Checkpoint inhibitors in the treatment of glioblastoma multiforme

Abstract

The present invention concerns the use of specific proteins which are effective in the treatment of cancer by inhibiting the immune escape mechanism in a subject in need thereof. Furthermore, the invention relates to a pharmaceutical kit comprising the specific proteins in combination with checkpoint inhibitors and their uses.

Description

[0001] CHECKPOINT INHIBITORS IN THE TREATMENT OF GLIOBLASTOMA MULTIFORME

[0002] ***** ***** *****

[0003] FIELD OF THE INVENTION

[0004] The present invention concerns the use of specific proteins which are effective in the treatment of cancer by inhibiting the immune escape mechanism in a subject in need thereof.

[0005] Furthermore, the invention relates to a pharmaceutical kit comprising the specific proteins in combination with checkpoint inhibitors and their uses.

[0006] STATE OF THE ART

[0007] Glioblastoma (GBM) is the most common primary malignant brain tumor with a median overall survival < 15 months and a 5-years survival rate 6,9%. Treatment failure is primarily attributed to the existence within this tumor of a small subpopulation of glioblastoma stem cells (GSCs), which is responsible for GBM resistance to therapy, recurrence and the complex inter- and intra-tumor heterogeneity, the latter encompassing different GBM subtypes and stromal cells within tumor microenvironment (TME).

[0008] The complexity of GBM is also ascribable to the harsh environment and the extremely intrinsic heterogeneity both at the cellular and molecular level. As a matter of fact, GBM embodies several different subsets of cells which sustain tumor development and progression such as tumor cells and non-neoplastic parenchymal cells, circulating progenitor cells and a variety of immune cells deeply integrated within the tumor mass.

[0009] The GBM TME plays a critical role in determining cellular behavior, driving GSCs adaptability and ultimately promoting therapeutic resistance through the production of cytokines and chemokines. The interaction of these factors with the extracellular matrix finally induces an extensive immunosuppressive phenotype, characterized by the inhibition and apoptosis of T cells and tumor associated microglia / macrophages (TAMs).

[0010] It has been very recently reported that also GSCs play a critical role within the immunosuppressive microenvironment of GBM through intrinsic and extrinsic mechanisms, thus triggering immune evasion and ineffective antitumor immune responses. Intrinsic immune evasion mechanisms are mainly characterized by low-level of antigen-presenting cells and high-level of immunosuppressive molecules, such as PD-L1 , TRAIL, TNF-alpha, IL-10. On the other hand, the crosstalk between GSCs and cells of the immune system, also characterized by the release of extracellular vesicles (EVs), mainly constitute the external evasion process.

[0011] Based on the crucial role played by the immunological compartment within GBM physiology, there is an absolute need to design and implement new and more effective experimental immunological approaches, also targeting GSC cells, which are responsible for GBM progression and recurrence, thus decreasing the immune-driven resistance and improving treatment benefits.

[0012] It is therefore object of the present invention the delineation of new therapeutic approaches able to reduce the GSCs tumorigenic potential through the activation of an immune response to tumor cells.

[0013] SUMMARY OF THE INVENTION

[0014] The problem underlying the present invention is that of making available compounds capable of activating and enhancing an immune response to tumor cells.

[0015] This problem is resolved by the present finding by the use of the Bone Morphogenetic Protein 4 (BMP4) capable of inhibiting the immune escape mechanism in a patient.

[0016] The present invention discloses the use of a Bone Morphogenetic Protein 4 (BMP4) in the treatment of cancer, wherein said BMP4 is a human recombinant BMP4 protein (hrBMP4). BMP4 inhibits the immune escape mechanism in a subject suffering from cancer.

[0017] In a second aspect the invention relates to a pharmaceutical kit comprising: a) Human recombinant Bone Morphogenetic Protein 4 (hrBMP4); and a b) Checkpoint inhibitor, for simultaneous, separate or sequential use.

[0018] In a third aspect, the use of a pharmaceutical kit is herein described, said pharmaceutical kit comprising: a) Human recombinant Bone Morphogenetic Protein 4 (hrBMP4); and a b) Checkpoint inhibitor, in the treatment of cancer.

