Methods of treatment using CAR cells in combination with S1P receptor modulators

Abstract

The present invention relates to a CAR cell composition for use in the treatment of a hematological malignancy in a subject in need thereof, wherein said CAR cells are immune cells that express a chimeric antigen receptor molecule that binds to a cancer associated antigen, wherein a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator, or a pharma- ceutically acceptable salt or phosphate derivative thereof.

Description

[Technical field]

[0001] The present invention relates to the field of cellular immunotherapy, and more particularly to a composition comprising CAR cells expressing a chimeric antigen receptor molecule that binds to a cancer associated antigen, for use in combination therapy with an S1P receptor modulator in the treatment of hematological malignancies in a subject in need thereof. [Background technology]

[0002] Many patients with hematological malignancies are incurable with standard therapy. Moreover, conventional treatment options often have severe side effects. Attempts have been made with cancer immunotherapy, but several barriers make this a very challenging goal to achieve clinical efficacy. Although hundreds of so-called tumor antigens have been identified, these are generally derived from the self and therefore have poor immunogenicity.

[0003] The presence of naturally occurring tumor-reactive T cells has been well documented in the peripheral blood and within tumors of cancer patients. However, although T cells recognize neoplastic cells, their presence is often insufficient to mediate clinical tumor regression. Tumors employ a myriad of mechanisms to neutralize or evade immune attack, particularly T cell-mediated responses. These mechanisms include, among others, downregulation of the expression of MHC molecules or disruption of the antigen processing and presentation machinery.

[0004] Adoptive transfer of autologous immune cells engineered ex vivo to express chimeric antigen receptors (CARs) has emerged as a novel therapeutic tool to circumvent some of these barriers. Such approaches rely on redirecting such immune effector cells to appropriate cell surface molecules on cancer cells, such as hematological malignancies. For example, in recent clinical trials, CAR-T cells targeting the CD19 molecule have shown remarkable activity in treating B-cell leukemia and B-cell lymphoma (Maude et al., 2014; Neelapu et al., 2017; Schuster et al., 2017; Park et al., 2018). Despite these promising results, it has been found that circulating tumor cells strongly prevent CAR-T cell trafficking to the bone marrow and therefore represent a considerable obstacle to CAR-T cell-mediated elimination of targeted tumor cells and persistence of CAR cells (M. Cazaux et al.; J Exp Med 6 May 2019; 216 (5): 1038-1049. doi: https: / / doi.org / 10.1084 / jem.20182375). Thus, besides the ability of the chimeric antigen receptor on genetically modified immune effector cells to recognize and destroy target cells, a successful therapeutic immune effector cell therapy must have the ability to proliferate, persist, remain functionally active, and be trafficked to the bone marrow over time to explore leukemia relapse.

[0005] Unfortunately, CAR-T cell therapy can also cause severe toxicity that affects various organs and limits the success of treatment (Brudno and Kochenderfer, Blood 127, 3321-3330 (2016)). CAR-T cells are highly activated upon tumor antigen recognition, which triggers cytokine release. This local cytokine storm can further activate bystander immune cells, which release more inflammatory cytokines and contribute to cytokine release syndrome (CRS) pathology, characterized by high systemic cytokine levels. Activated immune cells migrate to peripheral sites and cause a systemic inflammatory response in tissues (Lee et al. Blood 2014 124(2):188-195).

[0006] Even in clinical trials with the most dramatic response rates, severe, life-threatening events occur in patients. Specifically, in the case of acute lymphoblastic leukemia / lymphoma (ALL / LBL) patients treated with CAR-T cell therapy, while nearly all patients had at least some less severe signs of toxicity, 20-50% of patients showed severe supraphysiological cytokine production and massive in-vivo T cell expansion. These toxic levels of systemic cytokine release and severe immune cell cross-activation in some patients result in the following toxicities: (1) cytokine release syndrome (CRS), associated with supraphysiological cytokine production and massive in vivo T cell expansion; (2) CRS and hemophagocytic lymphohistiocytosis and / or macrophage activation syndrome (MAS), defined as a severe hyperinflammatory syndrome characterized by elevated serum ferritin in combination with absent hemophagocytosis, renal failure, liver enzymes, splenomegaly, pulmonary edema, and / or NK cell activity; and (3) immune effector cell-associated neurotoxicity syndrome (ICANS), characterized by elevated cerebrospinal fluid cytokine levels and blood-brain barrier disruption (Sheth, VS, Gauthier, J. Taming the beast: CRS and ICANS after CAR T-cell therapy for ALL. Bone Marrow Transplant 56, 552-566 (2021)).

[0007] Another challenge in CAR-T cell therapy is the loss of efficacy against hematological cancers, caused, for example, by inhibition and resistance, antigen escape, limited CAR persistence, poor CAR trafficking, and tumor infiltration in cancer cell malignancies, as well as an immunosuppressive microenvironment. For example, 70-90% of relapsed and / or refractory ALL patients show durable responses to CD19-targeted CAR-T cell therapy, but recent follow-up data suggest the occurrence of common disease resistance mechanisms, including downregulation / loss of CD19 antigen in 30-70% of patients with recurrent disease after treatment (Sterner, RC, Sterner, RM CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11, 69 (2021)).

[0008] Thus, there is an ongoing need for improved strategies to target hematological malignancies, and in particular, novel compositions and methods for improving CAR therapy are highly desirable. Summary of the Invention

[0009] Facilitating sequestration of CAR-T cells in lymphoid tissues such as bone marrow and / or lymph nodes may provide clinical benefits as a result of maintaining CAR-T cell regulation and CAR-T functional activation in an appropriate microenvironment. In normal T cells (Zinkernagel RM, Ehl S, Aichele P, Oehen S, Kundig T, Hengartner H. Antigen localisation regulates immune responses in a dose- and time-dependent fashion: a geographical view of immune reactivity. Immunol Rev. 1997 Apr;156:199-209.), such a lymphoid microenvironment is expected to extend CAR-T activation and thus CAR persistence, but also regulate the overshoot of immune responses that can lead to CRS and ICANS. Furthermore, sequestration of CAR-T from the periphery is expected to reduce peripheral tissue activation and damage associated with adverse events. FTY720 has been shown to enhance the persistence of allogeneic T bodies (Marcus et al., Blood 2011;118(4):975-983). Furthermore, promoting the isolation of CAR cells may reduce the risk of GVHD development in allogeneic CAR cell therapy (Depil et al., Nature Reviews Drug Discovery 19, 185-199(2020)).

[0010] Without limiting the invention to any particular mechanism, the invention described herein fulfills this need, as the described combination of compositions comprising CAR cells in combination with S1P receptor modulators should provide a synergistic therapeutic effect by preventing CAR cells from leaving lymphatic tissues, and / or by further activating CAR cells in killing mode, and / or by further directly killing cancer cells, thereby improving antitumor efficacy and / or preventing recurrence in patients with hematological malignancies given the combination of CAR cells and S1P receptor modulators. Sequestration of CAR cells in lymphatic tissues by S1P receptor modulators also has the potential to limit CRS and / or MAS after CAR cell therapy or other adverse events such as ICANS, which are clinically observed in all autologous cell therapies.

[0011] Accordingly, the present disclosure provides, at least in part, compositions and methods for treating disorders such as cancer (e.g., hematological cancers or other B cell malignancies) using immune effector cells (e.g., T cells or natural killer cells (NK cells)) that express a chimeric antigen receptor (CAR) molecule (e.g., a CAR- that binds a B cell antigen, e.g., CD19).

[0012] The method includes administering immune effector cells (e.g., T cells or NK cells) expressing a CAR (e.g., a CAR targeting B cells) in combination with an S1P receptor modulator. In some embodiments, the combination maintains CAR-T functional activity, has better clinical efficacy, and / or has lower toxicity (e.g., due to prevention of CRS) compared to either therapy alone. In some embodiments, the subject is at risk for or has CRS and / or MAS, or the subject has been identified as at risk for or at risk of developing CRS and / or MAS. In some embodiments, the combination improves or ameliorates the anti-tumor efficacy of CAR cell therapy, particularly CAR cell therapy for treating hematological malignancies.

[0013] The present disclosure further relates to the use of cells, e.g., immune effector cells (e.g., T cells or NK cells), engineered to express a CAR molecule that binds to a cancer associated antigen (e.g., a cancer associated antigen described herein, e.g., CD19), in combination with an S1P receptor modulator (e.g., moclavimod) to treat a hematological cancer associated with said expression of said cancer associated antigen.

[0014] Also provided herein are compositions and methods for preventing CRS and / or MAS in a subject by using a combination of an S1P receptor modulator (e.g., moclavimod) and a CAR-expressing cell (e.g., a CAR-expressing cell that targets B cells, e.g., an anti-CD19 CAR cell).

[0015] Also provided are compositions and methods for preventing CRS and / or MAS in a subject by using a combination of an S1P receptor modulator and a CAR-expressing cell (e.g., a CAR-expressing cell that targets B cells, e.g., a CD19CAR-expressing cell), e.g., a subject is at risk for or has CRS and / or MAS, or a subject has been identified as at risk for or at risk of developing CRS and / or MAS. In one embodiment, provided herein is a method of treating a subject, e.g., a human, having a disease associated with expression of an antigen, e.g., a cancer-associated antigen described herein. The method comprises administering to the subject an effective amount of a cell, e.g., an immune effector cell (e.g., a T cell or an NK cell), expressing a CAR molecule that binds to a cancer-associated antigen (e.g., a cancer-associated antigen described herein, e.g., CD19), in combination with an S1P receptor modulator, e.g., moclavimod.

[0016] In another aspect, provided herein is a method of providing anti-tumor immunity to a subject, e.g., a human, having a disease associated with expression of an antigen, e.g., a cancer associated antigen, e.g., a cancer associated antigen described herein. The method comprises administering to the subject an effective amount of a cell, e.g., an immune effector cell (e.g., a T cell or an NK cell), expressing a CAR molecule that binds to the antigen (e.g., a cancer associated antigen described herein, e.g., CD19), in combination with an S1P receptor modulator, e.g., moclavimod.

[0017] In another aspect, provided herein is a method of treating and / or preventing cytokine release syndrome (CRS), e.g., CRS associated with CAR therapy (e.g., a CAR-expressing cell described herein) and / or macrophage activation syndrome (MAS), in a subject in need thereof, comprising administering to the subject an S1P receptor modulator (e.g., moclavimod), in combination with CAR cell therapy, thereby treating and / or preventing CRS and / or MAS in the subject.

[0018] In another aspect, provided herein is a method of increasing the efficacy of CAR therapy (e.g., a CAR-expressing cell described herein) in a subject in need thereof, comprising administering to the subject an S1P receptor modulator (e.g., moclavimod) in combination with the CAR cell therapy, thereby activating the CAR-expressing cells in a killing mode and / or directly killing cancer cells, thus increasing the anti-tumor effect.

[0019] In embodiments, the subject is at risk for developing, has, or has been diagnosed with CRS and / or MAS. In embodiments, the subject has been, is being, or will be administered a CAR cell therapy, e.g., a CAR-expressing cell as described herein.

[0020] In embodiments, the method includes selecting a subject for administration of an S1P receptor modulator. In embodiments, the subject is selected based on (i) their risk of developing CRS and / or MAS, (ii) their diagnosis of CRS and / or MAS, and / or (iii) whether the subject has been, is being, or will be administered a CAR cell therapy (e.g., a CAR cell therapy described herein, e.g., an anti-CD19 CAR cell therapy).

[0021] In embodiments, a subject is selected for administration of an S1P receptor modulator if the subject has been diagnosed with CRS and / or MAS, e.g., severe (grade 3 or 4) or non-severe CRS and / or MAS. In embodiments, a subject is selected for administration of an S1P receptor modulator if the subject is at risk (e.g., identified as at risk) of developing CRS and / or MAS. In embodiments, a subject is selected for administration of an S1P receptor modulator if the subject has been, is being, or will be administered a CAR cell therapy (e.g., a CAR cell therapy described herein, e.g., an anti-CD19 CAR cell therapy).

[0022] Thus, in one aspect, the present disclosure provides a CAR cell composition for use in the treatment of a hematological malignancy in a subject in need thereof, wherein said CAR cell is an immune cell, preferably a T cell, that expresses a chimeric antigen receptor molecule that binds to a cancer associated antigen, and wherein a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0023] In another aspect, the present disclosure provides a CAR cell composition for use in treating a metastatic tumor, preferably lymphoid organ metastasis, more preferably lymph node metastasis, from a solid tumor cancer, in a subject in need thereof, wherein said CAR cells are immune cells, preferably T cells, that express a chimeric antigen receptor molecule that binds to a cancer associated antigen, and wherein a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0024] In another embodiment, a therapeutically effective amount of an S1P receptor modulator is administered prior to or concurrently with administration of a therapeutically effective amount of a CAR cell composition.

[0025] In another embodiment, the CAR cells are treated ex vivo or in vitro with an effective amount of an S1P receptor modulator or a phosphate derivative thereof prior to administration. Thus, the present disclosure also provides an active CAR cell composition for use in treating a hematological malignancy in a subject in need thereof, wherein the CAR cells are immune cells, preferably T cells, that express a chimeric antigen receptor molecule that binds to a cancer-associated antigen, and a therapeutically effective amount of the active CAR cell composition is administered to the subject, and the CAR cells have been treated in vitro or ex vivo with an effective amount of an S1P receptor modulator or a phosphate derivative thereof, such as moclavimod.

[0026] In another aspect, the disclosure provides for the use of a CAR cell composition, wherein the CAR cell is an immune cell, preferably a T cell, that expresses a chimeric antigen receptor molecule that binds to a cancer associated antigen, for the manufacture of a medicament for treating a hematological malignancy in a subject in need thereof, wherein a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0027] In another aspect, the disclosure provides a method of treating a hematological malignancy comprising administering a therapeutically effective amount of a CAR cell composition in combination with a therapeutically effective amount of an S1P receptor modulator, wherein said CAR cell is an immune cell, preferably an immune T cell, that expresses a chimeric antigen receptor molecule (e.g., CD19) that binds to a cancer associated antigen.

[0028] In one set of embodiments, the method comprises: 1) collecting immune cells, e.g., immune T cells, from a donor subject in need thereof by performing leukapheresis; 2) genetically modifying immune cells of the donor subject ex vivo to express a chimeric antigen receptor (CAR) molecule that binds to a cancer-associated antigen (e.g., CD19), thereby obtaining a CAR cell composition; 3) conditioning the recipient subject, for example, by treating the recipient subject with an effective amount of a lymphodepleting chemotherapeutic agent or by administering total body irradiation; 4) administering to the recipient subject a therapeutically effective amount of the composition comprising the CAR cells obtained in step 2); 5) administering to the recipient subject, e.g., before or after step 4), an effective amount of an S1P receptor modulator, preferably moclavimod of formula II, or a pharma- ceutically acceptable salt thereof, such as formula IIa or IIb, or a phosphate derivative thereof, preferably before step 4); 6) Optionally, administering to the recipient subject an effective amount of one or more immunosuppressive agents. Includes.

[0029] In another aspect, the disclosure provides a method of preventing or reducing cytokine release syndrome (CRS) and / or macrophage activation syndrome (MAS) in a subject in need thereof with CAR cell therapy (e.g., anti-CD19 CAR-T cell therapy), comprising administering to the subject an S1P receptor modulator (e.g., moclavimod), or a pharma- ceutically acceptable salt or phosphate derivative thereof, in combination with the CAR cell therapy, thereby preventing CRS and / or MAS in the subject.

[0030] In another aspect, the disclosure provides an S1P receptor modulator for use in preventing cytokine release syndrome in CAR cell therapy (e.g., anti-CD19 CAR-T cell therapy) in a subject in need thereof, the use comprising administering to the subject an S1P receptor modulator (e.g., moclavimod), or a pharma- ceutically acceptable salt or phosphate derivative thereof, in combination with the CAR cell therapy, thereby preventing CRS and / or MAS in the subject.

[0031] In another aspect, the disclosure provides for the use of an S1P receptor modulator for the manufacture of a medicament for preventing cytokine release syndrome and / or macrophage activation syndrome in CAR cell therapy (e.g., anti-CD19 CAR-T cell therapy) in a subject in need thereof, comprising administering to the subject an S1P receptor modulator (e.g., moclavimod), or a pharma- ceutically acceptable salt or phosphate derivative thereof, in combination with the CAR cell therapy, thereby preventing CRS and / or macrophage activation syndrome in the subject.

[0032] The present disclosure is provided in various aspects, as outlined below.

[0033] 1. A CAR cell composition for use in the treatment of a hematological malignancy in a subject in need thereof, wherein said CAR cells are immune cells, preferably immune T cells, that express a chimeric antigen receptor molecule that binds to a cancer associated antigen, and a therapeutically effective amount of said CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0034] 2. The CAR cell composition for use according to embodiment 1, wherein said cancer associated antigen is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, BCMA, and combinations thereof, preferably CD19.

[0035] 3. The CAR cell composition for use according to embodiment 1 or 2, wherein said S1P receptor modulator is selected from mocravimod, siponimod, fingolimod, ozanimod, ponesimod, etrasimod, AKP-11, cenerimod, amiselimod, CBP-307, OPL-307, OPL-002, BMS-986166, SCD-044, BOS-173717, CP-1050, preferably mocravimod.

[0036] 4. The CAR cell composition for use according to embodiment 3, wherein the S1P receptor modulator is an S1P receptor agonist.

[0037] 5. The S1P receptor agonist is of the following formula (I) or (II) or (IIa) or (IIb):

[0038] [ka] [In the formula, R2 is H, halogen, trihalomethyl, C 1~4 Alkoxy, C 1~7 alkyl, phenethyl, or benzyloxy; R3 is H, halogen, CF3, OH, C 1~7 Alkyl, C 1~4 Alkoxy, benzyloxy, phenyl, or C 1~4 is alkoxymethyl, Each of R4 and R5 is independently H or a residue of formula (a):

[0039] [ka] wherein each of R8 and R9 is independently H or C optionally substituted with halogen. 1~4 is alkyl, n is an integer from 1 to 4; R6 is hydrogen, halogen, C 1~7 Alkyl, C 1~4alkoxy, or trifluoromethyl; or

[0040] [ka] or a pharma- ceutically acceptable salt thereof or

[0041] [ka] 5. The CAR cell composition for use according to embodiment 3 or 4, wherein

[0042] 6. The CAR cell composition for use according to any one of embodiments 1 to 5, wherein the S1P receptor agonist is moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof.

[0043] 7. The CAR cell composition for use according to any one of embodiments 1 to 6, wherein the hematological malignancy is leukemia and / or lymphoma.

