Patent Description:
The invention is defined in the claims. Any subject-matter beyond the scope of the claims is not part of the invention and provided for information or reference only.

Any reference to a method of treatment of a human or animal body by therapy involving a certain compound or composition is to be interpreted as a reference to said compound or composition for use in said method of treatment.

In recent years, a therapy called chimeric antigen receptor (CAR)-expressing T cell (CAR-T cell) therapy or T cell receptor (TCR)-genetically engineered T cell (TCR-T cell) therapy has been attempted for cancer treatment. Moreover, this cancer treatment mediated by T cell activation has some features in common with cancer treatment using immune checkpoint inhibitors, in terms of immune system activation occurring in vivo. Cell therapy using CAR-T or TCR-T cells is an autologous T cell therapy designed such that T cells taken from a patient are genetically engineered to express targetable CAR or particular TCR and then returned into the patient's body. CAR- or TCR-expressing T cells not only have the ability to kill antigen-expressing tumor cells including cancer cells (killer activity), but also efficiently expand the proliferation of functional T cells as a result of being repeatedly exposed to antigens. In addition, tumor antigens released from the killed tumor cells will be presented on professional antigen-presenting cells to thereby stimulate endogenous T cells, then induce their activation and expansion. Thus, once CAR-T cells have been infused into a patient, they will be engrafted and expand in the patient's body, whereby immune surveillance can be facilitated.

Moreover, an immune checkpoint inhibitor is a drug which binds to an immune checkpoint molecule or a ligand thereof to inhibit immunosuppressive signaling and thereby cancel the immune checkpoint molecule-suppressed activation of cancer cell-recognizing T cells. Major immune checkpoint inhibitors applied in clinical practice include anti-cytotoxic T-lymphocyte associated antigen <NUM> (CTLA-<NUM>) antibody, anti-programmed cell death-<NUM> (PD-<NUM>) antibody, anti-programmed death-<NUM> ligand-<NUM> (PD-L1) antibody, etc..

With recent advances in studies, it has been indicated that abnormal activation of monocytes or macrophages and tissue damage associated therewith (also referred to as macrophage activation syndrome, MAS), bone lesions and skin lesions or systemic organ lesions, which are referred to as hemophagocytic lymphohistiocytosis (HLH), Langerhans cell histiocytosis (LCH), etc., would be caused upon activation of T cells.

CAR-T cell therapy causes a problem called cytokine release syndrome (CRS) (i.e., CRS toxicity). CRS is a condition where blood cytokine levels are significantly increased, along with fever, hypotension, hypoxia, cerebral edema, neurodegeneration, etc..

CRS toxicity will always occur upon transfer of CAR-T cells into cancer patients, and the regulation of CRS toxicity is extremely important in expanding the application or trial of CAR-T cells<NUM>) (Non-patent Document <NUM>).

The mechanisms by which CAR-T cells recognize and disrupt blood cancer cells or cancer tissues have been clarified, but there is a lot of uncertainty about the mechanism of CRS toxicity. The interleukin (IL)-6R antibody (tocilizumab) approved at the same time as CAR-T cell therapy can regulate CRS toxicity below the threshold of tolerance, so that the mechanism of CRS toxicity has been suggested to involve at least IL-<NUM> overproduction-induced inflammatory damage in normal tissue<NUM>) (Non-patent Document <NUM>).

Likewise, immune checkpoint inhibitors have also been reported to cause immune enhancement referred to as autoimmune-related adverse events (irAEs) along with inflammatory immune reactions in all organs in the body including skin, digestive system, endocrine system, nervous system, etc. (Non-patent Documents <NUM> and <NUM>). The principle of treatment lies in drug withdrawal and steroid administration, but prophylactic and therapeutic agents for these irAEs have now begun to be studied, and various candidates including existing drugs are deemed to be under consideration.

Against the background of the elevation of inflammatory cytokines (e.g., TNF-α, IL-<NUM>, IL-1β, MCP-<NUM>) except for those involved in the IL-<NUM> pathway in patients with CRS toxicity resistant to tocilizumab administration and the inapplicability of antibody drugs in such patients complicated with cerebral edema, there has been a demand for the development of drugs widely applicable to CRS toxicity and novel CAR genes. For example, on the basis of an idea that CRS toxicity can be regulated by controlling excessive activation of CAR-T cells, there has been an attempt to suppress excessive activation of CAR-T cells with BTK inhibitors<NUM>) (Non-patent Document <NUM>) or an attempt to regulate excessive activation of CAR-T cells by integration of a "drug susceptibility suicide gene" into the CAR gene<NUM>) (Non-patent Document <NUM>), etc..

For suppression of CRS toxicity, these drug candidates including currently used steroids are designed with a main focus on the suppression of CAR-T cell functions, and there is a serious concern that these drug candidates will lead to the result of cancelling the cancer regression effect by CAR-T cells, although it is temporary. Since severe CRS toxicity is lethal, the application of these drug candidates is under consideration as a passive alternative.

Under present circumstances where CAR-T cell therapy is limited mainly to blood cancers and no clear successful results have been obtained in solid cancer cases, more potent CAR genes and concomitant drugs are also under consideration. For example, strategies are also attempted to further enhance killer activity and achieve its long-term maintenance, as exemplified by the development of novel chimeric antigen receptors which rely on antigen stimulation to activate cytokine signals<NUM>) (Non-patent Document <NUM>), combined use with PD-<NUM>/CTLA4 antibody serving as an immune checkpoint inhibitor<NUM>) (Non-patent Document <NUM>), etc..

In addition, an attempt to improve the access of CAR-T cells to cancer tissues by increasing the number of CAR-T cells to be transferred or by pretreatment with an anticancer agent is also regarded as a practical strategy to enhance the regressive effects of solid cancer.

As a result of such an improvement in CAR-T cell therapy, the acceptable proper regulation of CRS toxicity which concurrently occurs or always occurs<NUM>) will be a more important problem in the future.

On the other hand, a compound referred to as JTE-<NUM>, i.e., ((-)-ethyl N-{<NUM>,<NUM>-dichloro-<NUM>-hydroxy-<NUM>-[<NUM>-(<NUM>-methylpiperazin-<NUM>-yl)ethoxy]benzoyl}-L-phenylalaninate) dihydrochloride (Patent Document <NUM>) was designed as a drug candidate suppressing inflammatory cytokine production.

JTE-<NUM> is a compound suppressing inflammatory cytokine production and having selectivity for myeloid lineage cells, and its phase II study was performed on patients with systemic inflammatory response syndrome (SIRS). In the later phase I clinical pharmacology study, JTE-<NUM> was found to suppress the production of IL-<NUM>, IL-<NUM> and other inflammatory cytokines under LPS loading, and effectively suppress the elevation of C-reactive protein (CRP) levels in a dose-dependent manner. Moreover, JTE-<NUM> was then found to selectively suppress the proliferation of acute myeloid leukemia (AML) cell lines in an in vitro system<NUM>) (Non-patent Document <NUM>).

