CAR-T-CELL THERAPY

Abstract

The present invention provides compounds and compositions for use in the treatment of cancer as part of a Chimeric Antigen Receptor T-cell (CAR-T) therapy. In particular, the present invention discloses IL-18 binding molecules such as, for example, the IL-18 binding protein (IL-18BP) for use in Chimeric Antigen Receptor T-cell (CAR-T) therapy as a modulator to avoid or minimize side effects.

Claims

1. An IL-18 binding molecule for use in a Chimeric Antigen Receptor T-cell (CAR-T) therapy of a patient diagnosed with a disease associated with expression of a tumor antigen to avoid or minimize side effects related to the CAR-T therapy.

2. The IL-18 binding molecule for use of claim 1 as a modulator of IL-18 levels in the patient to avoid or minimize side effects related to the CAR-T therapy.

3. The IL-18 binding molecule for use of claim 2, wherein the IL-18 levels are levels of free IL-18.

4. The IL-18 binding molecule for use of any one of claims 1 to 3, wherein the IL-18 binding molecule inhibits IFN-gamma secretion.

5. The IL-18 binding molecule for use of any one of claims 1 to 4, wherein the IL-18 binding molecule inhibits IL-18 mediated stimulation of IFN-gamma secretion, that is further elevated by IL-12.

6. The IL-18 binding molecule for use of any one of claims 1 to 5, wherein the disease associated with expression of a tumor antigen is a proliferative disease, a precancerous condition, a cancer, and/or a non-cancer related indication associated with expression of the tumor antigen.

7. The IL-18 binding molecule for use of any one of claims 1 to 6, wherein the patient has cancer, particularly a hematological cancer or a solid tumor, particularly a tumor expressing tumor-associated antigens.

8. The IL-18 binding molecule for use of claim 7, wherein the cancer is a hematologic cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.

9. The IL-18 binding molecule for use of any one of claims 1 to 8, wherein a population of cells is used in the CAR-T therapy, which are engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

10. The IL-18 binding molecule for use of claim 9, wherein the cells are further engineered to express IL-18.

11. The IL-18 binding molecule for use of any one of claims 1 to 10, wherein the side effects are due to an overshooting of the immune system and/or a cytokine release syndrome.

12. The IL-18 binding molecule for use of claim 11, wherein the side effects are selected from the group consisting of unremitting fever, hypotension, hypoxia (lung involvement), other end-organ damage and dysfunction including but not limited to liver and kidney, cytopenias, coagulopathy, hemophagocytosis and neurologic toxicities.

13. The IL-18 binding molecule for use of any one of claims 1 to 12, as a modulator of free IL-18 levels in the patient.

14. The IL-18 binding molecule for use of any one of claims 1 to 13 for decreasing the IL-18 levels, particularly the free IL-18 levels, in the patient.

15. The IL-18 binding molecule for use of any one of claims 1 to 14, wherein the patient is an animal, particularly a human.

16. The IL-18 binding molecule for use of any one of claims 1 to 15, which is an IL-18 binding protein (IL-18BP), including any functional equivalent or functional part thereof which retains the modulator activity.

17. The IL-18 binding molecule for use of any one of claims 1 to 15, which is an IL-18 binding antibody that specifically binds to free IL-18, but not to IL-18 bound in a complex, including any functional equivalent or functional part thereof which retains the modulator activity.

18. The IL-18 binding molecule for use of claim 16, which is a human IL-18BP (hIL-18 BP), preferably a recombinant human IL-18BP (rhIL-18 BP), including any functional equivalent or functional part thereof, which retains the modulator activity.

19. The IL-18 binding molecule for use of claim 18, wherein said human IL-18BP is selected from isoform a, b, c and d of human IL-18BP, particularly isoform a as in SEQ ID NO: 2, isoform b as in SEQ ID NO: 3, isoform c as in SEQ ID NO: 4 or isoform d as in SEQ ID NO: 5, including any functional equivalent or functional part of isoforms a, b, c and/or d, which retains the modulator activity.

20. The IL-18 binding molecule for use of claim 19, which is an IL-18BP as shown in SEQ ID NO: 2, including any functional equivalent or functional part thereof, which retains the modulator activity, preferably wherein the functional equivalent has a sequence identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to the sequence depicted in SEQ ID NO: 2 and retains the modulator activity.