[0044] 8. The CAR cell composition for use according to any one of embodiments 1 to 7, wherein said hematological malignancy is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, preferably ALL, DLBCL, PMBCL, and MCL, more preferably DLBCL.

[0045] 9. An effective amount of CAR cells, preferably CAR-T cells, is administered in a dose of 0.1×10 6 ~6×10 8 The CAR cell composition for use according to any one of embodiments 1 to 8, administered at a dosage of 1 CAR-positive viable immune cells / kg body weight.

[0046] 10. The CAR cell composition for use according to any one of embodiments 1 to 9, wherein the CAR cells, preferably CAR-T cells, are administered 2 to 14 days after completion of lymphodepleting chemotherapy.

[0047] 11. The CAR cell composition for use according to any one of embodiments 1 to 10, wherein the S1P receptor modulator is administered in a dosage of 0.05 mg to 40 mg per day, preferably 0.1 mg to 35 mg, more preferably 0.5 mg to 30 mg, even more preferably 1 mg to 15 mg, even more preferably 1.5 mg to 7 mg, even more preferably 2 mg to 5 mg, even more preferably about 3 mg or about 1 mg.

[0048] 12. The CAR cell composition for use according to any one of embodiments 1 to 11, wherein said S1P receptor modulator is administered daily, preferably from a starting date of 1 to 20 days prior to administration of said composition comprising CAR cells, more preferably 11 days prior to administration of CAR cells, for at least 1, 2, or 3 months, or longer.

[0049] 13. The CAR cell composition for use according to any one of embodiments 1 to 12, wherein the S1P receptor modulator is administered in an amount sufficient to prevent CAR cells from leaving the bone marrow, and / or promote the engraftment and persistence of CAR cells, and / or increase the efficacy of CAR cell therapy.

[0050] 14. A method of treating a hematological malignancy in a subject in need thereof comprising administering a therapeutically effective amount of a CAR cell composition, preferably autologous or syngeneic CAR cells, in combination with a therapeutically effective amount of an S1P receptor modulator, wherein said CAR cells are immune cells that express a chimeric antigen receptor molecule that binds to a cancer associated antigen.

[0051] 15. The method of embodiment 14, wherein the CAR cells are CAR-T cells, preferably anti-CD19 CAR-T cells.

[0052] 16. The method of embodiment 14 or 15, wherein the number of CAR cells in the subject's peripheral blood is reduced by about 1% to about 40%, preferably about 10% to about 30%, after 7, preferably 14 days of S1P receptor modulator treatment, as measured by flow cytometry, compared to the number measured without S1P receptor modulator treatment.

[0053] 17. 1) harvesting immune cells, e.g., immune T cells, from a donor subject by performing leukapheresis; 2) genetically modifying immune cells of the donor subject ex vivo to express a chimeric antigen receptor (CAR) molecule that binds to a cancer-associated antigen, thereby obtaining a CAR cell composition; 3) conditioning the recipient subject, for example, by treating the recipient subject with an effective amount of a lymphodepleting chemotherapeutic agent or by administering total body irradiation; 4) administering to the recipient subject a therapeutically effective amount of the composition comprising the CAR cells obtained in step 2); 5) administering to the recipient subject an effective amount of an S1P receptor modulator, preferably moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof; 6) Optionally, administering to the recipient subject an effective amount of one or more immunosuppressive agents. 17. The method of any one of embodiments 14 to 16, comprising:

[0054] 18. The method of embodiment 17, wherein said lymphodepleting chemotherapeutic agent is selected from the group consisting of melphalan, cytarabine, etoposide, busulfan, bendamustine, cyclophosphamide, fludarabine, dexamethasone, and alemtuzumab, and combinations thereof, such as combination administration of fludarabine / cyclophosphamide, cytarabine / etoposide.

[0055] 19. Step 3) conditioning regimen: - Administration of fludarabine and cyclophosphamide, or - Administration of cytarabine and etoposide, or - Bendamustine administration 17. The method of embodiment 16, comprising:

[0056] 20. CAR cells, preferably CAR-T cells, more preferably anti-CD19 CAR-T cells, at 0.1 × 10 6 ~6×10 8 The method of any one of embodiments 14 to 19, wherein the immune cell is administered at a dosage of live CAR-positive immune cells / kg of body weight.

[0057] 21. The method according to any one of embodiments 14 to 20, wherein the CAR cells, preferably CAR-T cells, more preferably anti-CD19 CAR-T cells, are administered 2 to 14 days after completion of lymphodepleting chemotherapy.

[0058] 22. The method of any one of embodiments 14 to 21, wherein the cancer associated antigen is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, BCMA, preferably CD19.

[0059] 23. The method according to any one of embodiments 14 to 22, wherein said S1P receptor modulator is selected from mocravimod, siponimod, fingolimod, ozanimod, ponesimod, etrasimod, AKP-11, cenerimod, amiselimod, CBP-307, OPL-307, OPL-002, BMS-986166, SCD-044, BOS-173717, CP-1050, preferably mocravimod.

[0060] 24. The method of embodiment 23, wherein said S1P receptor modulator is an S1P receptor agonist. 25. The S1P receptor agonist is of the following formula (I) or (II) or (IIa) or (IIb):

[0061] [ka] [In the formula, R2 is H, halogen, trihalomethyl, C 1~4 Alkoxy, C 1~7 alkyl, phenethyl, or benzyloxy; R3 is H, halogen, CF3, OH, C 1~7 Alkyl, C 1~4 Alkoxy, benzyloxy, phenyl, or C 1~4 is alkoxymethyl, Each of R4 and R5 is independently H or a residue of formula (a):

[0062] [ka] wherein each of R8 and R9 is independently H or C optionally substituted with halogen. 1~4 is alkyl, n is an integer from 1 to 4; R6 is hydrogen, halogen, C 1~7 Alkyl, C 1~4 alkoxy, or trifluoromethyl; or

[0063] [ka] or a pharma- ceutically acceptable salt thereof; or

[0064] [ka] 25. The method of embodiment 24, wherein

[0065] 26. The method of any one of embodiments 14 to 25, wherein the S1P receptor modulator is moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof.

[0066] 27. The method of any one of embodiments 14 to 26, wherein the S1P receptor modulator is administered in a dosage of 0.05 mg to 40 mg per day, preferably 0.1 mg to 35 mg, more preferably 0.5 mg to 30 mg, even more preferably 1 mg to 15 mg, even more preferably 1.5 mg to 7 mg, even more preferably 2 mg to 5 mg, even more preferably about 3 mg or about 1 mg.

[0067] 28. The S1P receptor modulator is mocravimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof, and the mocravimod is formulated as a solid dosage form, and the solid dosage form comprises: - 1 mg / unit of moclavimod or a pharma- ceutically acceptable salt or phosphate derivative thereof, mannitol, preferably with a content of 48 to 88 mg / unit, more preferably with a content of 58 to 78 mg / unit, even more preferably with a content of about 68 mg / unit, microcrystalline cellulose, preferably in a content of 5 to 45 mg / unit, more preferably in a content of 15 to 35 mg / unit, even more preferably in a content of about 25 mg / unit, sodium starch glycolate, preferably in a content of 1 to 8 mg / unit, more preferably in a content of 2 to 6 mg / unit, even more preferably in a content of about 4 mg / unit, magnesium stearate, preferably in a content of 0.025 to 4 mg / unit, more preferably in a content of 0.5 to 2 mg / unit, and even more preferably in a content of about 1 mg / unit, Colloidal silicon dioxide, preferably in a content of 0.125 to 2 mg / unit, more preferably in a content of 0.25 to 1 mg / unit, even more preferably in a content of about 0.5 mg / unit. 28. The method of any one of embodiments 14 to 27, comprising:

[0068] 29. The method of any one of embodiments 14 to 28, wherein the S1P receptor modulator is administered daily for at least 1, 2, 3 months, or longer, preferably starting from a starting date of 1 to 20 days prior to administration of the composition comprising CAR cells, more preferably 11 days prior to administration of the immune cells.

[0069] 30. The method of any one of embodiments 14 to 29, wherein the hematological malignancy is leukemia and / or lymphoma.

[0070] 31. The method of any one of embodiments 14 to 30, wherein the hematological malignancy is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia ALL, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, preferably ALL, DLBCL, PMBCL, and MCL, more preferably DLBCL.

[0071] 32. The method of any one of embodiments 14 to 31, wherein the one or more immunosuppressants are selected from the group consisting of cyclosporine A, sirolimus, tacrolimus, methotrexate, and mycophenolate, preferably cyclosporine A, or a combination of cyclosporine A and methotrexate.

[0072] 33. The method of any one of embodiments 14 to 32, wherein the S1P receptor modulator is administered in an amount sufficient to prevent CAR cells from leaving the marrow, and / or promote the engraftment and persistence of CAR cells, and / or increase the efficacy of CAR cell therapy.

[0073] 34. The method of any one of embodiments 14 to 33, wherein the S1P receptor modulator is administered in an amount sufficient to reduce the risk of cytokine release syndrome, in particular systemic cytokine release syndrome, in a subject receiving the CAR cells.

[0074] 35. The method of any one of embodiments 14 to 34, wherein the donor subject of the CAR cells is the recipient subject.

[0075] 36. The method of any one of embodiments 14 to 34, wherein the donor subject of the CAR cells is not the recipient subject.

[0076] 37. The method of embodiment 36, wherein the S1P receptor modulator is administered in an amount sufficient to reduce the risk of GVHD.

[0077] 38. The method of any one of embodiments 14 to 36, wherein the S1P receptor modulator is administered in an amount sufficient to reduce the risk of neuroinflammation, e.g. immune effector cell-associated neurotoxicity syndrome (ICANS).

[0078] 39. A method for preventing cytokine release syndrome (CRS) and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS) in a subject in need thereof with CAR cell therapy (e.g., anti-CD19 therapy), comprising administering to the subject an S1P receptor modulator (e.g., moclavimod) in combination with the CAR cell therapy, thereby preventing CRS and / or MAS and / or ICANS in the subject.

[0079] 40. A method for increasing the anti-tumor efficacy of CAR therapy (e.g., anti-CD19 therapy) in a subject in need thereof, comprising administering to the subject an effective amount of an S1P receptor modulator (e.g., moclavimod) in combination with CAR cell therapy, thereby activating CAR-expressing cells and / or directly killing cancer cells, thus increasing the anti-tumor efficacy of said CAR therapy.

[0080] 41. The method of embodiment 39 or 40, wherein the CAR cell therapy is a CAR-T cell therapy, preferably an anti-CD19 CAR-T cell therapy.

[0081] 42. 1) harvesting immune cells, e.g., immune T cells, from a donor subject by performing leukapheresis; 2) genetically modifying immune cells of the donor subject ex vivo to express a chimeric antigen receptor (CAR) molecule that binds to a cancer-associated antigen, thereby obtaining a CAR cell composition; 3) conditioning the recipient subject, for example, by treating the recipient subject with an effective amount of a lymphodepleting chemotherapeutic agent or by administering total body irradiation; 4) administering to the recipient subject a therapeutically effective amount of the composition comprising the CAR cells obtained in step 2); 5) administering to the recipient subject an effective amount of an S1P receptor modulator, preferably moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof; 6) Optionally, administering to the recipient subject an effective amount of one or more immunosuppressive agents. 42. The method of any one of embodiments 39 to 41, comprising:

[0082] 43. The method of embodiment 42, wherein said lymphodepleting chemotherapeutic agent is selected from the group consisting of melphalan, cytarabine, etoposide, busulfan, bendamustine, cyclophosphamide, fludarabine, dexamethasone, and alemtuzumab, and combinations thereof, such as combination administration of fludarabine / cyclophosphamide, cytarabine / etoposide.

[0083] 44. Step 3) conditioning regimen: - Administration of fludarabine and cyclophosphamide, or - Administration of cytarabine and etoposide, or - Bendamustine administration 43. The method of embodiment 42, comprising:

[0084] 45. The method according to any one of embodiments 39 to 44, wherein said S1P receptor modulator is selected from mocravimod, siponimod, fingolimod, ozanimod, ponesimod, etrasimod, AKP-11, cenerimod, amiselimod, CBP-307, OPL-307, OPL-002, BMS-986166, SCD-044, BOS-173717, CP-1050, preferably mocravimod.

[0085] 46. ​​The method of embodiment 45, wherein the S1P receptor modulator is an S1P receptor agonist.

[0086] 47. The S1P receptor agonist is of the following formula (I) or (II) or (IIa) or (IIb):

[0087] [ka] [In the formula, R2 is H, halogen, trihalomethyl, C 1~4 Alkoxy, C 1~7 alkyl, phenethyl, or benzyloxy; R3 is H, halogen, CF3, OH, C 1~7 Alkyl, C1~4 Alkoxy, benzyloxy, phenyl, or C 1~4 is alkoxymethyl, Each of R4 and R5 is independently H or a residue of formula (a):

[0088] [ka] wherein each of R8 and R9 is independently H or C optionally substituted with halogen. 1~4 is alkyl, n is an integer from 1 to 4; R6 is hydrogen, halogen, C 1~7 Alkyl, C 1~4 alkoxy, or trifluoromethyl; or

[0089] [ka] or a pharma- ceutically acceptable salt thereof; or

[0090] [ka] 47. The method of embodiment 46, wherein

[0091] 48. The method of any one of embodiments 39 to 47, wherein the S1P receptor modulator is moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof.

[0092] 49. The method of any one of embodiments 39 to 48, wherein the S1P receptor modulator is administered in a dosage of 0.05 mg to 40 mg per day, preferably 0.1 mg to 35 mg, more preferably 0.5 mg to 30 mg, even more preferably 1 mg to 15 mg, even more preferably 1.5 mg to 7 mg, even more preferably 2 mg to 5 mg, even more preferably about 3 mg or about 1 mg.

[0093] 50. The S1P receptor modulator is mocravimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof, and the mocravimod is formulated as a solid dosage form, and the solid dosage form comprises: mannitol, preferably with a content of 48 to 88 mg / unit, more preferably with a content of 58 to 78 mg / unit, even more preferably with a content of about 68 mg / unit, microcrystalline cellulose, preferably in a content of 5 to 45 mg / unit, more preferably in a content of 15 to 35 mg / unit, even more preferably in a content of about 25 mg / unit, sodium starch glycolate, preferably in a content of 1 to 8 mg / unit, more preferably in a content of 2 to 6 mg / unit, even more preferably in a content of about 4 mg / unit, magnesium stearate, preferably in a content of 0.025 to 4 mg / unit, more preferably in a content of 0.5 to 2 mg / unit, and even more preferably in a content of about 1 mg / unit, Colloidal silicon dioxide, preferably in a content of 0.125 to 2 mg / unit, more preferably in a content of 0.25 to 1 mg / unit, even more preferably in a content of about 0.5 mg / unit. 50. The method of any one of embodiments 39 to 49, comprising:

[0094] 51. The method of any one of embodiments 39 to 50, wherein the S1P receptor modulator is administered daily for at least 1, 2, 3 months, or longer, preferably starting from a starting date of 1 to 20 days prior to administration of the composition comprising CAR cells, more preferably 11 days prior to administration of the immune cells.

[0095] 52. CAR cells, preferably CAR-T cells, more preferably anti-CD19 CAR-T cells, at 0.1 × 10 6 ~6×10 8 The method of any one of embodiments 39 to 51, wherein the immune cell is administered at a dosage of live CAR-positive immune cells / kg of body weight.

[0096] 53. The method according to any one of embodiments 39 to 52, wherein the CAR cells, preferably CAR-T cells, more preferably anti-CD19 CAR-T cells, are administered 2 to 14 days after completion of lymphodepleting chemotherapy.

[0097] 54. The method of any one of embodiments 39 to 53, wherein the cancer-associated antigen is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, BCMA.

[0098] 55. The method of any one of embodiments 39 to 54, wherein the subject in need of CAR cell therapy is a subject suffering from a hematological malignancy, and the hematological malignancy is a hematological malignancy selected from the group consisting of leukemia and / or lymphoma, such as diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia ALL, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, preferably ALL, DLBCL, PMBCL, and MCL, more preferably DLBCL.

[0099] 56. An S1P receptor modulator for use in preventing cytokine release syndrome (CRS) and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS) in CAR cell therapy (e.g. anti-CD19 therapy) in a subject in need thereof, the use comprising administering an S1P receptor modulator (e.g. moclavimod) to the subject in combination with CAR cell therapy, thereby preventing CRS and / or MAS and / or ICANS in the subject.

[0100] 57. An S1P receptor modulator for use in increasing the effectiveness of CAR cell therapy (e.g. anti-CD19 therapy) in a subject in need thereof, the use comprising administering an S1P receptor modulator (e.g. moclavimod) to the subject in combination with the CAR cell therapy, thereby activating CAR cells and / or directly killing cancer cells, thus increasing the anti-tumor effect.

[0101] 58. Use of a CAR cell composition for the manufacture of a medicament for treating a hematological malignancy in a subject in need thereof, wherein the CAR cells are immune cells, preferably T cells, that express a chimeric antigen receptor molecule that binds to a cancer-associated antigen, and a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0102] 59. The use according to embodiment 58, wherein the cancer-associated antigen is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, BCMA, and combinations thereof, preferably CD19.

[0103] 60. The use according to embodiment 58 or 59, wherein the S1P receptor modulator is selected from mocravimod, siponimod, fingolimod, ozanimod, ponesimod, etrasimod, AKP-11, cenerimod, amiselimod, CBP-307, OPL-307, OPL-002, BMS-986166, SCD-044, BOS-173717, CP-1050, preferably mocravimod.

[0104] 61. The use according to embodiment 60, wherein the S1P receptor modulator is an S1P receptor agonist.

[0105] 62. The S1P receptor agonist is of the following formula (I) or (II) or (IIa) or (IIb):

[0106] [ka] [In the formula, R2 is H, halogen, trihalomethyl, C 1~4 Alkoxy, C 1~7 alkyl, phenethyl, or benzyloxy; R3 is H, halogen, CF3, OH, C 1~7 Alkyl, C 1~4 Alkoxy, benzyloxy, phenyl, or C 1~4 is alkoxymethyl, Each of R4 and R5 is independently H or a residue of formula (a):

[0107] [ka] wherein each of R8 and R9 is independently H or C optionally substituted with halogen. 1~4 is alkyl, n is an integer from 1 to 4; R6 is hydrogen, halogen, C 1~7 Alkyl, C 1~4 alkoxy, or trifluoromethyl; or

[0108] [ka] or a pharma- ceutically acceptable salt thereof or

[0109] [ka] The use according to embodiment 60 or 61, wherein

[0110] 63. The use according to any one of embodiments 58 to 62, wherein the S1P receptor agonist is moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof.