There are reports showing that abnormal activation of monocytes/macrophages may be involved in the development of CRS toxicity and cerebral edema in CAR-T cell therapy<NUM>), <NUM>) (Non-patent Documents <NUM> and <NUM>). However, this is merely a "hypothesis" inferred from the findings that CRS toxicity development is synchronized with IL-<NUM> and other inflammatory cytokine production in clinical cytokine production profiles and that tocilizumab has an ameliorative effect on CRS toxicity. The verification of this hypothesis is expected not only to recognize the characteristics of CRS toxicity and thereby facilitate the development of appropriate drugs, but also to lead to the research and development of ameliorative and/or prophylactic agents for CRS toxicity without causing CAR-T cell suppression (immunosuppression), which is a matter of concern in the steroid (prednisolone: PSL) prescription used in tocilizumab-resistant patients.

CAR-T cells are positioned as "anticancer T effector cells for use in massive transfer into the body" which are obtained once T cells have been processed and expanded in vitro.

The overproliferation of these T effector cells is an event which also occurs endogenously, as exemplified by "overexpansion of endogenous anticancer T effector cells" caused by administration of immune checkpoint inhibitors and "overexpansion of antiviral T effector cells" induced upon virus infection and/or multiplication. These irAEs concurrently occurring upon treatment and disease development as well as abnormal macrophage activation and cytokine overproduction in HLH and MAS can be regarded as events common with CRS toxicity concurrently occurring in CAR-T cell therapy. In fact, tocilizumab has been tested for its effect on irAEs and reported to have a certain level of effect (Non-patent Document <NUM>).

Summing up events common to these side effects and diseases, there arises a hypothesis that individual groups of cells would mutually related and affect each other to cause over-proliferation of T effector cells and concurrently occurring abnormal activation of macrophages, etc. Thus, the elucidation of CRS toxicity and the development of therapy would lead to a new understanding of these irAEs, HLH and MAS symptoms and the development of appropriate therapeutic agents.

Patent Document <NUM>: <CIT> (Example <NUM>).

Under these circumstances, in CRS, irAEs, HLH, MAS and LCH, etc., there has been a demand for the development of compounds for use in a method for ameliorating these diseases or symptoms.

As a result of extensive and intensive efforts made to solve the problems stated above, the inventors of the present invention have found that JTE-<NUM> ameliorates the above diseases or symptoms. This finding led to the completion of the present invention.

Namely, the present invention is as follows.

In one embodiment, the compound according to any one of (<NUM>) to (<NUM>) above suppresses at least one of cytokine production and macrophage activation.

In one embodiment, the cytokine is at least one selected from the group consisting of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-1RA, IL-2Rα, IL-<NUM>, IL-<NUM>, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), insulin growth factor (IGF), interferon (IFN)-γ, IFN-γ-induced protein <NUM> (IP-<NUM>), monocyte chemotactic protein (MCP)-<NUM>, vascular endothelial growth factor (VEGF), osteopontin (OPN), Receptor activator of NF-κB ligand (RANKL), cytokine receptor gp130, soluble IL-<NUM> receptor (sIL-1R)-<NUM>, sIL-1R-<NUM>, soluble IL-<NUM> receptor (sIL-6R), soluble receptor for advanced glycation end products (sRAGE), soluble TNF receptor (sTNFR)-<NUM>, sTNFR-<NUM>, monokine induced by IFN-γ (MIG), macrophage inflammatory protein (MIP)-1α and MIP-1β.

In one embodiment, the cytokine is at least one selected from the group consisting of TNF-α, IL-1β, IL-<NUM>, IL-<NUM>, IL-<NUM>, IFN-γ, MCP-<NUM> and OPN.

The present invention provides a medicament for at least one selected from the group consisting of CRS, irAEs, HLH, MAS and LCH. The medicament comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof has the potential to suppress the release of inflammatory cytokines to thereby ameliorate or prevent these symptoms and, in turn, to relatively enhance the effects of CAR-T cell therapy and immune checkpoint inhibitors.

The present invention will be described in more detail below.

The present invention is directed to a medicament for use in the treatment of at least one selected from the group consisting of cytokine release syndrome (CRS), autoimmune-related adverse events (irAEs), macrophage activation syndrome (MAS), hemophagocytic lymphohistiocytosis (HLH) and Langerhans cell histiocytosis (LCH), wherein the medicament comprises a compound represented by the following formula II:
<CHM>
or a pharmaceutically acceptable salt thereof, and this medicament can be used as a prophylactic or ameliorative agent for these diseases or symptoms and also can be used in the form of a pharmaceutical composition comprising various additives.

In a compound represented by formula II or a pharmaceutically acceptable salt thereof (also collectively referred to as "the compound of the present invention"), the inventors of the present invention have now constructed an evaluation system capable of reproducing CRS toxicity through target cell recognition by CD19-CAR-T cells and CD8+ T cell activation induced by magnetic beads covalently attached with anti-CD3 and anti-CD28 antibodies (i.e., T-cell stimulation beads) to investigate the effect of this compound (e.g., JTE-<NUM>) in comparison with the effect of an existing drug, PSL. The results have indicated that PSL predominantly suppresses T cell functions (immunosuppression: suppression of CAR-T cell functions and CD8+ functions) but shows a weak suppressive effect on the production of IL-<NUM> and other inflammatory cytokines, whereas JTE-<NUM> is characterized by strongly suppressing the production of IL-<NUM> and other inflammatory cytokines in a dose-dependent manner, and its effects on CAR-T cells, CD4+ functions and CD8+ functions (e.g., cytotoxicity against target cancer cells, IFN-γ production ability) are limited and not dose-dependent. Moreover, it has been indicated that JTE-<NUM> suppresses not only IL-<NUM> production but also IL-<NUM> and Osteopontin (OPN) production in a dose-dependent manner, whereas tocilizumab has no suppressive effect on the production of these cytokines.

<FIG> shows the mechanism of action for the compound of the present invention. In <FIG>, the excessive reaction of endogenous T effector cells induces a series of diseases or symptoms along with monocyte/macrophage activation (<FIG>, the area below the broken line). The compound of the present invention can be used for prevention or amelioration (including treatment) of these diseases or symptoms.

In view of the foregoing, the compound of the present invention as a medicament for at least one disease or symptom selected from the group consisting of CRS, irAEs, HLH, MAS and LCH may serve, for example, as a therapeutic tool effective in the amelioration and/or prevention of CRS toxicity concurrently occurring in CAR-T cell therapy. Thus, the present invention provides a prophylactic method or an ameliorative or therapeutic method for at least one disease or symptom selected from the group consisting of CRS, irAEs, HLH, MAS and LCH, wherein the method comprises the step of administering the compound of the present invention or a medicament comprising the compound of the present invention to a patient with the disease or symptom.

Moreover, also in the case of hypercytokinemia-like symptoms concurrently occurring upon administration of immune checkpoint inhibitors such as bi-specific antibody and PD1/CTLA4 antibody which serve as enhancers for potential T cell functions, these CRS-like symptoms irAEs can be ameliorated without significantly affecting the enhanced T cell functions.

