Immunotherapy for the treatment of cancer

Abstract

The invention relates to improved immunogenicity of tumor cells by treating them ex vivo or directly in vivo with a combination of (i) a BET inhibitor and (ii) an RIG-I-like receptor agonist (RLR agonist) or a compound that induces expression of an endogenous RLR agonist (RLR agonist inducer).

Description

[0001] IMMUNOTHERAPY FOR THE TREATMENT OF CANCER

[0002] Field of the invention

[0003] The invention relates to the field of immunotherapy, in particular to methods, compounds and compositions for use in increasing the immunogenic potential of tumor cells.

[0004] Background of the invention

[0005] Recent years have seen a large increase in immunotherapeutic approaches for cancer, with notable clinical success being achieved by the use of antibodies blocking immune checkpoints molecules.1Highly encouraging results have also been obtained in cancer patients undergoing other types of immunotherapy, such as adoptive transfer of ex vivo activated T cells and various therapeutic vaccination strategies.2-7

[0006] Despite these substantial advances, many immunotherapies fail to induce clinical benefit in large groups of cancer patients, which creates an urgent need for ways to improve their efficacy. A major reason for the limited efficacy of cancer immunotherapies is that anti-tumor immunity is often rendered ineffective by immune evasion mechanisms employed by tumors. In this respect, a number of tumor-intrinsic deficiencies are key underlying causes of poor clinical responses to immunotherapy. Prominent deficiencies are defects in antigen processing and presentation by HLA class I molecules to tumor-specific CD8+ T cells.8-17Evidence indicates that such deficiencies, which can also be caused by defects in interferon signaling pathways, not only pre-exist in tumors and contribute to their tumorigenicity, but can also be acquired by tumors and lead to delayed relapses in cancer patients that initially experience clinical benefit in response to immunotherapy.17Thus, there is an urgent need for novel approaches aimed at promoting HLA class I-mediated antigen processing and presentation by tumor cells, as these would pave the way to more efficacious cancer immunotherapies.

[0007] Importantly, clinical benefit induced by cancer immunotherapies is associated with T cell-mediated recognition of tumor-specific antigens, in particular of neoantigens presented to CD8+ T cells.18Therefore, much effort is spent on development of immunotherapies that aim to direct immune responses at such neoantigens.19-22However, such immunotherapies strongly depend on procedures to identify, validate and select neoantigens in cancer cells that are very laborious, timeconsuming and costly. Moreover, it has proven challenging to accurately predict the immunogenicity of identified neoantigens as well as their ability to facilitate a functional and clinically relevant T cells response, due to the fact that the prediction algorithms used for this are suboptimal and not all factors that determine HLA- mediated presentation of neoepitopes and their functional immunogenicity towards T cells are known. Therefore, it would be highly desirable to develop therapeutic approaches that do not depend on such procedures for prediction and validation of tumor-specific (neo)epitopes. One attractive way of doing this is to use autologous tumor cells to directly stimulate the selective expansion of tumor-reactive T cells, and in particular CD8+ T cells. Such approaches would make use of the patient's own tumor cells as a direct source of relevant tumor antigens, in a manner that is antigen-agnostic (i.e. without knowing the relevant tumor antigens). The use of autologous tumor cells as a compound for the direct stimulation of anti-tumor immunity in cancer immunotherapy applications has been described before.23-28However, the ability of these approaches to provide adequate T-cell stimulation and promote clinical benefit has been very limited due to poor tumor immunogenicity, which is strongly determined by tumor antigenicity, and in particular by HLA class I-restricted presentation of tumor antigens. Indeed, it is long known that a mere increase in the peptide concentration presented by HLA class I on human tumor cells allows for the induction of a response by cytotoxic CD8+ T cells specific for that same peptide in an HLA class Il-independent manner.29In line with this, it has been shown that the strength of in vitro responsivity by tumor antigen-specific CD8+ T cells is correlated with the clinical efficacy of cancer immunotherapy.30,31Importantly, it has also been shown in vivo that merely enhancing the expression of MHC class I can lead to an increased clonal diversity of tumorinfiltrating lymphocytes (TIL), which suggests that this automatically leads to the presentation of a broader range of cancer antigens to T cells on the tumor cell surface.32Interestingly, the same observation was made in tumor cells in which antigen presentation by MHC class II to tumor-specific CD4+ T cells was lost or inhibited by targeting cathepsin S.33Overall, this indicates that approaches aimed at increasing the concentration of peptides derived from tumor antigens on HLA class I can be used as an in vitro or in vivo immunization strategy to elicit tumor-specific CD8+ T cell responses in an HLA class Il-independent manner.

[0008] WO2019063829 describes ex vivo and in vivo methods that use BET inhibitors (inhibitors of Bromodomain and Extra-Terminal domain proteins) for increasing expression and / or HLA class I-restricted presentation of neoantigens and / or tumor- associated antigens in autologous tumor cells to promote anti-tumor immune responses by CD8+ T cells, while reducing HLA class Il-mediated antigen presentation by tumor cells.

[0009] Summary of the invention

[0010] The present invention aims at providing efficacious and safe methods for cancer immunotherapy which involve using primary autologous tumor cells and treating these cells ex vivo or directly in vivo with a combination of (i) a BET inhibitor and (ii) an RIG- I-like receptor agonist (RLR agonist) or a compound that induces expression of an endogenous RLR agonist (RLR agonist inducer). The combination treatment leads to selective activation of tumor-specific CD8+ T cells in vivo or ex vivo.

[0011] As discussed in detail below, the inventors have surprisingly discovered that contacting tumor cells with a combination of a BET inhibitor and an RLR agonist or RLR agonist inducer has effects on the tumor cells that are beneficial for therapeutic reasons. In particular, as shown in the accompanying Examples, the combination of compounds enhances the expression of key molecular markers of tumor immunogenicity, such as HLA class I, interferon -beta (IFNP), the interferon-induced chemokine CXCL10 and calreticulin. Importantly, pretreatment of tumor cells with the same combination of compounds and subsequent exposure to autologous TIL leads to a much stronger activation of tumor-specific CD8+ T cells than pretreatment of tumor cells with a BET inhibitor or RLR agonist alone.

[0012] The inventors' findings therefore provide an attractive method for cancer immunotherapy, by targeting tumor-intrinsic molecular pathways to enhance tumor immunogenicity towards CD8+ T cells.

[0013] The present invention therefore provides new uses, methods and compositions for treating cancer based on the inventors' surprising findings regarding the effect of these compounds on tumor cells. In some aspects, the invention provides the direct in vivo administration of BET inhibitors and RLR agonists or RLR agonist inducers to patients with cancer. In further aspects, the invention provides approaches in which primary cancer cells or patient-derived cancer cell lines are exposed ex vivo to BET inhibitors and RLR agonists or RLR agonist inducers to enhance their immunogenicity, thereby making these cells suitable for use as an immunogenic vehicle to promote anti-tumor immune responses, for example by autologous tumor-cell-based vaccination or by promoting ex vivo expansion of tumor-specific CD8+ T cells that can be used for adoptive cell therapy. Accordingly, in a first aspect, the invention relates to an ex vivo method for obtaining a composition suitable for the treatment of cancer in a subject, comprising the steps of: a) providing primary tumor cells derived from the subject, and b) ex vivo contacting the tumor cells with

[0014] (i) an inhibitor of a bromodomain and extra-terminal domain family member (BET inhibitor) and

[0015] (ii) an RIG-I-like receptor agonist (RLR agonist) or a compound that induces expression of an endogenous RLR agonist (RLR agonist inducer), with the proviso that if the RLR agonist is poly(I:C), said poly(I:C) is transfected into the tumor cells.

[0016] The invention also relates to compositions obtainable or obtained by such a method and their use in the treatment of cancer.

[0017] Alternatively, the combination of BET inhibitor and RLR agonist or RLR agonist inducer can be administered in vivo to one or more tumor cell locations.

[0018] Thus, in a further aspect, the invention relates to a method for the treatment of cancer in a subject, the method comprising administering (i) a BET inhibitor and (ii) an RLR agonist or RLR agonist inducer to the subject.

[0019] The invention also relates to the ex vivo use of a combination of a BET inhibitor and a RLR agonist or RLR agonist inducer to increase the expression of tumor-specific antigens and / or tumor-associated antigens and / or the presentation of tumor-specific antigens and / or tumor-associated antigens by HLA class I in tumor cells.

[0020] Legends to the figures

[0021] Figure 1. Differentiation status of melanoma tumor cell lines. (A) Melanoma cells can be defined as melanocytic (MITFhi, AXL-, NGFR-), transitory (MITFhi, AXLhi, NGFR-), neural-crest like (MITFl0W, NGFRhi, AXLhi) or undifferentiated (MITF|OW, NGFR|OW, AXLhi). (B) Protein expression of MITF, AXL and NGFR in A375, ANRU, A375VR4and KADA melanoma cells assessed by flow cytometry. Figure l.C. MITF / AXL ratio is used to profile the differentiation status of cells, ranging from least differentiated (A375) to most differentiated (KADA). (D-E) Baseline expression of surface HLA class I (Figure l.D)and PD-L1 (Figure l.E) in melanoma cell lines.

[0022] Figure 2. Prolonged JQ1 treatment sensitizes tumor cells to poly(I:C) induced inflammation. (A) Experimental setup. (B) Heatmap (left) and bar graphs (right) of selected genes from qPCR after 72h JQ1 treatment and subsequent poly(I:C) transfection for 24h in ANRU and KADA. N = 3 (ANRU) or n = 1 (KADA) biological replicate. (C) Cell surface expression of HLA class I assessed by flow cytometry after 48h poly(I:C) stimulation preceded by either DMSO or JQ1 treatment, n >= 3 biological replicates. (D) Representative histogram overlay of flow cytometry staining from Figure 2.C in A375 cells. (E) HLA class I expression in A375VR4after JQ1 pretreatment and poly(I:C) stimulation. (F) Cell surface expression of HLA class I assessed by flow cytometry after A375 cells were subjected to pre-treatment with JQ1 alone for either 24, 48 or 72h and / or stimulation with naked or transfected poly(I:C) for 48h. (G) Cell surface expression of HLA class I on A375 cells pre-treated with JQ1 alone for 72h and / or subsequently stimulated with the specific RIG-I agonist 3pRNA for 48h. (H) Cell surface PD-L1 expression as assessed by flow cytometry staining after treatments described in Figure 3A in various melanoma cells. N = 3 biological replicates except KADA where n = 1 biological replicate. (I) Representative histogram of PD-L1 expression from A375 data shown in Figure 3H. Statistical analysis was performed using a one-way or two-way Anova. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0023] Figure 3. JQ1 treatment enhances poly(I:C)-induced immunogenic cell death. (A) Bar graphs from flow cytometry experiment showing Calreticulin expression (geoMFI, left) or frequency of Calreticulinhicells (right) in A375VR4expression treated with DMSO or JQ1 (2pM) and subsequent poly(I:C) transfection. (B) Representative histogram of one experiment from Figure 3. A. Calreticulin expression was gated on single cells (live and dead). (C) Flow cytometry blots exhibiting Dead Cell Marker and Calreticulin expression on A375VR4' Statistical analysis from performed using one-way Anova. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0024] Figure 4. Tumor treated with JQ1 promotes activation of cytotoxic TIL in melanoma when combined with dsRNA agonist. (A) Bar graph displaying IFNy secretion measured by ELISA after coculture of autologous TIL with both ANRU (left panel) or KADA (right panel) tumor cells after they were treated with DMSO, JQ1, Poly(I:C) or JQ1 + Poly(I:C) as depicted in Figure 2. A. (B) Flow cytometry analysis from coculture of TIL with treated ANRU and KADA tumor cells. Violin plot displays frequency of CD3+CD8+IFNy+CD107a+ in ANRU and KADA TIL (left panel) and frequency of CD3+CD8+TNFa+ in KADA TIL (right panel).