21. The IL-18 binding molecule for use of claim 20, wherein the functional equivalent or functional part thereof includes a mutein of IL-18BP, a fragment, a peptide, a functional derivative, a functional fragment, a fraction, a circularly permuted derivative, a fused protein comprising IL-18BP, an isoform or a salt thereof which retains the modulator activity.

22. A composition comprising the IL-18 binding molecule, particularly an IL-18 binding molecule of any one of claims 16 to 21, for use in a Chimeric Antigen Receptor T-cell (CAR-T) therapy of a patient, diagnosed with a disease associated with expression of a tumor antigen, to avoid or minimize side effects related to the CAR-T therapy, optionally wherein the composition comprises a pharmaceutically acceptable carrier and/or solvent and/or excipient.

23. The composition for use of claim 22, as a modulator of IL-18 levels in the patient to avoid or minimize side effects related to the CAR-T therapy.

24. The composition for use of claim 22 or 23, wherein the side effects are due to an overshooting of the immune system and/or a cytokine release syndrome.

25. The composition for use of any one of claims 22 to 24, wherein the disease is a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor antigen.

26. The composition for use of any one of claims 22 to 25, wherein the patient has cancer, particularly a hematological cancer or a solid tumor, particularly a tumor expressing tumor-associated antigens.

27. The composition for use of claim 26, wherein the cancer is a hematologic cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative condition, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.

28. The composition for use of any one of claims 22 to 27, wherein the side effects are selected from the group consisting of unremitting fevers, hypotension, hypoxia (lung involvement), other end-organ damage and dysfunction including but not limited to liver and kidney, cytopenias, coagulopathy, hemophagocytosis and neurologic toxicities.

29. The composition for use of any one of claims 22 to 28 comprising (i) a population of cells engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and (ii) an IL-18 binding molecule, particularly the IL-18 binding molecule of any one of claims 15 to 20.

30. The composition for use of claim 29, wherein the population of cells of (i) are further engineered to express IL-18.

31. The composition for use of any one of claims 22 to 30, wherein the IL-18 binding protein (IL-18BP) further comprises N-terminal and/or C-terminal deletion variants of IL-18BP.

32. The composition for use of any one of claims 22 to 31, wherein the patient is an animal, particularly a human.

33. The composition for use of any one of claims 22 to 32, comprising N-terminal and/or C-terminal deletion variants of IL-18BP in an amount of up to 40%, particularly up to 30%, particularly up to 20%, particularly up to 15%, particularly up to 10%, particularly up to 7.5%, particularly up to 5%, particularly up to 2.5%, particularly up to 1%, particularly up to 0.5%, particularly up to 0.25%, particularly up to 0.1%, particularly up to 0.05%, particularly up to 0.01%.

34. The composition for use of claim 31 or 33, wherein said deletion variants comprise deletions of between 1 and 5 amino acid residues at the C-terminal end of the IL-18BP and/or between 1 and 30 amino acid residues at the N-terminal end of the IL-18BP.

35. The composition for use of any one of claims 31 to 34, wherein said N-terminal and/or C-terminal deletion variants of IL-18BP are present in an amount of up to 40%, particularly in an amount of between 2% and 35%.

36. The IL-18 binding molecule of any one of claims 1 to 21 or the composition of any one of claims 22 to 35 for controlling and/or modulating, particularly decreasing, IL-18 levels, particularly free IL-18 levels, in the patient.

37. A method for modulating IL-18 levels in a patient under a Chimeric Antigen Receptor T-cell (CAR-T) therapy to avoid or minimize side effects, comprising administering to a patient in need thereof a therapeutically effective amount of an IL-18 binding molecule, particularly an IL-18 binding molecule according to any one of claims 16 to 21, or a therapeutically effective amount of a composition of any one of claims 22 to 35.

38. The method for use of claim 37, for modulating, particularly decreasing, IL-18 levels, particularly free IL-18 levels, in the patient.

39. The method for use of claim 37 or 38, wherein the patient has a disease associated with expression of a tumor antigen, e.g., a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor antigen.