[0111] 64. The use according to any one of embodiments 58 to 63, wherein the hematological malignancy is leukemia and / or lymphoma.

[0112] 65. The use according to any one of embodiments 58 to 64, wherein the hematological malignancy is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, preferably ALL, DLBCL, PMBCL, and MCL, more preferably DLBCL.

[0113] 66. An effective amount of CAR cells, preferably CAR-T cells, is administered in a dose of 0.1 × 10 6 ~6×10 8 The use of any one of embodiments 58 to 65, wherein the use is administered at a dosage of live CAR-positive immune cells / kg of body weight.

[0114] 67. The use according to any one of embodiments 58 to 66, wherein the CAR cells, preferably CAR-T cells, are administered 2 to 14 days after completion of lymphodepleting chemotherapy.

[0115] 68. The use according to any one of embodiments 58 to 67, wherein the S1P receptor modulator is administered in a dosage of 0.05 mg to 40 mg per day, preferably 0.1 mg to 35 mg, more preferably 0.5 mg to 30 mg, even more preferably 1 mg to 15 mg, even more preferably 1.5 mg to 7 mg, even more preferably 2 mg to 5 mg, even more preferably about 3 mg or about 1 mg.

[0116] 69. The use according to any one of embodiments 58 to 68, wherein the S1P receptor modulator is administered daily, preferably from a starting date of 1 to 20 days prior to administration of the composition comprising CAR cells, more preferably 11 days prior to administration of CAR cells, for at least 1, 2, or 3 months, or longer.

[0117] 70. The use according to any one of embodiments 58 to 69, wherein the S1P receptor modulator is administered in an amount sufficient to prevent CAR cells from leaving the bone marrow, and / or promote the engraftment and persistence of CAR cells, and / or increase the efficacy of CAR cell therapy.

[0118] 71. Use of an S1P receptor modulator for the manufacture of a medicament for preventing cytokine release syndrome (CRS) and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS) in CAR cell therapy (e.g. anti-CD19 CAR-T cell therapy), preferably autologous or syngeneic CAR cell therapy, in a subject in need thereof, comprising administering an S1P receptor modulator (e.g. moclavimod), or a pharma- ceutically acceptable salt or phosphate derivative thereof, in combination with CAR cell therapy to the subject, thereby preventing CRS and / or MAS and / or ICANS in the subject.

[0119] 72. The use according to embodiment 72, wherein the CAR cell therapy is an autologous or allogeneic CAR-T cell therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0120] Detailed Description The present disclosure includes a method of treating a hematological malignancy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CAR cell composition in combination with a therapeutically effective amount of an S1P receptor modulator.

[0121] The present disclosure relates to a CAR cell composition for use in treating a hematological malignancy in a subject in need thereof, wherein the CAR cell is an immune cell, e.g., a T cell, that expresses a chimeric antigen receptor molecule that binds a cancer associated antigen (e.g., CD19), and a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0122] The present disclosure also relates to the use of a CAR cell composition for the manufacture of a medicament for treating a hematological malignancy in a subject in need thereof, comprising administering a therapeutically effective amount of the CAR cell composition in combination with a therapeutically effective amount of an S1P receptor modulator.

[0123] The present disclosure also relates to the use of a CAR cell composition in the preparation of a pharmaceutical composition for treating a hematological malignancy in a subject in need thereof, wherein a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

[0124] General definition 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.

[0125] As used herein, a "modulator" is a compound that, when administered to a subject, brings about a desired interaction with a target receptor, either by the compound acting directly on the receptor itself, or by the compound's metabolite acting on the receptor.When administered to a subject, an S1P receptor modulator, preferably moclavimod (also called KRP203), interacts with the S1P receptor by either activating or inhibiting or down-modulating the receptor, resulting in disrupted signal transduction.

[0126] As used herein, "S1P agonist" refers to a compound that initiates a physiological response when combined with S1P receptor.Preferably, the initiated physiological response is agonist-induced internalization of S1P receptor.The kinetics of agonist-induced internalization from cell membrane and recycling of S1P receptor to cell membrane after said compound shedding are compound dependent.Such S1P receptor agonists can also be called functional antagonists.The sustained internalization conditions the "functional antagonism" property of agonists.

[0127] The term "pharmaceutically acceptable salts thereof" includes both acid and base addition salts. Non-limiting examples of pharmaceutically acceptable acid addition salts include chloride, hydrochloride, bromide, sulfate, nitrate, phosphate, sulfonate, methanesulfonate, formate, tartrate, maleate, citrate, benzoate, salicylate, and ascorbate. Non-limiting examples of pharmaceutically acceptable base addition salts include sodium, potassium, lithium, ammonium (substituted and unsubstituted), calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Pharmaceutically acceptable salts can be obtained, for example, using standard procedures well known in the pharmaceutical field. For mocravimod, the pharmaceutically acceptable salt will typically be an acid addition salt, since mocravimod is itself a base. Preferably, the pharmaceutically acceptable salt is the hydrochloride salt.

[0128] The term "phosphate derivatives thereof" includes phosphate esters such as those of formula IIa or IIb.

[0129] The term "excipient" as used herein refers to an inactive substance that is added together with a drug substance and is part of the compounding mixture. Pharmaceutically acceptable excipients are, for example, fillers, solvents, diluents, carriers, adjuvants, dispersing agents and sensing agents, delivery agents, such as preservatives, disintegrants, wetting agents, emulsifiers, suspending agents, thickening agents, sweeteners, flavorings, flavorings, antibacterial agents, fungicides, lubricants, and sustained delivery control agents, antioxidants, glidants. Their selection and appropriate ratio depend on the nature and method of administration and dosage.

[0130] As used herein, "average particle size" refers to D50, meaning that 50% of the particles have a size equal to or less than the indicated value. For example, an average particle size of 8 μm or less refers to a D50 of 8 μm, i.e., 50% of the particles have a size equal to or less than 8 μm. The term D90 means that 90% of the particles have a size equal to or less than the indicated value. For example, a D90 of 25 μm or less means that 90% of the particles have a size equal to or less than 25 μm. D50 and D90 are determined by laser light diffraction using a liquid path, for example on a BECKMAN-COULTER laser diffraction particle size analyzer LS230 equipped with its small volume dispersion module (liquid path), following the technical manual and manufacturer's instructions.

[0131] The term "cytokine release syndrome" (CRS) refers to a prominent complication associated with CAR cell therapy, which is an acute inflammatory process characterized by a range of clinical symptoms and substantial but transient elevations of serum cytokines. In most patients, CRS occurs 1-14 days after CAR-T cell infusion and rarely occurs later than 17 days after infusion. Data suggest that CAR-T cell-associated CRS is dependent on the binding of T cells to the target antigen, followed by proliferation and functional responses. Other factors influencing CRS may include the type, nature, and degree of lymphodepletion of the disease, as well as in some cases the design of the CAR. Because CRS is associated with the binding of T cells to the target antigen, it is not limited to anti-CD19 therapy alone. CRS was observed in 4 of 7 (57%) evaluable pediatric or young adult patients with r / r ALL who received CD22-targeted CAR-T cells. Additionally, CRS has been observed in patients with multiple myeloma (MM) who received CAR-T cells targeting CD19 or B-cell maturation antigen (BCMA). The CRS grading scale developed by the University of Pennsylvania is preferred (D. Porter et al., J Hematol Oncol. 2018; 11: 35).

[0132] The term "severe CRS" in contrast to the term "nonsevere CRS" refers to grade 3 or 4 CRS. Severe grade 3 CRS refers to hospitalization necessary for management of symptoms related to organ failure, including grade 4 LFTs or grade 3 creatinine associated with CRS and not attributable to any other condition, including hypotension treated with intravenous fluids (defined as multiple fluid boluses for blood pressure support) or low-dose vasopressors, coagulopathy requiring fresh frozen plasma or cryoprecipitate or fibrinogen concentrate, and hypoxia requiring oxygenation (nasal cannula oxygen, high-flow oxygen, CPAP, or BiPAP). Severe grade 4 CRS covers life-threatening complications such as hypotension requiring high-dose vasopressors, hypoxia requiring mechanical ventilation.

[0133] The term "macrophage activation syndrome" (MAS) refers to MAS-like symptoms observed in patients treated with CAR cells, which may be life-threatening. MAS is sometimes synonymous with secondary hemophagocytic lymphohistiocytosis (HLH), a term used by rheumatologists to describe a life-threatening complication of a systemic inflammatory disorder most commonly seen in systemic juvenile idiopathic arthritis (sJIA) and its adult equivalent, adult-onset Still's disease. The traditional definition of MAS has been problematic to apply to patients receiving CAR-T cells due to overlapping signs and symptoms with lymphodepletion and CRS. However, CRS symptoms characterized by fever and elevated C-reactive protein (CRP) may occur first, followed by MAS (or MAS-like symptoms) characterized by decreased fibrinogen, rapid elevation of ferritin and lactate dehydrogenase.

[0134] The term "chimeric antigen receptor" or "CAR" refers to an artificial cell receptor, e.g., a T body receptor, a single chain immune receptor, a chimeric T cell receptor, or a chimeric immune receptor, including engineered receptors that graft artificial specificity onto specific immune effector cells. CARs can be used to confer the specificity of a monoclonal antibody to T cells, thereby allowing the generation of large numbers of specific T cells, e.g., for use in adoptive cell therapy. In specific embodiments, CARs direct the specificity of cells, e.g., to a tumor-associated antigen. In some embodiments, CARs include an intracellular activation domain (allowing the T cell to be activated upon binding of the targeting moiety to a target cell, e.g., a target tumor cell), a transmembrane domain, and an extracellular domain that is of various lengths and includes a disease or disorder-related, e.g., tumor-antigen binding region. In certain aspects, CARs include a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody fused to CD3-zeta, a transmembrane domain, and an internal domain. Other CAR design specificities may come from the ligand of the receptor (e.g., peptide) or pattern recognition receptors such as dectin. In certain cases, the spacing of the antigen recognition domain can be modified to reduce activation-induced cell death. In certain cases, the CAR contains additional costimulatory signaling domains such as CD3zeta, FcR, CD27, CD28, CD137, DAP10 / 12, and / or OX40, ICOS, TLR (e.g., TLR2). In some cases, molecules including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally cleave T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors can be co-expressed with the CAR. Furthermore, one skilled in the art will understand that the costimulatory domain does not have to be encoded solely by the full-length nucleotide sequence of the gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, which are arranged in various combinations to elicit a desired immune response.

[0135] As used herein, the term "anti-tumor effect" refers to a biological effect that may be manifested by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an amelioration of various physiological symptoms associated with a cancerous condition.

[0136] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve either the production of antibodies or the activation of specific immunologically competent cells, or both. Those skilled in the art will appreciate that virtually any macromolecule can serve as an antigen, including any protein or peptide.

[0137] The terms "cancer-associated antigen" or "tumor antigen" or "proliferative disorder antigen" or "antigen associated with a proliferative disorder" interchangeably refer to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cancer cells, either in whole or as a fragment (e.g., MHC / peptide), and is useful for targeting pharmacological agents to cancer cells. In some embodiments, the antigen tumor is a tumor-specific antigen (TSA) that is found only on cancer cells and not on healthy cells, or a tumor-associated antigen (TAA) that has elevated levels on tumor cells but is also expressed at lower levels on healthy cells. TSAs of interest are neoantigens that are specific novel cancer mutations. Other TAAs of interest are intracellular targets exposed on the cell surface by the MHC system (MHC peptide antigens), such intracellular TAAs associated with hematological cancers are, for example, WT1, MAGE-4, tyrosinase, PRAME, and survivin.

[0138] In some embodiments, the tumor antigen is a marker expressed by both normal and cancer cells, such as a lineage marker, such as CD19 on B cells. In certain aspects, the tumor antigen of the present invention is derived from cancer, including, but not limited to, leukemia, lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia ALL, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, preferably ALL, DLBCL, PMBCL, and MCL.

[0139] In some embodiments, the tumor antigen is an antigen that is common to a particular proliferative disorder.

[0140] In some embodiments, a cancer associated antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpressed, 2-fold overexpressed, 3-fold overexpressed, or more, compared to normal cells.

[0141] In some embodiments, a cancer-associated antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, a cancer-associated antigen is expressed exclusively on the cell surface of cancer cells, either in whole or as a fragment (e.g., MHC / peptide), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CAR of the present disclosure comprises a TCR-like CAR that includes an antigen-binding domain (e.g., an antibody or antibody fragment) that binds MHC-presented peptides. Typically, peptides derived from endogenous proteins fill the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.

[0142] Examples of preferred cancer associated antigens or combinations of cancer associated antigens that can be targeted by a CAR, typically via an scFv or engineered T cell receptor, include CD4, CD5, CD7, CD19, CD19 / CD20, CD19 / CD22; CD20, CD22, CD20 / CD22, CD30, CD33, CD37, CD38, CD39, CD70; CD123, CD44v6 (isoform variant 6), CD123 / CLL1, BCMA CD38 / BCMA, CD19 / BCMA, CD56, CD138, LeY, ROR1, SLAMF7, nFLT3, LMP1, BCMA / TACI, CS1, GPRC5D, CS1 / BCMA, NKG2D, CLL-1, GD2, MCSP, CD5, NKG2D ligand, MHC peptide antigens. More preferably, said cancer associated antigen or combination of cancer associated antigens is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, BCMA, GD2, MCSP, CD5, NKG2D, MHC peptide antigens, or combinations thereof, preferably CD19.

[0143] As used herein, "CAR cells" refers to cells expressing a CAR, and CAR cell compositions refer to compositions comprising CAR cells or a population of CAR cells. In some embodiments, the cells expressing a CAR can be T cells, B cells, macrophage cells, dendritic cells, or NK cells. Treatments that include administering a therapeutically effective amount of a CAR cell composition are referred to herein as CAR cell therapy. The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex or signaling through the appropriate NK receptor or signaling domain of the CAR. Stimulation can mediate altered expression of a particular molecule.

[0144] As used herein, "CAR cell activation" refers to the activation of CAR cells as detected by a significant increase in the cellular or intracellular expression of one or more activation markers in the CAR cells, which generally results in an increase in the killing activity of the CAR cells. CAR-T cell activation can be detected, for example, by a significantly increased expression of one or more of the following markers: surface PD1, intracellular granzyme B, and surface CD69.

[0145] The term "T cell" (or "T-cell" or "immune T cell") refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T cell receptor on the cell surface. T cells can be either isolated or obtained from commercial sources. "T cells" include all types of immune cells that express CD3, including T-helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T-regulatory cells (Tregs), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses. Non-limiting examples of commercially available T cell lines include the following lines: BCL2(AAA) Jurkat (ATCC® CRL-2902™), BCL2(S70A) Jurkat (ATCC® CRL-2900™), BCL2(S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL-2899™), Neo-Jurkat (ATCC® CRL-2898™), TALL-104 cytotoxic human T cell line (ATCC#CRL-11386).Further examples include, but are not limited to, mature T cell lines, such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1, and T34; and immature T cell lines, such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-Tl, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT- 16, MT-1, MT-ALL,P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1~T14, TALL-1, T ALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, T ALL-197, TK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaMl.6 (ATCC CRL-2063), RS4;11 (ATCC CRL- 1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines such as HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Null leukemia cell lines including, but not limited to, REH, NALL-1, KM-3, L92-221 are another commercial supplier of immune cells, as are cell lines derived from other leukemias and lymphomas such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Non-limiting exemplary sources of such commercially available cell lines include American Type Culture.

[0146] The term "T-reg" or "Treg" refers to T cells with immune suppressive function. As used herein, T cells are lymphocytes including any type of T cells, for example, alpha beta T cells (e.g., CD8 or CD4+), gamma delta T cells, memory T cells, Treg cells, etc. Suitably, immune suppressive function may refer to the ability of Treg to reduce or inhibit one or more of several physiological and cellular effects promoted by the immune system in response to stimuli such as pathogens, alloantigens, or autoantigens. Examples of such effects include proliferation of conventional T cells (Tconv) and increased secretion of proinflammatory cytokines. Any such effect may be used as an indicator of the strength of the immune response. A relatively weaker immune response by Tconv in the presence of Treg would indicate the ability of Treg to suppress immune responses. For example, a relative decrease in cytokine secretion would indicate a weaker immune response and thus the ability of Treg to suppress immune responses. Tregs can also suppress immune responses by modulating the expression of costimulatory molecules on antigen-presenting cells (APCs), such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to assess the suppressive potency of in vitro activated Tregs after co-culture.

[0147] The term "gamma delta T cells" refers to a subset of T cells that express a distinct T cell receptor (TCR), i.e., γδ TCR, on their surface, composed of one γ chain and one δ chain. The term "gamma-delta T cells" specifically includes all subsets of gamma-delta T cells, including, but not limited to, Vδ1 and Vδ2, Vδ3 γδ T cells, as well as naive, effector memory, central memory, and well-differentiated γδ T cells. Compositions and methods for making and using engineered and non-engineered γδ T-cells and / or subtypes thereof include, but are not limited to, those described in US Patent Publication No. 2016 / 0175358, WO 2017 / 197347, U.S. Patent No. 9,499,788, US Patent Publication No. 2018 / 0169147, U.S. Patent No. 9,907,820, US Patent Publication No. 2018 / 0125889, and US Patent Publication No. 2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including said compositions and methods for making and using engineered and non-engineered γδ T-cells and / or subtypes thereof. The application further contemplates T cells, or other engineered leukocytes or lymphocytes, that express a gamma chain or a delta chain, optionally in combination with a second polypeptide, to form a functional TCR. Such engineered leukocytes or lymphocytes expressing one gamma chain or one delta chain may be used in the methods or present in the compositions described herein.

[0148] As used herein, the term "NK cells", also known as natural killer cells, refers to a type of lymphocyte that originates from bone marrow and plays a key role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other stressed cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface. NK cells can be either isolated or obtained from commercial suppliers. Non-limiting examples of commercially available NK cell lines include the NK-92 (ATCC® CRL-2407™), NK-92MI (ATCC® CRL-2408™) lines. Further examples include, but are not limited to, the NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources of such commercially available cell lines include the American Type Culture Collection, or ATCC (www.atcc.org / ) and the German Collection of Microorganims and Cell Cultures (www.dsmz.de / ).

