibody that recognizes the extracellular region of a TNFR superfamily member or the extracellular region of a ligand of a TNFR superfamily member as an epitope. These antibodies may be polyclonal or monoclonal, and may be of any isotype, such as IgG, IgA, IgE, IgM, or IgY. A preferred example of these antibodies is a polyclonal IgG antibody. These antibodies may be chimeric antibodies (antibodies composed of a non-human variable region and a human constant region), humanized antibodies (antibodies composed of a non-human CDR, a human framework region, and a human constant region), or human antibodies (human-derived antibodies). Furthermore, the antibody fragment may be any fragment that has the ability to bind to a TNFR superfamily member or its ligand, and examples thereof include Fab fragments (antibody fragments consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, and a CH1 domain that is part of the heavy chain variable region), Fab' fragments (antibody fragments consisting of a light chain variable region, a light chain constant region, a heavy chain variable region, a CH1 domain that is part of the heavy chain variable region, and a hinge region), F(ab')2 fragments (antibody fragments in which two Fab' fragments are linked by a disulfide bridge at the hinge region), and Fv fragments (antibody fragments consisting of a heavy chain variable region and a light chain variable region). Techniques for producing antibodies and fragments thereof are known, and the antibodies and antibody fragments thereof can be produced by known methods.

[0025] An antagonist of a TNFR superfamily member may be any substance that inhibits the interaction between a TNFR superfamily member and its ligand by binding to the TNFR superfamily member in competition with the TNFR superfamily member's ligand, but does not itself have the ability to activate the TNFR superfamily member.

[0026] siRNAs, antisense nucleic acids, and ribozymes against TNFR superfamily members or their ligands can be prepared based on the base sequence of the mRNA of the target TNFR superfamily member or its ligand. The antisense nucleic acid may be either antisense DNA or antisense RNA.

[0027] More specifically, substances that inhibit the interaction between OX40 and OX40L include anti-OX40 antibodies (e.g., rocatinlimab, etc.), anti-OX40L antibodies (e.g., amlitelimab, etc.), fragments of these antibodies, OX40 antagonists, siRNA against OX40 or OX40L, antisense nucleic acids against OX40 or OX40L, and ribozymes against OX40 or OX40L.

[0028] Substances that inhibit the interaction between TNFR1 and TNF and / or LTα include anti-TNFR1 antibodies (e.g., atrosimab), anti-TNF antibodies, anti-LTα antibodies, fragments of these antibodies, TNFR1 antagonists, siRNA against TNFR1, TNF, or LTα, antisense nucleic acids against TNFR1, TNF, or LTα, and ribozymes against TNFR1, TNF, or LTα.

[0029] Substances that inhibit the interaction between TNFR2 and TNF and / or LTα include anti-TNFR2 antibodies (e.g., AN3025, etc.), anti-TNF antibodies, anti-LTα antibodies, fragments of these antibodies, TNFR2 antagonists, siRNA against TNFR2, TNF, or LTα, antisense nucleic acids against TNFR2, TNF, or LTα, and ribozymes against TNFR2, TNF, or LTα.

[0030] Substances that inhibit the interaction between GITR and GITRL include anti-GITR antibodies (e.g., LY3844583, etc.), anti-GITRL antibodies, antibody fragments thereof, GITR antagonists, siRNAs against GITR or GITRL, antisense nucleic acids against GITR or GITRL, and ribozymes against GITR or GITRL.

[0031] Substances that inhibit the interaction between CD27 and CD70 include anti-CD27 antibodies, anti-CD70 antibodies, antibody fragments thereof, CD27 antagonists, siRNA against CD27 or CD70, antisense nucleic acids against CD27 or CD70, and ribozymes against CD27 or CD70.

[0032] Substances that inhibit the interaction between 4-1BB and 4-1BBL include anti-4-1BB antibodies, anti-4-1BBL antibodies, antibody fragments thereof, 4-1BB antagonists, siRNA against 4-1BB or 4-1BBL, antisense nucleic acids against 4-1BB or 4-1BBL, and ribozymes against 4-1BB or 4-1BBL.

[0033] Substances that inhibit the interaction between DR3 and TL1A include anti-DR3 antibodies, anti-TL1A antibodies, antibody fragments thereof, DR3 antagonists, siRNA against DR3 or TL1A, antisense nucleic acids against DR3 or TL1A, and ribozymes against DR3 or TL1A.