40. The method for use of claim 39, wherein the patient has cancer, particularly a hematological cancer or a solid tumor, particularly a tumor expressing tumor-associated antigens.

41. The method for use of claim 40, wherein the cancer is a hematologic cancer selected from one or more of chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.

42. The method for use of any one of claims 37 to 41, wherein the patient is an animal, particularly a human.

43. The method of any one of claims 37 to 42, wherein the CAR-T-cells and the IL-18 binding molecule or the composition, respectively, are administered to the patient concomitantly.

44. The method of any one of claims 37 to 42, wherein the CAR-T-cells and the IL-18 binding molecule or the composition, respectively, are administered to the patient sequentially.

45. The method of any one of claims 37 to 44, wherein the IL-18 binding molecule or the composition, respectively, is administered to the patient as an acute intervention during the CAR-T therapy.

46. The IL-18 binding molecule for use of claim 7 or the composition for use of claim 26 or the method of claim 40 wherein the cancer is a solid tumor, particularly selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancer, combinations of said cancers, and metastatic lesions of said cancers.

47. The IL-18 binding molecule for use of any one of claim 1 to 21, 36 or 46 or the composition for use of any one of claim 22 to 35 or 46, or the method of any one of claims 37 to 46, wherein in a body fluid of the patient an abnormal level of free IL-18 has been determined, in particular wherein the level of free IL-18 exceeds the level of free IL-18 in body fluids of a healthy control subject, particularly by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, using an assay capable of detecting free IL-18 in body fluids, said assay comprising the IL-18 binding molecule as defined in any one of claims 16 to 21.

48. The IL-18 binding molecule for use, the composition for use or the method of claim 47, wherein the assay for quantifying the level of free IL-18 in the body fluid(s) comprises the following steps: a) bringing a sample of body fluid suspected to contain free IL-18 into contact with the IL-18 binding molecule as defined in any one of claims 16 to 21 as the capturing molecule for free IL-18; b) allowing the IL-18 binding molecule to bind to free IL-18; and c) detecting the binding of the IL-18 binding molecule and determining the amount of free IL-18 in the sample.

49. The IL-18 binding molecule for use, the composition for use or the method of any one of claim 47 or 48, wherein the body fluid is selected from the group consisting of broncho-alveolar lavage fluid (BALF) circulation fluids, secretion fluids, biopsy, and homogenized tissue, particularly serum, urine, tear, saliva, bile, sweat, exhalation or expiration, sputum, bronchoalveolar fluid, sebum, cellular, gland, mucosa and tissue secretion.

50. The IL-18 binding molecule for use of any one of claims 1 to 21, 36 or 46 to 49 or the composition for use of any one of claims 22 to 35 or 46 to 49, or the method of any one of claims 37 to 49, wherein the therapy of the patient further comprises the use of one or more check-point inhibitor(s).

51. The IL-18 binding molecule for use of claim 50 or the composition for use of claim 50, or the method of claim 50, wherein the check-point inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor or a PD-L1 inhibitor.

Description

BRIEF DESCRIPTION OF THE SEQUENCES

[0240]

TABLE-US-00001 SEQ ID NO 1: 13-amino acid Linker Sequence of hIL-18BP Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met SEQ ID NO 2: Amino Acid Sequence of IL-18 Binding Protein (hIL-18BP), isoform a TPVSQTTTAA TASVRSTKDP CPSQPPVFPA AKQCPALEVT WPEVEVPLNG TLSLSCVACS RFPNFSILYW LGNGSFIEHL PGRLWEGSTS RERGSTGTQL CKALVLEQLT PALHSTNFSC VLVDPEQVVQ RHVVLAQLWA GLRATLPPTQ EALPSSHSSP QQQG SEQ ID NO 3: Amino Acid Sequence of IL-18 Binding Protein (hIL-18BP), isoform b TPVSQTTTAA TASVRSTKDP CPSQPPVFPA AKQCPALEVT WPEVEVPL SWAEGNLAPH PRSPALQPQQ STAAGLRLST GPAAAQP SEQ ID NO: 4: Amino Acid Sequence of IL-18 Binding Protein (hIL-18BP), isoform c TPVSQTTTAA TASVRSTKDP CPSQPPVFPA AKQCPALEVT WPEVEVPLNG TLSLSCVACS RFPNFSILYW LGNGSFIEHL PGRLWEGSTS RERGSTGTQL CKALVLEQLT PALHSTNFSC VLVDPEQVVQ RHVVLAQLVW RSPRRGLQEQ EELCFHMWGK GGLCQSSL SEQ ID NO: 5: Amino Acid Sequence of IL-18 Binding Protein (hIL-18BP), isoform d TPVSQTTTAA TASVRSTKDP CPSQPPVFPA AKQCPALEVT WPEVEVPLNG TLSLSCVACS RFPNFSILYW LGNGSFIEHL PGRLWEGSTS RERGSTGWAE GNLAPHPRSP ALQPQQSTAA GRLSTGPAAA QP