[0149] As used herein, the term "B cell" or "B lymphocyte" or any term commonly used in the art refers to a type of lymphocyte that matures in the bone marrow and develops into a lymphocyte that functions in the humoral immune component of the adaptive immune system. B cells produce membrane-bound antibody molecules and do not secrete these antibodies. B cells differ from the other two classes of lymphocytes, T cells and natural killer cells, in that they express a B cell receptor (BCR) on their cell membrane. The BCR enables the B cell to bind to an antigen, which initiates an antibody response against it. Naive or memory B cells are capable of recruiting T cell helpers in response to an antigen. With or without help, they are activated by antigens, proliferate and differentiate into antibody-secreting effector cells known as plasmablasts or plasma cells. Furthermore, B cells present antigens and are therefore called professional antigen-presenting cells (APCs), secrete cytokines and thereby modulate the immune response. They are found in tumor-draining lymph nodes, tumor-associated tertiary lymphoid structures, and in the tumor microenvironment as active participants to promote antitumor responses, although a specific subset is polarized in tumor-promoting effects. Their general nature confers several advantages, making B cells amenable to antigen-specific activation, in Features that make them attractive as therapeutic cell platforms include in vivo persistence, memory pool formation, and the potential to secrete proteins in large quantities. CAR-transduced leukemic B cells have been reported, for example, using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-inducible homolog-directed repair to successfully introduce CAR-expression cassettes into B cells. CARs equipped with the CD79β signaling domain, a component of the B cell receptor (BCR) complex for activation, can be engineered in primary murine B cells to induce robust surface expression and antigen recognition independent of endogenous BCR. Thus, B cells are feasible carriers of CAR-based therapy by exploiting endogenous BCR signaling. CAR-B cells can be used to drive localized delivery of monoclonal antibodies at tumor sites by targeting specific TAAs.This introduces the potential of CAR-B cells as a safe and controllable vehicle for the delivery of effective therapeutic antibodies that confer severe toxicity upon systemic administration. Alternatively, CAR-B cells may be a novel platform for autoimmune diseases and preventive vaccines.

[0150] The term "macrophage cells" as used herein refers to innate myeloid cells that are professional phagocytes capable of orchestrating the homeostasis of the adaptive immune system. Given their high abundance in many solid tumors, tumor-associated macrophages (TAMs) occupy a special niche in the TME, where many mediators can tailor their phenotype. [Immunosuppressive TAMs (M2s) can dampen T cell responses and promote tumor progression. In contrast, M1 polarization encompasses a proinflammatory phenotype and possesses antitumor activity, thereby bringing about great interest in engineering macrophages in cancer to aid immune surveillance. CARs confer to macrophages specificity of response to tumor-associated antigens (TAAs) in parallel with enhanced effector functions against tumors. For example, macrophages can be engineered with CD19-CARs incorporating the cytosolic domains of Megf10 or FcRγ to mimic phagocytic signaling. As a result, this triggered antigen-specific phagocytosis and trogocytosis of lymphoma cells. CD3ζ-CAR macrophages also exhibit active phagocytosis comparable to FcRγ-CAR. Thus, redirected antigen-specific phagocytosis confers spatial control and precision in the elimination of cancer cells, ultimately contributing to therapeutic efficacy. Furthermore, macrophages can be transduced with conventional CARs via adenoviral vectors, polarizing them toward a proinflammatory M1 phenotype and stimulated T cell responses, resulting in significant tumor regression and prolonged survival.

[0151] The term "dendritic cells" (DCs) as used herein refers to a heterogeneous subset of professional antigen-presenting cells that prime naive T cells and reactivate memory responses. In cancer, DCs sense environmental cues in lymphoid organs or tumor microenvironment, and sensing of danger signals induces DC maturation, resulting in either immune tolerance or tumor-specific responses. Importantly, cytotoxic CD8+ T cells can be activated by DCs through cross-presentation of TAAs or neoantigens to promote stronger antitumor responses. These have major implications in cancer immunotherapy, and CARs represent an emerging strategy to engineer DCs for effective responses against tumors.

[0152] As used herein, the term "leukodepletion" refers to the art-recognized extracorporeal process by which a donor's or patient's blood is removed from the donor or patient and passed through an instrument that separates and removes selected specific components, such as white blood cells, and returns the remainder to the donor's or patient's circulation, e.g., by return transfusion.

[0153] The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition that induces a biological or medical response in a cell, tissue, system, or subject that is being sought by a researcher, veterinarian, physician, or other clinician. For example, the composition includes an effective amount of CAR cells, preferably anti-CD-19 CAR-T cells and / or moclavimod, effective to produce at least one therapeutic effect when administered to a subject, either as a single dose or as part of a series of doses, e.g., sufficient to prevent the occurrence of or reduce to some extent one or more of the signs or symptoms of the disorder or disease being treated (e.g., blood cancer). Effective amounts will vary, as will be understood by those skilled in the art, depending, for example, on the route of administration, the use of excipients, and the co-use with other active agents. In the case of the treatment of a particular disease or condition, the desired therapeutic effect is to inhibit the progression of the disease. This may involve only slowing the progression of the disease temporarily, but more preferably, it involves permanently halting the progression of the disease. This can be monitored by routine methods or can be monitored according to the diagnostic methods described herein. The desired response to the treatment of a disease or condition can be to delay the onset or even prevent the onset of the disease or condition. The exact amount of the composition comprising CAR cells to be administered can be determined by the physician, taking into account the individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject). In other embodiments, when referring to an S1P receptor modulator, an effective amount of the S1P receptor modulator (such as moclavimod) can refer to an amount sufficient to activate the CAR cell composition defined herein in vivo, in vitro, or ex vivo, and / or to produce a synergistic therapeutic response when administered in combination with an effective amount of the CAR cell composition in a subject in need thereof.

[0154] As used herein, the term "about" means that the following value may vary by ±20%, preferably ±10%, more preferably ±5%, even more preferably ±2%, and even more preferably ±1%.

[0155] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to a mammal, preferably a human. In some embodiments, a patient, subject, or individual in need of treatment includes those already suffering from a disease, condition, or disorder, e.g., a hematological malignancy.

[0156] The term "in combination" or "in combination therapy" as used herein means to deliver two (or more) different treatments to a subject during the course of the subject's suffering from a disorder, for example, to deliver two or more treatments after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or the treatment is terminated for other reasons. In some embodiments, the delivery of one treatment is still taking place when the delivery of the second treatment begins, so there is an overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In either case, in some embodiments, the treatment is more effective because of the combined administration. For example, the second treatment is more effective, for example, an equivalent effect is seen with the second treatment less, or the second treatment reduces symptoms to a greater extent than is seen when the second treatment is administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than would be observed if one treatment were delivered in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive. The delivery is such that the effect of the first treatment delivered is still detectable when the second is delivered. In one embodiment, CAR cells expressing a CAR molecule that binds to a cancer associated antigen are administered at a dose and / or dosing schedule described herein, and an S1P receptor modulator is administered at a dose and / or dosing schedule described herein. In some embodiments, "in combination with" is not intended to imply that the CAR cell therapy and the S1P receptor modulator (e.g., moclavimod, KRP203) must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of this disclosure.The CAR cell therapy can be administered contemporaneously with, prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks before) or, preferably, subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) a dose of an S1P receptor modulator, e.g., moclavimod. In certain embodiments, each agent is administered at a dose and / or time schedule determined for that particular agent.

[0157] As used herein, unless otherwise specified, the term "treating" or "treatment" refers to ameliorating, alleviating, inhibiting the progression of, or preventing the disorder or condition to which such term applies, or ameliorating, alleviating, inhibiting the progression of, or preventing one or more symptoms of the disorder or condition to which such term applies.

[0158] The term "derived from" as used herein indicates a relationship between a first and a second molecule. It generally refers to the structural similarity between the first and second molecules, and does not imply or include any process or source limitations on the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure so that it has the required function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include any limitations on the particular process that produces the intracellular signaling domain, and does not mean, for example, that one must start from the CD3 zeta sequence and delete or force mutations to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0159] As used herein, the term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through a defined signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers.

[0160] The term "genetically engineered" or "genetically modified" refers to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is an immune effector cell, e.g., a lymphocyte, e.g., a T cell, a B cell, or a NK cell, which can be obtained from either the patient or a donor. The cell can be modified to express an exogenous construct, e.g., a chimeric antigen receptor (CAR), incorporated into the genome of the cell.

[0161] The term "CD19" as used herein refers to cluster of differentiation 19 protein, which is an antigenic determinant expressed in all B lineage cells and detectable on leukemic precursor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001 178098. As used herein, "CD19" includes proteins that contain mutations of full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants. CD19 is expressed on most B lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other diseases associated with CD19 expression include, but are not limited to, hematological cancers, such as leukemia, lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia ALL, Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, preferably ALL, DLBCL, PMBCL, and MCL, more preferably DLBLC. It is also an early marker of B-cell precursors. See, for example, Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997). In one embodiment, the antigen-binding portion of CART recognizes and binds an antigen in the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed on a cancer cell.

[0162] The term "CD20" as used herein refers to an antigenic determinant known to be detectable on B cells. Human CD20 is also called transmembrane 4-domain, subfamily A, member 1 (MS4A1). Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD20 can be found under accession numbers NP_690605.1 and NP_068769.2, and the nucleotide sequences encoding the transcriptional variants 1 and 3 of human CD20 can be found under accession numbers NM_152866.2 and NM_021950.3, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD20 protein. In one embodiment, the CD20 protein is expressed on cancer cells.

[0163] The term "CD22" as used herein refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of isoforms 1-5 human CD22 can be found under accession numbers NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and the nucleotide sequences encoding variants 1-5 of human CD22 can be found under accession numbers NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one embodiment, the antigen binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD22 protein. In one embodiment, the CD22 protein is expressed on a cancer cell.

[0164] As used herein, the terms "alpha subunit of the IL-3 receptor," "IL3Ra," "CD123," "IL3Ra chain," and "IL3Ra subunit" refer interchangeably to antigenic determinants known to be detectable on leukemia progenitor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human IL3Ra can be found under accession number NP 002174, and the nucleotide sequence encoding human IL3Ra can be found under accession number NM 005191. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD123 protein. In one embodiment, the CD123 protein is expressed on cancer cells. As used herein, "CD123" includes proteins that contain mutations of the full-length wild-type CD123, such as point mutations, fragments, insertions, deletions, and splice variants.

[0165] The term "ROR1" as used herein refers to an antigenic determinant known to be detectable on leukemia precursor cells. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human ROR1 isoform 1 and 2 precursors can be found under accession numbers NP_005003.2 and NP_001077061.1, respectively, and the mRNA sequences encoding them can be found under accession numbers NM_005012.3 and NM_001083592.1, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen in the extracellular domain of the ROR1 protein. In one embodiment, the ROR1 protein is expressed on cancer cells.

[0166] The term "CD33" as used herein refers to cluster of differentiation 33 protein, which is an antigenic determinant detectable on leukemia cells and normal progenitor cells of myeloid lineage. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD33 can be found under UniProt / Swiss-Prot accession number P20138, and the nucleotide sequence encoding human CD33 can be found under accession number NM_001772.3. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD33 protein or a fragment thereof. In one embodiment, the CD33 protein is expressed on a cancer cell. As used herein, "CD33" includes proteins that contain mutations of full-length wild-type CD33, such as point mutations, fragments, insertions, deletions, and splice variants.

[0167] The term "CD38" as used herein refers to cluster of differentiation 38 protein, which is an antigenic determinant detectable on leukemia cells and normal progenitor cells of myeloid lineage. Human and murine amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD38 can be found under UniProt / Swiss-Prot accession number P28907, and the nucleotide sequence encoding human CD38 can be found under accession number NM_001775. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an epitope within the extracellular domain of the CD38 protein or a fragment thereof. In one embodiment, the CD38 protein is expressed on a cancer cell. As used herein, "CD38" includes proteins that contain mutations of full-length wild-type CD38, such as point mutations, fragments, insertions, deletions, and splice variants.

[0168] The term "CLL-1" as used herein refers to human C-type lectin-like molecule-1 (CLL-1), which is a type II transmembrane glycoprotein whose expression is restricted to myeloid cells and the majority of AML blast cells. Furthermore, CLL-1 is expressed in leukemia stem cells (LSCs) but not in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment (Wang, J., Chen, S., Xiao, W. et al. CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia. J Hematol Oncol 11, 7 (2018)).

[0169] The term "BCMA" as used herein refers to B cell maturation antigen. BCMA (also known as TNFRSF17, BCM, or CD269) is a member of the tumor necrosis factor receptor (TNFR) family and is expressed primarily on highly differentiated B cells, such as memory B cells, and plasma cells. Its ligands are called B cell activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL). BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. The gene for BCMA is encoded on chromosome 16 and gives rise to a 994 nucleotide long primary mRNA transcript (NCBI accession NM_001192.2) that codes for a 184 amino acid protein (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described, which may play a role in regulating BCMA expression. (Laabi Y. et al., Nucleic Acids Res., 1994, 22: 1147-1154).

[0170] The term "GD2" refers to disialoganglioside, a TAA found on NBL, melanoma, and sarcoma.

[0171] The term "MCSP" refers to melanoma-associated chondroitin sulfate proteoglycan, another TAA found in melanoma.

[0172] The term "CD5" refers to the receptor CD5, which is expressed on the surface of T cells and on a subset of murine B cells known as B-1a. CD5 serves to moderate the activation signal from the BCR, so that B-1 cells can be activated only by very strong stimuli (such as bacterial proteins) and not by normal tissue proteins.

[0173] The term "NKG2D" refers to the natural killer group 2D (NKG2D) receptor, which plays an important role in protecting the host from infection and cancer.By recognizing the ligand induced on infected or tumor cells, NKG2D modulates lymphocyte activation and promotes immunity to eliminate ligand-expressing cells.

[0174] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker and can be expressed as a single chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv as used herein may have the VL and VH variable regions in any order, e.g., with respect to the N-terminus and C-terminus of the polypeptide, and may comprise a VL-linker-VH or a VH-linker-VL.

[0175] As used herein, the term "recurrence" or "recurrent" has its ordinary meaning in the art and refers to the return of a hematological malignancy or signs and symptoms of a hematological malignancy after a period of complete remission due to treatment (e.g., initial complete remission).

[0176] The term "remission" as used herein has its usual meaning in the art, and refers to the reduction or disappearance of signs and symptoms of cancer.In partial remission, some, but not all, signs and symptoms of cancer have disappeared.In complete remission (CR), all signs and symptoms of cancer have disappeared, but cancer may still be present in the body.

[0177] The term "conditioning" as used herein refers to preparing a patient who needs immune effector cell therapy for the appropriate conditions. As used herein, conditioning includes, but is not limited to, reducing the number of endogenous lymphocytes, removing cytokine sinks, increasing the serum level of one or more homeostatic cytokines or proinflammatory factors, enhancing the effector function of T cells administered after conditioning, enhancing the activation and / or availability of antigen-presenting cells, or any combination thereof, prior to immune effector cell therapy. In one embodiment, "conditioning" includes lymphodepleting the patient.

[0178] The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into the individual. For example, the cell therapy methods described herein involve collecting lymphocytes from a patient, which are then manipulated to express, for example, a CAR construct, and then administered back into the same patient.

[0179] The term "syngeneic" refers to any material derived from an individual that is genetically identical to the one that is subsequently reintroduced into the individual, for example, the same individual or an identical twin.

[0180] The term "allogeneic" refers to any material derived from a different animal of the same species (e.g., the human species) as the individual into whom the material is introduced. Two or more individuals are said to be allogeneic to one another if their genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0181] Chimeric Antigen Receptor (CAR) Cells As used herein, a CAR cell composition is a compositing comprising a cell or population of cells that have been genetically modified to express one or more chimeric antigen receptor molecules.

[0182] In a specific embodiment, the CAR cell is a human immune effector cell or cell population (e.g., a human T cell, such as a conventional T cell or a regulatory T cell, or a human NK cell, e.g., a human T cell described herein or a human NK cell described herein). In one embodiment, the human T cell is a CD8+ T cell. In one embodiment, the cell is an autologous or syngeneic T cell. In one embodiment, the cell is an allogeneic T cell. It is understood that compositions and methods disclosed herein that refer to the term "cell" encompass compositions and methods comprising one or more cells, e.g., cell populations.

[0183] Methods for generating CAR-T cells are described, for example, in Feins et al. Am J Hematol. 2019;94:S3-S9. Methods for generating CAR NK cells are described, for example, in Xie et al. EBiomedicine 59 (2020) 102975. Methods for generating CAR Treg cells are described, for example, in Enrico Fritsche et al., Therapeutic Biomanufacturing, volume 38, issue 10, P1099-1112, October 2020.

[0184] Immune cells for generating CAR cells for use in accordance with the present invention may further comprise other genetic modifications (in addition to those required for expression of the CAR molecule), particularly for allogeneic use, i.e., the CAR cells are made from donor subject immune cells that are different from the recipient subject.

[0185] In a specific embodiment, the CAR construct comprises a transmembrane domain selected from the group consisting of CD28, CD3, CD4, CD8α, and CD16.

[0186] In a specific embodiment, the CAR construct comprises a cytoplasmic signaling domain selected from the group consisting of CD28, 4-1BB, ICOS, and CD27.

[0187] The term CAR as used herein further includes chimeric antigen receptor molecules or combinations of chimeric antigen receptor molecules that recognize multiple cancer-associated antigens, typically two distinct cancer-associated antigens (e.g., CD20 / CD22), particularly to avoid tumor escape and / or mitigate toxicity. Such CARs that recognize multiple cancer-associated antigens include, but are not limited to, dual CARs, tandem CARs, loop tandem CARs, and combinatorial CARs, which are disclosed, for example, in Guedan et al. 2019, Molecular Therapy: Methods & Clinical Development Vol. 12, pp 145-156.

[0188] CARs are classified according to the modules that fit into the cytoplasmic signaling domain. First generation CARs show a cytoplasmic signaling domain, with one signaling module derived from the ζ chain of the TCR / CD3 complex. Second and third generation CARs are designed to contain one or more costimulatory regions associated with the CD3ζ chain, respectively. Various costimulatory molecules, including OX40, CD27, and ICOS, have been evaluated in preclinical studies, but CD28 and 4-1BB are the most commonly used in clinical trials.