As used herein, the term "prevention" or "prophylactic" is intended to mean that the above diseases or symptoms are prevented from developing in a patient who may be predisposed to the above diseases or symptoms but has not yet been diagnosed as having them.

The term "treatment" or "therapeutic" is intended to mean that the above diseases or symptoms are inhibited, i.e., their progression is arrested or delayed or disappears.

The term "amelioration" or "ameliorative" is intended to mean that the above diseases or symptoms are alleviated, i.e., the recession of the above diseases or symptoms is caused or the progression of the symptoms is reversed.

In the present invention, a compound serving as an active ingredient, which is used as a medicament for at least one disease or symptom selected from the group consisting of CRS, irAEs, HLH, MAS and LCH (e.g., a prophylactic, therapeutic or ameliorative agent for these diseases), is represented by the following formula II. Among compounds falling within the present invention, a compound represented by formula II is also referred to as "compound (II).

A salt of compound (II) may be exemplified by salts with inorganic acids or organic acids (i.e., acid addition salts).

Examples of such an inorganic acid include hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, nitric acid and so on, while examples of such an organic acid include oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, succinic acid, citric acid, acetic acid, lactic acid, methanesulfonic acid, paratoluenesulfonic acid, benzoic acid, valeric acid, malonic acid, nicotinic acid, propionic acid and so on.

A preferred acid addition salt in the present invention is a salt with hydrochloric acid, more preferably a dihydrochloride salt.

Compound (II) may have one or two or more stereoisomers based on its asymmetric carbon. The present invention also encompasses these stereoisomers and a mixture thereof.

In the present invention, compound (II) is preferably exemplified by (-)-ethyl N-{<NUM>,<NUM>-dichloro-<NUM>-hydroxy-<NUM>-[<NUM>-(<NUM>-methylpiperazin-<NUM>-yl)ethoxy]benzoyl}-L-phenylalaninate represented by the following formula III:
<CHM>
(i.e., N-{<NUM>,<NUM>-dichloro-<NUM>-hydroxy-<NUM>-[<NUM>-(<NUM>-methylpiperazin-<NUM>-yl)ethoxy]benzoyl}-L-phenylalanine ethyl ester) or a pharmacologically acceptable salt thereof.

The compound to be used in the present invention is more preferably an acid addition salt (e.g., a dihydrochloride salt) of the above compound represented by formula III, and even more preferably a compound represented by formula IV:
<CHM>.

The compound represented by formula IV is (-)-ethyl N-{<NUM>,<NUM>-dichloro-<NUM>-hydroxy-<NUM>-[<NUM>-(<NUM>-methylpiperazin-<NUM>-yl)ethoxy]benzoyl}-L-phenylalaninate dihydrochloride (i.e., N-{<NUM>,<NUM>-dichloro-<NUM>-hydroxy-<NUM>-[<NUM>-(<NUM>-methylpiperazin-<NUM>-yl)ethoxy]benzoyl}-L-phenylalanine ethyl ester dihydrochloride), which is also known as "JTE-<NUM>.

The compound of the present invention suppresses the production of various inflammatory cytokines which are produced in animals (e.g., mammals such as rats, mice, rabbits, pigs, cats, dogs, cows, horses, monkeys, humans, etc.).

In the present invention, it is particularly effective as a medicament for one or more diseases or symptoms of CRS, irAEs, HLH, MAS and LCH.

For example, in human cytokine release syndrome, the compound of the present invention is particularly effective against CRS and cerebral edema in CAR-T cell therapy. Thus, the compound of the present invention can be used in combination with CAR-T cell therapy. The expression "used in combination" is intended to mean that the compound of the present invention is used concurrently with CAR-T cell therapy, but the timing for use in combination is not limited in any way and may be any timing of before CAR-T cell therapy, during CAR-T cell therapy and after CAR-T cell therapy. With regard to irAEs, HLH, MAS and LCH, the compound of the present invention can also be used concurrently with drugs or therapies used in the treatment of these diseases or symptoms, as in the case of CRS.

The compound of the present invention allows suppression of cytokine production, suppression of macrophage activation, or suppression of both cytokine production and macrophage activation. As used herein, the term "macrophage activation" is intended to mean the excessive release of various cytokines, cytotoxic proteases and/or active oxygen species, etc., as well as the enhancement of opsonization and/or phagocytic activity, etc..

The term "cytokine" is intended to include, for example, tumor necrosis factor (TNF)-α, IL-1β, IL-<NUM>, IL-<NUM>, IL-<NUM>, IL-1RA, IL-2Rα, IL-<NUM>, IL-<NUM>, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), insulin growth factor (IGF), interferon (IFN)-γ, IFN-γ-induced protein <NUM> (IP-<NUM>), monocyte chemotactic protein (MCP)-<NUM>, vascular endothelial growth factor (VEGF), osteopontin (OPN), Receptor activator of NF-κB ligand (RANKL), cytokine receptor gp130, soluble IL-<NUM> receptor (sIL-1R)-<NUM>, sIL-1R-<NUM>, soluble IL-<NUM> receptor (sIL-6R), soluble receptor for advanced glycation end products (sRAGE), soluble TNF receptor (sTNFR)-<NUM>, sTNFR-<NUM>, monokine induced by IFN-γ (MIG), macrophage inflammatory protein (MIP)-1α, MIP-1β and so on.

irAEs refer to a group of adverse events associated with immune checkpoint inhibitors whose administration results in excessive activation of overall immunity and thereby induces self-attack to cause various symptoms of autoimmune diseases. Major symptoms include interstitial lung disease, colitis, hypothyroidism, hepatopathy, exanthema, hypophysitis, diabetes, renal dysfunction, peripheral neuropathy, myasthenia gravis and so on, which have been reported in all organs throughout the body.

HLH refers to a disease caused by defects in genes which are involved in cytotoxic granules and their release. This disease is characterized by pathologies such as tissue damage and macrophage expansion associated with hypercytokinemia. As to inflammatory cytokines involved in HLH, there have been reported IFN-γ, IL-1β, IL-<NUM>, IL-<NUM>, TNF-α and so on.

MAS refers to a lethal pathological condition where inflammatory cytokines are in excessive levels, which is induced upon abnormal activation of macrophages in response to exogenous factors (e.g., viruses, bacteria, fungi and other infectious factors or drugs) or endogenous factors (e.g., debris generated upon apoptosis/necrosis of autologous cells), for example, proliferation and activation of virus-reactive T cell effectors. As to inflammatory cytokines involved in MAS, there have been reported IFN-γ, IL-1β, IL-<NUM>, IL-<NUM>, TNF-α and so on. HLH and MAS are similar to each other, but HLH is confirmed by pathological diagnosis, whereas MAS is confirmed by physiological diagnosis. In either pathological condition, macrophage activation plays an important role.