[0025] Statistical analysis from performed using one-way or two-way Anova. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. Figure 4 continued. Treatment of melanoma cells with JQ1 in combination with an RLR agonist strongly increases the tumor cell surface-expressed HLA class I / II ratio and coincides with a strongly elevated ratio of IFNy+ CD8 / CD4 T cells in autologous tumor-reactive TIL. (C) Bar graph displaying HLA class I (left graph), HLA class II (center graph) and HLA I / HLA II ratio (right graph) at the cell surface of ANRU tumor cells after DMSO or JQ1 pretreatment and subsequent IFNy or poly(I:C) stimulation for 48h (D) Ratio of HLA I / HLA II expression at the cell surface of ANRU tumor cells pretreated with DMSO or JQl and subsequently stimulated for 48 h with IFNy or poly(I:C). (E) Flow cytometry analysis from coculture of autologous TIL with treated ANRU tumor cells. Violin plot displays frequency of CD3+CD8+IFNy+ in ANRU TIL (left graph), frequency of CD3+CD4+IFNy+ in KADA TIL (center graph) and ratio of CD8+IFNy+ and CD4+IFNy+ TIL. (F) Proportion of CD8+ / CD4+ IFNy+ T cells in ANRU TIL after coculture with JQ1- and / or poly(I:C)- pretreated ANRU tumor cells. Statistical analysis was performed using one-way Anova. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0026] Figure 5. (A) Overview of experimental setup for using. DMSO was used as solvent for both JQ1 and iBET151. (B) Relative expression of genes involved in innate antiviral responses to cytosolic dsRNA after 72h JQ1 pretreatment followed by 24h of poly(I:C) transfection of human melanoma cell lines. Values indicate fold changes in gene expression relative to tumor cells pretreated with DMSO for 72h and subsequently left untreated for 24h. (C) Relative expression of genes involved in innate antiviral responses to cytosolic dsRNA after 72h JQ1 pretreatment followed by 24h of poly(I:C) transfection of the human melanoma cell line A375 in three independent experiments. Values indicate fold changes in gene expression relative to tumor cells pretreated with DMSO for 72h and subsequently left untreated for 24h. (D) Cell surface expression of HLA class I measured by flow cytometry on the human melanoma cell lines A375 and ANRU pretreated for 72h with iBET151 and / or transfected with poly(I:C) for 48h. (E) Cell surface expression of HLA class I and MHC class I on the human colorectal cancer cell line COLO320 and the murine colorectal cancer cell line MC38, respectively, after treatment for 72h with iBET151 and / or poly(I:C). (F) MAVS expression after 72h culture of tumor cell lines in the absence and presence of JQ1 only, as detected by flow cytometry. Abbreviations: pIC = poly(I:C); HLA I = HLA class I = HLA-ABC; MHC I = MHC class I; MSF = Myxofibrosarcoma; MPNST = Malignant Peripheral Nerve Sheath Tumor; *** p < 0.001; **** p < 0.0001. Figure 6. (A) Volcano plot showing a transcriptomic (RNAseq) analysis of ANRU tumor cells. The plot indicates genes significantly upregulated (right upper section) or downregulated (left upper section) in melanoma cells pretreated with JQ1 and subsequently transfected with poly(I:C) in comparison to cells transfected with poly(I:C) only. Note the upregulation of key genes involved in innate antiviral signaling induced by dsRNA, such as CXCL10, CXCL11, HLA-A and HLA-B, confirming our flow cytometry and qPCR data. (B) Plots showing highly significant correlations between expression of the top 100 significantly genes most strongly upregulated by JQ1 and poly(I:C) in the transcription analysis shown in figure 6A and expression of the 281 genes that are downregulated in immune-excluded tumors (as identified by Jerby- Anon et al.) in melanoma and all cancers. Each dot represents a cancer patient and its average expression level of the 100 genes most strongly upregulated genes in our analysis versus the average expression of 281 downregulated tumor-specific genes in the immune exclusions signature. TPM = Transcripts Per Million.

[0027] Figure 7. (A) Overview of experimental setups to assess the effect of combined JQ1 and poly(I:C) treated on activation of autologous tumor-specific tumor-infiltrating lymphocytes in an in vitro co-culture model. (B) IFNy levels, measured by an ELISA, in the supernatant of autologous TIL co-cultured with melanoma cells treated with the indicated conditions. The co-culture was done in the absence and presence of an antibody blocking all HLA class I types on the tumor cell surface. (C) and (D) Frequencies of CD8+ TIL positive for IFNy, TNFa and CD107a. (E) IFNy levels, measured by an ELISA, in the supernatant of autologous TIL co-cultured with melanoma cells treated with the indicated conditions. The co-culture was done in the absence and presence of an antibody blocking all HLA class I types. (F) and (G) Frequencies of CD8+ TIL positive for IFNy, TNFa and CD107a.

[0028] Figure 8. (A) Flow cytometry staining using MHC dextramers and antibodies for basic T cell markers of unstimulated ANRU TIL indicating the presence of a clear subpopulation of CD8+ T cells specific for the melanoma-associated antigen MART-1 and the neoantigen NUP210. (B) Flow cytometry staining as used in figure 8A treatment of tumor cells with JQ1 and / or poly(I:C) and subsequent coculture with autologous TIL. (C) Quantification of the frequencies of IFNy+ CD107a+ CD8+ TIL specific for either MARTI or NUP210 from three independent experiments done as in figure 8B.

[0029] Figure 9. IFNy levels, measured by an ELISA, in the supernatant of autologous ANRU TIL co-cultured with ANRU tumor cells treated with the indicated conditions JQ1 and / or poly(I:C). The co-culture was done in the absence and presence of an antibody blocking PD-L1.

[0030] Figure 10. (A) Flow cytometry data indicating expression of HSP90, calreticulin and death cell marker (DCM) on A375 cells treated with JQ1 and / or poly(I:C). (B) Quantification of the frequencies of HSP90hiand calreticulinhicells in multiple independent experiments done as depicted in figure 10A. (C) Graphical overview of the experimental setup used for studying to study if combined JQ1 and poly(I:C) treatment of tumor cells could lead to improved DC activation by promoting immunogenic cell death or the secretion of soluble DC-activating factors. (D) and (E) Quantified expression from four independent experiments of DC activation markers expressed at the DC surface after being co-cultured with A375 cells treated with JQ1 and / or poly(I:C). (F) and (G) Quantified expression from four independent experiments of DC activation markers expressed at the DC surface after co-culture with supernatant from A375 cells treated with JQ1 and / or poly(I:C). iDC = immature dendritic cells; * p < 0.05; *** p < 0.001; **** p < 0.0001

[0031] Figure 11. Frequency of activated IFNy+ CD8+ TIL as determined by flow cytometry after coculture with autologous tumor cells treated with JQ1 and / or poly(I:C).

[0032] Detailed description of the invention

[0033] This invention addresses the problem that certain tumors cannot efficiently induce an immune response by cytotoxic tumor-specific CD8+ T cells. This problem is overcome by the present invention wherein a patient is treated with a combination of a BET inhibitor and a RLR agonist or RLR agonist inducer or wherein a primary tumor cell is isolated from the tumor, or a tumor cell line is derived from the tumor, and incubated in vitro or ex vivo with a combination of a BET inhibitor and a RLR agonist or RLR agonist inducer. The resulting cell composition can then induce an effective T-cell immune response in the donor of the primary tumor cells when administered to that subject in a subsequent step.

[0034] It will be appreciated by those skilled in the art that therapeutic immune responses can be induced by vaccination against various tumor antigens, even though initially (i.e. before treatment) the target antigen in untreated individuals is not recognized, or at least not recognized efficiently enough to elicit a protective antitumor immunity. This is supported by observation of the so-called abscopal effect, which is the phenomenon that localized treatment of a tumor in metastatic cancer not only leads to regression of the treated tumor, but also of tumors outside the scope of the localized treatment. This effect has been described after intratumoral application of a variety of immunotherapies, indicating that activation of tumor antigen-specific T cells locally can lead to systemic T cell-mediated tumor eradication.34-39Such treatment can greatly improve the immune response to tumor cells and provides a useful tool to replace or be used in combination with existing immunotherapies.

[0035] As the ex vivo method of the invention does not require the systemic or local administration of BET inhibitors to a subject, it eliminates any potential problems associated with toxicity or adverse responses to the inhibitors, as well the problem that the inhibitors may not reach the tumor effectively. At the same time, in vivo administration of BETi orally and / or intravenously and / or intratumorally and / or in any other way is still of high relevance as it may enhance the efficacy of other immunotherapies used to treat the patient, such as checkpoint blockade or adoptive transfer of TCR transgenic T cells or CAR T cells.

[0036] Thus, the invention relates to an ex vivo method for obtaining a composition suitable for the treatment of cancer in a subject, comprising the steps of: a) providing primary tumor cells derived from the subject, and b) ex vivo contacting the tumor cells with

[0037] (i) an inhibitor of a bromodomain and extra-terminal domain family member (BET inhibitor) and

[0038] (ii) an RIG-I-like receptor agonist (RLR agonist) or a compound that induces expression of an endogenous RLR agonist (RLR agonist inducer), with the proviso that if the RLR agonist is poly(I:C), said poly(I:C) is transfected into the tumor cells.

[0039] Treating the tumor cells derived from a subject in the way as described above, strongly enhances HLA class I-mediated antigen presentation by the autologous tumor cells. When such treated cells are administered to the subject, a tumor-specific CD8+ T-cell response is induced, making such cells very suitable to be used in a subject to elicit a tumor-specific immune response.

[0040] It will be appreciated that the term "contacting" in the present context implies that the cells are in contact with the compounds for a sufficiently long time to have a functional effect on the tumor cells. The contacting step typically includes incubating the cells with the compounds in an appropriate solution or medium.

[0041] Typically, the step of providing primary tumor cells does not include steps practiced on the human or animal body, such as a surgical step practiced on the human or animal body. It will be appreciated that the inventors' surprising finding that the combination of compounds is able to greatly enhance HLA class I-mediated antigen presentation in tumor cells, and that this effect is much stronger than that of either compound alone, is not only applicable to primary tumor cells directly derived from the subject, but can be used to enhance HLA class I-mediated antigen presentation in tumor cell lines, such as tumor cell lines derived from primary tumor cells of the subject. Accordingly, where the present invention refers to methods, uses and / or compositions involving primary tumor cells derived from a subject, this also includes tumor cell lines, tumor organoids and other 3D tumor cultures derived from the subject.

[0042] In one embodiment, the cells are cultured in vitro in order to increase the number of cells before step b) is performed.

[0043] In the context of the invention the term "composition suitable for the treatment of cancer in a subject", should be interpreted as a composition capable of triggering an anti-tumor immune response when administered to the subject. Preferably the antitumor response is the induction of a tumor-specific CD8+ T-cell response. Preferably such a response is induced by the increased HLA class I-mediated antigen presentation on the tumor cells of the composition according to the invention.

[0044] In one embodiment, the ex vivo method for obtaining a composition suitable for the treatment of cancer in a subject, comprises a step c) of obtaining the cell composition. In a further embodiment, the ex vivo method is followed by a step d) of administering the cell composition obtained to the subject.

[0045] It will be appreciated that the contacting of the tumor cells with the BET inhibitor and the RLR agonist (or RLR agonist inducer) in step b) may in principle be performed in any order - for example, the BET inhibitor may be added before and / or concurrently with and / or after, the RLR agonist (or RLR agonist inducer).

[0046] However, in a preferred embodiment, said tumor cells are first contacted with the BET inhibitor in step b) and subsequently with the RLR agonist or RLR agonist inducer.

[0047] In a further preferred embodiment, the BET inhibitor is removed from the cell culture, for example by washing, prior to contacting the cells with the RLR agonist or RLR agonist inducer.

[0048] Preferably, the BET inhibitor is provided to the tumor cells ex vivo for a time sufficient to allow the cells to upregulate the expression of tumor-specific antigens and / or tumor-associated antigens and / or to increase the presentation of tumorspecific antigens and / or tumor-associated antigens by HLA class I on their surface, such as for instance at least 6 hours, more preferable at least 12 hours, more preferably at least 24 hours, even more preferably at least 36, even more preferably at least 48 hours, even more preferably at least 72 hours. In some embodiments, the duration of the incubation with the BET inhibitor is less than 28 days, such as less than 14 days.

[0049] When used herein, the term "tumor-specific antigens" refers to antigens that are expressed on tumor cells, but not on healthy somatic cells, and that are derived from mutated self-antigens (neoantigens) or are derived from viruses, such as human papilloma virus or Epstein-Barr virus).

[0050] The term "tumor-associated antigens" refers to antigens that are preferentially, but not necessarily exclusively, expressed by tumor cells.

[0051] Preferably, the RLR agonist or RLR agonist inducer is provided to the tumor cells ex vivo for at least 4 hours, such as at least 8 hours, or at least 24 hours, at least 48 hours, at least 60 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days. In some embodiments, the duration of the incubation with the RLR agonist or RLR agonist inducer is less than 28 days, such as less than 14 days.

[0052] In a preferred embodiment, step b) comprises the following sub-steps: bl) contacting the tumor cells with the BET inhibitor for at least 24 hours, more preferable at least 48 hours, more preferably at least 72 hours, b2) removing the BET inhibitor from the cells, for example by washing, and b3) contacting the tumor cells with the RLR agonist or RLR agonist inducer is provided to the tumor cells ex vivo for at least 12 hours, such as at least 24 hours, at least 36 hours or at least 48 hours.

[0053] In one embodiment, the tumor cells further are contacted with an immunostimulatory compound, preferably interferon-alpha (IFNa), IFNp or IFNy, in step b), preferably prior to contacting the tumor cells with the RLR agonist or RLR agonist inducer, most preferably the tumor cells are contacted with the BET inhibitor and the immunostimulatory compound simultaneously.

[0054] In one embodiment, the method of the invention comprises a further step of transducing the tumor cells with one or more genes encoding co-stimulatory molecules, such as CD80, CD86 or CD70 or encoding immuno-stimulatory cytokines of the gamma C family, CD83 or dendritic cell or T-cell attracting chemokines. This would make the tumor cells even more similar to professional antigen-presenting cells (e.g. dendritic cells, macrophages and B cells) that can directly prime and activate CD8-positive T cells by cross-presenting antigens on HLA class I in the presence of adequate co-stimulation.

[0055] BET inhibitors

[0056] Any suitable BET inhibitor may be used in the present invention. BET inhibitors (BETi) are a class of drugs with anti-cancer, immunosuppressive, and other effects in clinical trials and are widely used in research. These molecules reversibly bind the bromodomains of Bromodomain and Extra-Terminal motif (BET) proteins, such as BR.D2, BR.D3, BR.D4, and BRDT. They are thought to prevent protein-protein interaction between BET proteins and acetylated histones and transcription factors. Since 2010, a number of molecules have been described that are capable of targeting BET bromodomains.

[0057] Tumor cells, like all cells, use the HLA class I complex to present antigens on the surface. As mutations accumulate in the tumor cells, some of these antigens will include neoantigens. Such neoantigens can be detected as non-self by T-cells, such as CD8+ T-cells, resulting in an immune response targeted specifically at the tumor cells. Tumors are known to develop ways to avoid such immune responses; one such methods is by upregulating PD-L1, which suppresses HLA-mediated T-cell activation.