[0034] Substances that inhibit the interaction between HVEM and LIGHT include anti-HVEM antibodies (e.g., JJP-1008, etc.), anti-LIGHT antibodies, antibody fragments thereof, HVEM antagonists, siRNA against HVEM or LIGHT, antisense nucleic acids against HVEM or LIGHT, and ribozymes against HVEM or LIGHT.

[0035] Substances that inhibit the interaction between Fas and FasL include anti-Fas antibodies, anti-FasL antibodies, antibody fragments thereof, Fas antagonists (e.g., Kp7-6 (CAS: 629628-53-1)), siRNA against Fas or FasL, antisense nucleic acids against Fas or FasL, and ribozymes against Fas or FasL.

[0036] Substances that inhibit the interaction between TRAILR1 and TRAIL include anti-TRAILR1 antibodies, anti-TRAIL antibodies, antibody fragments thereof, TRAILR1 antagonists, siRNA against TRAILR1 or TRAIL, antisense nucleic acids against TRAILR1 or TRAIL, and ribozymes against TRAILR1 or TRAIL.

[0037] Substances that inhibit the interaction between TRAILR2 and TRAIL include anti-TRAILR2 antibodies, anti-TRAIL antibodies, antibody fragments thereof, TRAILR2 antagonists, siRNA against TRAILR2 or TRAIL, antisense nucleic acids against TRAILR2 or TRAIL, and ribozymes against TRAILR2 or TRAIL.

[0038] These inhibitors may be used singly or in combination of two or more.

[0039] [Formulation] The T cell decline inhibitor of the present disclosure is formulated into a desired dosage form using a pharmaceutically acceptable carrier or additive, and provided as a pharmaceutical composition. Examples of pharmaceutically acceptable carriers or additives include sterile water, physiological saline, stabilizers, excipients, disintegrants, lubricants, thickeners, gelling agents, antioxidants, buffers, preservatives, emulsifiers, chelating agents, binders, and the like. The content of the inhibitor in the pharmaceutical composition may be appropriately determined depending on the type of inhibitor, the administration method, and dosage of the inhibitor. The dosage form of the pharmaceutical composition containing the inhibitor may be appropriately determined depending on the administration method, and examples include parenteral administration preparations such as injections, drips, and suppositories; and oral administration preparations such as capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, and emulsions.

[0040] Furthermore, when the administration method and timing of the T cell decline inhibitor of the present disclosure and bendamustine or a pharmaceutically acceptable salt thereof are the same, the pharmaceutical composition containing the inhibitor may also contain bendamustine or a pharmaceutically acceptable salt thereof. In this case, the content of bendamustine or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may be appropriately determined depending on the administration method, dosage, etc.

[0041] [Use] The T cell decline inhibitor of the present disclosure is a drug used to suppress the decline of T cells caused by bendamustine or a pharmaceutically acceptable salt thereof. In the T cell decline inhibitor of the present disclosure, the type of T cells to be suppressed by bendamustine or a pharmaceutically acceptable salt thereof is not particularly limited, and can be any type of T cell, including CD4 + T cells and CD8 + In a preferred embodiment of the agent for suppressing T cell decline disclosed herein, bendamustine or a pharmaceutically acceptable salt thereof is used to suppress the proliferation of CD4 + Bendamustine or a pharmaceutically acceptable salt thereof is used to suppress the decrease of CD4 T cells. + It has been reported that T cells are easily reduced, but the T cell reduction inhibitor of the present disclosure + It can effectively suppress the decline of T cells.

[0042] [Target Patients] The T cell decline inhibitor of the present disclosure is administered to patients undergoing treatment with bendamustine or a pharmaceutically acceptable salt thereof. The type of disease of the patient to whom the T cell decline inhibitor of the present disclosure is administered is not particularly limited, as long as it is one for which the administration of bendamustine or a pharmaceutically acceptable salt thereof is expected to have a therapeutic effect, and examples thereof include cancers such as B cell tumors, breast cancer, lung cancer, and soft tissue sarcoma.