[0019] In a fourth aspect the invention describes a method for treating a disease which involves the inhibition of the immune escape mechanism, said method comprising the step of administering a Bone Morphogenetic Protein 4 (BMP4) to a subject in need thereof.

[0020] In a fifth aspect the invention describes a method for treating cancer, said method comprising the step of administering a Bone Morphogenetic Protein 4 (BMP4) to a subject in need thereof. BMP4 may be administered with a checkpoint inhibitor.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Experimental section given for illustrative and non-limiting purposes, and from the annexed Figures 1-3, wherein:

[0023] Figure 1 : FACS analysis showing that GSCs exposure for 24h to I L-1 p, TNFa and IFN- y alone (top) or in combination (middle) strongly enhances PD-L1 protein level, as compared to untreated cells (control; top).

[0024] Figure 2: PD-L1 protein expression level in different subsets of GSC cells stimulated with cytokines and treated with BMP4. A) GSCs exposure to IL-1 cytokine in combination with TNFa and IFN-y enhances PD-L1 level (Increase %) as compared to unstimulated GSCs (CTRL). B) BMP4 treatment reduces the increase of PD-L1 protein expression (Increase %) observed after IL-1 p, TNFa and IFN-y treatment alone (shown in A) as compared to unstimulated GSCs (CTRL). C) GSCs treatment with BMP4 in combination with IL-1 p, TNFa and IFN-y lessens PD-L1 level (% Decrease) as compared to combo cytokines treatment alone. D) Histogram showing BMP4 effect on PD-L1 expression level in different subset of GSCs.

[0025] Figure 3: FACS analysis depicting the reduction of PD-L1 protein level (arrowed) in GSCs treated with BMP4 for 72h, as compared to control (CTRL).

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] As described above, the present invention discloses the use of a Bone Morphogenetic Protein 4 (BMP4) in the treatment of cancer. It was surprisingly seen that the BMP4 inhibis the immune escape mechanism in a subject suffering from cancer. The treatment with BMP4 is a novel target strategy for the treatment of those diseases characterized by immune resistance.

[0028] As will be evident from the Experimental section BMP4 has surprisingly shown an unexpected activity in the activation of an immune response, and in particular as a checkpoint inhibitor.

[0029] Immune checkpoint (IC) molecules are defined as ligand-receptor pairs that exert inhibitory or stimulatory effects on immune responses. Several evidences have shown that inhibitory or stimulatory IC molecules are expressed on a sizeable fraction of tumor types and other tumor microenvironment (TME) cells, playing crucial roles in the maintenance of many malignant behaviors, including epithelial-mesenchymal transition, CSCs self-renewal, metastasis, drug resistance, anti-apoptosis, angiogenesis or enhanced energy metabolism. Clinically most relevant immune checkpoints are the cytotoxic T lymphocyte antigen 4 (CTLA-4), the programmed death 1 receptor (PD-1) and its ligand (PD-L1) along with the two emerging targets T-cell immunoglobulin, mucin domain 3 (TIM-3) and indoleamine 2,3- dioxygenase-1 (IDO1).

[0030] Interestingly, blockade of these immune checkpoints with antibodies successfully demonstrated efficacy in various solid tumors, predominantly melanoma and non-small cell lung cancer and prolonged the survival of patients.

[0031] Unfortunately, immunotherapy strategies based on ICI blockade of PD-1 / PD-L1 and CTLA- 4 has failed to induce clinical benefit in GBM patients.

[0032] Nevertheless, it has been reported by the same clinical studies that neoadjuvant PD-1 blockade induces significant survival benefit and immune response in patients with recurrent GBM.

[0033] Moreover, the notion of a highly immune-restricted central nervous system has also evolved, further providing the rationale for testing therapies that promote immune trafficking to the CNS and infiltration into the tumor to counteract the immunosuppressive mechanisms that support GBM progression.