[0058] Small-molecular BET inhibitors are interesting in this respect, as they were recently found to strongly suppress constitutive and inducible PD-L1 expression in various tumor mouse models, as well as on various human cancer cell lines.40,41

[0059] BET inhibitors are a relatively new class of anti-cancer drugs that were originally developed for use as a targeted therapy to inhibit tumor growth. These effects were described first for the BET inhibitor JQ1 in NUT midline carcinoma, a rare malignancy characterized by overexpression of the BET protein BRIM, one of the main BET inhibitor targets, along with BRD2 and BRD3.42Later studies reported comparable effects of JQ1 and the BET inhibitors I-BET151 and I-BET762 in several other types of cancer, including Myc-driven hematological malignancies, melanoma and neuroblastoma.43-45

[0060] BRD4 is thought to be the main target in all these studies, while the effects on Myc-driven tumors are also attributed to Myc-downregulation.46,47As a consequence of these results, various BET inhibitors are now in early clinical development as antiproliferative agents for both solid tumors and hematological cancers. Thus far, this has indicated that BET inhibitors can indeed exert anti-tumor activity with manageable and reversible toxicity in patients.48However, despite the promising results obtained with BET inhibitors as a potential treatment for cancer, several disadvantages still need to be overcome. For example, systemic administration of a BET inhibitor may be toxic, or result in unwanted effects such as autoimmune responses. Further, depending on the tumor type, BET inhibitors may not reach the tumor in sufficient amounts to exert their effect. Lastly, it is known that not all tumor types respond to treatment with BET inhibitors.

[0061] To this effect, the inventors have developed a method of isolating primary tumor cells from a subject with cancer, and treating those primary tumor cells ex vivo with a combination of a BET inhibitor and an RLR agonist or RLR agonist inducer. In this way, primary tumor cells with strongly increased antigen presentation by HLA class I are obtained. Such cells are useful as they may be administered to the subject to generate an effective tumor-specific CD8+ T-cell response.

[0062] In the context of the invention, the term tumor-specific T-cell response should be interpreted as a T-cell mediated immune response in vivo specifically directed at the tumor cells. As treatment with the combination of compounds results in increased antigen presentation by HLA class I in the tumor cells in vitro or ex vivo, this results in an increased presentation of tumor-specific antigens. Such tumor specific antigens have been demonstrated to elicit an immune response which is mediated by T-cells recognizing non-native tumor specific antigens.

[0063] In the context of the invention, the term "ex vivo" is to be understood as taking place outside of the subject from which the tumor cells are derived from. The subject may be an animal or a human, the subject may also be a non-human mammal. The subject may have a cancer or a carcinoma, a solid tumor or a hematological cancer. As used herein, the term "patient" refers to a subject undergoing treatment or examination by a medical or veterinary professional.

[0064] Primary tumor cells may be isolated by identifying a subject with cancer and removing a subset or a substantial part, or even all of the tumor cells from the subject. Methods for diagnosing a subject with cancer, identifying the tumor in said subject and removing part or all of the tumor are generally known to the skilled person. Nonlimiting examples of suitable methods for isolating tumor cells from a subject are by surgery, biopsy or blood withdrawal. The skilled person will be aware of other methods of obtaining tumor cells and the most suitable method for doing so, depending on the tumor type. Once tumor cells are isolated from the subject, they may optionally be separated from impurities such as non-tumor cells or extracellular material. Preferably, the tumor cells are kept in a suitable culture medium once isolated from the subject. However, it is also envisioned in the invention that the tumor cells are stored for later use, for example by freezing the cells or keeping the cells in a suitable storage medium.

[0065] In the context of the invention, the term "primary tumor cells" should be interpreted as cells that are isolated from a tumor in a subject and prepared for ex vivo treatment without substantially modifying the tumor cells, such as modifying the genome to generate immortal cell lines.

[0066] It is to be understood that the primary tumor cells may be expanded after isolation from the subject and before the step of contacting them with the combination of a BET inhibitor and an RLR agonist or RLR agonist inducer. Methods for expanding primary tumor cells ex vivo and in vitro are well known to the skilled person.

[0067] BET inhibitors are known to the skilled person to be able to block, inhibit or down regulate the function of BET motif containing proteins. Non-limiting examples of BET domain containing proteins are the BRD2, BRD3, BRD4, and BRDT proteins. BET inhibitors have been described in the art and are well known to the skilled person. When used herein therefore the term BET inhibitor refers to any compound, molecule or composition capable of blocking, inhibiting or down regulating BET domain containing proteins. Non-limiting examples of classes of BET inhibitors according to the invention are small molecules, antisense nucleotides and antibodies.

[0068] Small molecule BET inhibitors are generally able to reversibly bind the bromodomains of BET proteins, and prevent protein-protein interaction between BET proteins and acetylated histones and transcription factors.

[0069] Preferred examples of small molecule BET inhibitors according to the invention are BET inhibitors selected from the group consisting of: JQ1, MK-8628, BMS-986158, ABBV- 075, CPI-0610, FT-1101, GS-5829 (Alobresib), GSK525762, PLX51107,

[0070] R06870810 / Ten-010, OTX-015, CPI-0610, 3-methyl-l,2,3,4-tetrahydroquinazolin-2- ones, 2-thiazolidinones, CPI-203, I-BET762 (GSK525762A), I-BET151 (GSK1210151A), AZD5153, AZD-5153 6-hydroxy-2-naphthoic acid, BET-d246, Dinaciclib, BAY1238097, CC-90010, ODM-207, BI 894999, ZL0420, ZL0454, ARV-771, ARV-825, A1874, BET-d260, Thalidomide-NH-C4-NH-Boc, GSK046, GSK620, GSK778, BRD4 inhibitor-10, Y06036, MS436, XMD8-92, BRD4 Inhibitor-14, BRD4 Inhibitor-16, BET-IN-1, BET-IN-2, BET-IN-7, BET-IN-8, BET-IN-9, BET-IN-10, BET-IN-12, BET-IN- 13, BET-IN-14, BET-IN-15, BET-IN-16, BET-IN-17, BET-IN-19, BET-IN-20, BET-IN-21, RVX-297, NEO2734, Bromosporine, dBET57, BI 2536, RVX208 (Apabetalone), PFI, ZEN-3694, ZEN-3219, ZEN-3411, ZEN-3862 dBETl, dBET6, MZ-1, UNC6349, BET bromodomain inhibitor 1, BET bromodomain inhibitor 2, BET-BAY 002, NVS-BET-1, I- BET-432, PROTAC BET degrader-1, PROTAC BET degrader-2, PROTAC BET degrader- 3, PROTAC BET Degrader-10, I-BET282, I-BET282E, GSK040, I-BET762 carboxylic acid, I-BET726 (GSK1324726A), Physachenolide C, SDR-04, PROTAC BRD2 / BRD4 degrader-1, DW71177, OXFBD04, SJ1461, (S)-GNE-987, SB-284851-BT,

[0071] Amredobresib, GNE-987, INCB054329, INCB054329 Racemate, TD-428, INCB- 057643, RX-37, CF-53, ET-JQ1-OH, GSK023, Bromodomain inhibitor-8, QCA570, dBRD4-BDl, MT1, CD161, CD235, Rac)-BAY1238097, XD14, GSK217, GSK737, GSK852, TC AC 28, HJB97, Y06137, PROTAC BRD4 ligand-1, PROTACT GNE-987, PNZ5, DDO-8926, (+)-JQ-l-aldehyde, OARV-771, GS-626510, BAY1238097, PROTAC BRD3 / BRD4-L degrader-2, BY27, (+)-JQl PA, JQ1-TCO, SIM1, GSK097, BRD4 Dl-IN- 1, BRD4 Dl-IN-12, MS645, MS417, (R)-BAY1238097, GSK620, ABBV-744, dBET23, (E / Z)-ZL0420, ZEN-2759, LT052, SNIPER(BRD)-1, I-CBP112 hydrochloride, XP-524, XY153, MS402, GXH-II-052, GSK973, NC-III-49-1, AT 1. Preferably, the BET inhibitor is selected from the group consisting of JQ1, OTX-015, I-BET151 (GSK1210151A), I- BET762 (GSK525762A) and MZ1.

[0072] Specially preferred are BET inhibitors selected from the group consisting of JQ1, I-BET151 (GSK1210151A), and I-BET762 (GSK525762A). It is further understood that the invention also includes chemical modifications of the small molecule BET inhibitors as described herein. In a more preferred embodiment of the invention the small molecule BET inhibitor is selected from the group consisting of JQ1, MK-8628, BMS- 986158, INCB054329, ABBV-075, CPI-0610, FT-1101, GS-5829, GSK525762, PLX51107, Ten-010, OTX-015, and CPI-0610. However, it is further envisioned that the BET inhibitor may be a novel or previously unknown small molecule BET inhibitor. BET inhibitory activity of a compound can easily be assessed by the skilled person, for example using pull-down techniques with a BET motif protein and detection of binding of the compound to the BET motif protein.

[0073] Alternatively, the BET inhibitor according to the invention may be an antisense gene silencer, such as but not limited to siRNA, miRNA, CRISPRs and TALENs. An antisense oligonucleotide may comprise deoxyribonucleic acids and / or ribonucleic acids and / or artificial nucleotides such as a nucleotide analogue, the nucleotides may comprise natural bases, adenine, cytosine, guanine, thymine, uracil, or non-natural bases. The oligonucleotide may also comprise a modified backbone. Non-limiting examples of nucleic acid analogous with a modified backbone include peptide nucleic acid (PNA), Morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). The skilled person will be aware of protocols and design strategies in order to effectively achieve downregulation of BET domain containing proteins.

[0074] Ex vivo contacting the primary tumor cells with a BET inhibitor in the context of the method of the invention is to be understood as providing the BET inhibitor to the cells in an amount and for a time period sufficient to induce a response, the response preferably being increased antigen presentation by HLA class I.

[0075] Preferably, the BET inhibitor is provided to the tumor cells ex vivo for a time sufficient to allow the cells to express tumor-specific antigens and / or tumor-associated antigens on their surface, such as for instance at least 6 hours, more preferable at least 8 hours, more preferably at least 10 hours, even more preferably at least 12, even more preferably at least 18 hours, even more preferably at least 24 most preferably at least 30 hours.

[0076] Preferably, the BET inhibitor is provided to the tumor cells ex vivo in a concentration between 1 and 10.000 times the IC50 value, preferably between 5 and 2000 times the IC50 value, more preferably between 10 and 1000 times the IC50 value, most preferably the concentration is between 25 and 250 times the IC50 value of said BET inhibitor for the BET domain containing protein.

[0077] In the case of JQ1, the IC50 values for the first and second bromodomain are 77nM and 33nM respectively, therefore, the concentration of JQ1 when used as a BET inhibitor in the method of the invention is preferably between 50 nM and 500 pM, more preferably between 250 nM and 100 pM, more preferably between 500 nM and 50 pM, most preferably the concentration of JQ1 when used in the method according to the invention is between 1 pM and 10 pM. Even more preferably the concentration is between 0.1 pM and 10 pM.

[0078] It is to be understood that the concentration or duration of treatment with the BET inhibitor may be adjusted for each individual BET inhibitor, the skilled person will be aware that he may do so for example using the individual IC50 or Ki values as a starting point to adjust the concentration of the BET inhibitor used in the method of the invention.

[0079] RLR agonists and RLR agonist inducers

[0080] Any suitable RLR agonist or RLR agonist inducer may be used in the present invention.

[0081] In some embodiments, step b) of the ex vivo method of the invention comprises contacting the tumor cells with an RLR agonist. When used herein the term "RLR agonist" refers to a compound or molecule that is capable of activating a human RIG-I-like receptor. RIG-I-like receptors include RIG-I itself (UniProt: 095786 ■ RIGI_HUMAN), MDA5 (also termed IFIH1 (UniProt: Q9BYX4 ■ IFIH1_HUMAN)) and LGP2 (also termed DHX58 (UniProt: Q96C10 ■ DHX58_HUMAN)).

[0082] In one embodiment, the RLR agonist is a RIG-I agonist. In one embodiment, the RLR agonist is an MDA5 agonist. In one embodiment, the RLR agonist is an LGP2 agonist. The RLR agonist may also be an agonist of two or more RIG-I-like receptors, for example be a RIG-I agonist and an MDA5 agonist.

[0083] In one embodiment, the RLR agonist is transfected into the tumor cells. When used herein, the term "transfection" also encompasses introduction of nanoparticles into cells as well as other methods of delivering exogenous agonists to receptors located in the cytosol. Transfection can for example be carried out using calcium phosphate (i.e. tricalcium phosphate), by electroporation, by cell squeezing, or by mixing a cationic lipid with the material to produce liposomes that fuse with the cell membrane and deposit their cargo inside. In one embodiment, the RLR agonist is transfected into the cytosol of tumor cells.

[0084] In one embodiment, the RLR agonist is introduced into the tumor cells via nanoparticles, such as lipid nanoparticles or polymer-based nanoparticles.

[0085] The RLR agonist may for example be double-stranded polyribonucleotide, double-stranded RNA, single-stranded RNA, or an oncolytic RNA virus, optionally in live-attenuated form or in the form of a virus-like particle. In one embodiment, the RLR agonist is a polyinosinic-polycytidylic acid (poly(I:C)), 5' triphosphate doublestranded RNA (also called 3pRNA or 5'ppp-dsRNA), 5' triphosphate hairpin RNA (3p- hpRNA), poly(deoxyadenylic-deoxythymidylic) (poly(dA:dT)) or a triphosphorylated stem-loop RNA (e.g. SLR10). The RLR agonist may for example also be a small molecule (e.g. KIN1148).