[0241] The invention is also characterized by the following figures, figure legends and the following non-limiting examples.

[0242] FIG. 1: IL-18 dependent and independent pathways of CAR-T-cell-induced CRS.

[0243] FIG. 2: IL-18 increases IFN-γ secretion from CAR-T-cells. Kinetics of IFN-γ secretion from CAR-T-cells over three days. Statistical significance of variations in IFN-γ levels versus levels observed with CAR-T-cells alone are reported (*: p<0.05; **: p<0.005).

[0244] FIG. 3: IL-18-mediated IFN-γ secretion is abolished by rhIL-18BP. Cumulative increase of IFN-γ levels in culture medium after each day normalized to levels at Day 1 with Tumor+CAR-T-cells. Statistical significance of variations in IFN-γ increase versus increase observed with Tumor+CAR-T-cells are reported (ns: not significant; *: p<0.05; ***: p<0.0005).

[0245] FIG. 4: IL-12-boosted IFN-γ secretion is drastically diminished by rhIL-18BP. Cumulative increase of IFN-γ levels in culture medium after each day normalized to levels at Day 1 with Tumor+CAR-T-cells. Statistical significance of variations in IFN-γ increase versus increase observed with Tumor+CAR-T-cells are reported (*: p<0.05; ***: p<0.0005).

EXAMPLES

Example 1 Generation of Transgenically Encoded Mouse IL-18 in CAR-T-Cells in the Treatment of Murine B16F10 Melanoma. Efficacy of rhIL-18BP in Mitigating the IL-18 Driven Cytokine Release Syndrome Induced by CAR-T-Cells Treatment

[0246] IL-18 signaling enhances TCR signaling and therefore it was concluded that it could also potentiate the CAR-T activation/proliferation since the CAR-T contains the TCR signaling domain for T-cell activation and proliferation. A note of caution though is that the increased CAR-T activation may lead to exaggerated antigen-specific T-cell activation and also to Ag-nonspecific activation mimicking a hyperinflammatoy status similar to cytokine releases syndrome.

Methods

[0247] Murine T-cells are engineered to express murine IL-18 and a fragment of antibody directed against murine CD19, a specific tumor associated antigen (murine CD19-IL-18). Murine CD19-IL-18 CAR-T-cells are adoptively transferred to C57BL/6 mice bearing B16F10 melanoma tumors expressing the surface antigen CD19. Co-expression of IL-18 in CAR-T-cells results in the development of a T-bet.sup.high phenotype, polarizing the CAR-T-cells towards Th1 effectors and in the stimulation of a systemic acute inflammatory response. It has been previously observed that one week after CAR-T-cells transfer the mice show very high levels of IL-18 in blood coincident with the CAR-T-cell expansion, and that it decreases afterwards. Coincident with the high levels of IL-18 the mouse body weight decreased as the main manifestation of cytokine release syndrome together with mild manifestations of systemic inflammation.

[0248] Strategies to minimize detrimental side effects represent an appealing approach to use this therapy in solid tumors without harmful complications in humans.

[0249] The administration of rhIL-18BP in the days following the CAR-T-cell transfer surprisingly represents a safe way to avoid detrimental toxic effects. In this regard, rhIL-18BP may be administered at a dose of 1 mg/kg to 3 mg/kg i.v. or s.c or i.v. for a loading dose and thereafter s.c. in a saline solution upon development of side effects or, e.g., upon determining elevated circulating IL-18 levels in the patient.