[0043] Suitable examples of patients to whom the T cell decline inhibitor of the present disclosure is administered include patients receiving treatment for B cell tumors by administration of bendamustine or a pharmaceutically acceptable salt thereof. The type of B cell tumor is not particularly limited, but examples include leukemia (including acute and chronic), myeloma, and lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma). Specific examples of mature B-cell tumors include B-cell acute lymphoblastic leukemia (acute lymphoblastic leukemia / lymphoblastic lymphoma), chronic lymphocytic leukemia, diffuse large B-cell lymphoma, mantle cell lymphoma, monoclonal B-cell lymphocytosis, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic B-cell lymphoma / leukemia, lymphoplasmacytic lymphoma, monoclonal gammopathy, μ heavy chain disease, γ heavy chain disease, α heavy chain disease, multiple myeloma, and solitary bone disease. Examples of B-cell tumors include plasmacytoma, extraskeletal plasmacytoma, monoclonal immunoglobulin deposition disease, mucosa-associated lymphoid tissue extranodal marginal zone lymphoma, nodal marginal zone lymphoma, follicular lymphoma, pediatric follicular lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, Burkitt lymphoma, and high-grade B-cell lymphoma. Among these B-cell tumors, preferred examples include non-Hodgkin's lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia.

[0044] The bendamustine administered to a patient may be in the free form or in the form of a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts of bendamustine include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, and phosphate; acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, lactate, and methylsulfonate. Among bendamustine and its pharmaceutically acceptable salts, bendamustine or its hydrochloride is preferred, and bendamustine hydrochloride is more preferred.

[0045] Bendamustine or a pharmaceutically acceptable salt thereof to be administered to a patient is formulated together with a pharmaceutically acceptable carrier or additive, etc., and prepared into a desired dosage form and provided. Examples of pharmaceutically acceptable carriers or additives include sterile water, physiological saline, stabilizers, excipients, disintegrants, lubricants, thickeners, gelling agents, antioxidants, buffers, preservatives, emulsifiers, chelating agents, binders, etc. The content of bendamustine or a pharmaceutically acceptable salt thereof in the pharmaceutical composition may be appropriately determined depending on the type of target disease, the administration method, the dosage, etc. The dosage form of a pharmaceutical composition containing bendamustine or a pharmaceutically acceptable salt thereof may be appropriately determined depending on the administration method, etc., and examples include parenteral administration preparations such as injections, drip infusions, and suppositories; and oral administration preparations such as capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewable preparations, buccal preparations, pastes, syrups, suspensions, elixirs, and emulsions.

[0046] The method of administration of bendamustine or a pharmaceutically acceptable salt thereof is not particularly limited, and examples thereof include oral administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intravesical administration, pulmonary administration, nasal administration, etc. Among these, intravenous administration is preferred.

[0047] The dose of bendamustine or a pharmaceutically acceptable salt thereof may be appropriately determined depending on the type of disease to be treated, the age and body weight of the patient, etc. For example, the daily dose of bendamustine or a pharmaceutically acceptable salt thereof may be set to 50 to 200 mg / m 2 (body surface area) and can be administered once a day or in divided doses several times a day. The administration cycle of bendamustine or a pharmaceutically acceptable salt thereof can be appropriately determined depending on the type of disease to be treated, the age and body weight of the patient, etc. For example, one cycle can consist of administration for only one day or administration every two to four days (preferably two days) followed by a 15 to 30 day drug holiday, and the cycle can be repeated.

[0048] The method of administration of the T cell decline inhibitor of the present disclosure is not particularly limited and may be appropriately determined depending on the type of inhibitor used, and examples include oral administration, rectal administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intravesical administration, pulmonary administration, nasal administration, etc. Among these, intravenous administration is preferred.

[0049] The timing of administration of the T cell decline inhibitor of the present disclosure may be appropriately determined depending on the type of inhibitor used, the type of disease to be treated, the age and weight of the patient, etc., and the T cell decline inhibitor of the present disclosure may be administered before, simultaneously with, or after administration of bendamustine or a pharmaceutically acceptable salt thereof. More specifically, the administration timing of the T cell decline inhibitor of the present disclosure may be about 3 hours before to immediately before, simultaneously with, or within about 3 hours after administration of bendamustine or a pharmaceutically acceptable salt thereof. The administration timing of the T cell decline inhibitor of the present disclosure is preferably before administration of bendamustine or a pharmaceutically acceptable salt thereof, and preferably about 3 hours before to immediately before administration of bendamustine or a pharmaceutically acceptable salt thereof.