[0086] In one embodiment, the poly(I:C) is provided in the form described in WO17085228 (incorporated by reference), e.g. in the form of an aqueous composition comprising particles wherein

[0087] (i) each of said particles comprises a complex of at least one poly(l:C), or a salt or solvate thereof, and at least one linear polyalkyleneimine, or a salt and / or solvate thereof, wherein the average molecular weight of said linear polyalkyleneimine is between 17 and 23 kDa; (ii) at least 90 percent of said particles has a mono-modal diameter distribution below 300 nm;

[0088] (iii) said particles have a z-average diameter of less than or equal to 150 nm, as measured according to ISO 22412; and

[0089] (iv) said composition has a zeta potential equal or superior to 30 mV, according to ISO 13099.

[0090] In one embodiment, the RLR agonist is not a TLR3 agonist. In another embodiment, the RLR agonist is not a TLR agonist.

[0091] Preferably, the tumor cells are incubated for at least 4 hours, such as at least 8 hours, or at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days with the RLR agonist.

[0092] In some embodiments, step b) of the ex vivo method of the invention comprises contacting the tumor cells with an RLR agonist inducer.

[0093] When used herein the term "RLR agonist inducer" refers to a compound or molecule that is capable of inducing expression of an endogenous RLR agonist. It is well-known that endogenous dsRNAs may be derived from endogenous retroviral elements and that such expression of such endogenous dsRNAs may be induced by contacting cells with compounds, such as DNMT inhibitors (e.g. 5-aza-deoxycitidine), HDAC inhibitors (e.g. entinostat), CDK4 / 6 inhibitors (e.g. Palbociclib), p53 activators (e.g. Nutlin).49-54

[0094] In one embodiment, the RLR agonist inducer is transfected into the tumor cells. In one embodiment, the RLR agonist inducer is introduced into the tumor cells via nanoparticles, such as lipid nanoparticles or polymer-based nanoparticles.

[0095] Preferably, the tumor cells are incubated for at least 4 hours, such as at least 8 hours, or at least 24 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days with the RLR agonist inducer.

[0096] It is envisioned that the method according to the invention may be used to obtain a composition suitable for the treatment of cancer, wherein the cancer is a solid tumor or a hematological cancer. Preferably, the cancer is a melanoma or a colorectal cancer.

[0097] Solid tumors are also referred to as neoplasms and encompass the vast majority of tumor types, typically all tumors that are not considered hematological cancers. Hematological cancers are also known to the skilled person as tumors of the hematopoietic and lymphoid tissues, and comprise leukemias, lymphomas and myelomas.

[0098] The method of the invention may be used to or provide a composition suitable for treating cancer in a subject wherein the cancer is selected from the list consisting of Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers Kaposi Sarcoma, AIDS-Related Lymphoma, Primary CNS Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid / Rhabdoid Tumor, Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Tumors, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Childhood Carcinoid Tumors, Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System Atypical Teratoid / Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Childhood Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumors, Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Heart Tumors, Hepatocellular (Liver) Cancer, Langerhans Cell Histiocytosis, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma (Soft Tissue Sarcoma), Kidney (Renal Cell) Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (Non-Small Cell and Small Cell), Lymphoma, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Cancer, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplastic Syndromes, Chronic Myelogenous Leukemia (CML), Acute Myeloid Leukemia (AML), Chronic Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Lip and Oral Cavity Cancer, Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm / Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Recurrent Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Rhabdomyosarcoma, Childhood (Soft Tissue Sarcoma), Salivary Gland Cancer (Head and Neck Cancer), Sarcoma (such as myxofibrosarcoma or a malignant peripheral nerve sheath tumor), Childhood Vascular Tumors (Soft Tissue Sarcoma), Ewing Sarcoma (Bone Cancer), Kaposi Sarcoma (Soft Tissue Sarcoma), Osteosarcoma (Bone Cancer), Uterine Sarcoma, Sezary Syndrome (Lymphoma), Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Nasopharyngeal Cancer, Oropharyngeal Cancer, Hypopharyngeal Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Ureter and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer, Endometrial Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Vascular Tumors (Soft Tissue Sarcoma), Vulvar Cancer, and Wilms Tumor.

[0099] In order to effectively exploit the method according to the invention, care may need to be taken that the composition is not tumorigenic when administered to the subject.

[0100] Hence, the invention relates to a method as described above, wherein the composition is treated to prevent it from being tumorigenic in vivo before administering it to the subject.

[0101] Preferably therefore the composition comprising the primary tumor cells is treated in such a way that the cells contained in the composition cannot proliferate when administered to a subject in vivo. Examples of such treatment method are subjecting the cells to ionizing radiation, or subjecting the cells to mitotic inhibitors or cell cycle inhibitors, but other methods are known to the skilled person.

[0102] In a preferred embodiment of the invention, a method is provided wherein the composition is treated to prevent it from being tumorigenic in vivo by subjecting them to ionizing radiation. Preferably, the cells are subjected to at least 20 Gy, more preferably to at least 30 Gy, even more preferably at least 40 Gy, most preferably 50 Gy or more.

[0103] In an even further preferred embodiment of the invention, a method is provided wherein the method additionally comprises a step of ex vivo contacting the tumor cells with T-cells or dendritic cells obtained from the subject.

[0104] It is envisioned that the tumor cells treated according to the invention may be used for ex vivo expansion of T-cells (such as autologous T-cells) or dendritic cells, or to promote presentation of tumor antigens on dendritic cells. An advantage of this preferred method is that the T-cells or dendritic cells are activated in the composition and the method can therefore be used for treating cancer in the subject.

[0105] This method can also be used to generate T-cells and / or dendritic cells suitable for immunotherapy of a patient, or it can also be used to obtain a combination of tumor cells and / or T cells and / or dendritic cells suitable for the immunotherapy of cancer.

[0106] Therefore, in a further preferred embodiment of the invention, an ex vivo method is provided for obtaining a composition suitable for the treatment of cancer in a subject, wherein the method additionally comprises a step of ex vivo contacting the tumor cells with T-cells or dendritic cells obtained from the subject, and wherein the composition comprises T-cells, ex vivo expanded T-cells, or dendritic cells or a combination thereof, ex vivo treated with a the combination of a BET inhibitor with an RLR agonist or RLR agonist inducer.

[0107] The method according to the invention may be particularly advantageous for tumor cells that HLA class I-negative tumor cells and / or PD-Ll-negative tumor cells and / or HLA class Il-positive tumor cells.

[0108] In the context of the invention, the term "HLA class I negative or PD-L1 negative" should be interpreted as that the protein expression of HLA class I or PD-L1 (for example, when using immunostaining with an antibody that specifically recognizes these proteins, for example to perform immunohistochemistry or flow cytometry), is not upregulated and / or is not detectable.

[0109] In the context of the invention, the term "HLA class II positive" should be interpreted as that the expression of HLA class II is detectable, for example, when using immunostaining with an antibody that specifically recognizes these proteins, for example to perform immunohistochemistry or flow cytometry. In an embodiment, a tumor is considered to be HLA-negative or PD-Ll-negative if the expression level does not significantly exceed that of non-tumor cells or of a normal tumor cell. The expression may refer to mRNA levels or protein levels.

[0110] Additionally, the methods and uses of the invention may be particularly advantageous for patients having tumors with disrupted or poorly functioning molecular signaling downstream of the IFNy receptor, and / or patients having tumors with high constitutive or IFNy-induced expression of indoleamine 2,3-dioxygenase, and / or HLA class II and / or PD-L1.

[0111] In an even further preferred embodiment of the invention, a method is provided wherein the tumor cells in step a) are grown to organoids. It is commonly known in the art that not all primary tumor cells are easily kept in culture ex vivo. One way to overcome this is to grow organoids from the primary tumor cells to establish a stable cell culture. Such organoids then can be treated with the BET inhibitors and RLR agonists or RLR agonist inducers according to the method of the invention. Protocols for growing organoids from tumor cells are known to the skilled person.

[0112] In one embodiment, the ex vivo method of the invention, further comprises contacting the tumor cells with a Myc inhibitor in step b). Such a Myc-inhibitor may be a direct MYC inhibitor (e.g. MYCMI7, TOM, F4, MYC-Ribotac) or an indirect MYC inhibitor (e.g. GSK-3Bi, AURKAi, WNT pathway inhibitors). In one such embodiment, a 24h-72h pretreatment with both a BET inhibitor and a MYC inhibitor is followed by stimulation with an RLR agonist, such as poly(I:C), or an RLR agonist inducer. Another non-limiting option is pretreatment with a BET inhibitor for at least 72h during which a MYC inhibitor is added only during the last 24h, before the drugs are washed away and cells are stimulated with an RLR agonist or RLR agonist inducer. In another embodiment, cells are simultaneously treated with a BET inhibitor, a Myc inhibitor and an RLR agonist or an RLR agonist inducer.

[0113] In a further aspect of the invention, a composition is provided obtainable by a method according to the invention.

[0114] In a further preferred embodiment of the invention, a composition is provided obtained by a method according to the invention.

[0115] In a further aspect of the invention, a pharmaceutical composition is provided comprising the composition as described above and a pharmaceutically acceptable carrier or excipient.

[0116] Such compositions are characterized by an increased expression and presentation of tumor-specific antigens and / or tumor-associated antigens wherein the term "increased" refers to the level of expression and presentation of tumor-specific antigens and / or tumor-associated antigens as compared to normal, non-tumor cells and / or non-treated tumor cells in vivo or ex vivo.

[0117] In a further aspect, the invention provides a composition or a pharmaceutical composition of the invention for use in medicine.

[0118] In a further aspect, the invention provides a composition for use in the treatment of cancer wherein the treatment comprises administering the composition obtainable or obtained using the ex vivo method of the invention to a subject, preferably wherein said administering comprises intradermal, subcutaneous, intramuscular, intravenous, or intratumoral administration or a combination thereof.

[0119] In an alternative embodiment the invention provides a method for treating cancer comprising administering the composition obtainable or obtained using the ex vivo method of the invention to a subject wherein said administering preferably comprises intradermal, subcutaneous, intramuscular, intravenous, or intratumoral administration or a combination thereof.

[0120] In another alternative embodiment the invention provides the use of the composition obtainable or obtained using the ex vivo method of the invention in the manufacture of a medicament for the treatment of cancer.

[0121] In a further preferred embodiment the invention provides a composition for use in the treatment of cancer, wherein the treatment with the compositions of cells as described above is combined with the systemic or localized administration in vivo or ex vivo of a drug selected from the group consisting of immunomodulatory compound, angiogenesis inhibitors, chemotherapeutics or Myc inhibitors, preferably wherein the drug is an immunomodulatory compound selected from the group consisting of BET inhibitors, immunostimulatory cytokines, natural, endogenous or synthetic ligands of Toll like receptors, ligands of other immunostimulatory receptors, modulators of immune checkpoints, or agonistic modulators, epigenetic drugs (e.g. HDAC inhibitors and DNMT inhibitors) or compounds for targeted therapy (e.g. BRAF inhibitors and CDK4 / 6 inhibitors).

[0122] Non-limiting examples of chemotherapeutics include: Alkylating agents, Anthracyclines, Cytoskeletal disruptors (Taxanes), Epothilones, Histone Deacetylase Inhibitors, Inhibitors of Topoisomerase I, Inhibitors of Topoisomerase II, Kinase inhibitors, Nucleotide analogs and precursor analogs, Peptide antibiotics, Platinumbased agents, Retinoids, Vinca alkaloids and derivatives. Non-limiting methods to inhibit or downregulate Myc, such as c-Myc, include using small-molecular compounds, RNA interference, or antibodies.

[0123] Non-limiting examples of immunostimulatory cytokines according to the invention are type I and II interferons (e.g. IFNa, IFN , IFNy), IL-2, TNFa, GM-CSF, and Gamma C family of cytokines, e.g. IL-2, IL-7, IL-15 and IL-35.

[0124] Non-limiting examples of natural, endogenous or synthetic ligands of toll-like receptors include natural, endogenous or synthetic ligands for:

[0125] - TLR1 (e.g. triacylated lipopeptides, Pam3Cys)

[0126] - TLR.2 (e.g. Lipopolysaccharide, Heat Killed Listeria monocytogenes)

[0127] - TLR.3 (e.g. nexavant)

[0128] - TLR.4 (e.g. Lipopolysaccharide, MPLA)

[0129] - TLR.5 (e.g. Flagellin)

[0130] - TLR6 (e.g. FSL1)

[0131] - TLR7 (e.g. imiquimod)

[0132] - TLR8 (e.g. R848)

[0133] - TLR9 (e.g. CpG-oligonucleotides)

[0134] - TLR10

[0135] - Any TLR agonist based on attenuated and / or fragmented viruses or bacteria

[0136] - Any endogenous ligand (e.g. heat-shock proteins)

[0137] Non-limiting examples of ligands of other immunostimulatory receptors include natural, endogenous or synthetic ligands for Receptor for advanced glycation endproducts (RAGE) and Stimulator of interferon genes (STING).