Example 2: IL-18 Dependent Stimulation of CAR-T-Cell-Induced CRS and Mitigation with IL-18BP

[0250] CAR-T-cell therapy is regarded as a revolutionary cancer therapy; the curative potential of CAR-T-cell therapies represents a paradigm shift in cancer treatment. Over the last years, there have been rapid advancements in the field and CD19-targeted CAR-T-cells are now approved by the FDA for the treatment of B-cell leukemias and lymphomas; see e.g. Maude SL, Laetsch T W, Buechner J, et al. (2018) “Tisagenlecleucel in children and young adult with B-cell lymphoblastic leukemia.” N Engl J Med. 378:439-448 or Neelapu S S, Locke F L, Bartlett N L, et al. (2017) “Axicabtagene ciloleucel CAR-T-cell therapy in refractory large B-cell lymphoma.” N Engl J Med. 377:2531-2544.

[0251] Despite promising results in rates of remission, patients undergoing CAR-T-cell infusion often experience an uncontrolled immune activation in the form of a Cytokine Release Syndrome (CRS). This hyperinflammatory response is a life-threatening treatment-related toxicity that limits the full therapeutic potential of CAR-T-cells therapies. Approximately 45%-50% of patients enrolled in the early CAR-T-cell trials required intensive care management. With more experience and earlier interventions in CRS management, this rate may be diminished.

[0252] The management of the CRS following CAR-T-cell infusion is currently dominated by the use of Tocilizumab to provide IL-6R blockade. This treatment has improved the management of the CRS symptomatology. Nevertheless, the impact of IL-6R blockade on the neurologic sequela following CAR-T-cells is still unknown today. One of the working hypotheses to decipher the molecular bases of CRS is that it results from the induction of two distinct cytokine signaling pathways, the IL-1β/IL-6 pathway and the IL-18/IFN-γ pathway. So far, therapeutic approaches focusing on the first pathway did not provide fully satisfying efficacy/safety profiles.

[0253] IL-18 is a pro-inflammatory cytokine belonging to the IL-1 family, first identified for its IFN-γ-inducing properties. By binding to IL-18, IL-18 binding protein (IL-18BP) neutralizes IL-18 and acts as a key regulator of associated immune responses. Under healthy conditions, all IL-18 is bound by IL-18BP and there are no or little amounts of free active IL-18. In patients suffering from a systemic inflammatory disorder, both IL-18 and IL-18BP levels are drastically increased, but due to the higher elevation of IL-18, IL-18BP is not able to efficiently neutralize the active free form of IL-18, which leads to a pathological presence of free IL-18. Free IL-18 which is not inactivated by IL-18 BP, induces the immune system constantly and can lead to severe autoinflammatory situations and CRS with a potentially fatal outcome.

[0254] A recombinant human IL-18BP (rhIL-18BP, Tadekinig alfa) for use in the treatment of severe rare autoinflammatory diseases (hemophagocytic lympho-histiocytosis (HLH)) is currently being developed. HLH are hyperferritinemic systemic inflammatory disorders, characterized by the uncontrolled proliferation of activated lymphocytes and macrophages, which trigger secretion of high amounts of inflammatory cytokines. High levels of IL-18, and specifically of free IL-18, have been found in serum of HLH patients when their condition evolves into a macrophage activation syndrome (MAS); see Weiss E S, Girard-Guyonvarc'h C, Holzinger D, et al. (2018) “Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome.” Blood. 131(13): 1442-1455. Treatment with Tadekinig alfa neutralizes circulating free IL-18 and thereby, reduces and controls the IL-18 mediated inflammation.