[0050] The dosage of the T cell decline inhibitor of the present disclosure may be appropriately determined depending on the type of inhibitor used, the type of disease to be treated, the age and weight of the patient, etc. For example, examples of the dosage of the T cell decline inhibitor of the present disclosure include a single dose of the inhibitor of about 1 to 1000 mg / individual, about 10 to 800 mg / individual, or about 100 to 600 mg / individual.

[0051] 3. Therapeutic Drug Set In another embodiment of the present disclosure, there is provided a pharmaceutical set used for treating cancer, the pharmaceutical set comprising a first pharmaceutical composition comprising bendamustine or a pharmaceutically acceptable salt thereof, and a second pharmaceutical composition comprising a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells.

[0052] The therapeutic drug set disclosed herein is a therapeutic drug set for treating a disease with bendamustine or a pharmaceutically acceptable salt thereof, as well as for inhibiting T cell decline with bendamustine or a pharmaceutically acceptable salt thereof, and contains the T cell decline inhibitor and bendamustine or a pharmaceutically acceptable salt thereof, each formulated as a separate pharmaceutical composition.

[0053] In the therapeutic drug set of the present disclosure, the dosage form of the first pharmaceutical composition, the content of bendamustine or a pharmaceutically acceptable salt thereof, the type and content of the inhibitor used in the second pharmaceutical composition, the dosage form of the second pharmaceutical composition, the administration method and dosage of the first pharmaceutical composition and the second pharmaceutical composition, the type of disease to which the therapeutic drug set of the present disclosure is applicable, etc. are as described in the section "2. T cell decline inhibitor" above.

[0054] 4. Screening Method In yet another embodiment of the present disclosure, there is provided a method for screening test substances for a candidate substance that is effective in suppressing T cell reduction caused by bendamustine or a pharmaceutically acceptable salt thereof, the screening method comprising the following steps: a first step of incubating T cells in the presence of at least one ligand of a TNFR superfamily member expressed in T cells, bendamustine or a pharmaceutically acceptable salt thereof, and the test substance, and determining the survival rate of the T cells; and a second step of selecting, as the candidate substance, a test substance that results in a high survival rate of T cells determined in the first step.

[0055] [Step 1] In step 1, T cells are incubated in the presence of at least one ligand of a TNFR superfamily member expressed in T cells, bendamustine or a pharmaceutically acceptable salt thereof, and a test substance, and the survival rate of the T cells is determined.

[0056] The ligand used in step 1 may be at least one ligand of a TNFR superfamily member expressed in T cells, and a suitable example is OX40L. The ligand used in step 1 may be in an isolated or purified state, or may be in the state of cells expressing the ligand.

[0057] The test substance is a substance to be confirmed for its ability to suppress the decrease in T cells caused by bendamustine or a pharmaceutically acceptable salt thereof, and the type of substance is not particularly limited, but examples include peptides, proteins, nucleic acids, low molecular weight organic compounds, inorganic compounds, cell extracts, plant extracts, and mixtures thereof.

[0058] The T cells used in the first step are CD4 + T cells and CD8 + At least one of the T cells is sufficient, but at least CD4 + Preferably, it comprises T cells.

[0059] In the incubation in step 1, T cells may be cultured in a medium containing the ligand (including cells expressing the ligand), bendamustine or a pharmaceutically acceptable salt thereof, and a test substance in a medium in which T cells can survive. Those skilled in the art can appropriately set conditions, such as the amount of the ligand, bendamustine or a pharmaceutically acceptable salt thereof, and the test substance to be added, the seeding amount of T cells, and the incubation time, within the scope of ordinary creative ability.

[0060] After the incubation, the survival rate of T cells is determined. The number of live or dead T cells can be measured by a known method. The survival rate of T cells is calculated from the number of live T cells before the incubation in the first step and the number of live T cells after the incubation.