[0138] Non-limiting examples of modulators of immune checkpoints may be selected from the group consisting of: CTLA-4, PD-1 , PDL-1 , PDL-2, TIM3, LAG3, B7-H3, B7- H4, BTLA, GAL9, and A2aR. The modulators can be a peptide, antibody, interfering RNA, or small molecule, preferably an antibody that binds PD-1 or PD-L1 and inhibits the binding of PD-L1 to PD-1, such as pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab or durvalumab. In some cases, the immune modulator is a monoclonal antibody, or an Ig fusion protein.

[0139] Non-limiting examples of agonistic modulators include modulators directed to a stimulatory immune molecule, e.g. 4-IBB (CD137), CD137L, 0X40, OX40L, ICOS, CD40, CD40L, CD70, CD27, CD28, CD80, CD86, B7RP1, or HVEM. The modulators can be a peptide, antibody, interfering RNA, or small molecule. In some cases, the immune modulator is a monoclonal antibody, or an Ig fusion protein. Non-limiting examples of epigenetic drugs include DNA methyltransferase inhibitors (DNMTi), histone deacetylase inhibitors (HDACi),

[0140] Non-limiting examples of drugs for targeted cancer therapy include APR-246.

[0141] As discussed above, and as demonstrated in the accompanying Examples, the inventors have shown that the combination of BET inhibitors with RLR agonists or RLR agonist inducers enhances the expression of key molecular markers of tumor immunogenicity, such as HLA class I, IFN , the interferon-induced chemokine CXCL10 and calreticulin. Accordingly, in a further aspect, the invention provides the use of a combination of a BET inhibitor with an RLR agonist or an RLR agonist inducer to increase HLA class I antigen presentation in one or more tumor cell- and / or suppress constitutive and IFNy-induced HLA class II expression in one or more tumor cell. It will be appreciated by those skilled in the art that causing those effects in tumor cells will be beneficial in the generation of new therapeutic methods, uses and compositions.

[0142] In a preferred embodiment, the invention provides the use, e.g. ex vivo use, of a combination of a BET inhibitor and an RLR agonist or RLR agonist inducer to increase the expression of tumor-specific antigens and / or tumor-associated antigens and / or the presentation of tumor-specific antigens and / or tumor-associated antigens by HLA class I in tumor cells.

[0143] The inventors surprising findings of the beneficial effects of combination of a BET inhibitor and an RLR agonist or RLR agonist inducer identifies new uses of such compounds, and identifies new sub-groups of patients with cancer that could be treated using said compounds. As discussed above, BET inhibitors were previously described for use as an anti-tumor therapy for inhibiting tumor growth. However, the inventors' findings now demonstrate that the combination of compounds can be used to treat cancer in another way - specifically, by increasing the expression of tumorspecific antigens and / or tumor-associated antigens and / or the presentation of tumorspecific antigens and / or tumor-associated antigens by HLA class I on tumor cells, which activates tumor-specific CD8+ T cells in the patient and induces an anti-tumor immune response.

[0144] The inventors' findings identify several new sub-groups of patients with cancer that could be treated the combination of a BET inhibitor and an RLR agonist or RLR agonist inducer, in particular: (i) patients with cancer which comprises tumor cells having no or low levels of expression and / or HLA class I-restricted presentation of tumor-specific antigens and / or shared antigens - in such patients, the combination of compounds enhances (IFNy-induced) presentation of antigens by HLA class I, leading to better recognition and eradication of the tumor by CD8+ T cells; and / or

[0145] (ii) patients with cancer which comprises HLA class I-low or -negative tumor cells and / or PDL-l-negative tumor cells - in such patients, the combination of compounds enhances (IFNy-induced) expression of HLA class I, leading to better recognition and eradication of the tumor by CD8+ T cells; and / or

[0146] (iii) patients with cancer which comprises tumor cells having low or absent HLA class I expression - in such patients, the combination of compounds enhances (IFNy- induced) expression of HLA class I, leading to better recognition and eradication of the tumor by CD8+ T cells; and / or

[0147] (iv) patients with cancer which comprises tumor cells having poor IFNy- responsiveness and -signaling - in such patients, the combination of compounds increases responsiveness of the tumors to IFNy, leading to better antigen presentation by HLA class I and subsequently to better recognition and eradication of the tumor by CD8+ T cells; and / or

[0148] (v) patients with cancer with comprises tumor cells with high indoleamine 2,3- dioxygenase (IDO) expression12,55'56- in such patients, the combination of compounds suppresses (IFNy-induced) expression of IDO, leading to reduced immunosuppression of T cells, and thus better T cell-mediated eradication of the tumor; and / or

[0149] (vi) patients with cancer which comprises tumor cells having high HLA class II expression57,58- in such patients, the combination of compounds suppresses (IFNy- induced) expression of HLA class II. As will be appreciated, in healthy conditions, HLA class II is primarily expressed on professional antigen-presenting cells of the immune system (e.g. dendritic cells), to promote immune responses - however, in pathological conditions, HLA class II can be aberrantly expressed or induced on tumors, and thought to suppress immune responses in several ways, for example by being a ligand for immune checkpoint molecule LAG3 that, like PD1, negatively regulates T cells and by activating immunosuppressive subsets of CD4+ T cells; and / or

[0150] (vii) patients with cancer which comprises tumor cells having elevated and / or high CD73 expression, for example compared to the normal level of expression of CD73 in a healthy cell.59in such patients, the combination of compounds suppresses (IFNy-induced) expression of CD73, leading to reduced immunosuppression of T cells, and thus better T cell-mediated eradication of the tumor. CD73 catabolize the breakdown of extracellular ATP into adenosine, which suppresses T cells functioning.

[0151] Methods for identifying and characterizing such tumor cells and the relevant molecular markers, and thereby identifying the patient sub-groups to be tested, are known to those skilled in the arts of medicine and molecular biology.

[0152] As mentioned above, in a further aspect, the invention relates to a method for the treatment of cancer in a subject, the method comprising administering (i) a BET inhibitor and (ii) an RLR agonist or RLR agonist inducer to the subject.

[0153] Similarly, the invention relates to a BET inhibitor for use in the treatment of cancer in a subject, wherein the treatment comprises administering the BET inhibitor and an RLR agonist to the subject.

[0154] Furthermore, the invention relates to an RLR agonist for use or an RLR agonist inducer for use in the treatment of cancer in a subject, wherein the treatment comprises administering (i) the RLR agonist or RLR agonist inducer and (ii) a BET inhibitor to the subject.

[0155] In one embodiment, the BET inhibitor is administered systemically. In another embodiment, the BET inhibitor is administered locally, such as intratumorally. In one embodiment, the RLR agonist or RLR agonist inducer is administered systemically. In another embodiment, the RLR agonist or RLR agonist inducer is administered locally, such as intratumorally.

[0156] In one embodiment, the administration of the BET inhibitor and the RLR agonist or RLR agonist inducer is performed intratumorally or systemically and the treatment comprises the further steps of isolating tumor infiltrating lymphocytes from tumor tissue of the subject, expanding the tumor infiltrating lymphocytes ex vivo and transferring the expanded the tumor infiltrating lymphocytes into the subject.

[0157] In one embodiment, the administration is intradermal, subcutaneous, intramuscular, intravenous, or intratumoral or a combination thereof.

[0158] In one embodiment, the cancer in the subject comprises HLA class I-negative tumor cells and / or PD-Ll-negative tumor cells and / or HLA class Il-positive tumor cells.

[0159] In one embodiment, the cancer in the subject comprises tumor cells that exhibit no or low levels of expression and / or HLA class I-restricted presentation of tumorspecific antigens and / or tumor-associated antigens. In the context of this invention, no or low levels of expression and / or presentation of tumor-specific antigens and / or tumor-associated antigens are levels that induce no or only a weak CD8+ T cellresponse and in any case do not elicit a clinically effective CD8+ T cell-mediated antitumor response. If necessary, cell lines that express single HLA class I alleles of a patient (e.g. Single Antigen expressing Lines (SALs)) and that are loaded with autologous tumor antigens could be exposed to patient-derived tumor antigen-specific CD8+ T cells to assess whether patient-derived tumor cells indeed only induce a lower response by the same tumor antigen-specific CD8+ T cells.60

[0160] Appropriate administration approaches, dosages, and regimens of BET inhibitors are known to those skilled in the art. For example, a BET inhibitor may be administered orally and / or by intracutaneous or subcutaneous injection. In one embodiment, the administration is intratumoral. Preferred dosages of a BET inhibitor are O.Olmg per day, O.lmg per day, lmg per day, or 2mg per day, or 5mg per day, or lOmg per day, or 20mg / per day, or 30mg per day, or 40mg per day, or 50mg per day, or 60mg per day, or 70mg per day, or 80mg per day, or 90mg per day, or lOOmg per day, or 150mg per day, or 200mg per day, or more. Preferably a BET inhibitor is administered in such dosages and / or by such routes once daily for 1 day or more, such as for 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 21 or 28 days.

[0161] Appropriate administration approaches, dosages, and regimens of RLR agonists are known to those skilled in the art. For example, an RLR agonist may be administered orally and / or by intracutaneous or subcutaneous injection. In one embodiment, the administration is intratumoral. Preferred dosages of a RLR agonist are O.Olmg / kg, O. lmg / kg, 0.5mg / kg, lmg / kg, 2mg / kg, 2.5mg / kg, 5mg / kg, lOmg / kg or 20 mg / kg. Preferably an RLR agonist is administered in such dosages and / or by such routes once daily for 1 day or more, such as for 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 21 or 28 days.

[0162] For example, the BET inhibitor R06870810 (a BET inhibitor based on JQ1) may be administered once daily (at escalating doses) via subcutaneous injection in either 28-day cycles (continuous 28 days dosing or 21 days dosing followed by 7 days off drug) or in 21-day cycle (14 days dosing followed by 7 days off drug).

[0163] The BET inhibitor OTX015 / MK-8628 (a BET inhibitor) may be administered at a starting dose of 10 mg, orally (PO) once per day (QD) continuously for 21 days per cycle.

[0164] The BET inhibitor ZEN003694 may be administered orally once daily in 28-day cycles. The BET inhibitor GSK525762 may be administered in a 5 milligram (mg) starting dose, orally, once per day. Dose escalations and / or dose adjustments may be performed to address tolerability and safety issues. Dose escalation may continue up to a dose of 200 mg per day is reached. GSK525762 may be provided in 1 mg, 10 mg and 30 mg tablets and / or administered with 240 milliliter (mL) water.

[0165] Preferably in the methods and uses of the invention, administration of the combination of compounds increases the expression and presentation of tumor-specific antigens in the tumor cells of the cancer in the subject. Additionally, and in line with the inventors' findings, administration of the BET inhibitor increases IFNy-signaling; increases antigen presentation; suppresses constitutive and interferon-gamma- induced HLA class II expression; suppresses constitutive and interferon-gamma- induced indoleamine 2,3-dioxygenase (IDO) production; and suppresses constitutive and interferon-gamma-induced PD-L1 expression.

[0166] It will be appreciated that the administration of the BET inhibitor and the RLR agonist (or RLR agonist inducer) may in principle be performed in any order - for example, the BET inhibitor may be administered before and / or concurrently with and / or after, the RLR agonist (or RLR agonist inducer).

[0167] However, in a preferred embodiment, the BET inhibitor is administered before the RLR agonist or RLR agonist inducer.

[0168] Preferably in the methods and uses of the invention, the combination of the BET inhibitor and RLR agonist or RLR agonist inducer is further combined with the systemic or localized administration of a drug selected from the group consisting of immunomodulatory compound, angiogenesis inhibitors, chemotherapeutics or Myc inhibitors, preferably wherein the drug is an immunomodulatory compound selected from the group consisting : immunostimulatory cytokines, natural, endogenous or synthetic ligands of Toll like receptors, ligands of other immunostimulatory receptors, modulators of immune checkpoints, or agonistic modulators.

[0169] Furthermore, the combination of the BET inhibitor and RLR agonist or RLR agonist inducer may be administered in combination with other anti-cancer therapies, including one or more of: radiation, surgery, chemotherapy, targeted therapy and / or checkpoint blockade therapy.

[0170] In a further embodiments, the combination of the BET inhibitor and RLR agonist or RLR agonist inducer may be administered to the subject in combination with a drug selected from the group consisting of immunomodulatory compound, angiogenesis inhibitors, chemotherapeutics or Myc inhibitors, preferably wherein the drug is an immunomodulatory compound selected from the group consisting of BET inhibitors, immunostimulatory cytokines, natural, endogenous or synthetic ligands of Toll like receptors, ligands of other immunostimulatory receptors, modulators of immune checkpoints, or agonistic modulators, epigenetic drugs (e.g. HDAC inhibitors and DNMT inhibitors) or compounds for targeted therapy (e.g. BRAF inhibitors and CDK4 / 6 inhibitors).

[0171] Non-limiting examples of chemotherapeutics include: Alkylating agents, Anthracyclines, Cytoskeletal disruptors (Taxanes), Epothilones, Histone Deacetylase Inhibitors, Inhibitors of Topoisomerase I, Inhibitors of Topoisomerase II, Kinase inhibitors, Nucleotide analogs and precursor analogs, Peptide antibiotics, Platinumbased agents, Retinoids, Vinca alkaloids and derivatives,

[0172] Non-limiting methods to inhibit or downregulate Myc, such as c-Myc, include using small-molecular compounds, RNA interference, or Antibodies.

[0173] Non-limiting examples of immunostimulatory cytokines according to the invention are type I and II interferons: IFNa, IFN , IFNy, IL-2, TNFa, GM-CSF, and Gamma C family of cytokines, e.g. IL-2, IL-7, IL-15 and IL-35.