[0255] Patients treated with CD19-specific CAR-T-cells who are experiencing a CRS, present increased levels of circulating cytokines, with IFN-γ having the largest relative change from baseline; see Kalos M, Levine B L, Porter D L, et al. (2011) “T-cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.” Sci Transl Med. 3(95):95ra73. Recognition of tumor cells by specific CAR-T-cells leads to activation of these Th1 cells and secretion of IFN-γ and other soluble mediator molecules. This first signaling involves cell to cell contact and is IL-18-independent, see Rooney C and Sauer T. (2018) “Modelling cytokine release syndrome.” Nat Med. 24(6):705-706, whereas the following activation of macrophages, by the secreted mediators or by cell to cell contact with activated Th1 cells, releases IL-18, which will in turn triggers the secretion of IFN-γ from Th1 cells (FIG. 1). This signaling is IL-18-dependent and could be inhibited by IL-18BP. Moreover, IL-18-mediated stimulation of Th1 cells is optimized by an accessory molecule, IL-12, which induces the expression of IL-18Rβ on the surface of Th1 cells: IL-18 presents a higher affinity for the heterodimeric IL-18Rαβ than for the monomeric IL-18Ra receptor, its binding to Th1 cells is thereby stabilized and the production of IFN-γ is boosted; see Tominaga K, Yoshimoto T, Torigoe K, et al. (2000) “IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T-cells.” Int Immunol. 12(2):151-160.

[0256] Both pathways contribute to secretion of high levels of pro-inflammatory cytokines, including IL-18, and their activation may lead to a CRS. Management of CAR-T-cell-induced CRS with Tadekinig alfa as exogenous source of IL-18BP would restore the IL-18/IL-18BP balance and provide an alternative approach for patients presenting high levels of IL-18.

[0257] This study aims at recapitulating the IL-18-dependent stimulation of CAR-T-cells and evaluating the mitigator effect of rhIL-18BP on CRS via monitoring of secretion of IFN-γ from activated CAR-T-cells. The goal is to collect evidence that Tadekinig alfa represents a therapeutic option to limit the toxicity associated with CAR-T-cell-induced CRS.

[0258] The study is designed to follow the CAR-T-cells stimulation by monitoring IFN-γ secreted from activated CAR-T-cells. As IFN-γ is one of the major components of the CAR-T-cell-induced CRS, these experimental conditions provide a tool to evaluate the effect of rhIL-18BP on CRS following CAR-T-cell therapy.

[0259] CAR-T-cells are activated by cell to cell contact and by the pro-inflammatory cytokine IL-18. Therefore, reduction of free IL-18 via binding of IL-18BP is anticipated to reduce IL-18-dependent CAR-T-cells stimulation, and thus to reduce IFN-γ secreted from these CAR-T-cells. This would bring evidence that efficient neutralization of free IL-18 mitigates CAR-T-cells-mediated CRS and that Tadekinig alfa constitutes a therapeutic approach to limit toxicity experienced by CAR-T-cell patients presenting high levels of IL-18.

[0260] CD19-specific CAR-T-cells (CD19SCFV-CD28-4-1BB-CD3 CAR-T-cells, #PM-CAR1003-10M, ProMAB) were co-cultured with Raji lymphoma target-cells (#CCL-86, ATCC) with or without rhIL-18 (IL-18, 100 ng/mL, #9124-IL-010, R&D Systems), rhIL-12 (IL-12, 10 ng/mL, #219-IL-005, R&D Systems) or rhIL-18BP (IL-18BP, Tadekinig alfa, AB2 Bio) in 20-fold molar excess over rhIL-18 and combinations of these factors at an E:T ratio of 5:1. Each condition was assessed in triplicates.

[0261] Aliquots (60 μL) of cell culture supernatants were collected at 24, 48 and 72-hour time points from each well, analyzed for IFN-γ secretion by ELISA (#EHIFNG, Invitrogen) and replaced by fresh medium containing the same combination of reagents. The concentrations of IFN-γ in were extrapolated from the calibration curve and the cumulative concentrations were calculated.

[0262] The measured concentration in IFN-γ, together with the derived calculations are reported in Table 1.

[0263] CAR-T-cells alone do not produce significant amounts of IFN-γ when compared to the background levels (tumor cells only) with 27.49±0.73 pg/mL versus 15.84±2.42 pg/mL, respectively after three days of culture.

[0264] Once in presence of tumor cells, CAR-T-cells produce large amounts of IFN-γ (6910.5±478.0 pg/mL at day 1 and 32675.1±1449.3 pg/mL at day 3). Presence of rhIL-18 in the culture triggers a greater IFN-γ production, reaching over 70 ng/mL after three days, at a higher rate (FIG. 2).