[0061] [Step 2] In Step 2, test substances that have a high T cell viability determined in Step 1 are selected as candidate substances effective in suppressing T cell reduction caused by bendamustine or a pharmaceutically acceptable salt thereof. In Step 2, the level of T cell viability can be determined by comparing it with the T cell viability after Step 1 performed without the test substance (hereinafter referred to as control viability). That is, test substances that show a high T cell viability higher than the control viability are determined to have a high T cell viability and are selected as the candidate substances.

[0062] In the screening method of the present disclosure, it is desirable to specifically verify the inhibitory effect of bendamustine or a pharmaceutically acceptable salt thereof on the reduction of T cells in the candidate substance selected as the candidate by animal testing, etc., and to confirm its clinical feasibility.

[0063] The invention of the present disclosure will be explained in more detail below with reference to examples. The present disclosure is not limited to the explanation of the examples shown below. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of the literature and the like shown in this disclosure are hereby incorporated by reference in their entirety.

[0064] 1. Verification of the Effect of Bendamustine on T Cell Death in the Presence of OX40L Stimulation 1-1. Study Overview The molecular mechanisms by which bendamustine produces therapeutic effects and side effects distinct from those of other anticancer drugs remain largely unknown. However, bendamustine has been reported to potently inhibit LUBAC, which is expressed in B cell tumors (Non-Patent Document 3). Furthermore, OX40 expressed in T cells is known to be involved in the survival and proliferation of normal T cells. The inventors hypothesized that bendamustine's LUBAC inhibitory effect alters OX40 signaling in T cells, resulting in a decrease in T cell numbers. Therefore, in this study, we examined the effect of bendamustine on T cell death in co-cultures of T cells and cells expressing OX40L, the OX40 ligand.

[0065] 1-2. Methods: The human OX40L gene was introduced into a CS vector expressing GFP (green fluorescent protein) to generate an OX40L expression vector. The OX40L expression vector was then introduced into mouse L cells (NCTC clone 929), a fibroblast cell line, and the GFP-positive group transfected with the OX40L expression vector was collected using a cell sorter to obtain OX40L-expressing L cells. More than 95% of the resulting OX40L-expressing L cells were confirmed to express OX40L on the cell surface. Separately, a mock (empty CS vector) was introduced into mouse L cells, and the mock-transfected GFP-positive group was collected using a cell sorter to obtain mock L cells.

[0066] OX40L-expressing L cells and mock L cells were irradiated with 55 Gy to suppress proliferation activity, and then 1 × 10 5 300 μL of cells / mL were seeded into each well of a 48-well plate. After 24 hours of culture, the supernatant was removed, and 5 × 10 cells of DEL cells (human anaplastic large cell lymphoma, DSMZ no. ACC 338), a T cell line expressing OX40, were added. 5 300 μL of the cells / mL were added to each well and co-cultured with human OX40L-expressing L cells or mock L cells. After 4 hours, bendamustine hydrochloride (Cat. No. S1212, Selleck) was added to the cells at the designated concentrations of 1-75 μM. After 24 hours, CaspACE, a fluorescent apoptosis marker for measuring caspase activity in living cells, was added. TM FITC-VAD-FMK (Promega) was added to each well to a final concentration of 5 μM, and after 20 minutes of incubation, propidium iodide (PI) solution (Biolegend) was added. DEL cells were harvested and washed with FACS buffer (D-PBS containing 0.1% FBS), and caspase activity was analyzed by flow cytometry.

[0067] 1-3. Results When T cells were co-cultured with OX40L-expressing L cells in the presence of bendamustine, the proportion of apoptotic cells (PI-negative, caspase-positive T cells) was higher than when co-cultured with mock L cells (Figure 1). This indicates that, under conditions in which OX40 signaling is present due to ligand binding to OX40 on T cells, the LUBAC inhibitory effect of bendamustine converts this signaling into a signal that induces apoptosis. In other words, these results confirmed that bendamustine converts OX40 signaling into a cell death signal, which is one of the factors causing the decrease in T cell numbers.

[0068] 2. Verification of the effect of anti-OX40L antibodies on the antitumor effect of bendamustine against murine B-cell lymphoma 2-1. Study overview The results of the study suggested that inhibition of OX40 signaling can suppress the bendamustine-induced decrease in T cell numbers. Therefore, in this study, we first verified the effect of anti-OX40L antibody administration on the antitumor effect of bendamustine in a B-cell lymphoma mouse model using the HM876 cell line.