LCH<NUM>) refers to a pathological condition showing abnormal expansion of Langerhans cells in the skin, bone, lymph nodes and other organs. In lesion sites, there are observed not only Langerhans cells, but also inflammatory infiltrates including eosinophils, lymphocytes, macrophages, osteoclast-like giant cells, etc., and these cells have been known to activate each other to thereby cause oversecretion of inflammatory cytokines/chemokines, as typified by OPN, RANKL, IL-<NUM>, C-C motif chemokine <NUM> (CCL2), etc., which in turn causes tissue destruction.

These diseases or symptoms are due to the use of immune checkpoint inhibitors, chimeric antigen receptor-expressing T cell therapy, engineered T cell receptor-expressing T cell therapy, etc., or are induced by any of infections, cancers and autoimmune diseases or any combination thereof.

An immune checkpoint inhibitor refers to a drug which binds to an immune checkpoint molecule (a group of molecules which suppress autoimmune responses and also suppress excessive immune reactions) or a ligand thereof to thereby inhibit immunosuppressive signaling and thus cancel the immune checkpoint molecule-suppressed activation of T cells. Examples include anti-CTLA-<NUM> antibody, anti-PD-<NUM> antibody, anti-PD-L1 antibody and so on.

CAR-T cell therapy is an autologous T cell therapy designed such that T cells taken from a patient are genetically engineered to express a targetable chimeric antigen receptor (CAR) and then returned into the patient's body.

Engineered T cell receptor-expressing T (TCAR-T) cell therapy is a therapy designed such that a particular TCR gene which recognizes a cancer antigen epitope in a particular human leukocyte antigen (HLA)-restricted manner is introduced into and amplified in effector T cells in vitro and then administered in vivo, by way of example.

This cell therapy is similar to CAR-T cell therapy.

Preferred embodiments of the medicament according to (<NUM>) above are as listed below.

A medicament for use in the prevention, treatment or amelioration of at least one selected from the group consisting of cytokine release syndrome macrophage activation syndrome and hemophagocytic lymphohistiocytosis, each being due to chimeric antigen receptor-expressing T cell therapy, as further defined in the claims.

A medicament for use in the prevention, treatment or amelioration of cytokine release syndrome due to chimeric antigen receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of macrophage activation syndrome due to chimeric antigen receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of hemophagocytic lymphohistiocytosis due to chimeric antigen receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of at least one selected from the group consisting of cytokine release syndrome macrophage activation syndrome and hemophagocytic lymphohistiocytosis for use in combination with chimeric antigen receptor-expressing T cell therapy, wherein the medicament comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof.

A medicament for use in the prevention, treatment or amelioration of at least one selected from the group consisting of cytokine release syndrome macrophage activation syndrome and hemophagocytic lymphohistiocytosis, each being due to engineered T cell receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of cytokine release syndrome due to engineered T cell receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of macrophage activation syndrome due to engineered T cell receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of hemophagocytic lymphohistiocytosis due to engineered T cell receptor-expressing T cell therapy.

A medicament for use in the prevention, treatment or amelioration of at least one selected from the group consisting of cytokine release syndrome macrophage activation syndrome and hemophagocytic lymphohistiocytosis for use in combination with engineered T cell receptor-expressing T cell therapy, wherein the medicament comprises a compound represented by formula I or a pharmaceutically acceptable salt thereof.

For use as a "medicament for cytokine release syndrome due to chimeric antigen receptor-expressing T cell therapy" and as a "medicament for cytokine release syndrome due to engineered T cell receptor-expressing T cell therapy," preferred are medicaments suppressing the production of at least one, more preferably a plurality of cytokines selected from the group consisting of TNF-α, IL-1β, IL-<NUM>, IL-<NUM>, IFN-γ and MCP-<NUM>.

A medicament for use in the prevention, treatment or amelioration of macrophage activation syndrome.

Preferred for this purpose is a medicament suppressing the production of at least one, more preferably a plurality of cytokines selected from the group consisting of TNF-α, IL-1β, IL-<NUM>, IL-<NUM>, IFN-γ and MCP-<NUM>.

A medicament for use in the prevention, treatment or amelioration of hemophagocytic lymphohistiocytosis.

Preferred for this purpose is a medicament suppressing the production of at least one, more preferably a plurality of cytokines selected from the group consisting of TNF-α, IL-1β, IL-<NUM>, IL-<NUM>, IL-<NUM>, IFN-γ and MCP-<NUM>.

A medicament for use in the prevention, treatment or amelioration of autoimmune-related adverse events due to the use of immune checkpoint inhibitors. Preferred for this purpose is a medicament suppressing the production of at least one, more preferably a plurality of cytokines selected from the group consisting of TNF-α, IL-1β, IL-<NUM>, IL-<NUM> and IFN-γ.

A medicament for use in the prevention, treatment or amelioration of Langerhans cell histiocytosis induced by autoimmune diseases. Preferred for this purpose is a medicament suppressing the production of at least one, more preferably a plurality of cytokines selected from the group consisting of IL-<NUM>, MCP-<NUM> and OPN.

Moreover, for use as a "medicament for at least one selected from the group consisting of cytokine release syndrome, autoimmune-related adverse events, macrophage activation syndrome, hemophagocytic lymphohistiocytosis and Langerhans cell histiocytosis, wherein the medicament comprises a compound represented by formula.

II or a pharmaceutically acceptable salt thereof," preferred is a medicament suppressing the production of at least one, more preferably a plurality of cytokines selected from the group consisting of IL-<NUM>, MCP-<NUM> and OPN.

The compound of the present invention may be administered directly as a medicament (e.g., a prophylactic, therapeutic or ameliorative agent) for at least one selected from the group consisting of CRS, irAEs, HLH, MAS and LCH, although it is
administered in the form of a pharmaceutical composition in one embodiment of the present invention. For use as a pharmaceutical composition containing the compound of the present invention as an active ingredient, this pharmaceutical composition may usually be formulated in any dosage form such as tablets, pills, powders, granules, suppositories, injections, eye drops, solutions, capsules, troches, aerosols, elixirs, suspensions, emulsions, syrups and so on in admixture with pharmacologically acceptable additives, as exemplified by pharmacologically acceptable carriers, excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, flavorings, colorants, sweeteners, thickeners, correctives, solubilizers and/or other additives (e.g., water, vegetable oils, alcohols (e.g., ethanol or benzyl alcohol), polyethylene glycol, glycerol triacetate, gelatin, lactose, carbohydrates, magnesium stearate, talc, lanolin, white petrolatum, etc.).

For oral administration, the pharmaceutical composition may comprise a diluent, a dispersant and/or a surfactant in the form of powder or granules. For example, it may be present in water, in a syrup, in a dry state in a capsule or sachet, in a non-aqueous solution or suspension which may contain a suspending agent, or in a tablet which may contain a binder and a lubricant. The pharmaceutical composition may also comprise a sweetener, a corrective, a preservative (e.g., an antimicrobial preservative), a suspending agent, a thickener and/or an emulsifier.