TABLE-US-00002 TABLE 1 Concentrations of IFN-γ in culture medium over three days (D1; D2 and D3) and derived calculations. Increase in cumulative concentration IL-18BP- Cumulative Mean SD vs basal levels mediated Concentration concentration concentration concentration (Tumor + inhibition Culture condition (pg/mL) (pg/mL) (pg/mL) (pg/mL) CAR-T) (%) D1-Tumor 4.48 4.48 5.82 3.38 D1-Tumor 3.31 3.31 D1-Tumor 9.66 9.66 D2-Tumor 10.71 13.40 13.44 2.80 D2-Tumor 8.68 10.66 D2-Tumor 10.47 16.26 D3-Tumor 4.56 13.68 15.84 2.42 D3-Tumor 8.18 15.38 D3-Tumor 6.38 18.45 D1-CAR-T 10.47 10.47 11.79 1.23 D1-CAR-T 12.01 12.01 D1-CAR-T 12.90 12.90 D2-CAR-T 8.10 14.38 15.21 0.71 D2-CAR-T 8.35 15.55 D2-CAR-T 7.94 15.68 D3-CAR-T 17.09 28.24 27.49 0.73 D3-CAR-T 15.24 27.45 D3-CAR-T 14.27 26.78 D1-Tumor + CAR-T 7462 7462 6910 478 1.0 D1-Tumor + CAR-T 6633 6633 1.0 D1-Tumor + CAR-T 6637 6637 1.0 D2-Tumor + CAR-T 16075 20552 21483 947 2.8 D2-Tumor + CAR-T 18466 22445 3.4 D2-Tumor + CAR-T 17471 21453 3.2 D3-Tumor + CAR-T 20132 34254 32675 1449 4.6 D3-Tumor + CAR-T 17306 32367 4.9 D3-Tumor + CAR-T 16940 31405 4.7 D1-Tumor + CAR-T + IL-18 14274 14274 14120 2461 1.9 D1-Tumor + CAR-T + IL-18 11585 11585 1.7 D1-Tumor + CAR-T + IL-18 16500 16500 2.5 D2-Tumor + CAR-T + IL-18 32865 41430 40337 1489 5.6 D2-Tumor + CAR-T + IL-18 31691 38641 5.8 D2-Tumor + CAR-T + IL-18 31039 40939 6.2 D3-Tumor + CAR-T + IL-18 39457 67740 71068 4826 9.1 D3-Tumor + CAR-T + IL-18 42932 68897 10.4 D3-Tumor + CAR-T + IL-18 48093 76617 11.5 D1-Tumor + CAR-T + IL-18 + IL-12 469713 469713 434054 33200 62.9 D1-Tumor + CAR-T + IL-18 + IL-12 404037 404037 60.9 D1-Tumor + CAR-T + IL-18 + IL-12 428412 428412 64.6 D2-Tumor + CAR-T + IL-18 + IL-12 1194333 1476161 1328014 130718 197.8 D2-Tumor + CAR-T + IL-18 + IL-12 1036554 1278976 192.8 D2-Tumor + CAR-T + IL-18 + IL-12 971859 1228906 185.2 D3-Tumor + CAR-T + IL-18 + IL-12 1031172 2029000 1853355 159306 272.0 D3-Tumor + CAR-T + IL-18 + IL-12 946503 1610858 273.0 D3-Tumor + CAR-T + IL-18 + IL-12 679444 1719607 259.1 D1-Tumor + CAR-T + IL-18 + IL-18BP 5305 5305 5792 422 0.7 100 D1-Tumor + CAR-T + IL-18 + IL-18BP 6015 6015 0.9 D1-Tumor + CAR-T + IL-18 + IL-18BP 6055 6055 0.9 D2-Tumor + CAR-T + IL-18 + IL-18BP 19343 22526 23051 1232 3.0 92 D2-Tumor + CAR-T + IL-18 + IL-18BP 18560 22169 3.3 D2-Tumor + CAR-T + IL-18 + IL-18BP 20825 24458 3.7 D3-Tumor + CAR-T + IL-18 + IL-18BP 25218 40007 40981 4430 5.4 79 D3-Tumor + CAR-T + IL-18 + IL-18BP 22375 37119 5.6 D3-Tumor + CAR-T + IL-18 + IL-18BP 29690 45617 6.9 D1-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 63463 63463 81757 15849 8.5 82 D1-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 91349 91349 13.8 D1-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 90458 90458 13.6 D2-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 202872 240950 287234 48586 32.3 79 D2-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 228109 282918 42.7 D2-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 283560 337835 50.9 D3-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 256494 416295 474688 59349 556 75 D3-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 281146 472821 71.3 D3-Tumor + CAR-T + IL-18 + 1L-12 + IL-18BP 310536 534949 80.6