[0034] Surprisingly, as will be further demonstrated, BMP4 has shown to be able to reduce the tumorigenic potential through the activation of an immune response to tumor cells, and is a promising new therapeutic approach for the treatment of malignancies.

[0035] The terms “hrBMP4” or “BMP4” as used herein are intended to include members of the bone morphogenetic protein (BMP) family, and in particular Bone Morphogenetic Protein 4 and human recombinant Bone Morphogenetic Protein 4. hrBMP4 is a naturally occurring protein that together with its family of related ligands is involved in nearly all processes of development and also maintenance of a number of adult tissues.

[0036] The expression “inhibiting the immune escape mechanism” or “blockade of immune escape” as used herein intends to comprise all the steps that tumors undergo in order to evade an antitumor immune response. Particularly, inhibitory IC upregulation could hijack the immune defense of the host to promote T cell exhaustion and the acquisition of immune resistance. In a preferred aspect, in the use of the BMP4 protein 1 , said BMP4 protein is a human recombinant BMP4 protein (hrBMP4).

[0037] Surprisingly, the use of the BMP4 protein appears to influence the modulation of immune checkpoint molecules and in particular a downregulation of cell surface expression of PD- L1 is obtained by activity of BMP4.

[0038] Surprisingly the use of the BMP4 protein was observed in the modulation of immune checkpoint molecules, and in particular the immune escape block is obtained by activity of BMP4 on the PD-1 / PD-L1 immune checkpoint inhibitors (ICI).

[0039] These findings demonstrated that BMP4, which promotes the commitment of GSC cells to differentiation, plays a critical role within GBM microenvironment through the modulation of the level of PD-L1 protein, that is one of the major and relevant components of the GBM TME.

[0040] In a more preferred aspect, in the use of the BMP4 protein said immune-driven inhibition of the immune escape mechanism or blockade of immune escape is in subject suffering from cancer, in particular from a Central Nervous System Tumor or a solid tumor.

[0041] High levels of PD-L1 expression are often associated with ineffective immunogenic therapies ad poor patient prognosis. The influence of the BMP4 protein on the expression of PD-L1 therefore suggests the possibility of a double action:

[0042] 1) direct, relating to the downregulation of PD-L1 and

[0043] 2) indirect as the downregulation of PD-L1 would make immunotherapies more effective.

[0044] In a further more preferred aspect, said cancer is selected from the group consisting of Central Nervous System or glial cancer or a solid tumor a-and more preferably said cancer is glioblastoma multiforme (GBM).

[0045] In a further preferred aspect in the use of the BMP4 protein, the glioblastoma multiforme is a grade 3 or 4 glioma and is a subtype selected from the group consisting of classical, mesenchymal and proneural.

[0046] More preferably the use of the BMP4 protein in cancer is driven by stem cells, in particular when the cancer is GBM, the use is driven by GBM stem cells.

[0047] Despite advances in conventional treatments, the prognosis for most patients with GBM remains poor and this has led to an intensive search for alternative treatments. The discovery of GBM stem cells (GSCs), which are responsible for tumor initiation and relapse and are also involved in the GBM tumor microenvironment (TME), has opened new frontiers for the development of potential new therapeutic approaches for brain cancer.

[0048] In a second aspect the invention relates to a pharmaceutical kit comprising: a) Human recombinant Bone Morphogenetic Protein 4 (hrBMP4); and a b) Checkpoint inhibitor, for simultaneous, separate or sequential use.

[0049] In a preferred aspect, in the pharmaceutical kit for use according to the invention, BMP4 can be administered by intramuscular, intravenous, intrathecal, oral, enteral, parenteral, intraventricular, intra-tumor and intra-parenchyma by Convection Enhanced Delivery (CED) or by inhalation. hrBMP4 can be safely and effectively intra-parenchymally infused in human GBM recurrencies. The understanding of the complex multidirectional interactions between CSCs and immune microenvironment could be the key factors to develop of new multimodal strategies to interrupt or reverse the GBM progression.