[0189] First-generation CAR-T cells showed cytotoxic activity in vitro but suboptimal persistence in vivo, limiting their therapeutic potential. Indeed, second-generation CARs showed better T cell persistence after infusion compared to first-generation CARs. However, 4-1BB-containing CARs have been reported to show greater persistence in vivo, in xenograft models, and in patients compared to those carrying the CD28 domain. CD28-based CAR-T cells showed constitutive proliferation, effector memory differentiation, and susceptibility to exhaustion, whereas 4-1BB-based CAR-T cells were reported to show central memory features with enhanced survival and inhibition of exhaustion. Because the costimulatory domain improved CAR-T cell function, survival, and persistence, third-generation CARs were created that combine two costimulatory domains. By linking the epitope specificity of monoclonal antibodies with the killing capacity of specific T cells, CARs bypass the requirement for antigen presentation by MHC molecules. In addition, CAR-T cells can also recognize non-classical TCR targets such as lipids and carbohydrates, giving CAR-T cells the ability to recognize a broader range of target antigens. Unlike TCR-transgenic T cells, CAR-T cells do not require antigen processing and are not HLA restricted, allowing this therapy to reach a larger patient subset. Furthermore, since CAR-T cell infusion products are obtained from peripheral blood, patients with no resectable tumors or low T cell infiltration into tumors (ineligible for TIL therapy) can benefit from CAR therapy.

[0190] Thus, in one preferred embodiment, a CAR cell according to the present disclosure is a T cell expressing a CAR (i.e., a CAR-T cell), said CAR comprising at least: (i) an antigen-binding domain, typically an extracellular domain comprising a single chain variable fragment having binding specificity for a cancer-associated antigen (e.g., CD19); (ii) a transmembrane domain; and (iii) a cytoplasmic signaling domain, preferably comprising one signaling module derived from the zeta chain of the TCR / CD3 complex, and optionally one or more costimulatory domains, typically a CD28 or 4-1BB costimulatory domain; Includes.

[0191] Adoptive transfer of CAR-T cells has shown impressive results in the treatment of B-cell derived malignancies. As a result, the US Food and Drug Administration (FDA) approved the first two gene therapies for B-cell malignancies based on CAR-T cells in 2017. Kymriah (tisagenrecucel) is a second-generation CAR targeting CD19, containing a costimulatory domain derived from 4-1BB, and is approved for the treatment of pediatric acute B-cell lymphoblastic leukemia. Yescarta (axicabtagene silolucel) also targets the CD19 antigen and is indicated for patients with diffuse large B-cell lymphoma. Unlike Kymriah, Yescarta contains a costimulatory module derived from CD28. In July 2020, the FDA approved Tecartus (brexcavtagene autreucel), also targeting the CD19 antigen, for patients with mantle cell lymphoma that has not responded to or relapsed to other treatments. In February 2021, the FDA approved Breyanzi (lysocabtagenemaraleucel), which targets the CD19 antigen, for patients with relapsed or refractory large B-cell lymphoma. In March 2021, the FDA approved Abecma (idecbutagenviraleucel), the first CAR-T cell targeted to BCMA, to treat adult patients with multiple myeloma.

[0192] As used in this disclosure, a CAR cell comprises a nucleic acid encoding a CAR that comprises an extracellular domain that includes an antigen-binding domain that specifically binds to a cancer-associated antigen.

[0193] In a specific embodiment, said cancer-associated antigen is selected from the group consisting of cell surface markers of hematopoietic cells, preferably immune cells, e.g., B or T cells.

[0194] In one particular embodiment, a CAR cell for use in the present disclosure comprises a CAR having an extracellular domain that comprises an antigen binding domain that specifically binds to a cancer associated antigen selected from the group consisting of CD4, CD5, CD7, CD19, CD19 / CD20, CD19 / CD22; CD20, CD22, CD20 / CD22, CD30, CD33, CD37, CD38, CD39, CD70; CD123, CD44v6 (isoform variant 6), CD123 / CLL1, BCMA CD38 / BCMA, CD19 / BCMA, CD56, CD138, LeY, ROR1, SLAMF7, nFLT3, LMP1, BCMA / TACI, CS1, GPRC5D, CS1 / BCMA, NKG2D, CLL-1 ligand. More preferably, said cancer-associated antigen or combination of cancer-associated antigens is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, GD2, MCSP, CD5, NKG2D, MHC peptide antigen, BCMA, and combinations thereof, preferably CD19. In other specific embodiments, said cancer-associated antigen is selected from cancer-associated antigens typical of hematological malignancies or cancer-associated antigens of lymphoid organ metastases, preferably lymph node metastases, which may be from solid tumor cancers, particularly breast cancer, typically HER2 or HER3. Said cancer-associated antigens include antigens expressed on the surface of metastatic cancers or antigens expressed on the surface of metastatic cancers in the context of major histocompatibility complex molecules.

[0195] In a preferred embodiment, said preferred cancer associated antigens are selected from the group consisting of CD19, CD30, CD33, CD123, CD20, CD22, CD38, LeY, ROR1, CLL-1, GD2, MCSP, CD5, NKG2D, MHC peptide antigens, BCMA, preferably CD19.

[0196] In a specific embodiment, the CAR cells for use according to the present disclosure comprise as extracellular antigen binding domains: CD4, CD5, CD7, CD19, CD19 / CD20, CD19 / CD22; CD20, CD22, CD20 / CD22, CD30, CD33, CD37, CD38, CD39, CD70; CD123, CD44v6 (isoform variant 6), CD123 / CLL1, BCMA The CAR expresses a single chain variable fragment (scFv) that specifically binds to a cell surface marker selected from the group consisting of CD38 / BCMA, CD19 / BCMA, CD56, CD138, LeY, ROR1, SLAMF7, nFLT3, LMP1, BCMA / TACI, CS1, GPRC5D, CS1 / BCMA, NKG2D ligand, more preferably selected from the group consisting of CD19, CD30, CD33, CD123, CD20, CD22, CD38, LeY, ROR1, CLL-1, GD2, MCSP, CD5, NKG2D, MHC peptide antigen, BCMA, preferably CD19.

[0197] Examples of CAR constructs that bind to cancer-associated antigens as described herein are described in WO2019227003, which is incorporated by reference in its entirety.

[0198] Other exemplary CAR constructs are those used in the following CAR-T commercial or developmental products: Kymriah (tisagenleucel), Yescarta (axicabtagene siloleucel), Tecartus (brexcabtagene autolucel), Breyanzi (lysocabtagene maralucel), Abecma (idecabtagene biculeucel).

[0199] The compositions comprising the CAR cells described herein are used in combination with S1P receptor modulators.

[0200] S1P receptor modulators S1P receptors are classified into five subtypes related G-coupled protein receptors (ie, S1P1, S1P2, S1P3, S1P4, and S1P5) that are expressed in a wide variety of tissues and display different cellular specificities.

[0201] In certain embodiments, the modulators of S1P receptors for use according to the methods of the present disclosure are intended to inhibit the activity of the five S1P receptor types 1-5 (S1PRs) by activating or internalizing or inhibiting the receptor for signal transduction. 1~5 ) are compounds that modulate one or more of the S1P agonists and S1P inhibitors, respectively, herein.

[0202] In specific embodiments, said S1P receptor modulator for use in the treatment methods of the present disclosure is selected from KRP203 (moclavimod), FTY720 (fingolimod, Gilenya™), BAF312 (siponimod, Mayzent®), ozanimod (Zeposia®), ponesimod, etrasimod, AKP-11, cenerimod, amiselimod, CBP-307, OPL-307, OPL-002, BMS-986166, SCD-044, BOS-173717, CP-1050. Preferably, said S1P receptor modulator for use in the treatment methods of the present disclosure is selected from KRP203 (mocravimod), FTY720 (fingolimod), and Mayzent® (siponimod), most preferably mocravimod.

[0203] In one embodiment, the S1P receptor modulator for use in the treatment method of the present disclosure is an S1P agonist. Examples of such S1P agonists include KRP203 (moclavimod), an S1PR1 selective agonist, or FTY720 (fingolimod), an S1PR1, 3~5or siponimod, an S1PR3 agonist, or a pharma- ceutically acceptable salt or phosphate derivative thereof.Preferably, in certain embodiments, the S1P agonist is selected from S1P agonists that selectively activate S1PR1.

[0204] In a preferred embodiment, the S1P receptor modulator for use according to the present disclosure is a compound of formula (I):

[0205] [ka] [In the formula, R2 is H, halogen, trihalomethyl, C 1~4 Alkoxy, C 1~7 alkyl, phenethyl, or benzyloxy; R3 is H, halogen, CF3, OH, C 1~7 Alkyl, C 1~4 Alkoxy, benzyloxy, phenyl, or C 1~4 is alkoxymethyl, Each of R4 and R5 is independently H or a residue of formula (a):

[0206] [ka] wherein each of R8 and R9 is independently H or C optionally substituted with halogen. 1~4 is alkyl, n is an integer from 1 to 4; R6 is hydrogen, halogen, C 1~7 Alkyl, C 1~4 alkoxy, or trifluoromethyl. It is.

[0207] In a specific embodiment, the compound of formula (I) is an S1P agonist, preferably an S1PR1 selective agonist. Typically, in a preferred embodiment, R3 is chlorine. More preferably, R2 is H, R3 is chlorine, and R6 is hydrogen. For example, R2 is H, R3 is chlorine, R6 is hydrogen, and each of R3 and R5 is independently H.

[0208] In a more preferred embodiment, the S1P receptor modulator, preferably an S1P agonist, preferably an S1PR1 selective agonist for use according to the present disclosure is 2-amino-2-[4-(3-benzyloxyphenylthio)-2-chlorophenyl]ethyl-propane-1,3-diol (also called moclavimod or KRP203) of formula (II):

[0209] [ka] Or a pharma- ceutically acceptable salt thereof.

[0210] Other S1P receptor modulators, preferably S1PR1 selective agonists, for use according to the present disclosure include phosphate derivatives of the following formula:

[0211] [ka] Includes.

[0212] Said compounds and methods for their synthesis are also disclosed in WO 03 / 029205, WO 2004 / 074297, WO 2006 / 009092, WO 2006 / 041019, and WO 2014128611 A1, the disclosures of which are incorporated herein by reference.

[0213] Mocravimod or its pharmacologic acceptable salt or its phosphate derivative is especially preferred. In fact, the comparison of the pharmacodynamic effects of various S1P modulators, such as mocravimod, FTY720, and BAF312, established in healthy volunteers, reveals the difference in the effectiveness of lymphocyte sequestration. The measurable parameter that determines the maintenance of the mechanism of action, i.e., lymphocyte sequestration in secondary lymphoid organs and bone marrow, is the reduction in peripheral lymphocyte count. After a single 1 mg dose of FTY720, multiple doses of BAF312 for 28 days, or a single 3 mg dose of KRP203, the recovery of absolute lymphocyte count to 80% of normal count was achieved after 8, 7, and 10 more days, respectively. Thus, the lymphocyte recovery time of KRP203 was significantly longer than BAF312 and FTY720.

[0214] Particle size of S1P receptor modulators In the pharmaceutical industry, particle characterization of powder materials is one of the critical aspects in drug product development and quality control of solid oral dosage forms. Particle size distribution of drug substances can have a significant effect on the performance of the final drug product (e.g., dissolution, bioavailability, content uniformity, stability, etc.). In addition, particle size distribution of drug substances can affect the manufacturability of the drug product, such as flowability, blend uniformity, and compactibility, and has a prominent impact on almost every step of the manufacturing process of solid oral dosage forms, including premixing / blending, granulation, drying, milling, blending, coating, encapsulation, and compression. Thus, the particle size of drug substances ultimately affects the safety, efficacy, and quality of the drug product.

[0215] In one embodiment, the S1P receptor modulators of the present disclosure have a mean particle size (D50) of 8 μm or less, preferably 6 μm, more preferably 5 μm. In another embodiment, the S1P receptor modulators of the present disclosure have a D90 of 25 μm or less, preferably 22 μm, more preferably 19 μm. In fact, it has been found by the inventors that a mean particle size (D50) of more than 8 μm, preferably more than 6 μm, more preferably more than 5 μm and / or a D90 of more than 25 μm, preferably more than 22 μm, more preferably more than 19 μm impairs drug dissolution by reducing surface contact with the solvent, resulting in reduced bioavailability and in vivo performance.

[0216] Pharmaceutical Compositions Comprising S1P Receptor Modulators The present disclosure also relates to a pharmaceutical composition of said S1P receptor modulator, preferably moclavimod or a pharma- ceutically acceptable salt or phosphate derivative thereof, as described above, particularly for its use in the disclosed methods of treatment.

[0217] In one embodiment, the pharmaceutical composition of the present disclosure comprises an S1P receptor modulator, preferably moclavimod or a pharma- ceutically acceptable salt or phosphate derivative thereof, and one or more pharma- ceutically acceptable excipients.

[0218] Any suitable excipient known to those of ordinary skill in the art for use in pharmaceutical compositions may be used in the compositions described herein.

[0219] The pharmaceutical composition may be administered in any manner suitable for the disease or disorder to be treated, as determined by a medical artisan.The appropriate dose and the appropriate duration and frequency of administration are determined by factors such as those described herein, including the patient's condition, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration.In general, the appropriate dose (or effective dose) and treatment regimen provide the pharmaceutical composition in an amount sufficient to provide a therapeutic effect, for example, improved clinical outcome, such as more frequent complete or partial remission, or longer disease-free and / or overall survival, or reduced severity of symptoms, or other benefits detailed herein.

[0220] The pharmaceutical compositions described herein can be administered to a subject in need thereof by any of several routes that can effectively deliver an effective amount of the compound.The pharmaceutical compositions can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically, bucally, or as an oral or nasal spray.In a preferred embodiment, the pharmaceutical composition is suitable for oral administration.

[0221] In another embodiment, the pharmaceutical composition can be in a solid dosage form suitable for oral administration.Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.In a preferred embodiment, the pharmaceutical composition is a capsule or tablet.The capsule can be a soft or hard gelatin capsule, preferably a hard gelatin capsule.For example, the capsule is HGC crushed or HPMC crushed capsule.

[0222] In one embodiment, the release of the contents of the capsule or tablet can be immediate or modified, such as delayed, targeted, or extended, etc. In a preferred embodiment, the solid dosage form is an immediate release dosage form.

[0223] In one embodiment, the pharmaceutical composition comprises an S1P receptor modulator, preferably moclavimod, and one or more pharma- ceutically acceptable excipients, in particular at least one filler and mixtures thereof, disintegrants, lubricants, and glidants.

[0224] Filler A filler (also called a diluent, dilutant, or thinning agent) is a substance added to a drug substance to make it suitable for oral administration (e.g., capsule, tablet). The filler itself should not produce any pharmacological effect in humans. Examples of fillers include mannitol, microcrystalline cellulose, lactose monohydrate, anhydrous lactose, corn starch, xylitol, sorbitol, sucrose, dicalcium phosphate, maltodextrin, and gelatin. The pharmaceutical composition of the present disclosure comprises at least one filler selected from mannitol, microcrystalline cellulose, and mixtures thereof. In a preferred embodiment, the pharmaceutical composition of the present disclosure comprises a mixture of mannitol and microcrystalline cellulose.

[0225] Disintegrants Disintegrants are added to oral solid dosage forms to aid in their deagglomeration. Disintegrants are formulated to cause rapid disintegration of solid dosage forms when in contact with moisture. Disintegration is typically seen as the first step in the dissolution process. Examples of disintegrants include modified starches such as sodium starch glycolate, sodium carboxymethyl starch, and pregelatinized starch, cross-linked polymers such as cross-linked polyvinylpyrrolidone (crospovidone) or cross-linked sodium carboxymethylcellulose (croscarmellose sodium), and calcium silicate. The pharmaceutical composition of the present disclosure includes sodium starch glycolate as a disintegrant.

[0226] Lubricants Lubricants are substances that we use in tablet and capsule formulations to reduce friction. Lubricants can facilitate the extrusion of the tablet from the matrix and thus prevent the formation of scratches on its surface. In nature, lubricants can be divided into two groups: a) fat and fat-like substances, b) powdery substances. Powdery substances are more applicable than fat-like ones, since fat-like substances affect the solubility and chemical stability of the tablet. Powdery lubricants are introduced by powdering the granules. They provide a constant rate of outflow of mass for tableting from the hopper to the matrix, which ensures the accuracy and constancy of the drug substance dosage.

[0227] Examples of lubricants include magnesium stearate, hydrogenated castor oil, glyceryl behenate, calcium stearate, zinc stearate, mineral oil, silicone fluid, sodium lauryl sulfate, L-leucine, and sodium stearyl fumarate. The pharmaceutical composition of the present disclosure includes magnesium stearate as a lubricant.

[0228] Glidants Glidants are mixed with the compound to enhance the flow properties of tablet core blend materials. During the initial period of compression, glidants are mixed into the particle arrangement of the tablet powder blend to improve flow and uniformity in the die cavity of the tablet press. Glidants encourage the flow of tablet granulation by reducing friction between particles. The effect of glidants on the flow of granules depends on the particle size and shape of the granules and the glidant. Above a certain concentration, glidants act rather to hinder flow. In tablet manufacturing, glidants are usually added just before compression. Examples of glidants include colloidal silicon dioxide, starch, magnesium stearate, and talc. The pharmaceutical composition of the present disclosure includes colloidal silicon dioxide as a lubricant.

[0229] The pharmaceutical compositions of the present disclosure comprise an S1P receptor modulator, at least one filler selected from mannitol, microcrystalline cellulose, and mixtures thereof, sodium starch glycolate as a disintegrant, magnesium stearate as a lubricant, and colloidal silicon dioxide as a glidant.

[0230] Any suitable excipient known to one skilled in the art of pharmaceutical compositions may further be used in the compositions described herein.

[0231] In one embodiment, the dosage strength of the S1P receptor modulator, preferably the hydrochloride salt of formula (I) or the phosphate derivative of formula IIa or IIb, in a solid dosage form is from 0.05 mg to 15 mg / unit, preferably from 0.1 mg to 10 mg / unit, for example about 0.1 mg / unit, or about 0.4 mg / unit, or about 1 mg / unit, or about 10 mg / unit, more preferably about 1 mg / unit.