[0265] At each tested time point, rhIL-18 triggered about 2-fold stimulation of the IFN-γ produced by the co-culture of CAR-T and tumor cells (FIG. 3). Over three days, the IFN-γ levels increased in average 10-fold compared to the basal levels (i.e. without rhIL-18). Co-treatment of the CAR-T/tumor cells with rhIL-18BP led to a full inhibition (day 1) to a 92% and 79% reduction (day 2 and day 3, respectively) of the IL-18-mediated IFN-γ production.

[0266] Co-treatment of the CAR-T and tumor cells with rhIL-18 and rhIL-12 drastically boosted the secretion of IFN-γ, reaching over 250-fold the basal levels over three days (FIG. 4). Addition of rhIL-18BP provokes a 82%, 79% and 75% reduction of these levels at day 1, day 2 and day 3, respectively.

[0267] IFN-γ, a T-cell mediator, is not secreted from CAR-T-cells when they are individually cultured. In presence of tumor cells, CD19-specific-CAR-T-cells produce significant levels of IFN-γ, reflecting its activation. This T-cell stimulation is initiated by a cell to cell contact, through recognition of the CD19 surface antigen on B cells and is IL-18-independent.

[0268] Upon addition of rhIL-18, production of IFN-γ was further increased, reflecting the responsiveness of T-cells to this pro-inflammatory cytokine. The production rate was greater than in the absence of rhIL-18, as seen by a steeper slope in the kinetics, and did not reach a plateau after a three day-culture. Even though IFN-γ production from Tumor/CAR-T-cells co-culture gradually increased over three days, rhIL-18 maintained a constant 2-fold stimulation. This strongly supports that rhIL-18 triggers a parallel signalling, complementary to the one initiated by the cell to cell contact.

[0269] Co-treatment with rhIL-18BP led to the complete inhibition of IL-18-mediated stimulation of IFN-γ production at day 1, and still to 79% reduction at day 3.

[0270] IL-12 has been described in the literature as an accessory molecule of IL-18, acting in vivo in synergy with it by stabilizing the IL-18 binding to further increase the activation of T-cells. The experiment revealed that addition of rhIL-12 to rhIL-18 in the Tumor/CAR-T-cells co-culture drastically boosts the production of IFN-γ, and that this stimulation is drastically reduced (80% in average) upon addition of rhIL-18BP. Therefore, in conditions that are closer to the physiological ones, treatment with rhIL-18BP provokes similar reduction of T-cells stimulation than in the absence of rhIL-12.

[0271] The experimental conditions of this study recapitulate the IL-18-dependent stimulation of CAR-T-cells and enable to evaluate the mitigating effect of rhIL-18BP on CRS induced by CAR-T-cells activation. It has been designed to involve the main key players of the CAR-T-cell-mediated CRS, namely CD19-specific-CAR-T-cells, Raji B lymphomas, T-cell mediator IFN-γ, pro-inflammatory cytokine IL-18 and accessory molecule IL-12, both secreted by activated macrophages.

[0272] The generated results revealed that beside the IL-18-independent low stimulation of CAR-T-cells by tumor cells, there is a huge activation of T-cells that is triggered by rhIL-18 and further elevated by rhIL-12. Therefore, presence of high levels of free IL-18 secreted from activated macrophages further stimulates CAR-T-cells and aggravates the extent of CRS. These findings provides a rationale for neutralizing overshooting levels of free IL-18 when present in serum of cancer patients following a CAR-T-cell therapy. This study demonstrates that co-treatment with rhIL-18BP abrogates the IL-18-mediated activation of CAR-T-cells, bringing evidence for a therapeutic use of Tadekinig alfa in patients experiencing a CRS following a CAR-T-cell therapy, in the aim of neutralizing unbalanced levels of circulating IL-18.