[0050] In a third aspect, the use of a pharmaceutical kit is herein described, said pharmaceutical kit comprising: a) Human recombinant Bone Morphogenetic Protein 4 (hrBMP4); and a b) Checkpoint inhibitor, in the treatment of cancer.

[0051] In a preferred aspect, said cancer is selected from the group consisting of Central Nervous System or glial cancer or a solid tumor a-and more preferably said cancer is glioblastoma multiforme (GBM).

[0052] In a further preferred aspect in the use of the pharmaceutical kit, the glioblastoma multiforme is a grade 3 or 4 glioma and is a subtype selected from the group consisting of classical, mesenchymal and proneural.

[0053] More preferably the use of the pharmaceutical kit for treating cancer is driven by stem cells, in particular when the cancer is GBM, the use is driven by GBM stem cells.

[0054] In a fourth aspect the invention describes a method for treating a disease which involves the inhibition of the immune escape mechanism, said method comprising the step of administering a Bone Morphogenetic Protein 4 (BMP4) to a subject in need thereof.

[0055] In a fifth aspect the invention describes a method for treating cancer, said method comprising the step of administering a Bone Morphogenetic Protein 4 (BMP4) to a subject in need thereof. BMP4 may be administered with a checkpoint inhibitor. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following experimental section. EXPERIMENTAL SECTION

[0056] Reference is now made to the following experiments, which together with the above descriptions illustrate some embodiments of the invention.

[0057] Results

[0058] In order to identify immunological features of GSCs during inflammation, GSC stable lines were isolated and established from adult human GBM fresh tumor tissues and exposed to different cytokines (IL-1 p, TNFa, IFN-y) for 24h alone or combination, in order to mimic the tumor microenvironment. The latter is characterized by high levels of the ligand PD-L1 , which binds to its receptor PD-1 , expressed by T-lymphocytes, thus contributing to the induction of the GBM immunosuppressive phenotype.

[0059] As shown in Figure 1 , FACS analysis reveals that the expression level of PD-L1 is strongly increased in the presence of TNFa or IFN-y, while a smaller increase is obtained in the presence of IL-1 p, as compared to untreated cells. The data of Figure 1 were obtained on GSC cells exposed to IL-1 p, TNFa or IFN-y alone at the concentration of 10ng / ml for 24h. While the exposure to TNFa in combination with IFN-y under the same conditions (concentration and time) reported a similar effect, the highest level of PD-L1 protein was reached when GSCs were exposed to the combination of the three cytokines.

[0060] These results confirm that I L-1 p in combination with TNFa and IFN-y is able to mimic the GBM microenvironment.

[0061] As show in Figure 2, BMP4 treatment strongly reduces the PD-L1 protein expression level as compared to untreated cells, when cells were not exposed to pro-immunoinflanmmatory citokines. This data demonstrates that BMP4 plays a critical role on the regulation of PD / PD- L1 axis immune response.

[0062] The effective role of BMP4 on GSCs exposed to the combination of the three cytokines was investigated, thus miming GBM TME.

[0063] Given the high intrinsic heterogeneity which characterizes GBM cases, three distinct sets of GSCs, isolated from three different GBM patients, were treated with IL-1 p cytokine in combination with TNFa and IFN-y.

[0064] While a heterogeneous sensitivity of GSC lines to the combination of TNFa with IFN-y and IL-1 p treatment was reported, enhanced PD-L1 levels as compared to untreated cells were observed (Figure 3a).

[0065] Importantly, when GSCs, irrespective of their subset, were exposed to IL-1 p in combination with TNFa and IFN-y and then treated for 48h with hrBMP4 (100ng / ml) PD-L1 protein expression was significantly inhibited, as compared to controls (Figure 3b-d). These findings demonstrated that BMP4, which promotes the commitment of GSC cells to differentiation, plays a critical role within GBM microenvironment through the modulation of the level of PD- L1 protein, that is one of the major and relevant component of the GBM TME.

[0066] Materials and Methods

[0067] GSC primary cell line culture

[0068] Fresh tissues from patients affected by GBM were obtained in accordance with research ethics board approval from the National Neurological Institute "C. Besta" and classified according to the World Health Organization guidelines.