For parenteral administration, the pharmaceutical composition is in the dosage form of a solution or suspension and may contain the compound of the present invention (e.g., compound (III)) and purified water. Additional ingredients which may be contained in a solution or suspension include a preservative (e.g., an antimicrobial preservative), a buffer, a solution thereof and a mixture thereof. The ingredients of the pharmaceutical composition may exert one or more functions. The pharmaceutical composition may be filled into single-dose or multiple-dose containers, e.g., sealed vials and ampules for storage in a lyophilized state, and may be added to a sterile liquid carrier (e.g., water or physiological saline) before use. In a preferred embodiment, the compound of the present invention (e.g., compound (III) or a pharmaceutically acceptable salt thereof) is formulated into a lyophilized formulation containing the same together with D-mannitol. Such a lyophilized formulation is preferably diluted with physiological saline before use.

Such a pharmaceutical composition containing the compound of the present invention may be prepared in a manner well known in the field of medicaments, for example, by the method described in <NPL>, particularly Part <NUM>: "Pharmaceutical Preparation and their Manufacture. " This method comprises the step of associating the compound of the present invention with the other ingredients of the pharmaceutical composition.

The medicament of the present invention may be administered in an appropriate manner, and the route of its administration is not limited in any way. Examples include oral, buccal, nasal, percutaneous, injection, sustained release, controlled release, iontophoresis and sonophoresis. The route of injection is not limited in any way but includes parenteral routes such as intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial and other routes of injection. For parenteral administration, the pharmaceutical composition may be formulated as a liquid or non-liquid sterile injection formulation, with an intravenous injection formulation being preferred.

The suitable dose of the compound of the present invention will vary depending on the patient's type and symptoms, the route of administration, sexual difference, body weight, etc. For oral administration in adults, for example, the daily dose of the compound of the present invention (particularly compound (III) or a pharmaceutically acceptable salt thereof) is usually about <NUM> to <NUM>,<NUM> (e.g., <NUM> to <NUM>, preferably <NUM> to <NUM>).

For intravenous administration in adults, for example, the daily dose of the compound of the present invention (particularly compound (III) or a pharmaceutically acceptable salt thereof) is usually about <NUM> to <NUM>/kg (e.g., about <NUM> to <NUM>/kg, preferably about <NUM> to <NUM>/kg), which may be given as a single dose or in divided doses (e.g., <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more doses). Alternatively, the compound of the present invention (particularly compound (III) or a pharmaceutically acceptable salt thereof) may be administered by continuous intravenous administration over the selected period of time (e.g., several hours to one or more days). The total daily dose for continuous administration is usually the same as the daily dose used in non-continuous intravenous administration.

The present invention will be further described in more detail below by way of the following illustrative examples. The scope of the present invention is defined by the claims.

Cryopreserved PBMCs were quickly thawed in a <NUM> thermostat and added to a tube containing medium. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the cells were suspended in the medium, a portion of which was then mixed with a trypan blue solution to count the number of viable cells. The cell suspension was centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, followed by addition of MACS Buffer in a volume of <NUM>µL per <NUM> × <NUM><NUM> cells to suspend the cells, to which Human CD8 MicroBeads (<NUM>µL) were further added. After being allowed to stand on ice for <NUM> minutes, the cell suspension was diluted with MACS Buffer (<NUM>) and centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant.

MACS Buffer (<NUM>µL) was added to the tube, and the cells were suspended and then applied to a separatory column to separate CD8+ cells. The CD8+ cell fraction was diluted to <NUM> with the medium and then centrifuged (at room temperature at <NUM> for <NUM> minutes). The supernatant was removed and the cells were suspended in the medium (<NUM>), followed by counting the number of viable cells. The cell suspension adjusted with the medium to <NUM> × <NUM><NUM> cells/mL was seeded in a <NUM>-well plate in a volume of <NUM>µL/well (<NUM> × <NUM><NUM> cells/well) and allowed to stand in a CO<NUM> incubator for <NUM> to <NUM> hours.

After addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) in a volume of <NUM>µL/well, T-cell stimulation beads (<NUM>µL/well; which corresponds to <NUM>µL of T-cell stimulation bead stock solution) and the medium (<NUM>µL/well) were added and the cells were cultured in a CO<NUM> incubator for <NUM> days to collect the culture supernatants.

The collected culture supernatants were quantified for IFN-γ using Human IFN-γ DuoSet ELISA.

Cryopreserved PBMCs were quickly thawed in a <NUM> thermostat and added to a tube containing medium. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the cells were suspended again in the medium, followed by counting the number of viable cells. The cell suspension was centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, followed by addition of MACS Buffer to suspend the cells, to which Human CD14 MicroBeads were further added. After being allowed to stand on ice for <NUM> minutes, the cell suspension was diluted with MACS Buffer and centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant.

MACS Buffer was added to the tube, and the cells were suspended and then applied to a separatory column to separate CD14+ cells. The CD14+ cell fraction was diluted with the medium and then centrifuged (at room temperature at <NUM> for <NUM> minutes). The supernatant was removed and the cells were suspended in the medium (<NUM>), followed by counting the number of viable cells. The cell suspension was adjusted with the medium to <NUM> × <NUM><NUM> or <NUM> × <NUM><NUM> cells/mL and seeded in a <NUM>-well plate (<NUM> × <NUM><NUM> cells/well), which was then allowed to stand in a CO<NUM> incubator for <NUM> to <NUM> hours.

After addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>), a LPS solution (final concentration: <NUM> ng/mL) was further added and the <NUM>-well plate was allowed to stand in a CO<NUM> incubator for <NUM> days to collect the culture supernatants.

The collected culture supernatants were quantified for IL-<NUM> using Human IL-<NUM> DuoSet ELISA.

Cryopreserved PBMCs were quickly thawed in a <NUM> thermostat and added to a tube containing medium. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the cells were suspended again in the medium (<NUM>), followed by counting the number of viable cells. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the medium was added to suspend the cells at <NUM> × <NUM><NUM> cells/mL. This cell suspension was seeded in a <NUM>-well plate in a volume of <NUM>µL/well (<NUM> × <NUM><NUM> cells/well). After addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) in a volume of <NUM>µL/well, a LPS solution was added in a volume of <NUM>µL/well (final concentration: <NUM> ng/mL). The cells were cultured in a CO<NUM> incubator for <NUM> days to collect the culture supernatants.

Cryopreserved PBMCs were quickly thawed in a <NUM> thermostat and added to a tube containing medium. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the cells were suspended again in the medium (<NUM>), followed by counting the number of viable cells. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the medium was added to suspend the cells at <NUM> × <NUM><NUM> cells/mL. This cell suspension was seeded in a <NUM>-well plate in a volume of <NUM>µL/well (<NUM> × <NUM><NUM> cells/well) and allowed to stand in a CO<NUM> incubator for <NUM> to <NUM> hours.

After addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) in a volume of <NUM>µL/well, T-cell stimulation beads (<NUM>µL/well; which corresponds to <NUM>µL of T-cell stimulation bead stock solution) and a LPS solution (final concentration: <NUM> ng/mL; <NUM>µL/well) were added. The cells were cultured in a CO<NUM> incubator for <NUM> days to collect the culture supernatants.