[0174] Non-limiting examples of natural, endogenous or synthetic ligands of toll-like receptors include natural, endogenous or synthetic ligands for:

[0175] - TLR1 (e.g. triacylated lipopeptides, Pam3Cys)

[0176] - TLR2 (e.g. Lipopolysaccharide, Heat Killed Listeria monocytogenes)

[0177] - TLR3 (e.g. poly I : C)

[0178] - TLR4 (e.g. Lipopolysaccharide, MPLA)

[0179] - TLR5 (e.g. Flagellin)

[0180] - TLR6 (e.g. FSL1)

[0181] - TLR7 (e.g. imiquimod)

[0182] - TLR8 (e.g. R848)

[0183] - TLR9 (e.g. CpG-oligonucleotides)

[0184] - TLR10

[0185] - Any TLR agonist based on attenuated and / or fragmented viruses or bacteria

[0186] - Any endogenous ligand (e.g. heat-shock proteins)

[0187] Non-limiting examples of ligands of other immunostimulatory receptors include natural, endogenous or synthetic ligands for Receptor for advanced glycation endproducts (RAGE) and Stimulator of interferon genes (STING).

[0188] Non-limiting examples of modulators of immune checkpoints may be selected from the group consisting of: CTLA-4, PD-1 , PDL-1 , PDL-2, TIM3, LAG3, B7-H3, B7- H4, BTLA, GAL9, and A2aR. The modulators can be a peptide, antibody, interfering RIMA, or small molecule, preferably an antibody that binds PD-1 or PD-L1 and inhibits the binding of PD-L1 to PD-1, such as pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab or durvalumab. In some cases, the immune modulator is a monoclonal antibody, or an Ig fusion protein.

[0189] Non-limiting examples of agonistic modulators include modulators directed to a stimulatory immune molecule, e.g. 4-IBB (CD137), CD137L, 0X40, OX40L, ICOS, CD40, CD40L, CD70, CD27, CD28, CD80, CD86, B7RP1, or HVEM. The modulators can be a peptide, antibody, interfering RNA, or small molecule. In some cases, the immune modulator is a monoclonal antibody, or an Ig fusion protein.

[0190] Non-limiting examples of epigenetic drugs include DNA methyltransferase inhibitors (DNMTi), histone deacetylase inhibitors (HDACi),

[0191] Non-limiting examples of drugs for targeted cancer therapy include APR-246.

[0192] All references, articles, publications, patents and patent applications cited herein are incorporated by reference in their entireties for all purposes.

[0193] Examples

[0194] Example 1

[0195] Material and methods

[0196] Cell lines

[0197] The A375 melanoma cell line was bought from the American Type Culture Collection, which was also used to create the vemurafenib-resistant A375 subclone (A375VR4) as previously described.61Briefly, vemurafenib resistant the A375VR4 cell line was generated by repeated exposures of A375 cells to increasing concentrations of vemurafenib. Both A375 and A375VR4 cells were cultured in RPMI 1640 medium (Gibco™, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Gibco™, Life Technologies), 1% penicillin-streptomycin (Gibco™, Life Technologies), 5 pg / ml Plasmocin (invivoGen). The early-passage melanoma cell lines ANRU and KADA were established from patients at the oncology clinic at Karolinska University Hospital (ethical permit: #2011 / 143-32 / 1) based on a published protocol, and they were cultured in IMDM (Gibco™, Life Technologies) supplemented with 10% FBS, 1% penicillin-streptomycin, 5 pg / ml Plasmocin.62These melanoma cell lines were characterized for their differentiation status, as this is known to be a determinant of resistance to T cell-mediated eradication, immunotherapy and targeted therapy.10,63'64 We used the well-established melanoma differentiation markers MITF, NGFR and AXL for this, which can be used to determine to what extent the melanoma cells resemble one of the four major types of differentiation phenotypes identified by others, as indicated in figure 1A to c.63,65-67In this way, we ensured that we included melanoma cell types that reflect the heterogeneity in tumor differentiation that can be expected in patients. Overall, flow cytometry for these markers indicated that A375 and ANR.U cells had a comparatively differentiated phenotype, while A375VR4 and KADA cells were more de-differentiated (figure IB and C). We also compared these cell lines for baseline expression of two important molecular determinants of tumor immunogenicity: HLA class I and PD-L1. We found that KADA was highly positive for HLA class I, while the other cell lines had a comparatively low baseline HLA class I expression (figure ID). KADA also expressed high levels of PD-L1, whereas the other cell lines were completely PD-L1 negative (figure IE).

[0198] Culture and expansion of tumor-infiltrating lymphocytes

[0199] Culture and expansion of TIL was done as published before.4A piece of the tumor (approximately 0.5 cm3) was minced into 1 mm3 pieces. Each piece was placed in one well of a 24-well plate containing 1 mL / well CellGro medium supplemented with 2 % autologous plasma (generated by centrifugation and heat-inactivation of plasma collected on the day of surgery) and 6,000 lU / mL IL-2 (Proleukin, Novartis, Basel, Switzerland). On day 1 of culture, half of the medium volume in each well was replaced with fresh medium supplemented with plasma and IL-2. After 3-4 days of culture, lymphocytes could usually be seen emerging from the tumor piece. Cultures were monitored regularly and split before reaching confluence. After approximately 2 weeks, expanding wells were pooled and counted. To generate large T cell numbers, TIL were cultured for approximately 14 days in presence of autologous, irradiated feeders (40 Gy, TIL:feeder ratio 1 :50-1 :80) with 300 IU / mL IL-2 and 30 ng / mL anti-CD3 antibody (OKT3, Cilag, Zug, Switzerland). Cells were split or medium exchanged as necessary. Cultures were initiated in standing T75 flasks, but transferred to VueLife cell culture bags 1 week before harvest. At the end of culture, cells were harvested, pooled, characterized, counted and frozen.

[0200] Drug treatment

[0201] For 3-day treatments, cells were seeded on flat bottom polystyrene 6-well plates (TPPTM) at a density of from 20.000 to 300.000 cells / well for all cell lines. These cell densities were chosen based on growth rate to adequately transfect when wells were 70%-90% confluent. Cells were then treated 24h after initial seeding with 0.4 pM JQ1 for 72 h, unless stated otherwise. The small-molecular drug JQ1 purchased from Tocris was reconstituted in DMSO according to manufacturer instructions. DMSO treated cells were used as control.

[0202] Poly(I:C) and 3pRNA transfection

[0203] Poly(I:C) (HMW; Invivogen) or 3pRNA (Invivogen) was transfected using Lipofectamine 2000 (ThermoFisher Scientific) according to the manufacturer's instructions. The transfection medium consisted of Opti-MEM medium (Gibco) with 1 pg / ml poly(I:C) or 50 ng / ml 3pRNA, and 10 pg / ml Lipofectamine 2000. After JQ1 pretreatment, the culture medium was replaced with 500 pl of transfection medium per well in a 6-well plate for 4h, after which it was removed and replaced with fresh culture medium (2ml in 6 well plates) and incubated for an additional 24 h to 48 h.

[0204] Gene expression analysis by quantitative real-time PCR

[0205] For gene expression analysis, treated tumor cells were washed with PBS and lysed by adding 750 pl of Trizol reagent (ThermoFisher Scientific) directly into the culture well. Total RNA was then purified using the PureLink™ RNA Mini Kit (Thermo Scientific) according to the manufacturer's instructions. Total RNA quality and concentration was determined using a Nanodrop 2000. Total RNA was then reverse transcribed using the iScript cDNA Synthesis Kit (BioRad) and stored at -20°C until further use. Quantitative real-time polymerase chain reaction (qPCR) was done using the iTaq Universal SYBR Green Supermix (BioRad) with a QuantStudio™ 6 Flex Real-Time PCR System (ThermoFisher Scientific). The following cycle conditions were used: 50°C for 2 min, 95°C for 3 min, 40 cycles at 95°C for 30 s, and 60°C for 1 min. The amount of cDNA used per reaction was based on an input of 500 ng RNA in a final volume of 10 pL. All reactions were run in triplicates. All primers used are listed in Table 1. Relative gene expression was calculated with the AACT method. Transcript levels of gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used for normalization. Relative gene expression was calculated as a fold-change by dividing the normalized gene expression of the treated sample by the normalized gene expression of the untreated sample. The obtained fold changes were visualized using GraphPad Prism (version 9.0).

[0206] Table 1: Primers used for used for qPCR.

[0207] Flow cytometry

[0208] Cells were dispensed into a 96-well V-bottom plate (Costar) at concentration of 0.8- 1.5 x 105cells per well. Cells were washed with PBS and incubated with a dead cell marker (LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit, Thermo Fisher Scientific) for 15 min at room temperature. Cells were then washed again with PBS. Antibodies for cell surface-expressed molecules listed in table 2 (tumor cells) and table 3 (T cells) were diluted in FACS buffer (PBS + 1% BSA) and incubated for 30 min at 4°C in dark. For intracellular staining cells were washed with PBS and fixed with Cytofix / Cytoperm™ fixation permeabilization solution (BD Biosciences), or with the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) for intranuclear staining according to manufacturer's instruction. Cells were subsequently washed with kit buffers and stained for 30-40 min at room temperature in dark. Cells were then resuspended in FACS buffer and samples ran on the Novocyte Quanteon (Agilent). Compensation matrixes were generated from compensation beads (AbC™ Total compensation beads and ArC™ reactive beads, Invitrogen) according to manufacturer's protocol.

[0209] Table 2: Antibodies and viability dye used for flow cytometry of tumor cells.

[0210] Tumor stainings

[0211] Tumor-TIL co-culture

[0212] KADA and ANRU tumor cells treated with JQ1 and / or poly(I:C) were plated on a 96- well U-bottom sterile plate (TPP at a density of 40.000 cells / well). Where indicated, HLA class I was blocked by pre-incubating tumor cells with 10 pg / ml anti-HLA-ABC antibody (clone W6 / 32, Biolegend) for 30-60 minutes at 37°C. Subsequently, 200.000 TIL were added per well, leading to a tumor:TIL ratio of 1 :5. As a positive and negative control for their activation, also included were wells with TIL alone cultured in respectively the presence and absence of CD3 / CD28 Dynabeads (Life Technologies). After adding TIL to the culture plate, cells were for incubated for 24h at 37°C in AIM- V medium (Gibco) supplemented with 2% human AB serum. After incubation, culture plates stored at -20°C for later downstream analysis by ELISA. Finally, an ELISA was performed to determine levels of IFNy in the co-culture supernatant, which is a well- established indicator of T cell activation.

[0213] Enzyme-linked immunosorbent assay (ELISA)

[0214] Supernatant from the tumor-TIL co-culture was used for sandwich ELSA (human IFNy ELISABASIC kit - HRP, MABTECH) to detect secreted IFNy, which is a well-established indicator of T cell activation. ELISAs were done following the manufacturer's protocol. All samples were diluted 1 :20 in incubation buffer (PBS containing 0,05% Tween 20 and 0,1% BSA) while the positive control TIL+ CD3 / CD28 activating Dynabeads (Life technologies) positive control were diluted 1 :50. Dilution factors were then corrected upon analysis. Analysis was done in GraphPad Prism software (version 9.0).

[0215] Assay to detect degranulation and cytokine production by autologous TIL

[0216] To assess degranulation and cytokine secretion by autologous tumor-specific CD4+ and CD8+ T cells, KADA and ANRU tumor cells were co-cultured with autologous TILs in AIM-V medium (Gibco) supplemented with 2% human AB serum in a 96-well U- bottom sterile plate (TPP), using 40.000 tumor cells / well and 200.000 TIL / well respectively. As a negative and positive control for their activation, also included were wells with TIL alone cultured in respectively the absence and presence of CD3 / CD28 Dynabeads (Life Technologies). At the start of the co-culture, anti-lysosomal- associated protein 1 (LAMP1, CD107a) antibody was added to the wells. After 1 h of incubation at 37°C BD GolgiStop and Golgi plug were mixed (1 : 1.5 respectively) and added to wells, followed by another 3 h of incubation at 37°C. Ultimately, cells were transferred to a 96-well V-bottom plate (Costar) and stained for relevant extracellular or intracellular markers. Flow cytometry was performed as mentioned previously. The flow cytometry antibodies used in this assay are shown in table 3.

[0217] Table 3: Antibodies and viability dye used for flow cytometry of T cells

[0218] TIL stainings

[0219] Dendritic cell stainings Statistical analysis GraphPad Prism software was also used for both statistical and graphical analysis. Data are presented as mean ±SEM. For statistical analysis, a RM one-way ANOVA was performed to test whether there were any statistical differences between all treatment conditions. If the one-way ANOVA turned out to be significant, Sidak multiple comparison tests were performed to test the difference between individual treatment conditions. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; NS, not significant

[0220] Results

[0221] JQ1 pretreatment sensitizes tumor cells to poly(I:C)-induced inflammation.