[0069] Human GBM specimens after surgery resection were dissociated to a single cell by enzymatic digestion in papain at 37°C for 45 / 60 minutes (Worthington cat# LS003127). The cell suspension obtained was seeded in NeuroCult medium (Stemcell Technologies Inc. cat # 05751) supplement with 20ng / ml of EGF (Peprotech Inc. cat # PEP-AF-100-15) and 10 ng / ml of FGF2 (Peprotech Inc. cat # PEP-100-18B-C) and cultured in a humidified atmosphere at 37°C, 5% O2 and 5% CO2.

[0070] FACS analysis

[0071] To determine PD-L1 expression, 5x105cells / sample were cultured in the presence of IL- i p / TNFa / IFN-y (10ng / ml) for 24h and sequentially treated with hrBMP4 (100ng / ml) for 48h were collected and centrifuged. The following primary conjugated antibodies were employed for 30 min in the dark at 4°C: mouse anti-PD-L1 (CD274-APC). Cells were washed and exposed for 30 min at 4°C to goat anti-mouse R-PE-labeled (1 :500; Jackson Immunoresearch). After extensive washing, cells were analyzed by FACS. For quantification, a rainbow calibration particle mixture (8 peaks), 3.0-3.4 m (BD Biosciences) was employed, and the intensity of cell labeling was expressed as molecules of equivalent phycoerythrin (MEPE) or molecules of equivalent fluorescin (ME-FITC). Analyses were performed on a FACS Canto at 3 laser (BD Bioscience) and data were analyzed using Summit 4.3 software (Beckman Coulter, Brea, CA, USA). Background fluorescence was estimated by substituting primary antibodies with specific isotype controls. Measurement of autofluorescence was also routinely conducted for each condition tested.

[0072] From the above description and the above-noted examples, the advantage attained by the product described and obtained according to the present invention are apparent.

Claims

CLAIMS1 . A Bone Morphogenetic Protein 4 (BMP4) for use in the treatment of cancer, wherein said BMP4 is a human recombinant BMP4 protein (hrBMP4).

2. The BMP4 protein for use according to claim 1 , wherein said BMP4 inhibits the immune escape mechanism in a subject in need thereof.

3. The BMP4 protein for use according to claim 2, wherein said immune escape mechanism is due to the PD-1 / PD-L1 immune checkpoint inhibitors blockade.

4. The BMP4 protein for use according to any one of claims 1 to 3, wherein said cancer is selected from the group consisting of a Central Nervous System Tumor, glial cancer or a solid tumor.

5. The BMP4 protein for use according to any one of claims 4 or 5, wherein said cancer is glioblastoma multiforme (GBM).

6. The BMP4 protein for use according to any one of claims 4 to 6, wherein said glioblastoma multiforme is a grade 3 or 4 glioma and is a subtype selected from the group consisting of classical, mesenchymal and proneural.

7. The BMP4 protein for use according to any one of claims 4 to 7, wherein said cancer is driven by stem cells.

8. A pharmaceutical kit comprising: a) Human recombinant Bone Morphogenetic Protein 4 (hrBMP4); and a b) Checkpoint inhibitor, for simultaneous, separate or sequential use.

9. A pharmaceutical kit comprising: a) Human recombinant Bone Morphogenetic Protein 4 (hrBMP4); and a b) Checkpoint inhibitor,for use in the treatment of cancer.

10. The pharmaceutical kit for use according to claim 9, wherein said cancer is selected from the group consisting of a Central Nervous System Tumor, glial cancer or a solid tumor.

11. The pharmaceutical kit for use according to claims 9 or 10, wherein said cancer is glioblastoma multiforme (GBM).

12. The pharmaceutical kit for use according to any one of claims 9 to 11 , wherein said glioblastoma multiforme is a grade 3 or 4 glioma and is a subtype selected from the group consisting of classical, mesenchymal and proneural.

13. The pharmaceutical kit for use according to any one of claims 9 to 12, wherein said cancer is driven by stem cells.

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