The collected culture supernatants were quantified for IFN-γ and IL-<NUM> using Human IFN-γ DuoSet ELISA and Human IL-<NUM> DuoSet ELISA.

Cryopreserved human PBMCs were quickly thawed in a <NUM> thermostat and added to a tube containing medium. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the cells were suspended again in the medium (<NUM>), followed by counting the number of viable cells. The remainder of the cell suspension was centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, followed by addition of MACS Buffer in a volume of <NUM>µE and Human CD4 MicroBeads in a volume of <NUM>µL per <NUM> × <NUM><NUM> cells to suspend the cells. After being allowed to stand on ice for <NUM> minutes, the cell suspension was diluted with MACS Buffer (<NUM>) and centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant. MACS Buffer (<NUM>µL) was added to the tube, and the cells were suspended and then applied to a separatory column to separate CD4- cells (a pass-through fraction of CD4+ cells). The CD4- cell fraction was diluted to <NUM> with the medium and then centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, and the cells were then suspended again in the medium (<NUM>), followed by counting the number of viable cells. The cell suspension adjusted with the medium to <NUM> × <NUM><NUM> cells/mL was seeded in a <NUM>-well plate in a volume of <NUM>µL/well (<NUM> × <NUM><NUM> cells/well) and allowed to stand in a CO<NUM> incubator for <NUM> to <NUM> hours.

After addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) in a volume of <NUM>µL/well, T-cell stimulation beads (<NUM>µL/well; which corresponds to <NUM>µL of T-cell stimulation bead stock solution) and a LPS solution (final concentration: <NUM> ng/mL; <NUM>µL/well) were added and the cells were cultured in a CO<NUM> incubator for <NUM> days to collect the culture supernatants.

The collected culture supernatants were quantified for MCP-<NUM> using Human CCL2/MCP-<NUM> DuoSet ELISA.

The culture supernatants collected in <NUM>. <NUM> were quantified for TNF-α using Human TNF-α DuoSet ELISA.

Cryopreserved PBMCs were quickly thawed in a <NUM> thermostat and added to a tube containing medium. After centrifugation (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, the cells were suspended again in the medium, followed by counting the number of viable cells. The cell suspension was centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant, followed by addition of MACS Buffer to suspend the cells, to which Human CD14 MicroBeads were further added. After being allowed to stand on ice for <NUM> minutes, the cell suspension was diluted with MACS Buffer and centrifuged (at room temperature at <NUM> for <NUM> minutes) to remove the supernatant. MACS Buffer was added to the tube, and the cells were suspended and then applied to a separatory column to separate CD14+ cells. The CD14+ cell fraction was diluted with the medium and then centrifuged (at room temperature at <NUM> for <NUM> minutes). The supernatant was removed and the cells were suspended in the medium (<NUM>), followed by counting the number of viable cells. The cell suspension was adjusted with the medium to <NUM> × <NUM><NUM> or <NUM> × <NUM><NUM> cells/mL and seeded in a <NUM>-well plate (<NUM> × <NUM><NUM> cells/well), which was then allowed to stand in a CO<NUM> incubator for <NUM> to <NUM> hours.

After addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>), a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or tocilizumab (final concentration: <NUM>, <NUM> and <NUM> ng/mL), aLPS solution (final concentration: <NUM> ng/mL) was further added and the <NUM>-well plate was allowed to stand in a CO<NUM> incubator for <NUM> days to collect the culture supernatants.

The collected culture supernatants were quantified for OPN using Human OPN DuoSet ELISA.

The culture supernatants collected in <NUM>. <NUM> were quantified for IL-1β using Human IL-1β DuoSet ELISA.

MACS Buffer was added to the tube, and the cells were suspended and then applied to a separatory column to separate CD14+ cells. The CD14+ cell fraction was diluted with the medium and then centrifuged (at room temperature at <NUM> for <NUM> minutes). The supernatant was removed and the cells were suspended in the medium (<NUM>), followed by counting the number of viable cells. The cell suspension adjusted with the medium to <NUM> × <NUM><NUM> cells/mL was seeded in a <NUM>-well plate (<NUM> × <NUM><NUM> cells/well) and allowed to stand in a CO<NUM> incubator for <NUM> to <NUM> hours. The CD14+ cells (<NUM> × <NUM><NUM> cells/well) were seeded in a <NUM>-well flat bottom plate, followed by addition of a <NUM>% DMSO solution, a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>), a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or tocilizumab (final concentration: <NUM>, <NUM> and <NUM> ng/mL) and incubation for <NUM> minutes at <NUM>. The cells were then stimulated with LPS (final concentration: <NUM> ng/mL) for <NUM> hours at <NUM> and an ATP solution (final concentration: <NUM>) was added thereto, and the <NUM>-well plate was further allowed to stand in a CO<NUM> incubator for <NUM> hours to collect the culture supernatants.

CD14+ cells were cultured in <NUM> ng/mL M-CSF-containing RPMI medium at <NUM> for <NUM> days to induce differentiation into macrophages (during which the medium was replaced once every two days with fresh <NUM> ng/mL M-CSF-containing RPMI medium). The macrophages were seeded in a <NUM>-well plate at <NUM> × <NUM><NUM> cells/mL, followed by addition of a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>), a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or tocilizumab (final concentration: <NUM>, <NUM> and <NUM> ng/mL) and incubation at <NUM> for <NUM> minutes. The macrophages were then stimulated with LPS (final concentration: <NUM> ng/mL) or RPMI medium alone at <NUM> for <NUM> days, followed by collecting the culture supernatants.

The collected culture supernatants were quantified for VEGF using Human VEGF DuoSet ELISA.

FMC63 scFv, the hinge, transmembrane and cytoplasmic domains of CD28 and the cytoplasmic domain of CD3-ζ were used to construct anti-CD19 CAR on the basis of the method of Kochenderfer, et al. The sequence was obtained from GenBank (HM852952) and an IDT codon optimization tool (http://sg. com/CodonOpt) was used to remove BamHI recognition sequences within FMC63. The optimized FMC63-28z sequence was chemically synthesized and amplified by polymerase chain reaction (PCR) with the following primer set.

The PCR product was inserted into the multicloning site (EcoRI and BamHI) of lentivirus plasmid CSII-EF-MCS-2A-eGFP by using an In-Fusion HD cloning kit to obtain CSII-EF-FMC63-28z-2A-eGFP. To prepare a lentivirus vector, CSII-EF-FMC63-28z-2A-eGFP and a packaging plasmid (pMDLg/p. RRE, pRSV-rev and pMD. G) were co-transfected into Lenti-X293T cells. The prepared lentivirus vector was infected into peripheral blood mononuclear cells stimulated with anti-CD3 antibody, anti-CD28 antibody and IL-<NUM>, followed by FACS sorting to obtain a CD3-positive and eGFP-positive fraction as CAR-T cells.

On the basis of the method of Kochenderfer, et al. <NUM>), the open reading frame of human CD19 was synthesized and amplified by PCR with the following primers.