[0222] We set out to test whether JQ1 would be able to sensitize tumor cells to mimics of viral dsRNA that serve as agonists of RIG-I-like receptors (RLR) in the cytosol. Therefore, melanoma cells were pretreated with JQ1 for 72 h and subsequently transfected with poly(I:C), which was followed by harvest of pretreated tumor cells for analysis by qPCR or flow cytometry, or for use in coculture assays with autologous TIL, as depicted in figure 2A. Transfection of tumor cells with poly(I:C) led to increased gene expression of RIG-I, MDA5, IFNp and CXCL10, and this effect was markedly enhanced when tumor cells were pretreated with JQ1 (figure 2B). Since treated tumors cells displayed elevated expression of IFNp, we wondered whether combination of JQ1 and poly(I:C) affected antigen presentation by HLA class I. Indeed, JQ1 pretreatment followed by poly(I:C) stimulation clearly enhanced HLA class I expression on KADA, ANRU and A375 cells (figure 2C and D). At the same time, JQ1 alone had no significant effect on the expression of genes depicted in figure 2B or on HLA I expression as shown in figure 2C and D. We also found that 72 h JQ1 pretreatment followed by poly(I:C) stimulation strongly enhanced HLA I expression on A375VR4 cells (figure 2E). In contrast to other cells, higher concentrations of JQ1 were needed during pretreatment to achieve a stronger effect, most likely due to the de-differentiated nature of the A375VR4 cells associated with resistance to the targeted therapeutic vemurafenib. The enhanced effect of poly(I:C) on HLA class I expression in A375 cells was quantitatively dependent on incubation times (fig 2F). Figure 2F also shows that no immunogenicity-enhancing effects on tumor cells occurred if tumors cells were treated with naked (i.e. nontransfected) poly(I:C) or lipofectamine only, which is in line with our observations on all other cell lines (data not shown). This indicates that the specific immunogenicityenhancing effects of poly(I:C) revealed by these experiments require poly(I:C) to be transfected and that it should be preceded by a prolonged JQ1 pretreatment. To confirm these effects, we also used the specific RIG-I agonist 5' triphosphate double- stranded RNA (3pRNA) for transfection, instead of poly(I:C), after a 72 h JQ1 pretreatment in the same experimental setup as shown in figure 2A. As expected, this phenocopied the boosting effect on tumor immunogenicity, as indicated by enhanced 3pRNA-induced HLA class I expression on the tumor cell surface (figure 2G). Overall, these data indicate that a JQ1 pretreatment followed by poly(I:C) stimulation leads to an unexpected synergistic induction of key immunogenicity markers in melanoma cells.

[0223] Lastly, type I interferon signalling has been described to convert "cold" tumours "hot" and promote checkpoint molecule expression on tumour cells, rendering them sensitive to checkpoint blockade.68-70However, PD-L1 expression has also been described to be promoted by BRD4, and JQ1 has been shown to suppress both constitutive and IFNy induced PD-L1 by our group and others.71,72Interestingly, in all tumor cells that were PD-L1 negative at baseline (figure IE), treatment with JQ1 or poly(I:C) alone led to PD-L1 upregulation, while enhanced effects were seen after treatment with a combination of JQ1 and poly(I:C) (figure 2H-I). Overall, these data indicate that prolonged BETi-treatment of melanoma cells strongly sensitizes them to the immunogenicity-enhancing effects of RLR agonists, irrespective of their differentiation status, phenotype associated with resistance to targeted therapeutics.

[0224] Enhanced dsRNA signaling through BETi promotes immunogenic cell death in melanoma.

[0225] Several studies reported that RIG-I signaling promotes immunogenic cell death (ICD) in tumor cells, an important mechanism for activation of cross-presenting conventional type I dendritic cells (cDCl) that facilitate anti-tumor immunity.73,74To investigate this, we analyzed tumor cells for cell surface expression of calreticulin, a well-known ICD marker that serves as an 'eat me' signal for myeloid cells. While both JQ1 treatment and poly(I:C) stimulation increased expression of calreticulin and an increased calreticulin13' population in A375VR4cells, this was further increased when combined (figure 3A-C). In line with our previous findings, these data indicate that JQ1 can boost multiple aspects of tumor immunogenicity that pertain to activation of both T cells and DCs.

[0226] Enhanced antiviral signaling in melanoma promotes activation of cytolytic autologous TIL in an antigen-dependent manner. Next, we wondered whether the sensitizing effect of JQ1 on dsRNA signaling would have any functional effect on tumor recognition by T cells. Indeed, it has been reported that activation of the RIG-I / MDA-5 pathway promotes CD8+ T cell-mediated antitumor immunity68,75,76. Therefore, JQ1- or DMSO-pretreated ANRU and KADA tumor cells were transfected with poly(I:C) and subsequently co-cultured with autologous TIL, after which an ELISA for IFNy was performed on the supernatant to assess T cell activation (figure 4A). Interestingly, treatment of tumor cells with poly(I:C) alone, but not with JQ1 alone, strongly induced activation of KADA TIL, while the opposite was observed in ANRU cells. However, combined treatment of tumor cells with JQ1 and poly(I:C) clearly led to the strongest recognition by TIL in both ANRU and KADA. This recognition was HLA class I / peptide dependent as incubation of tumors cells with an HLA class I-blocking antibody prior to the addition of TIL to the co-culture strongly reduced their IFNy production. TIL cultured in the absence of tumor cells did not produce any IFNy (data not shown), which indicates that the activation observed is completely specific to tumor-reactive T cells. To further characterize the observed IFNy production by the TIL, flow cytometry analysis was performed of ANRU and KADA TIL for production of IFNy, TNFa, and CD107a on bulkTIL. In line with the ELISA, combined treatment of tumor cells with JQ1 and poly(I:C) induced a higher frequency of activated CD107a+ / IFNy+ CD8+ TIL as opposed to tumor cells subjected to single treatment with these agents (figure 4B). Notably, TNFa production by KADA TIL was significantly enhanced when they were exposed to autologous tumor cells treated with JQ1 and poly(I:C) (figure 4B). Overall, the data in figure 4A and B show that treatment of tumor cell with a BET inhibitor and subsequent treatment with a dsRNA agonist strongly enhances the activation of autologous CD8+ TIL in a mannerthat is dependent on HLA class I recognition.

[0227] Importantly, JQ1 treatment can suppress both constitutive and IFNy-induced HLA class II expression on melanoma cells (see WO2019063829). This is of potential clinical relevance, since the expression of HLA class II on tumor cells has been associated with suppression of anti-tumor immunity and resistance to apoptosis.57'58'77 78Therefore, we wondered how combined treatment with JQ1 and poly(I:C) would impact on HLA class II expression and activation of CD4+ TIL. Indeed, JQ1 not only enhanced poly(I:C)-induced HLA class I expression, but also decreased expression levels of HLA class II in ANRU cells when used alone or in combination with poly(I:C) (figure 4C, left and center). This dichotomous effect of JQ1 and poly(I:C) on HLA class I and II expression becomes even more apparent when assessing the HLA class I / II ratio of treated cells. This indicates that the combined use of JQ1 and poly(I:C) leads to a strong increase in the HLA class I / II ratio (figure 4C, right). Importantly, that the increase in the HLA class I / II ratio induced by combined JQ1 and poly(I:C) treatment is exceptionally strong becomes even more clear when comparing it with the HLA class I / II ratio induced by combined treatment of tumor cells with JQ1 and IFNy (figure 4D). The strongly increased HLA class I / II ratio at the tumor cell surface induced by combined JQ1 and poly(I:C) treatment suggests that it favors activation of tumorspecific CD8+ T cells over that of CD4+T cells. Indeed, in line with this observation, combined treatment of tumor cells with JQ1 and poly(I:C) strongly enhanced IFNy production by autologous CD8+ TIL in a coculture setting, while CD4+ TIL were less activated (figure 4E and F). Overall, these data show that BET inhibition in combination with RLR agonist skews tumor cells towards a phenotype that, based amongst others on enhanced IFN production and a high HLA class I / II ratio, seems to reflect an antiviral state and that selectively improves activation of tumor-specific CD8+ T cells in a direct manner.

[0228] Example 2

[0229] We set out to reproduce and somewhat extend the data shown in figure 2B, which indicate that, also in comparison to 24h stimulation with transfected poly(I:C) only, 72h JQ1 pretreatment followed by 24h stimulation with transfected poly(I:C) significantly enhanced expression of key genes involved in innate antiviral responses to cytosolic RNA species. Indeed, the data in figure 5B and C clearly confirm our earlier findings depicted in figure 2B and indicate that JQ1 also strongly enhances the ability of transfected poly(I:C) to induce the important T cell-attracting chemokine CXCL11 in multiple human cancer cells.

[0230] After that, we verified if other BET inhibitors would boost immunogenicity-enhancing effects of transfected poly(I:C) on tumor cells in a similar way as observed for JQ1. Therefore, we pretreated the human melanoma cell lines A375 and ANRU for 72h with iBET151, a well-known BET inhibitor, followed by 48h exposure to transfected poly(I:C) and flow cytometry to determine cell surface expression of HLA class I. Importantly, we observed that combining iBET151 and transfected poly(I:C) indeed in a synergistic manner increased HLA class I cell surface expression on both tumor cell lines, as shown in figure 5D. Next, we analyzed cell surface expression of HLA class I and MHC class I on the human colorectal cancer cell line COLO320 and the murine colorectal cancer cell lines MC38, respectively, after 72h treatment with JQ1 and / or poly(I:C) transfection. As depicted in figure 5E, JQ1 and transfected poly(I:C) synergistically boosted cell surface expression of HLA class I on both cell lines, which indicates that these immunogenicity-enhancing effects are present in multiple cancer types from distinct tissue origins. This is further corroborated by the fact that 72h treatment with JQ1 alone consistently upregulated protein expression of the mitochondrial antiviralsignaling protein (MAVS) in melanoma cells as well as in tumor cell lines (MFS1 and MPNST1) derived from two different subtypes of sarcoma and in the non-small cell lung cancer cell line A549 (figure 5F). Importantly, MAVS is indispensable for antiviral innate immunity, as it serves as an critical signaling hub that RIG-I-like receptors bind to directly upon detection of immunogenic RIMA species in order to induce downstream antiviral responses.

[0231] Example 3

[0232] To validate in an unbiased manner if combining JQ1 with RLR activation by poly(I:C) does indeed lead to a significant upregulation of antiviral and pro-inflammatory genes, we performed transcriptomic analysis, by RNA sequencing (RNAseq), of ANRU tumor cells pretreated for 72h with JQ1 and subsequently stimulated for 24h with transfected poly(I:C). Figure 6A shows that this revealed a strong and significant upregulation of multiple pro-inflammatory genes as well as genes involved in innate antiviral responses upon treatment with JQ1 and poly(I:C), when compared to cells treated with poly(I:C) only. In accordance with our previous flow cytometry and qPCR data, multiple HLA class I genes and chemokines such as CXCL10 and CXCL11 were among the genes significantly enhanced by combining JQ1 and poly(I:C). In a paper by Jerby- Anon and co-workers an 'immune exclusion signature' of gene expression was identified using single-cell RNAseq data from melanoma biopsies with low immune infiltration.1Using publicly available databases, we show in figure 6B that the average expression of thelOO genes most strongly upregulated by our combination treatment with JQ1 and poly(I:C) strongly correlates with expression of the downregulated set of genes in the immune exclusion signature in cancer patients. Overall, this suggests that our approach could serve to promote immune infiltration by amplifying expression of tumor-derived genes typically downregulated in cold tumors, both in melanoma as well as in other cancers. l.Jerby-Arnon, L. et al. A Cancer Cell Program Promotes T Cell Exclusion and Resistance to Checkpoint Blockade. Cell 175, 984-997. e24 (2018).

[0233] Example 4

[0234] Figure 7A shows the experimental setup used for generating data included in the same figure. We did additional experiments and analysis to verify activation of autologous tumor-infiltrating T cells (TIL) cocultured with tumor cells treated with JQ1 and / or poly(I:C). As shown before in figure 4A, combined JQ1 and poly(I:C) treatment of the human melanoma cell line KADA in a synergistic manner enhances recognition by autologous TIL, as indicated by IFNy levels measured by an ELISA in the coculture supernatant. Moreover, figure 7C and D highlight that combined JQ1 and poly(I:C) treatment of KADA cells and subsequent coculture with autologous TIL lead to an increased frequency of activated CD8+ T cells that are positive for IFNy, TNFa and CD107a, the latter being a marker for degranulation of cytotoxic effector molecules by T cells that reflect their tumor-killing potential. Figure 7E, F and G depict data from the same type of experiment as shown, respectively in figure 7B, C and D, but now using the human melanoma cell line ANRU and matched (autologous) TIL. Importantly, using an antibody to block HLA class I (HLA-A, B and C) at the tumor cell surface almost completely abrogated recognition by autologous TIL, as shown in figure 6B and E.

[0235] Example 5

[0236] We decide to investigate how the combined treatment of tumor cells with JQ1 and poly(I:C) would impact on CD8+ T cell-mediated recognition of the melanoma- associated tumor antigen MART-1 and the neoantigen NUP210, the latter having been identified earlier by our group in the human melanoma cell lines ANRU. Using MHC- dextramers we found that ANRU TIL contained clear subpopulations of both MART- specific and NUP210-specific CD8+ T cells (figure 8A). In line with our previous data, combined JQ1 and poly(I:C) treatment of ANRU tumor cells followed by coculture with autologous TIL in a synergistic manner enhanced the overall frequency of activated IFNy+ CD107a+ CD8+ TIL (figure 8B, top row), but this effect was even more pronounced for NUP210-specific CD8+ TIL (figure 8B, bottom row). In contrast, treatment of tumor cells with JQ1 led to the strongest increase in activated IFNy+ CD107a+ MARTl-specific CD8+ TIL were most strongly activated by, while their activation was somewhat reduced by stimulation with poly(I:C), either alone or in combination with JQ1 (figure 8B middle row). This effect was very reproducible, as indicated by figure 8C showing data from three independent experiments. Importantly, the same figure clearly shows that JQ1 and poly(I:C) enhance the activation of NUP210 -specific CD8+ TIL only when used in combination, but not when used on their own.