[0069] 2-2. Methods The present inventors previously established the HM876 cell line from B cell lymphoma that developed in genetically modified C57BL / 6 mice (Jo T Blood 2020;136:684-697). In this study, a mouse model of B cell lymphoma was created using the HM876 cell line. Eight- to ten-week-old wild-type C57BL / 6 female mice (CLEA Japan, Inc.) were irradiated with 4 Gy and the day after, 5 × 10 HM876 cells were injected per mouse. 6Two OX40L cells were subcutaneously inoculated into the left and right flanks, and 14 days later, lymphomas of 6 mm or greater in diameter were observed at each inoculation site. At this time, mice were divided into two groups (10 mice per group). One group received 100 μg of anti-OX40L antibody (antibody that recognizes the extracellular domain of OX40L and blocks OX40-OX40L binding; rat IgG2bκ, clone RM134L, BioLegend) intraperitoneally, while the other group received 100 μg of a control antibody (rat IgG2bκ, clone RTK4530, BioLegend). Three hours later, both groups received an intravenous injection of 40 mg / kg of bendamustine hydrochloride (Cat. No. S1212, Selleck) (day 1). Four days later, the antibody and bendamustine were administered again in the same manner (day 5), and changes in tumor size were monitored over time. The size of each tumor was measured as V (mm 3 ) = (W 2 Calculated using the approximate formula (V: volume (mm 3 ), W: short diameter (mm), L: long diameter (mm), and the sum of the tumor sizes on both sides was considered to be the tumor volume for each individual.

[0070] 2-3. Results In both the group administered the control antibody followed by bendamustine and the group administered the anti-OX40L antibody followed by bendamustine, lymphoma disappeared after 15 days, and the group administered the anti-OX40L antibody demonstrated a therapeutic effect equivalent to that of the group administered the control antibody (Figure 2). In other words, these results confirmed that administration of the anti-OX40L antibody does not interfere with the therapeutic effect of bendamustine on B-cell lymphoma.

[0071] 3. Verification of the effects of administration of bendamustine and anti-OX40L antibody on T cell counts and T cell subsets 3-1. Study overview Next, this study verified the effects of administration of bendamustine and anti-OX40L antibody on T cell counts and T cell subsets.

[0072] 3-2. Methods: Nine- to ten-week-old wild-type C57BL / 6 female mice (CLEA Japan, Inc.) were divided into two groups (nine mice per group) after peripheral blood collection. One group received 100 μg of anti-OX40L antibody (rat IgG2b κ, clone RM134L, BioLegend) intraperitoneally, while the other group received 100 μg of a control antibody (rat IgG2b κ, clone RTK4530, BioLegend) intraperitoneally. Three hours later, both groups received an intravenous injection of 40 mg / kg of bendamustine (day 1). Four days later, the mice were re-administered with the antibody and bendamustine in the same manner (day 5), and peripheral blood was collected on days 17 and 31.

[0073] The peripheral blood samples were subjected to blood count and flow cytometry analysis of lymphocyte differentials. Data were acquired using FACSLyric (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.). After gating on live cells in the cytogram, CD4 + T cells are CD3 + CD4 + Differential, CD8 + T cells are CD3 + CD8 + Fraction, B cells are B220 + The fractional percentage was evaluated and the cell number was calculated. The antibodies used were as follows: FITC-anti-CD8a (clone 53-6.7, Cat. No. 11-0081-82; Invitrogen) and PerCP-Cy. TM 5.5-anti-CD4 (clone RM4.5, Cat. No. 550954; BD Biosciences) ・APC-Cy TM 7-anti-CD3 (clone 17A2, Cat. No. 560590; BD Biosciences) ・APC-anti-B220 (clone RA3-6B2, Cat. No. 553092; BD Biosciences)

[0074] 3-3. Results Mice administered with anti-OX40L antibody had significantly lower CD4 in peripheral blood than mice administered with control antibody. +T cells and CD8 + The number of T cells was significantly higher (Fig. 3A, B). On the other hand, there was no significant difference in the number of B cells in the peripheral blood between anti-OX40L antibody and control antibody administration, confirming that anti-OX40L antibody does not adversely affect B cell numbers (Fig. 3C). These results confirm that anti-OX40L antibody is effective in suppressing the decrease in T cells caused by bendamustine and is effective in maintaining T cells.