The PCR product was inserted into lentivirus backbone plasmid CSII-EF-MCS via the EcoRI and XbaI sites by using an In-Fusion HD cloning kit to obtain CSII-EF-hCD19. In addition, to prepare a firefly luciferase (fLuc) expression plasmid, fLuc cDNA was amplified by PCR with the following primer set using the pmirGLO plasmid (Promega) as a template.

The PCR product was cleaved with EcoRI and BamHI and then inserted into the multicloning site of CSII-EF-MCS-2A-eGFP to obtain CSII-EF-fLuc-2A-eGFP. To prepare a lentivirus vector, CSII-EF-hCD19 or CSII-EF-fLuc-2A-eGFP and a packaging plasmid (pMDLg/p. RRE, pRSV-rev and pMD. G) were co-transfected into Lenti-X293T cells. The prepared lentivirus vectors were infected into K562 cells, followed by FACS sorting to obtain a CD19- and eGFP-positive fraction as target cells.

The target cells (<NUM> × <NUM><NUM> cells/well) were seeded in a <NUM>-well plate, to which the CAR-T cells were then added to give an E/T ratio of <NUM>,<NUM>, <NUM>, <NUM>, <NUM> and <NUM>. This culture system was incubated for <NUM> days under conditions where CD14+ cells (<NUM> × <NUM><NUM> cells/well) were added or not added thereto. Subsequently, luciferase activity was determined by detection of emission intensity with an EnSpire <NUM>.

CAR-T cells (<NUM> × <NUM><NUM> cells/well) and target cells (<NUM> × <NUM><NUM> cells/well) were seeded in a <NUM>-well plate and incubated under conditions where CD14+ cells (<NUM> × <NUM><NUM> cells/well) were added or not added thereto. The supernatants were collected at <NUM>, <NUM>, <NUM>, <NUM> and <NUM> hours after the initiation of culture, and IFN-γ, TNF-α, MCP-<NUM>, IL-<NUM> and IL-<NUM> in each supernatant were measured by FACS multiplex assay.

CAR-T cells (<NUM> × <NUM><NUM> cells/well), target cells (<NUM> × <NUM><NUM> cells/well) and CD14+ cells (<NUM> × <NUM><NUM> cells/well) were seeded in a <NUM>-well plate, to which a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) or a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) was then added, followed by incubation for <NUM> days. Subsequently, luciferase activity was determined by detection of emission intensity with an EnSpire <NUM>.

CAR-T cells (<NUM> × <NUM><NUM> cells/well), target cells (<NUM> × <NUM><NUM> cells/well) and monocytes (<NUM> × <NUM><NUM> cells/well) were seeded in a <NUM>-well plate, to which a JTE-<NUM> solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>), a PSL solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>) or a tocilizumab solution (final concentration: <NUM>, <NUM>, <NUM> and <NUM>µg/mL) was then added, followed by incubation. The supernatants were collected at <NUM> hours after the initiation of culture, and various cytokines in each supernatant were measured by FACS multiplex assay.

JTE-<NUM> showed no significant suppressive effect on IFN-γ production from CD8+ T cells stimulated with T-cell stimulation beads (IC<NUM> value = ><NUM>). On the other hand, PSL suppressed this IFN-γ production in a concentration-dependent manner (IC<NUM> value = <NUM>). JTE-<NUM> was found to have a weak direct effect on CD8+ T cells (<FIG>, <FIG> and Table <NUM>).

JTE-<NUM> and PSL were evaluated for their effect on IL-<NUM> production from CD14+ cells. JTE-<NUM> and PSL both showed suppressive activity and had IC<NUM> values of <NUM> and <NUM>, respectively. As previously reported, JTE-<NUM> was found to have an effect on myeloid lineage cells (<FIG>, <FIG> and Table <NUM>).

JTE-<NUM> and PSL were evaluated for their effect on IL-<NUM> production from PBMCs. JTE-<NUM> and PSL both showed suppressive activity and had IC<NUM> values of <NUM> and <NUM>, respectively (<FIG> and Table <NUM>).

JTE-<NUM> and PSL were evaluated for their effect on IL-<NUM> and IFN-γ production from PBMCs co-stimulated with T-cell stimulation beads and LPS. JTE-<NUM> suppressed the co-stimulated IL-<NUM> and IFN-γ production in a concentration-dependent manner and had IC<NUM> values of <NUM> for IL-<NUM> and <NUM> for IFN-γ. Moreover, the ratio of IC<NUM> values for IL-<NUM> and IFN-γ (ratio) was <NUM>. On the other hand, PSL more strongly suppressed IFN-γ production than IL-<NUM> production. JTE-<NUM> was found to be different from PSL which strongly acts on T cell functions (<FIG> and Table <NUM>).

To clarify the involvement of CD4+ T cells in the system co-stimulated with T-cell stimulation beads and LPS, CD4+ T cell-depleted PBMCs (i.e., PBMCs processed to remove CD4+ T cells) were used to evaluate the effects of JTE-<NUM> and PSL. JTE-<NUM> suppressed the co-stimulated IL-<NUM> and IFN-γ production in a concentration-dependent manner and had similar IC<NUM> values for both (IC<NUM> value for IL-<NUM> = <NUM>, IC<NUM> value for IFN-γ = <NUM>). Moreover, the ratio of IC<NUM> values for IL-<NUM> and IFN-γ (ratio) was <NUM>, and JTE-<NUM> showed an effect directed rather toward IL-<NUM> production inhibition. On the other hand, PSL suppressed IFN-γ production in a concentration-dependent manner (IC<NUM> value = <NUM>) but did not suppress IL-<NUM> production (<FIG> and Table <NUM>). Similar results were able to be obtained in the studies using PBMCs and CD4+ T cell-depleted PBMCs, thus suggesting that the involvement of CD4+ T cells in the system co-stimulated with T-cell stimulation beads and LPS would be low.

JTE-<NUM> and PSL were evaluated for their effect on MCP-<NUM> production from CD14+ cells. JTE-<NUM> and PSL both showed suppressive activity and had IC<NUM> values of <NUM> and <NUM>, respectively. JTE-<NUM> was suggested to exert a strong inhibitory effect on macrophage activation through MCP-<NUM> production inhibition (<FIG> and Table <NUM>).

JTE-<NUM> was found to have a weaker suppressive effect on TNF-α production from CD8+ T cells stimulated with T-cell stimulation beads (IC<NUM> value = <NUM>) when compared to the suppressive effect of PSL (IC<NUM> value = <NUM>) (<FIG> and Table <NUM>).

<NUM> Effects of JTE-<NUM> and PSL on LPS stimulation-induced TNF-α production in CD14+ cells.

PSL was found to have a suppressive effect on TNF-α production from CD14+ cells stimulated with LPS (IC<NUM> value = <NUM>). On the other hand, JTE-<NUM> showed a suppressive effect in a manner dependent on its concentration added, but its IC<NUM> value was not able to be calculated. In general, it has been known that TNF-α production from CD14+ cells transiently increases from immediately after stimulation. The measurement was made under evaluation conditions optimal for gradually increasing cytokines such as IL-<NUM>, and the culture supernatants used in this measurement were therefore evaluated under conditions where the production level of TNF-α was reduced, so that JTE-<NUM> would not show any clear suppression (<FIG> and Table <NUM>).