[0232] More particularly, the pharmaceutical compositions of the present disclosure, particularly those containing 1 mg / unit of moclavimod or a pharma- ceutically acceptable salt or phosphate derivative thereof, contain the following components: mannitol, preferably with a content of 48 to 88 mg / unit, more preferably with a content of 58 to 78 mg / unit, even more preferably with a content of about 68 mg / unit, microcrystalline cellulose, preferably in a content of 5 to 45 mg / unit, more preferably in a content of 15 to 35 mg / unit, even more preferably in a content of about 25 mg / unit, sodium starch glycolate, preferably in a content of 1 to 8 mg / unit, more preferably in a content of 2 to 6 mg / unit, even more preferably in a content of about 4 mg / unit, magnesium stearate, preferably in a content of 0.025 to 4 mg / unit, more preferably in a content of 0.5 to 2 mg / unit, and even more preferably in a content of about 1 mg / unit, Colloidal silicon dioxide, preferably in a content of 0.125 to 2 mg / unit, more preferably in a content of 0.25 to 1 mg / unit, even more preferably in a content of about 0.5 mg / unit. Further includes:

[0233] In another embodiment, the pharmaceutical composition of the present disclosure comprises the following components: an S1P receptor modulator, preferably moclavimod or a pharma- ceutically acceptable salt or phosphate derivative thereof, preferably in a content of 0.05% to 15%, more preferably 0.1% to 10% mg / unit, even more preferably about 0.1% or 0.4% or 1% or 10%, mannitol, preferably with a content of between 48% and 88%, more preferably between 58% and 78%, even more preferably about 68%; microcrystalline cellulose, preferably with a content of between 5% and 45%, more preferably between 15% and 35%, even more preferably about 25%; sodium starch glycolate, preferably with a content between 1% and 8%, more preferably between 2% and 6%, even more preferably about 4%; magnesium stearate, preferably with a content of between 0.025% and 4%, more preferably between 0.5% and 2%, even more preferably about 1%; Colloidal silicon dioxide, preferably in a content of 0.125 to 2 mg / unit, more preferably in a content of 0.25 to 1 mg / unit, even more preferably in a content of about 0.5 mg / unit. and percentages are expressed as mg / mg of dry weight of total composition.

[0234] In one embodiment, the pharmaceutical composition of the present disclosure is stable at 50° C. for at least 1 month, preferably for 2 months.

[0235] In another embodiment, the pharmaceutical composition of the present disclosure is stable at 5° C. for at least 24 months.

[0236] In another embodiment, the pharmaceutical composition of the present disclosure is stable at 25° C. / 60% relative humidity for at least 24 months.

[0237] As used herein, the stability of a composition is measured according to the following method, i.e., high pressure liquid chromatography (HPLC), which is well known in the art: A composition is stable if the sum of the impurities is less than or equal to 0.7% with a [98-102]% confidence interval.

[0238] Process for preparing a pharmaceutical composition Another aspect of the present disclosure is a process for preparing the above-mentioned pharmaceutical composition, comprising: a. blending an S1P receptor modulator, or a pharma- ceutically acceptable salt or phosphate derivative thereof, with microcrystalline cellulose, colloidal silicon dioxide, preferably at 22 rpm for 18 minutes; b. adding mannitol and blending the resulting mixture, preferably at 22 rpm, for 9 minutes; c. adding sodium starch glycolate and blending the resulting mixture, preferably at 22 rpm, for 5 minutes; d. adding magnesium stearate and blending the resulting pharmaceutical composition, preferably at 22 rpm, for 5 minutes; e. Recovering the pharmaceutical composition of the present disclosure. The present invention relates to a process comprising:

[0239] In a preferred embodiment, the process comprises: f. filling the resulting pharmaceutical composition into a capsule; g. Recovering the resulting capsule filled with the pharmaceutical composition. Further includes:

[0240] Patient populations preferably targeted by combination therapy The treatment method disclosed herein is suitable for patients with hematological malignancies or lymphatic organ metastases, preferably lymph node metastases.Hematological malignancies are types of cancers that affect the blood, bone marrow, and lymphatic system, such as leukemia, lymphoma, and malignant lymphoproliferative conditions.Lymphatic organ metastases, preferably lymph node metastases, can be from solid tumor cancers, particularly breast cancer.

[0241] In one embodiment, the hematological malignancy is leukemia. Leukemia can be classified into acute leukemia and chronic leukemia. Acute leukemia can be further classified into acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL). Chronic leukemia includes chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (CLL). Other related conditions include myelodysplastic syndrome (MDS, formerly known as "preleukemia"), a diverse collection of hematological conditions united by ineffective production (or dysplasia) of bone marrow blood cells and the risk of malignant transformation to AML.

[0242] In other embodiments, the hematological malignancy is lymphoma. Lymphomas are a group of blood cell tumors that arise from lymphocytes. Exemplary lymphomas include non-Hodgkin's lymphoma and Hodgkin's lymphoma.

[0243] Non-Hodgkin's lymphoma (NHL) is a group of cancers of lymphocytes that form from either B or T cells. NHL can occur at any age and is often characterized by larger than normal lymph nodes, weight loss, and fever. Various types of NHL are classified as aggressive (fast-growing) and indolent (slow-growing) types. B-cell non-Hodgkin's lymphomas include Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. Examples of T-cell non-Hodgkin's lymphomas include mycosis fungoides, anaplastic large cell lymphoma, and precursor T-lymphoblastic lymphoma. Lymphomas that arise after bone marrow or stem cell transplantation are typically B-cell non-Hodgkin's lymphomas. See, e.g., Maloney. NEJM. 366.21(2012):2008-16. Diffuse large B-cell lymphoma (DLBCL) is a form of NHL that arises from B cells.

[0244] Acute lymphoblastic leukemia (ALL) Acute lymphocytic leukemia (ALL) is a B-cell malignancy characterized by neoplastic cell proliferation and accumulation in the bone marrow, blood, lymph nodes, and spleen. ALL can occur in adult or pediatric populations, can progress rapidly, and can be fatal if left untreated. ALL includes relapsed and / or refractory ALL (r / r ALL). In relapsed and / or refractory ALL, treatment options include high-dose chemotherapy followed by allogeneic stem cell transplantation (SCT), standard chemoimmunotherapy, targeted treatment with small molecule pathway inhibitors, or supportive care with non-curative symptomatic goals. Allogeneic SCT is the only potentially curative option for r / r pediatric ALL, but outcomes are suboptimal. Among patients with relapsed and / or refractory pediatric ALL who underwent allogeneic SCT in third or subsequent remission, for active disease, or after relapse from a prior allogeneic SCT, 1-year overall survival (OS) rates range from 25% to 55%, and 5-year OS rates are generally between 20% and 45%.

[0245] For patients with ALL who are Philadelphia chromosome positive (Ph+), dasatinib (Sprycel) was approved in 2006 for the treatment of adult patients with resistance or intolerance to previous therapy. Ponatinib (Iclusig) was approved in 2013 for the treatment of adult patients with Ph+ ALL who are resistant or intolerant to dasatinib. Blincyto (blinatumomab), a bispecific anti-CD3 / CD19 monoclonal antibody, has been approved for the treatment of adults with Ph- relapsed or refractory B-precursor ALL. Despite current treatment modalities, maintaining remission in relapsed ALL patients is difficult and patients are hospitalized for long periods of time with poor quality of life. Prognosis for patients with relapsed and / or refractory disease remains poor.

[0246] Diffuse large B-cell lymphoma (DLBCL) DLBCL is an aggressive lymphoma that may arise in lymph nodes or outside the lymphatic system, for example, in the gastrointestinal tract, testes, thyroid, skin, breast, bone, or brain. Three cellular morphological variants are commonly observed in DLBCL: centroblastic, immunoblastic, and anaplastic. Centroblastic morphology is the most common, with the appearance of medium- to large-sized lymphocytes with minimal cytoplasm. There are several subtypes of DLBCL. Most patients with DLBCL are adults, although the disease occasionally occurs in children. Treatment of DLBCL includes chemotherapy (e.g., cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide), antibodies (e.g., Rituxan), radiation, or autologous stem cell transplantation (ASCT). However, approximately half of patients who are relapsed and / or refractory to first-line therapy are not eligible for ASCT due to advanced age and / or comorbidities. Furthermore, of patients suitable for high-dose therapy and autologous stem cell transplantation (HD-ASCT), only about half have a sufficient response to salvage therapy to proceed to HD-ASCT. Furthermore, of those who proceed to HD-ASCT, 60% of patients relapse after transplant. Clinical investigations, supportive chemotherapy, and in rare cases, a second HD-ASCT or allogeneic stem cell transplantation (AlloSCT) are some of the options available to these patients.

[0247] Primary mediastinal large B-cell lymphoma (PMBCL) Primary mediastinal large B-cell lymphoma (PMBCL) has distinct clinical, pathological, and molecular features compared to DLBCL. PMBCL is thought to arise from B cells in the thymus (medulla oblongata) and represents approximately 3% of patients diagnosed with DLBCL. PMBCL is typically identified in a younger adult population in their 40s with a slight female predominance. Gene expression profiling suggests deregulated pathways in PMBCL overlap with Hodgkin's lymphoma. Initial therapy for PMBCL generally includes anthracycline-containing regimens and rituximab, such as infusion-adjusted etoposide, doxorubicin, and cyclophosphamide and vincristine, prednisone, and rituximab (DA-EPOCH-R), with or without involved-field radiation therapy. Treatment options for relapsed / refractory PMBCL are similar to those in DLBCL. Patients with chemotherapy-refractory disease have a prognosis similar to or worse than those with refractory DLBCL.

[0248] Mantle cell lymphoma (MCL) MCL is an aggressive cancer that is poorly responsive to currently available treatments, i.e., essentially incurable. Mantle cell lymphoma occurs in B cells, a type of white blood cell. In general, these patients have a poor prognosis, and they go through cycles of remission and relapse until the disease eventually becomes fatal. Mantle cell lymphoma has historically had one of the worst prognoses of any B cell lymphoma, as the disease can be very aggressive and chemotherapy does not provide a cure.

[0249] In one embodiment, the treatment method disclosed herein is suitable for patients with metastatic tumors from solid tumor cancer. The solid tumor may be breast cancer, triple negative breast cancer cells, ER positive breast cancer, ER+ and ER- breast cancer, ER+ breast cancer, prostate cancer, pancreatic cancer, urothelial cancer, non-muscle invasive urothelial cancer cells, ovarian cancer, hypoxic ovarian cancer, bladder cancer, melanoma, lung cancer such as non-small cell lung cancer (NSCL), colorectal cancer, nephroblastoma such as Wilms' tumor, thyroid cancer, thyroid cancer, hepatocellular carcinoma, cholangiocarcinoma neuroblastoma, or glioblastoma multiforme. In fact, these types of solid tumors may be drained into lymphatic organs, preferably lymph nodes, in such a manner that metastases can be found in such lymphatic organs, preferably lymph nodes. Therefore, the treatment method disclosed herein is suitable for patients with lymphatic organ metastases, preferably lymph node metastases. Typically, the disclosed treatment method will help eliminate metastases found in lymphatic organs, especially lymph nodes. In some embodiments, the hematological malignancy is selected from the group consisting of leukemia, lymphoma, diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma.

[0250] Thus, the methods of the present disclosure are particularly suitable for subjects with ALL, DLBCL, PMBCL, and MCL.

[0251] In a preferred embodiment, the hematological malignancy is diffuse large B-cell lymphoma (DLBCL).

[0252] In a specific embodiment, the subject is a subject eligible for CAR cell therapy to treat hematological disorders.Typically, the subject has been administered, is administered, or will be administered CAR cell therapy, such as CAR cell therapy described herein.In an embodiment, the subject has been administered, is administered, or will be administered anti-CD19 CAR cell therapy.

[0253] In a specific embodiment, the subject is at risk of developing cytokine release syndrome (CRS) and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS) following CAR cell therapy, e.g., CAR-T cell therapy, e.g., anti-CD19 CAR-T cell therapy.

[0254] In a specific embodiment, the subject has or has been diagnosed with CRS and / or MAS and / or ICANS following CAR cell therapy, e.g., CAR-T cell therapy, e.g., anti-CD19 CAR-T cell therapy.

[0255] In a specific embodiment, the subject is at risk for GVHD following allogeneic CAR cell therapy.

[0256] Combination Therapy Compositions comprising CAR cells for use in combination therapy with S1P receptor modulators as described herein are useful as a method for treating hematological malignancies in subjects in need thereof, more particularly as a drug in the patient populations and disease indications defined above.

[0257] In one embodiment, the CAR cells, preferably CAR-T cells, more preferably CAR-T cells targeting the CD19 antigen, are administered in a dose of 0.1×10 6 ~6×10 8In another embodiment, the CAR cells, preferably CAR-T cells, more preferably CAR-T cells targeting the CD19 antigen, are administered 2-14 days after completion of lymphodepleting chemotherapy.

[0258] In one embodiment, the number of CAR cells in the peripheral blood of a subject is reduced by about 1% to about 40%, preferably about 10% to about 30%, as measured by flow cytometry after 7, preferably 14 days of S1P receptor modulator treatment, compared to the number measured without S1P receptor modulator treatment.

[0259] In another embodiment, the amount of S1P receptor modulator can be administered in a fixed amount per day. Preferably, said fixed daily dosage is between 0.05 mg and 40 mg per day, preferably between 0.1 mg and 35 mg per day, more preferably between 0.5 mg and 30 mg, even more preferably between 1 mg and 15 mg, even more preferably between 1.5 mg and 7 mg per day, even more preferably between 2 mg and 5 mg, even more preferably about 3 mg, or about 1 mg per day.

[0260] For example, S1P receptor modulator can be mocravimod, and said mocravimod can be administered at a daily dose of about 1mg per day.Alternatively, mocravimod can be administered at a dose of about 3mg per day, preferably as three solid dosage forms of about 1mg or as one solid dosage form of about 3mg.Alternatively, mocravimod can be administered at a dose of about 2mg per day, preferably as two solid dosage forms of about 1mg or as one solid dosage form of about 2mg.

[0261] In some embodiments, the S1P receptor modulator is administered daily for at least 1, 2, or 3 months, or longer, preferably starting 1-20 days prior to the composition comprising CAR cells, and more preferably 11 days prior to the T cell therapy.

[0262] The composition comprising CAR cells, preferably CAR-T cells, expressing a chimeric antigen receptor molecule that binds a cancer associated antigen, more preferably CAR-T cells targeting the CD19 antigen (CD19CAR-T cells), more preferably moclavimod, for use in combination therapy with an S1P receptor modulator as described herein, is particularly useful for preventing CAR cells from leaving the bone marrow and / or promoting the engraftment and persistence of CAR cells, thereby improving CAR cell therapy.

[0263] Detailed embodiments of such methods are disclosed herein below.

[0264] In a specific embodiment, the method of the present disclosure comprises the following steps: 1) collecting immune cells, e.g., immune T cells, from a donor subject in need thereof by performing leukapheresis; 2) genetically modifying immune cells of the donor subject ex vivo to express a chimeric antigen receptor (CAR) molecule that binds to a cancer-associated antigen, thereby obtaining a CAR cell composition; 3) conditioning the recipient subject, e.g., by treating the recipient subject with an effective amount of a lymphodepleting chemotherapeutic agent or by subjecting the recipient subject to body irradiation; 4) administering to the recipient subject a therapeutically effective amount of the composition comprising the CAR cells obtained in step 2); 5) administering to the recipient subject an effective amount of an S1P receptor modulator, preferably moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof; 6) Optionally, administering to the recipient subject an effective amount of one or more immunosuppressive agents. Includes.

[0265] Detailed embodiments of each step are disclosed herein below.

[0266] (1) harvesting immune cells from a donor subject by leukapheresis; To perform the treatment methods disclosed herein, immune cells from a donor subject are collected by leukapheresis, and white blood cells are collected, enriched, or depleted ex vivo to select and / or isolate cells of interest, e.g., T cells. A physician collects the subject's blood by using a catheter placed in the subject's vein. A portion of the subject's white blood cells are separated from the patient's blood, and the remainder of the patient's blood is returned to the patient's vein. This procedure may take 3-6 hours and may need to be repeated to obtain enough white blood cells to perform step 2). The white blood cells are then frozen and sent for genetic modification.

[0267] In a preferred embodiment, the donor subject is the same as the recipient subject in need of treatment, i.e., the composition comprising CAR cells administered in step 4) comprises autologous or syngeneic CAR cells.

[0268] In other embodiments, the donor subject is not the same as the recipient subject in need of treatment, i.e., the composition comprising CAR cells administered in step 4) comprises allogeneic CAR cells.

[0269] In a preferred embodiment, said immune cell is an immune effector cell selected from the group consisting of a lymphocytic T cell or a natural killer cell, more preferably a lymphocytic T cell.

[0270] In certain embodiments, immune cells, such as T cells, can be obtained from a blood draw of 10cc to 400cc. In certain embodiments, immune cells, such as T cells, are obtained from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc.

[0271] Step 2: Genetically modifying the plant In a specific embodiment, frozen white blood cells may be thawed and the cells may be sorted to preferentially select immune cells of interest, such as T lymphocytes or NK cells. Once isolated, these immune cells may be expanded by methods known in the art and processed so that one or more CAR constructs can be introduced.

[0272] The immune cells are genetically modified to express a CAR molecule that binds to a cancer-associated antigen as disclosed above. In a preferred embodiment, said cancer-associated antigen is selected from the group consisting of CD19, CD30, CD33, CD123, CD20, CD22, CD38, LeY, ROR1, CLL-1, BCMA, GD2, MCSP, CD5, NKG2D, MHC peptide antigens, more preferably CD19.

[0273] This procedure may take approximately at least two weeks.

[0274] Steps in Lymphatic Depletion (3) To prepare the patient's body for CAR cell therapy, the patient is conditioned or undergoes a conditioning regimen, unless the patient's white blood cell count is below 1,000 cells / μL within one week prior to infusion. Conditioning includes treating the patient with an effective amount of a lymphodepleting chemotherapeutic agent before, after, or concurrently with administration of the treatment described herein.

[0275] As used herein, lymphodepleting chemotherapy is also referred to as lymphodepletion, lymphodepleting treatment, or lymphodepleting regimen.

[0276] In some embodiments, lymphodepleting chemotherapy is administered to the subject prior to administration of a CAR cell therapy, e.g., a CAR-T cell therapy, as described herein, e.g., in combination with an S1P receptor modulator. In embodiments, lymphodepletion includes administration of one or more (e.g., all) of melphalan, cytarabine, etoposide, busulfan, bendamustine, cyclophosphamide, fludarabine, dexamethasone, and alemtuzumab, preferably cyclophosphamide, fludarabine, dexamethasone, and alemtuzumab.

[0277] In some embodiments, a subject is administered lymphodepleting chemotherapy, e.g., in combination with an S1P receptor modulator, after administration of a CAR cell therapy, e.g., a CAR-T cell therapy, as described herein, particularly in cases where, e.g., CAR cell therapy must be administered one or more additional times.

[0278] In a specific embodiment, if the delay between step 3) and step 4) is longer than 4 weeks and the patient's white blood cell count is greater than 1,000 cells / μL, step 3) may be performed one or more times before performing step 4).

[0279] In one embodiment, said lymphodepleting chemotherapeutic agent is selected from the group consisting of cyclophosphamide, cytarabine, etoposide, bendamustine, busulfan, melphalan, fludarabine, and combinations thereof, such as combination administration of fludarabine / cyclophosphamide, cytarabine / etoposide.