[0075] 4. Overall Discussion These results demonstrate that blocking the binding of OX40 and OX40L expressed on T cells and inhibiting OX40-mediated signaling during bendamustine treatment of B-cell malignancies can suppress the decline in T-cell numbers without interfering with the therapeutic efficacy of bendamustine. OX40 is a member of the TNFR superfamily, and given that a common signaling pathway exists downstream of TNFR superfamily receptors expressed on T cells, it is likely that inhibiting signaling mediated by not only OX40 but also other receptors in the TNFR superfamily can similarly suppress the decline in T-cell numbers without interfering with the therapeutic efficacy of bendamustine for B-cell malignancies.

Claims

1. A T cell decline inhibitor used to suppress the decline of T cells caused by bendamustine or a pharmaceutically acceptable salt thereof, the T cell decline inhibitor comprising a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells.

2. The T cell reduction inhibitor according to claim 1, which is used in patients with B cell tumors to which bendamustine or a pharmaceutically acceptable salt thereof is administered.

3. The T cell decline inhibitor according to claim 1 or 2, wherein the substance inhibits the interaction between at least one TNFR superfamily member and its ligand.

4. The T cell decline inhibitor according to claim 3, wherein the substance is at least one selected from the group consisting of an antibody that binds to a TNFR superfamily member or its ligand, an antibody fragment thereof, an antagonist of a TNFR superfamily member, an siRNA against a TNFR superfamily member or its ligand, an antisense nucleic acid against a TNFR superfamily member or its ligand, and a ribozyme against a TNFR superfamily member or its ligand.

5. A T cell reduction inhibitor according to claim 1 or 2, wherein the substance inhibits signal transduction mediated by OX40.

6. The T cell reduction inhibitor according to claim 5, wherein the substance inhibits the interaction between OX40 and OX40L.

7. The T cell reduction inhibitor according to claim 6, wherein the substance is at least one selected from the group consisting of an anti-OX40L antibody, an anti-OX40 antibody, an antibody fragment thereof, an OX40 antagonist, an siRNA against OX40 or OX40L, an antisense nucleic acid against OX40 or OX40L, and a ribozyme against OX40 or OX40L.

8. A therapeutic drug set comprising a first pharmaceutical composition containing bendamustine or a pharmaceutically acceptable salt thereof, and a second pharmaceutical composition containing a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells.

9. The therapeutic drug set described in claim 8, used for the treatment of B-cell tumors.

10. Use of a substance that inhibits at least one signal transduction mediated by TNFR superfamily members expressed in T cells for the manufacture of a T cell decline inhibitor used to inhibit T cell decline caused by bendamustine or a pharmaceutically acceptable salt thereof.

11. A substance that inhibits at least one signal transduction mediated by TNFR superfamily members expressed in T cells, used in treatment to suppress T cell depletion caused by bendamustine or a pharmaceutically acceptable salt thereof.

12. A method for suppressing the decline of T cells caused by bendamustine or a pharmaceutically acceptable salt thereof, comprising administering to a patient requiring treatment with bendamustine or a pharmaceutically acceptable salt thereof a substance that inhibits at least one signal transduction mediated by TNFR superfamily members expressed in T cells, and bendamustine or a pharmaceutically acceptable salt thereof.

13. A method for treating a disease to which bendamustine or a pharmaceutically acceptable salt thereof is administered, comprising administering to a patient seeking treatment with bendamustine or a pharmaceutically acceptable salt thereof a substance that inhibits at least one signal transduction mediated by a TNFR superfamily member expressed in T cells, and bendamustine or a pharmaceutically acceptable salt thereof.

14. A method for screening test substances for candidate substances effective in suppressing the decrease in T cells caused by bendamustine or a pharmaceutically acceptable salt thereof, the screening method comprising: a first step of incubating T cells in the presence of at least one ligand of a TNFR superfamily member expressed in T cells, bendamustine or a pharmaceutically acceptable salt thereof, and the test substance, and determining the survival rate of the T cells; and a second step of selecting, as the candidate substance, a test substance that results in a high survival rate of T cells determined in the first step.

15. The screening method according to claim 14, wherein the ligand is OX40L.

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