JTE-<NUM> and PSL both showed suppressive activity and had IC<NUM> values of <NUM> and <NUM>, respectively (<FIG> and Table <NUM>). In PBMCs containing various cells, these cells mutually stimulate each other to allow continuous production. For this reason, JTE-<NUM> would show a suppressive effect.

JTE-<NUM>, PSL and tocilizumab were evaluated for their effect on OPN production from CD14+ cells. JTE-<NUM> showed suppressive activity and had an IC<NUM> value of <NUM>. On the other hand, PSL and tocilizumab both showed no suppressive effect on OPN production (<FIG> and Table <NUM>). JTE-<NUM> was suggested to inhibit osteoclast activation seen in LCH lesions through OPN production inhibition.

JTE-<NUM> showed suppressive activity on IL-1β production and had an IC<NUM> value of <NUM>. On the other hand, PSL and tocilizumab both showed no suppressive effect on IL-1β production (<FIG> and Table <NUM>). JTE-<NUM> was suggested to have a different effect from that of existing drugs such as PSL and tocilizumab.

JTE-<NUM>, PSL and tocilizumab were evaluated for their effect on IL-<NUM> production from LPS-pretreated CD14+ cells. JTE-<NUM> showed suppressive activity and had an IC<NUM> value of <NUM>. On the other hand, PSL and tocilizumab both showed no suppressive effect on IL-<NUM> production (<FIG> and Table <NUM>). JTE-<NUM> was suggested to inhibit IL-<NUM> production to thereby ameliorate pathological conditions in a group of diseases characterized by high IL-<NUM> production. IL-<NUM> is an inflammatory cytokine whose production is maintained high independently of IL-<NUM>, and JTE-<NUM> was suggested to have an effect on an inflammatory pathological condition masked upon tocilizumab administration.

PSL showed a strong effect on VEGF production from macrophages, and its IC<NUM> value was not able to be calculated because <NUM>% or more inhibition was observed even at the lowest evaluation concentration, i.e., <NUM>. On the other hand, JTE-<NUM> tended to suppress VEGF production from macrophages, but its effect was marginal. Tocilizumab showed no suppressive effect on VEGF production (<FIG> and Table <NUM>).

To detect cytokine release leading to CRS toxicity, a culture system for target cell recognition by CAR-T cells (killer activity) and a culture system further containing peripheral blood CD14+ cells were compared with each other. The killer activity on target cells was elevated in culture with CD14+ cells when compared to culture without CD14+ cells (<FIG>).

Subsequently, the production of various cytokines in culture with and without CD14+ cells was quantified over time. There was no great difference in the production of IFN-γ, a cytokine derived from CAR-T cells. On the other hand, in the production of IL-<NUM> and MCP-<NUM>, a several-fold to <NUM>-fold or more difference was observed in each case, and this result would reflect that the activation state of CD14+ cells was significant (<FIG>). Incidentally, IL-<NUM> and MCP-<NUM> have both been reported as biomarkers for predicting and determining the severity of CRS toxicity<NUM>). In view of the foregoing, such a mixed culture system of three types of cells, i.e., "CAR-T cells, target cells and CD14+ cells" would be an in vitro culture system allowing simultaneous evaluation of "target cancer cell killer activity" and "CRS toxicity" in CAR-T cell therapy.

PSL showed a suppressive effect on the killer activity of CAR-T cells in a concentration-dependent manner, whereas JTE-<NUM> did not affect the killer activity of CAR-T cells (<FIG>).

The culture supernatants used for killer activity evaluation were evaluated for each cytokine. JTE-<NUM> and PSL were found to have similar suppressive activity on IFN-γ production. JTE-<NUM> had an IC<NUM> value of <NUM> for IL-<NUM> production. On the other hand, PSL showed no suppressive effect (<FIG> and Table <NUM>). JTE-<NUM> showed a concentration-dependent suppressive effect on MCP-<NUM> production and had an IC<NUM> value of <NUM>. Likewise, PSL had an IC<NUM> value of <NUM>. On the other hand, tocilizumab showed no suppressive effect (<FIG> and Table <NUM>). JTE-<NUM> showed a suppressive effect on IL-<NUM> production and had an IC<NUM> value of <NUM>. On the other hand, PSL and tocilizumab both showed no suppressive effect (<FIG> and Table <NUM>). JTE-<NUM> showed suppressive effects on TNF-α and IL-1β and had IC<NUM> values of <NUM> for TNF-α and <NUM> for IL-1β. On the other hand, PSL and tocilizumab showed no suppressive effect (<FIG>, <FIG> and Table <NUM>). IL-<NUM> was not able to be evaluated because almost no production was observed under the conditions used in this evaluation (<FIG> and Table <NUM>).

<FIG> shows a "schematic diagram illustrating the development of various side effects and symptoms (e.g., irAEs, HLH, MAS) associated with the overactivation of endogenous T effector cells.

T effector cells whose overexpansion is induced, e.g., by immune checkpoint inhibitors and/or virus infection mutually interact with co-existing tissue-resident macrophages and antigen-presenting cells, etc., to cause not only anticancer and antiviral actions but also autoimmune-related adverse events and so on. The immunosuppressive effect of prednisolone (PSL) ameliorates these side effects and symptoms and also entirely inhibits anticancer and antiviral T effector functions.

On the other hand, tocilizumab is used in an attempt to inhibit IL-<NUM> which is overproduced upon macrophage activation, and is expected to reduce and ameliorate autoimmune-related adverse events. JTE-<NUM> is expected to more widely inhibit abnormal macrophage activation and is also shown to cause limited suppression of T effector cells. The discovery underlying the presentation of this diagram is shown in the results obtained for T cell reaction (CD3 + CD28 stimulated reaction system) and monocyte/macrophage reaction (LPS stimulated reaction system) in the single culture system and the combined culture system which is novel.

In view of the foregoing, <FIG> shows that JTE-<NUM> is expected to exert the effect of suppressing macrophage activation to ameliorate organ damage without greatly affecting the major functions of T effector cells.

Claim 1:
A compound which is represented by the following formula II or is a pharmaceutically acceptable salt thereof for use in the treatment of patients with cytokine release syndrome toxicity resistant to tocilizumab administration or for use in the prevention, treatment or amelioration of at least one selected from the group consisting of autoimmune-related adverse events, macrophage activation syndrome, hemophagocytic lymphohistiocytosis and Langerhans cell histiocytosis wherein the autoimmune disease-related adverse events are a group of adverse events associated with immune checkpoint inhibitors whose administration results in excessive activation of overall immunity and thereby induces self-attack to cause various symptoms of autoimmune diseases, wherein the symptoms are selected from interstitial lung disease, colitis, hypothyroidism, hepatopathy, exanthema, hypophysitis, diabetes, renal dysfunction, peripheral neuropathy and myasthenia gravis;
<CHM>