[0280] In another embodiment, the conditioning regimen of step 3) consists of: - Administration of fludarabine and cyclophosphamide, or - Administration of cytarabine and etoposide, or - Administration of bendamustine.

[0281] In one embodiment, fludarabine is administered at a dose of 25 to 30 mg / m 2daily for 3 or 4 days, and cyclophosphamide is administered at a dose of 250 to 500 mg / m, starting with the first dose of fludarabine. 2 is given intravenously daily for 2 or 3 days at a dosage of

[0282] In another embodiment, cytarabine is administered at 500 mg / m 2 daily for 2 days, and etoposide was administered intravenously at a dose of 150 mg / m starting with the first dose of cytarabine. 2 is administered intravenously daily for three days.

[0283] In another embodiment, bendamustine is administered at 90 mg / m 2 is administered intravenously daily for two days at a dosage of

[0284] Administering a composition comprising CAR cells (4). To carry out the treatment methods disclosed herein, an effective amount of a composition comprising the CAR cells obtained in step (2) is administered to a subject in need of such treatment as described herein.

[0285] In a specific embodiment, the CAR cells, preferably CAR-T cells obtained in step (2), may be activated in vitro or ex vivo by an effective amount of an S1P receptor modulator. Thus, one aspect of the present disclosure also relates to a method of activating or pretreating CAR cells, preferably CAR-T cells, particularly for use in the treatment methods described herein. In a particular method, the method comprises (i) pretreating CAR cells, ex vivo or in vitro, with an effective amount of an S1P receptor modulator to activate such CAR cells (preferably CAR-T cells), thereby obtaining activated CAR cells (preferably activated CAR-T cells), and then (ii) administering a therapeutically effective amount of the obtained activated CAR cells to a subject in need of such treatment as described herein. In a specific embodiment of the latter method, no S1P receptor modulator is administered directly to the subject in combination with the activated CAR cell composition (except for trace amounts of S1P receptor modulators, possibly used to activate the CAR cell composition (in vitro or ex vivo)).

[0286] The pharmaceutical composition comprising the CAR cells can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will depend on factors such as the condition of the patient and the type and severity of the patient's disease, but the appropriate dosage can be determined by clinical trials.

[0287] In a preferred embodiment, the CAR cells of step 3) administered are CAR-T cells, such as anti-CD19 CAR-T cells.

[0288] The composition comprising said CAR cells may further comprise one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In one embodiment, the composition of the present invention is formulated for intravenous administration, preferably for infusion. Cells can be administered by using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).

[0289] In one embodiment, the pharmaceutical composition is substantially free, e.g., free of detectable levels, of contaminants selected from the group consisting of endotoxins, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3 / anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cells or plasmid components, bacteria, and fungi. In one embodiment, the bacteria is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.

[0290] In one embodiment, the CAR cells, e.g., CAR-T cells, particularly anti-CD19 CAR-T cells, in step 3) are 0.1×10 6 ~6×10 8 , 0.5×10 6 ~5.0×10 8 , 1.0×10 6 ~4.0×10 8 , 2.0×10 6 ~3.0×10 8 , 3.0×10 6 ~2.0×10 8 , 3.0×10 6 ~1.0×10 8 , 4.0×10 6 ~9.0×10 7 , 5.0×10 6 ~8.0×10 7 , 6.0×10 6 ~7.0×10 7 , 7.0×10 6 ~6.0×10 7 , 8.0×10 6 ~5.0×10 7 , 9.0×10 6 ~4.0×10 7 , 1.0×10 7 ~3.0×10 7 In a preferred embodiment, the dose of CAR cells (e.g., CAR-T cells, particularly anti-CD19 CAR-T cells) in step 3) is 1.2×10 6 ~6×10 8 In another embodiment, the dose of CAR cells in step 3) is 0.4×10 8 ~2×10 8 CAR-positive live immune cells / kg body weight.

[0291] In another embodiment, the CAR cells (e.g., CAR-T cells, particularly anti-CD19 CAR-T cells) in step 3) are administered 2-14 days or 3-14 days after completion of lymphodepleting chemotherapy.

[0292] The administration of the composition comprising CAR cells can be performed in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions described herein can be administered to patients intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In one embodiment, the composition comprising CAR cells is administered to patients by intradermal or subcutaneous injection. In a preferred embodiment, the T cell composition of the present invention is administered by intravenous injection. The compositions of the present invention can be injected into lymph nodes or directly into the site of infection.

[0293] In one embodiment, the CAR is introduced into the immune cells according to step 2), and the subject receives an initial administration of the CAR cells, and eventually one or more subsequent administrations of the CAR cells, where the one or more subsequent administrations are administered less than 15 days after the previous administration, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.

[0294] In one embodiment, the subject is administered multiple doses of CAR cells per week, e.g., 2, 3, or 4 doses of the CAR cells of the invention per week. In one embodiment, the subject receives multiple doses of CAR cells per week (e.g., 2, 3, or 4 doses per week) (also referred to herein as a cycle), followed by a week without CAR cell administration, after which one or more additional doses of CAR cells (e.g., multiple doses per week of CAR cells) are administered to the subject.

[0295] In another embodiment, the subject receives multiple cycles of CAR cells, with the time between each cycle being less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR cells are administered every other day, three doses per week. In one embodiment, the CAR cells of the present invention are administered for at least 2, 3, 4, 5, 6, 7, 8 weeks, or longer.

[0296] A potential problem that may arise in patients being treated with CAR cells (especially with murine scFv carrying CAR) is anaphylaxis after multiple treatments. Without being bound by this theory, it is believed that such an anaphylactic response may be caused by the patient developing a humoral anti-CAR response, i.e., the anti-CAR antibodies have an anti-IgE isotype. It is believed that if there is a 10-14 day hiatus in exposure to antigen, the patient's antibody-producing cells undergo a class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype. If the patient is at high risk of developing an anti-CAR antibody response during the course of a transient CAR therapy (such as one generated by RNA transduction), the hiatus in CAR-T cell infusion may not last longer than 10-14 days.

[0297] In certain embodiments, it may be desirable to administer CAR cells to a subject, then subsequently redraw blood (or perform apheresis), genetically modify the immune cells collected according to step (1), and reinfuse the patient with these CAR cells. This process can be performed multiple times, every few weeks.

[0298] Administering an S1P receptor modulator (5). To carry out the methods of treatment disclosed herein, an effective amount of an S1P receptor modulator as described herein, preferably moclavimod, is administered to a subject in need of such treatment.

[0299] In some embodiments, the amount of S1P receptor modulator, preferably moclavimod, or a pharma- ceutically acceptable salt or phosphate derivative thereof, administered per day is a fixed amount. In some embodiments, the fixed daily dosage is 0.05 mg to 40 mg per day, preferably 0.1 mg to 35 mg per day, more preferably 0.5 mg to 30 mg, even more preferably 1 mg to 15 mg, even more preferably 1.5 mg to 7 mg per day, even more preferably 2 mg to 5 mg, even more preferably about 3 mg, or about 1 mg per day.

[0300] For example, S1P receptor modulator can be mocravimod, and said mocravimod can be administered at a daily dose of about 1mg per day.Alternatively, mocravimod can be administered at a dose of about 3mg per day, preferably as three solid dosage forms of about 1mg or as one solid dosage form of about 3mg.Alternatively, mocravimod can be administered at a dose of about 2mg per day, preferably as two solid dosage forms of about 1mg or as one solid dosage form of about 2mg.

[0301] As used herein, the amount of a pharma- ceutically acceptable salt or phosphate derivative of mocravimod refers to the amount of mocravimod base.

[0302] The compositions comprising the CAR cells described herein and / or the S1P receptor modulators can be administered simultaneously, in the same or separate compositions, or sequentially.

[0303] For sequential administration, in one embodiment, a composition comprising a CAR cell as described herein can be administered first, and an S1P receptor modulator can be administered second. In another specific embodiment of sequential administration, administration of the S1P receptor modulator (typically as a daily dosage) begins prior to the first administration of the CAR cell composition.

[0304] In one embodiment, the S1P receptor modulator is administered prior to administration of the composition comprising the CAR cells. In an embodiment, the S1P receptor modulator is administered at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days prior to the start of administration of the CAR cell composition, preferably 11 days prior to the start of administration of the CAR cell composition.

[0305] In some embodiments, the S1P receptor modulator is mocravimod, and said mocravimod is administered daily, preferably starting 1-20 days prior to the first administration of said composition comprising CAR cells (CAR cell therapy), more preferably starting 11 days prior to the initiation of CAR cell therapy, for 1, 2, 3 months, or longer.

[0306] The daily dosage of S1P receptor modulator can be administered as one dose per day or multiple doses during a single day.In a preferred embodiment, the daily dosage is administered once a day.In some embodiments, the dosage is administered several times a day, preferably three times a day.In some embodiments, the minimum dosage that is sufficient to provide effective treatment can be used.

[0307] In another embodiment, the S1P receptor modulator is mocravimod, and the mocravimod is administered in a daily dose of 3 mg, for example, in three 1 mg solid dosage forms per day. Indeed, three 1 mg solid dosage forms, for example capsules or tablets, administered in a spaced apart manner are easier to swallow than a single 3 mg solid dosage form.

[0308] In some embodiments, CAR cell therapy is administered to a subject with a hematological malignancy as described herein. CAR cell therapy can be continued (e.g., if some cancer-associated antigen-expressing cancer cells are still detectable in the subject) or interrupted (e.g., if a risk-benefit analysis supports interrupting treatment).

[0309] In CAR cell therapies in which multiple administrations of a composition comprising CAR cells are desired, the S1P receptor modulator regimen is initiated or completed prior to administration of the composition comprising CAR cells.

[0310] In some embodiments, the patient is further administered one or more immunosuppressants, including but not limited to any one or all of the following agents: cyclosporine A, sirolimus, tacrolimus, methotrexate, and mycophenolate, preferably cyclosporine A, or a combination of cyclosporine A and methotrexate.

[0311] In a preferred embodiment of the disclosed method, said S1P receptor modulator is administered in an amount sufficient to prevent CAR cells (typically CAR-T cells) from leaving bone marrow and / or lymph nodes, and / or promote the engraftment and persistence of CAR cells (typically CAR-T cells), and / or increase the efficacy of CAR cell therapy activation (typically CAR-T cells).Indeed, increasing the efficacy of CAR cell therapy means activating such CAR cells, which allows improving the ability of CAR cells to kill tumor cells and thus better treat hematological malignancies in a subject in need thereof, i.e. increasing the anti-tumor effect.

[0312] In another embodiment, the S1P receptor modulator is administered in an amount sufficient to reduce the risk of cytokine release syndrome (CRS), particularly systemic cytokine release syndrome and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS), in a subject receiving CAR cells. Indeed, reducing the risk of cytokine release syndrome and / or macrophage activation syndrome and / or immune effector cell-associated neurotoxicity syndrome allows for improved patient response to CAR cell treatment. CRS is the result of the extensive activation of administered CAR cells, particularly CAR-T cells, resulting in the release of large amounts of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, and IL-17A, and preventing cytokine release helps prevent CRS and therefore improves patient response to CAR cell treatment. MAS is a severe hyperinflammatory syndrome characterized by CRS and elevated serum ferritin in combination with absent hemophagocytosis, renal failure, liver enzymes, splenomegaly, pulmonary edema, and / or NK cell activity; preventing the release of cytokines, serum ferritin, and liver enzymes will help prevent MAS and thus improve patient response to CAR cell treatment.

[0313] ICANS is characterized by elevated cerebrospinal fluid cytokine levels and blood-brain barrier disruption, and preventing the release of cytokines in the cerebrospinal fluid would help prevent ICANS and thus improve patient response to CAR cell treatment.

[0314] As a result, treatment of hematological malignancies, particularly diffuse large B-cell lymphoma (DLBCL), in subjects in need thereof is improved.

[0315] In one embodiment, the S1P receptor modulator is administered in an amount sufficient to reduce the risk of cytokine release syndrome, particularly systemic cytokine release syndrome, in a subject receiving allogeneic CAR cells.

[0316] In a preferred embodiment, said S1P receptor modulator is administered in an amount sufficient to reduce the risk of cytokine release syndrome, particularly systemic cytokine release syndrome, in a subject receiving autologous or syngeneic CAR cells.

[0317] Thus, the present disclosure also relates to S1P receptor modulators for use in preventing or reducing the risk of cytokine release syndrome (CRS), in particular systemic cytokine release syndrome and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS), in a subject in need thereof receiving CAR cell therapy, typically receiving a CAR cell therapy as disclosed above.

[0318] In specific embodiments of the methods where the recipient is not the donor of the CAR cells, e.g., where the CAR cells are allogeneic or universal CAR cells, the S1P receptor modulator is administered in an amount sufficient to reduce the risk of GVHD. [Brief description of the drawings]

[0319] [Figure 1] Figure 1 shows the experimental scheme of Example 1. Sublethal irradiation (4Gy) and 0.5x106 leukemia injection IV on day -7 in C57BI / 6 mice. Injection of 3mg / kg of KRP203 or PBS injection is represented by black arrows depending on the day of injection. + and - indicate administration or non-administration of CAR T cells on day 0. [Diagram 2]Figure 1 shows a summary graph showing the percentage of tumor cells recovered from lymph node, spleen, bone marrow and blood.Each point represents one mouse.From left to right, light gray represents CAR8+PBS group, medium gray represents CAR8+KRP203 -day 1 group, and dark gray represents CAR8+KRP203 +day 2 group. [Diagram 3] Quantification of surface CD69, intracellular granzyme B, and surface PD-1 expression on CAR-T cells in lymph nodes, spleen, bone marrow, and blood. Each dot represents one mouse. [Figure 4] FIG. 2 shows an experimental scheme of Example 2. [Diagram 5] Moclavimod (dark grey) and fingolimod (medium grey) reduced the levels of IFN-γ induced by CAR-T cell injection. (A) Levels of IFN-γ were measured in plasma 5 days after CAR-T injection. Individual values ​​and mean + / - SEM are shown. n=6-8 mice / group. Comparisons between all treatment groups were performed using one-way ANOVA followed by Dunnett's post-test. ***p<0.001. [Figure 6] Figure 1: Mocravimod (black) reduced the levels of IFN-γ, TNF-α, and IL-2 induced by CAR-T cell injection. INF-g, TNF-a, and IL-2 levels were measured in plasma 5 days after CAR-T injection. Mean + / - SD is shown. n=5 mice / group. Unpaired t-test was used to compare vehicle and mocravimod groups at each time point. **p<0.005, ***p<0.001, ****p<0.0001 [Figure 7] Figure 1: Mocravimod reduced the absolute number of CAR-T cells in the blood. Absolute CAR-T cell numbers were measured in plasma 1-5 days after CAR-T injection. Mean + / - SD is shown. n=5 mice / group. Mocravimod was compared to the vehicle group on day 5 using an unpaired t-test. *p<0.05. [Figure 8]FIG. 1 shows that mocravimod increased the percentage of CAR-T cells in the bone marrow compared to the untreated arm. CAR-T cells were measured in the bone marrow 4 days after CAR-T injection. Individual values ​​and mean + / - SEM are shown. n=3 mice / group. Mocravimod was compared to control using an unpaired t-test. *p<0.05. [Figure 9] Figure 1: Mocravimod kills tumor cells in vitro. No mocravimod was compared to 7.5 μM mocravimod using an unpaired t-test. **p<0.005, ***p<0.001, ****p<0.0001. [Figure 10] Figure 1 shows the survival benefit of moclavimod in combination with CAR-T cells compared to CAR-T cells alone and untreated. *p<0.05, **p<0.01, ***p<0.001 [Figure 11] FIG. 1 shows that mocrabimod in combination with CAR-T cells increases tumor elimination. EXAMPLES

[0320] [Example 1] S1P modulators increase CAR-T activation material and method Mice and cell lines C57BL / 6J male mice aged 6–8 weeks were obtained from Envigo. Ubi-GFP RAG1- / -OT-I TCR mice were bred and crossed under specific pathogen-free conditions in our animal facility. All experiments were approved by the Institut Pasteur Safety Committee in accordance with French and European guidelines (CETEA2017-0038). Immortalized pro-B cells were generated by infecting bone marrow cells with a retrovirus encoding the viral Abelson kinase (v-abl). This tumor cell line was then retrovirally transduced to express a fluorescence resonance energy transfer (FRET)-based reporter for caspase 3 activity. Mice were examined daily and sacrificed if they exhibited collapse, disheveled hair, weakness, or nodal tumor masses >1 cm. Cells were cultured in RPMI medium 1640-GlutaMAXTM supplemented with 10% heat-inactivated fetal bovine serum, 50 U.mL-1 penicillin, 50 μg.mL-1 streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 50 μM 2-mercaptoethanol and maintained at 37°C and 5% CO2. Cells were routinely tested for the absence of Mycoplasma contamination (Venor-GeM Advance Mycoplasma Detection Kit, Minerva Biolabs).

[0321] Generation and adoptive transfer of CAR-T cells The tCD34.2A.amCD19.CD28IEVζ retroviral vector encoding the anti-CD19 CAR is composed of the anti-murine CD19 single-chain fragment variable domain derived from the 1D3 rat hybridoma, the transmembrane and intracellular domains of CD28, and the CD3z intracellular domain. This retroviral vector also encodes a truncated human CD34 molecule used to identify and purify CAR-T cells. CD8+ T cells were isolated from lymph nodes of Ubi-GFP RAG1- / -OT-I TCR mice. T cells were activated in the presence of 2.5 μg.ml-1 soluble anti-CD28 mAb (clone 37.51, BioLegend) and 10 ng.ml-1 murine IL-12 (I8523, Sigma-Aldrich) in plates coated with 2.5 μg.ml-1 anti-CD3 mAb (clone 17.A2, BioLegend). Two rounds of spin-infection were performed 24 and 48 h after T cell activation using retroviral particles supplemented with 8 μg.ml-1 polybrene (Merck). T cells were cultured for an additional 4 days in the presence of 10 ng.ml-1 hIL-2 (202-IL, R&D Systems). CAR transduction efficiency was typically >80%. If lower, purification of transduced cells was performed using a hCD34 positive selection kit (Miltenyi Biotec). B cell tumors were cultured at 0.5 × 10 ng / ml after sublethal irradiation (4 Gy). 6 10 transformed pro-B cells were injected into mice and used as a conditioning regimen for CAR-T cell engraftment. Tumors developed primarily in the bone marrow and were detectable in the blood by day 7, at which point CAR-T cells (5 × 10 6 cells) were injected intravenously (iv).