[0237] Example 6

[0238] The data shown figure 2H indicate that inhibiting PD-L1-PD1 interaction with a blocking antibody could further improve the activation of autologous tumor-specific T cells by combined JQ1 and poly(I:C) treatment of tumor cells. Therefore, we treated ANR.U tumor cells with JQ1 and / or poly(I:C) and subsequently cocultured them in vitro with autologous TIL in the absence and presence of a PD-L1 blocking antibody. As expected, we found that PD-L1 blockade further improved the recognition of tumor cells treated with a combination of JQ1 and poly(I:C), whereas this no effect of PD-L1 blockade was observed for TIL cocultured with tumor cells treated with poly(I:C) only (figure 9).

[0239] Example 7

[0240] To extend the data shown in figure 3, we performed studied A375 cells treated with JQ1 and / or poly(I:C) for expression levels HSP90 and calreticulin, which are both markers for immunogenic cell death known to promote uptake of cell debris by dendritic cells (DC) as well as DC activation. We found that combined JQ1 and poly(I:C) treatment synergistically enhanced both HSP90 and calreticulin expression on A375 cells (figure 10 A and B), suggesting that this approach does indeed strongly promote immunogenic cell death on tumor cells. To verify if combined JQ1 and poly(I:C) treatment would indeed lead to improved DC activation by promoting immunogenic cell death or the secretion of soluble DC-activating factors, we used the experimental setup shown in figure 10C (monocyte-derived DC were generated as described in Lbvgren, T. et al. Cancer Immunol. Immunother. 66, 1333-1344 (2017)). This enables us to study if the tumor cells or their culture supernatant could (better) enhance DC maturation if they were first subjected to combined JQ1 and poly(I:C) treatment. Indeed, if we exposed A375 cells to JQ1 and / or poly(I:C) and subsequently cocultured them with immature human monocyte-derived DC, only cells subjected to the combined JQ1 and poly(I:C) treatment enhanced expression of the established DC maturation markers CD80, CD86, HLA class II and CDla (figure 10D and E). In line with this, when adding supernatant from A375 cells treated with JQ1 and / or poly(I:C) to immature DC, only cells subjected to the combined JQ1 and poly(I:C) treatment enhanced expression of the established DC maturation markers CD80, CD86, HLA class II, HLA class I and of PD-L1 (figure 10 F and G)

[0241] Example 8

[0242] We generated data with experiments in which ANR.U and KADA tumor cells were treated with JQ1 and / or poly(I:C) and subsequently cocultured with autologous TIL. Flow cytometry was then used to determine the frequencies of activated IFNy+ CD8+ TIL. Importantly, combined JQ1 and poly(I:C) treatment in both cell lines clearly enhanced the frequency of activated IFNy+ CD8+ TIL (figure 11).

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Claims

Claims1) Ex vivo method for obtaining a composition suitable for the treatment of cancer in a subject, comprising the steps of: a) providing primary tumor cells derived from the subject, and b) ex vivo contacting the tumor cells with(i) an inhibitor of a bromodomain and extra-terminal domain family member (BET inhibitor) and(ii) an RIG-I-like receptor agonist (RLR agonist) or a compound that induces expression of an endogenous RLR agonist (RLR agonist inducer), with the proviso that if the RLR agonist is poly(I:C), said poly(I:C) is transfected into the tumor cells.2) Method according to claim 1, wherein in step b) said tumor cells are first contacted with the BET inhibitor and subsequently with the RLR agonist or RLR agonist inducer.3) Method according to claim 2, wherein in step b) the BET inhibitor is removed from the cell culture, for example by washing, prior to contacting the cells with the RLR agonist or RLR agonist inducer.4) Method according to claim 1 - 3, wherein the tumor cells are incubated for at least 4 hours, such as at least 8 hours, or at least 24 hours, at least 48 hours, at least 60 hours, at least 3 days, at least 4 days, at least 5 days, at least 6 days or at least 7 days with the RLR agonist or RLR agonist inducer.5) Method according to claim 1 - 4, wherein the cells are cultured in vitro in order to increase the number of cells before step b) is performed.6) Method according to claim 1 - 5, wherein the tumor cells further are contacted with an immunostimulatory compound, preferably interferon alpha, interferon beta or interferon gamma, in step b), preferably prior to contacting the tumor cells with the RLR agonist or RLR agonist inducer, most preferably wherein the tumor cells are contacted with the BET inhibitor and the immunostimulatory compound simultaneously.7) Method according to claim 1 - 6, wherein the composition is treated so that it is not tumorigenic in vivo after step b).8) Method according to any one of claims 1 - 7, wherein the method additionally comprises a step of ex vivo contacting the tumor cells with T-cells or dendritic cells obtained from the subject.9) Method according to any one of claims 1 - 8, wherein the tumor cells are HLA class I-negative tumor cells and / or PD-Ll-negative tumor cells and / or HLA class Il-positive tumor cells.10) Method according to any one of claims 1 - 9, wherein the cancer is a solid tumor or a hematological cancer.11) Method according to any one of claims 1 - 10, wherein the cancer is a melanoma.12) Method according to any one of claims 1 - 10, wherein the cancer is a colorectal cancer.13) Method according to any one of claims 1 - 12, wherein the BET inhibitor is selected from the list consisting of: JQ1, MK-8628, BMS-986158, ABBV-075, CPI-0610, FT- 1101, GS-5829 (Alobresib), GSK525762, PLX51107, R06870810 / Ten-010, OTX-015, CPI-0610, 3-methyl-l,2,3,4-tetrahydroquinazolin-2-ones, 2-thiazolidinones, CPI-203, I-BET762 (GSK525762A), I-BET151 (GSK1210151A), AZD5153, AZD-5153 6- hydroxy-2-naphthoic acid, BET-d246, Dinaciclib, BAY1238097, CC-90010, ODM-207, BI 894999, ZL0420, ZL0454, ARV-771, ARV-825, A1874, BET-d260, Thalidomide-NH- C4-NH-Boc, GSK046, GSK620, GSK778, BRD4 inhibitor-10, Y06036, MS436, XMD8- 92, BRD4 Inhibitor-14, BRD4 Inhibitor-16, BET-IN-1, BET-IN-2, BET-IN-7, BET-IN-8, BET-IN-9, BET-IN-10, BET-IN-12, BET-IN-13, BET-IN-14, BET-IN-15, BET-IN-16, BET- IN-17, BET-IN-19, BET-IN-20, BET-IN-21, RVX-297, NEO2734, Bromosporine, dBET57, BI 2536, RVX208 (Apabetalone), PFI, ZEN-3694, ZEN-3219, ZEN-3411, ZEN- 3862 dBETl, dBET6, MZ-1, UNC6349, BET bromodomain inhibitor 1, BET bromodomain inhibitor 2, BET-BAY 002, NVS-BET-1, I-BET-432, PROTAC BET degrader-1, PROTAC BET degrader-2, PROTAC BET degrader-3, PROTAC BET Degrader-10, I-BET282, I-BET282E, GSK040, I-BET762 carboxylic acid, I-BET726(GSK1324726A), Physachenolide C, SDR-04, PROTAC BRD2 / BRD4 degrader-1, DW71177, OXFBD04, SJ1461, (S)-GNE-987, SB-284851-BT, Amredobresib, GNE-987, INCB054329, INCB054329 Racemate, TD-428, INCB-057643, RX-37, CF-53, ET-JQ1- OH, GSK023, Bromodomain inhibitor-8, QCA570, dBRD4-BDl, MT1, CD161, CD235, Rac)-BAY1238097, XD14, GSK217, GSK737, GSK852, TC AC 28, HJB97, Y06137, PROTAC BRD4 ligand-1, PROTACT GNE-987, PNZ5, DDO-8926, (+)-JQ-l-aldehyde, OARV-771, GS-626510, BAY1238097, PROTAC BRD3 / BRD4-L degrader-2, BY27, (+)- JQ1 PA, JQ1-TCO, SIM1, GSK097, BRD4 Dl-IN-1, BRD4 Dl-IN-12, MS645, MS417, (R)-BAY1238097, GSK620, ABBV-744, dBET23, (E / Z)-ZL0420, ZEN-2759, LT052, SNIPER(BRD)-1, I-CBP112 hydrochloride, XP-524, XY153, MS402, GXH-II-052, GSK973, NC-III-49-1, AT 1 preferably wherein the BET inhibitor is selected from the group consisting of JQ1, OTX-015, I-BET151 (GSK1210151A), I-BET762 (GSK525762A) and MZ1.14) Method according to any one of claims 1 - 13, wherein step b) comprises contacting the tumor cells with an RLR agonist and wherein the RLR agonist is a RIG- I agonist and / or an MDA5 agonist.15) Method according to claim 14, wherein the RLR agonist is a double-stranded RNA.16) Method according to claim 14, wherein the RLR agonist is a poly(I:C).17) Method according to any one of claims 1 - 16, wherein the RLR agonist or RLR agonist inducer is transfected into the tumor cells.18) Method according to any one of claims 1 - 17, wherein the RLR agonist or RLR agonist inducer is introduced into the tumor cells via nanoparticles, such as lipid nanoparticles or polymer-based nanoparticles.19) Method according to any one of claims 1 - 18, further comprising contacting the tumor cells with a Myc inhibitor in step b).20) Method according to any one of claims 1 - 19, further comprising contacting the tumor cells with an immune checkpoint inhibitor, such as an antibody that binds PD-1 or PD-L1 and inhibits the binding of PD-L1 to PD-1.21) Method according to any one of claims 1 - 20, wherein the tumor cells in step a) are in vitro grown to organoids.22) Method according to any one of claims 1 - 21, comprising a further step of transducing the tumor cells with one or more genes encoding co-stimulatory molecules, such as CD80, CD86 or CD70 or encoding immuno-stimulatory cytokines of the gamma C family, CD83 or dendritic cell or T-cell attracting chemokines.23) Composition obtainable by a method according to any one of claims 1 - 22.24) Composition obtained by a method according to any one of claims 1 - 23.25) Pharmaceutical composition comprising the composition according to claim 23 or 24 and a pharmaceutically acceptable carrier or excipient.26) A composition as defined in claim 23 or 24, or a pharmaceutical composition as defined in claim 25, for use in medicine.27) A composition as defined in claim 23 or 24, or a pharmaceutical composition as defined in claim 25, for use in the treatment of cancer wherein the treatment comprises administering of the composition to the subject, preferably wherein said administering comprises intradermal, subcutaneous, intramuscular, intravenous, or intratumoral administration or a combination thereof.28) Use of a composition as defined in claim 23 or 24, or a pharmaceutical composition as defined in claim 25, in the manufacture of a medicament for the treatment of cancer, wherein the treatment comprises administering of the composition to the subject, preferably wherein said administering comprises intradermal, subcutaneous, intramuscular, intravenous, or intratumoral administration or a combination thereof.29) A method for the treatment of cancer in a subject, the method comprising the step of administering to the subject a composition as defined in claim 23 or 24, or a pharmaceutical composition as defined in claim 25, and wherein preferably said administering comprises intradermal, subcutaneous, intramuscular, intravenous, or intratumoral administration or a combination thereof.30) Composition for use according to claim 27, or the use according to claim 28, or the method for the treatment of cancer according to claim 29, wherein the treatment is combined with the systemic or localized administration of a drug selected from the group consisting of immunomodulatory compounds, angiogenesis inhibitors, chemotherapeutics or Myc inhibitors, preferably wherein the drug is an immunomodulatory compound selected from the group consisting of BET inhibitors, immunostimulatory cytokines, natural, endogenous or synthetic ligands of Toll like receptors, ligands of other immunostimulatory receptors, modulators of immune checkpoints, or agonistic modulators.31) Composition for use according to claim 27, or the use according to claim 28, or the method for the treatment of cancer according to claim 29, wherein the treatment is combined with the systemic or localized administration of an antibody that binds PD- 1 or PD-L1 and inhibits the binding of PD-L1 to PD-1, such as pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab or durvalumab.32) The ex vivo use of a combination of a BET inhibitor and a RLR agonist or RLR agonist inducer to increase the expression of tumor-specific antigens and / or tumor- associated antigens and / or the presentation of tumor-specific antigens and / or tumor- associated antigens by HLA class I in tumor cells.33) A BET inhibitor for use in the treatment of cancer in a subject, wherein the treatment comprises administering the BET inhibitor and an RLR agonist to the subject.34) An RLR agonist for use or an RLR agonist inducer for use in the treatment of cancer in a subject, wherein the treatment comprises administering (i) the RLR agonist or RLR agonist inducer and (ii) a BET inhibitor to the subject.35) A method for the treatment of cancer in a subject, the method comprising administering (i) a BET inhibitor and (ii) an RLR agonist or RLR agonist inducer to the subject.36) The BET inhibitor for use according to claim 33, the RLR agonist for use or RLR agonist inducer for use according to claim 34 or the method according to claim 35,wherein the administration of the BET inhibitor and the RLR agonist or RLR agonist inducer is performed intratumorally or systemically and wherein the treatment comprises the further steps of isolating tumor infiltrating lymphocytes from tumor tissue of the subject, expanding the tumor infiltrating lymphocytes ex vivo and transferring the expanded the tumor infiltrating lymphocytes into the subject.37) The BET inhibitor for use according to claim 33 or 36, the RLR agonist for use or the RLR agonist inducer for use according to claim 34 or 36 or the method according to claim 35 or 36, wherein the cancer in the subject comprises HLA class I-negative tumor cells and / or PD-Ll-negative tumor cells and / or HLA class Il-positive tumor cells.

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