[0322] KRP203 Treatment Mice received continuous KRP203 (3 mg.kg-1 body weight) intraperitoneally every 2 days. KRP203 treatment was started either 1 day before or 2 days after injection of CAR-T cells. Control animals received PBS intraperitoneally. The experimental scheme is depicted in Figure 1.

[0323] Flow cytometry and antibodies For ex vivo analysis, bone marrow cells were isolated by flushing femurs and tibias from tumor-bearing mice followed by filtration through a 70 μm cell strainer. Single cell suspensions from spleens and lymph nodes were prepared by filtering cells through a 70 μm cell strainer. Blood was collected by cardiac puncture after mouse sacrifice and red blood cells were removed using red blood cell lysis buffer (eBiosciences). Single cell suspensions were Fc-blocked using anti-CD16 / 32 mAb (clone 93, BioLegend) and 1% normal murine serum. Staining was performed using the following mAbs: hCD34-Alexa Fluor 647 (clone 561, BioLegend), CD69-BUV737 (clone H1.2F3, BD Biosciences), CD8a-BUV395 (clone 53-6.7, BD Biosciences), CD19-APC-fire750 (clone 6D5, BioLegend), and PD-1-PE / Cy7 (clone 29F.1A12, BioLegend). Intracellular staining was performed using the Cytofix / Cytoperm kit (BD Biosciences) according to the manufacturer's guidelines, and PE-conjugated anti-granzyme B mAb (clone QA18A28, BioLegend). Analysis was performed using a Cytoflex LX (Beckman Coulter) flow cytometer and analyzed using FlowJo version 10.8.0 (BD).

[0324] statistical analysis All statistical tests were performed using Prism version 9.2.0 (GraphPad). Data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) and two-way ANOVA tests were used to correct for multiple comparisons. All statistical tests were two-sided with a significance level of 0.05. ns, non-significant; * p<0.05, ** p<0.01, *** p<0.001.

[0325] result Leukemic tumor cells in distinct lymphoid organs were evaluated. The results are presented in Figure 2. Tumor burden, measured by the absolute number of tumor cells in lymph nodes, was reduced in mice receiving CAR8+PBS versus mice receiving tumor cells only (untreated group). It has been demonstrated that when CAR-T cells and KRP203 are administered on day -1 or +2, the absolute tumor number in lymph nodes is significantly further reduced compared to the control (CAR8+PBS) and untreated arms. This means that when KRP203 is administered in combination with CAR-T cells to leukemic mice, a reduction in the absolute tumor number in lymph nodes can be achieved. The absolute tumor number in lymph nodes is further reduced when KRP203 is administered on days -1, 1, and 3, compared to administration of KRP203 on day 2 only.

[0326] Activation and elimination of CAR-T cells were evaluated. The results are presented in Figure 3. The results are consistent with Figure 2, with a reduced tumor burden in the lymph nodes. Indeed, it has been demonstrated that a higher percentage of CAR-T cells express activation markers (CD69, GrB, PD-1) when combined with KRP203 treatment. In lymph nodes, the percentage of activation of CAR-T cells is significantly increased by 2-3 times when KRP203 is administered from day -1 compared to the control. When administered on day +2, there is again a trend for increased activation of CAR-T cells compared to the control, but to a lower extent compared to administration on day -1. The increase in activation markers (CD69, PD-1) indicates that CAR-T cells are more responsive to tumor cells, and the increase in granzyme B (GrB) content indicates an enhanced ability of CAR-T cells to kill cancer cells. Furthermore, sequestration of CAR-T cells in lymph nodes using S1P modulators may result in such CAR-T cells surviving longer.

[0327] Additionally, we investigated mouse survival and how it was affected by administration of KRP203 in combination with CAR T cells. Mice treated with the combination of CAR T cells and KRP203 showed a statistically significant survival benefit compared to mice that were left untreated or received CAR T cells alone (Figure 10).

[0328] [Example 2] Combining S1P modulators with CAR-T cells prevents CRS material and method animal The study was performed using female NOD / SCID / IL-2Rγ null immunodeficient mouse strain (NCG). All procedures described in the study were reviewed and approved by the local ethical committee (CELEAG). Mice were housed in the TCS BSL-2 animal facility in groups of 2 / 6 individuals. Each mouse was uniquely identified.

[0329] Tumor cell engraftment JEKO-1-luc-GFP tumor cells were expanded in vitro according to ATCC recommendations. 5 × 10 6 Tumor cells were cultured in RPMI-1640 containing 10% SVF and 1% penicillin / streptomycin at 37 °C in a water-saturated and sterile atmosphere and 5% CO. 2 After confirmation of viability, tumor cells in logarithmic growth phase were injected into selected animals.

[0330] JEKO-1-luc-GFP tumor cells were cultured at 50 × 10 6 The cells were suspended in PBS at a concentration of 10 cells / mL.

[0331] Tumor cells were injected intravenously into 24 immunodeficient mice using 100 μL of cell suspension (5.106 JEKO-1 / mouse). Tumor engraftment was defined as day 0.

[0332] Randomization, groups, and treatments Mice were distributed across four groups according to their body weight and treated as follows: #1 vehicle-vehicle, #2 CAR-T-vehicle, #3 CAR-T-moclavimod, and #4 CAR-T-fingolimod.

[0333] Mocravimod and fingolimod (FTY720) were administered by intraperitoneal injection at a dose of 3 mg / kg. Mocravimod and vehicle groups (groups #1, #2, and #3) were treated every 2 days, while the fingolimod group (#4) was treated daily starting on day 6. CAR-T cells were administered once on day 7 at a dose of 0.75 × 10 6 The cells were administered at a dose of 1000 cells / mouse (see table below).

[0334] [Table 1]

[0335] The experimental scheme is depicted in FIG.

[0336] Bleeding and organ collection Blood was collected retro-orbitally on day 12 for plasma isolation and CBA assay, and on day 25, the day of euthanasia, for flow cytometry analysis.

[0337] Spleens and bone marrow were also harvested, mechanically disaggregated according to TransCure bioServices standard operating procedures (SOPs), and red blood cell lysis was performed with RBC lysis buffer for 1 min at room temperature followed by flow cytometry analysis according to internal TransCure bioServices protocols. Prior to disaggregation, a small portion of the spleen was fixed in 4% PFA and paraffin embedded for further IHC analysis.

[0338] The resulting isolated cells were stained for immunophenotyping by flow cytometry according to internal TransCure bioServices protocols.

[0339] Cytokine assays Cytokines (TNF-α, INF-γ, IL-10, IL-6, IL-4, IL-2, and IL-17a) were analyzed on plasma samples (25 μL) by customized cytometric bead array (CBA) kits according to the manufacturer's instructions (BD Biosciences) and TransCure standard procedures.

[0340] Levels of several human cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, and IL-17A) were measured by cytometric bead array (CBA) assays on day 12 (5 days after CAR-T cell injection) (Figure 5). No or very low levels of IL-2, IL4, IL-6, IL-10, IL-17A, or TNF-α were detected in the blood of mice, regardless of treatment group.

[0341] Cytokine profile analysis in blood 5 days after CAR-T cell administration showed that IFN-γ was only detected in mice treated with CAR-T cells. Intermediate levels of IFN-γ were found in the CAR-T-moclavimod and CAR-T-fingolimod treated groups (less than 10 pg / mL), and significantly higher levels were found in the CAR-T-vehicle group compared to the vehicle-vehicle group (Figure 5). Moreover, the levels of IFN-γ were significantly lower in the CAR-T-moclavimod and CAR-T-fingolimod groups compared to the CAR-T-vehicle treated group.

[0342] conclusion CAR-T cells induced proinflammatory IFN-γ release, which was significantly reduced upon moclavimod or fingolimod treatment. These results suggest that S1P modulator treatment may be useful for preventing cytokine release syndrome (CRS) induced by CAR-T cell therapy.

[0343] [Example 3] Combining S1P modulators with CAR-T cells prevents CRS material and method Cytokine assays To determine the kinetics of cytokine secretion and evaluate the efficacy of moclavimod in reducing circulating cytokine levels when administered in combination with CAR T cells, NOD CRISPR Prkdc Il2r gamma (NCG) mice were administered 5 × 10 6 JEKO-1-luc-GFP tumor cells were injected intravenously. Treatment with either mocravimod or vehicle was initiated 6 days after tumor cell engraftment. Mocravimod (3 mg / kg) was administered every 2 days. 0.75 × 10 6 CAR T cells were injected intravenously 7 days after tumor cell engraftment. Mice were bled 24, 48, 72, 96, and 120 hours after CAR T cell injection and plasma was collected. Interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), and interleukin-2 (IL-2) were analyzed in plasma samples using the BD CBA human Th1 / Th2 / Th17 cytokine assay. CAR T cells were phenotyped and enumerated in the blood.

[0344] Cytokine profile analysis in the blood 5 days after CAR-T cell administration showed that the levels of IFN-g, TNFa, and IL-2 were significantly lower in the CAR-T-moclavimod-treated group (approximately 100 pg / mL, approximately 0 pg / mL, and approximately 2 pg / mL, respectively) compared to the CAR-T-vehicle group (approximately 250 pg / mL, approximately 6 pg / mL, and approximately 3 pg / mL, respectively) (Figure 6).

[0345] CAR-T cell engraftment The method for analyzing CAR-T cells in bone marrow was carried out using the same protocol as in Example 1, and the method for analyzing CAR-T cells in blood was carried out using the same protocol as in the cytokine assay.

[0346] It has been demonstrated that when administration of mocravimod is started on day -1 prior to CAR-T cell injection, the absolute number of CAR-T cells in the blood is significantly decreased compared to the control (vehicle) on day 5 (Figure 7). When comparing the combination of mocravimod and CAR-T cells with the control, the absolute number of CAR-T cells in the blood on day 1 was the same (about 500 cells). However, while the absolute number of CAR-T cells in the blood tends to increase from day 3 to day 5 in the control (from about 500 cells to about 1500 cells), the absolute number of CAR-T cells in the blood tends to decrease in the combination of mocravimod and CAR-T cells (from about 500 cells to about 100 cells).

[0347] These results are consistent with the percentage of CAR-T cells in the bone marrow on day 4. When mocravimod was administered together with CAR-T cells, the percentage of CAR-T cells in the bone marrow was significantly increased compared to the control (CAR-T+PBS) (Figure 8). This result indicates the depletion of CAR-T cells in the blood and the increase of CAR-T cells in the bone marrow in the combination of mocravimod and CAR-T cells, indicating the sequestration of CAR T cells into lymphoid organs. Thus, the combination of mocravimod and CAR-T cells prevents cells from leaving the bone marrow, promotes the engraftment of CAR-T cells, and increases the efficacy of CAR-T cell therapy.

[0348] conclusion CAR-T cells secreted the proinflammatory cytokines IFN-γ, TNF-α, and IL-2, which were significantly reduced upon mocrabimod treatment. These results support that S1P modulator treatment, more preferably mocrabimod, may be useful in preventing cytokine release syndrome (CRS) induced by CAR-T cell therapy.

[0349] CAR-T cells tend to leave the bone marrow and be found in the blood. These results support that S1P modulator treatment would be useful in preventing CAR-T cells from leaving the bone marrow, promoting the engraftment and persistence of CAR cells, and thus increasing the efficacy of CAR-T cell therapy.

[0350] [Example 4] S1P modulators induce apoptosis material and method To investigate the cytotoxicity (induction of cell death) of mocravimod on various hematological cancer cell lines, the cell lines were expanded and incubated for 2 and 24 h in the presence of 7.5 μM mocravimod. The following cell lines were used: MOLM-13, THP-1, Kasumi-1, Jurkat, Raji, JEKO-1, and MV-4-11. 5 Tumor cells / well were seeded in 96-well plates. After 2 and 24 hours of incubation at 37° C., 5% CO2, cells were stained with Annexin V and DAPI to assess cell death.

[0351] result The percentage of dead cells in each of the tumor cell lines, MOLM-13 (AML), THP-1 (AML), Kasumi-1 (AML), Jurkat (ALL), Raji (B cell lymphoma), JEKO-1 (mantle cell lymphoma), and MV-4-11 (AML), was higher when mocravimod was administered in the treatment arm compared to the control untreated arm (Figure 9). Thus, mocravimod induces tumor cell apoptosis in all MOLM-13, THP-1, Kasumi-1, Jurkat, Raji, JEKO-1, and MV-4-11 cell lines. Thus, administration of mocravimod in combination with CAR-T cells allows increasing the effectiveness of killing hematological malignancies.

[0352] [Example 5] Combining mocrabimod with CAR T cells increases tumor elimination material and method Scid-beige mice, 0.5 × 10 6 Nalm6-GFP +Tumor cells were injected via the tail vein (day 0). Tumor cells were pre-expanded in vitro. Some mice were left untreated while some were expanded with 1 × 10 cells 2 weeks after tumor injection (day 14). 7 CAR T cells and 1 × 10 6 Peripheral blood mononuclear cells (PBMCs) were received from each mouse. Half of the mice that received CAR T cells were treated with mocrabimod, while the other half received vehicle (DMSO). Mocrabimod treatment was started 3 days before CAR T cell injection at a dose of 3 mg / kg and continued to be administered every 2 days until the end of the experiment. Tumor cells were analyzed in bone marrow (BM) 4 days after CAR T cell administration (day 18).

[0353] result In all groups, Nalm6-GFP + Tumor cells were evaluated in the BM. Mice that did not receive CAR T cell treatment showed the highest tumor burden (Figure 11). Tumor cells were efficiently reduced by CAR T cell administration. Furthermore, when KRP203 was administered in combination with CAR T cells, tumor cells were further reduced. Thus, administration of moclavimod in combination with CAR-T cells allows for increased tumor elimination.

Claims

1. 1. A CAR cell composition comprising CAR cells for use in treating a hematological malignancy in a subject in need thereof, wherein the CAR cells are immune cells, preferably immune T cells, that express a chimeric antigen receptor molecule that binds to a cancer-associated antigen, and wherein a therapeutically effective amount of the CAR cell composition is administered in combination with a therapeutically effective amount of an S1P receptor modulator.

2. 2. The CAR cell composition of claim 1, wherein the cancer-associated antigen is selected from the group consisting of CD19, CD123, CD20, CD22, CD30, CD33, CD38, LeY, ROR1, CLL-1, BCMA, and combinations thereof, preferably CD19.

3. 2. The CAR cell composition of claim 1, wherein the S1P receptor modulator is selected from moclavimod, siponimod, fingolimod, ozanimod, ponesimod, etrasimod, AKP-11, cenerimod, amiselimod, CBP-307, OPL-307, OPL-002, BMS-986166, SCD-044, BOS-173717, CP-1050, preferably moclavimod.

4. The CAR cell composition of claim 3, wherein the S1P receptor modulator is an S1P receptor agonist.

5. The S1P receptor agonist is of the following formula (I) or (II) or (IIa) or (IIb): 【Chemistry 1】 [In the formula, R ２ is H, halogen, trihalomethyl, C １～４ Alkoxy, C １～７ alkyl, phenethyl, or benzyloxy; R ３ is H, halogen, CF ３ , O.H., C. １～７ Alkyl, C １～４ Alkoxy, benzyloxy, phenyl, or C １～４ is alkoxymethyl, R ４ and R ５ each independently represents H or a residue of formula (a): 【Chemistry 2】 [In the formula, R ８ and R ９ each independently represents H or C optionally substituted with halogen １～４ is alkyl, n is an integer from 1 to 4; R ６ is hydrogen, halogen, C １～７ Alkyl, C １～４ alkoxy, or trifluoromethyl; or 【Transformation 3】 or a pharmaceutically acceptable salt thereof or 【Chemistry 4】 The CAR cell composition of claim 4, wherein

6. 5. The CAR cell composition of claim 4, wherein the S1P receptor agonist is moclavimod, or a pharmaceutically acceptable salt or phosphate derivative thereof.

7. 7. The CAR cell composition of any one of claims 1 to 6, wherein the hematological malignancy is a leukemia and / or lymphoma preferably selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, non-Hodgkin's lymphoma, mantle cell lymphoma (MCL), primary mediastinal large B-cell lymphoma (PMBCL), or multiple myeloma, more preferably ALL, DLBCL, PMBCL, and MCL, even more preferably DLBCL.

8. A therapeutically effective amount of CAR cells, preferably CAR-T cells, is 0.1 x 10 ６ ~6 x 10 ８ 7. The CAR cell composition of any one of claims 1 to 6, administered at a dosage of live CAR-positive immune cells / kg of body weight.

9. 7. The CAR cell composition of any one of claims 1 to 6, wherein the CAR cells, preferably CAR-T cells, are administered 2 to 14 days after completion of lymphodepleting chemotherapy.

10. 7. The CAR cell composition of any one of claims 1 to 6, wherein the S1P receptor modulator is administered at a dosage of 0.05 mg to 40 mg per day, preferably 0.1 mg to 35 mg, more preferably 0.5 mg to 30 mg, even more preferably 1 mg to 15 mg, even more preferably 1.5 mg to 7 mg, even more preferably 2 mg to 5 mg, and even more preferably about 3 mg or about 1 mg.

11. 7. The CAR cell composition of any one of claims 1 to 6, wherein the S1P receptor modulator is administered daily, preferably from a starting date of 1 to 20 days prior to administration of the composition comprising CAR cells, more preferably 11 days prior to administration of CAR cells, for at least 1, 2, or 3 months, or longer.

12. 7. The CAR cell composition of any one of claims 1-6, wherein the S1P receptor modulator is administered in an amount sufficient to prevent CAR cells from leaving the bone marrow, and / or promote CAR cell engraftment and persistence, and / or increase the efficacy of CAR cell therapy.

13. A CAR cell composition described in any one of claims 1 to 6, wherein the S1P receptor modulator is administered in an amount sufficient to reduce the risk of cytokine release syndrome, particularly systemic cytokine release syndrome, in a subject receiving, preferably, autologous or syngeneic CAR cells.

14. 1. A composition comprising an S1P receptor modulator for use in preventing cytokine release syndrome (CRS) and / or macrophage activation syndrome (MAS) and / or immune effector cell-associated neurotoxicity syndrome (ICANS) in a subject in need thereof with CAR cell therapy (e.g., anti-CD19 therapy), wherein the S1P receptor modulator (e.g., moclavimod), or a pharmaceutically acceptable salt or phosphate derivative thereof, is administered to the subject in combination with the CAR cell therapy, thereby preventing CRS and / or MAS and / or ICANS in the subject.

15. The composition of claim 14, wherein the CAR cell therapy is a CAR-T cell therapy, preferably an anti-CD19 CAR-T cell therapy.

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