WO2025063844A1 - Enhancement of t cell mediated therapies with a dna hypomethylating agent and a sumoylation inhibitor - Google Patents

Abstract

The present invention relates to combinations of a DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy for use in treating a patient. These combinations may be used to enhance a T cell mediated therapy in a patient. These combinations may be used to induce or enhance a T cell response in a patient. In some examples, the patient has cancer.

Description

ENHANCEMENT OF T CELL MEDIATED THERAPIES WITH A DNA HYPOMETHYLATING AGENT AND A SUMOYLATION INHIBITOR

FIELD OF THE INVENTION

The present invention relates to novel combinations for use in treating a patient. These novel combinations may be used to enhance a T cell mediated therapy in a patient. These novel combinations may be used to induce or enhance a T cell response in a patient. In some examples, the patient has cancer. Associated methods for treating such patients are also provided herein. BACKGROUND

Immunotherapies have significantly improved over the past decades. Immunotherapies include adoptive cell therapy based on expansion and administration of patient derived T cells. Two concepts have led to the advancement of adoptive cell therapy, firstly the identification of T cells in cancer patients that are capable of recognizing the tumour showed the potential of these T cells to be employed as a therapeutic strategy. Secondly the observation that the tumour microenvironment inhibits these T cells indicated that detailed understanding of inhibitory mechanisms is needed to overcome the inhibitory tumour microenvironment¹. T Cell Receptor (TCR) engineering of T cells is one of the strategies emerging from this concept²’³. Tumour specific TCR T cells either recognize neo-antigens derived from mutations which are unique to the tumour or tumour associated antigens (TAA) which are elevated in tumour tissues. These tumour specific antigens are presented to T cells by MHC class I. Specific engineering of the TCR in T cells allows these T cells to be redirected to the tumour and specifically target tumour cells 4,5,6

Despite this progress, challenges remain regarding efficacy and persistence of T cell therapy². The anti-tumour efficacy of adoptive T cell therapy is dependent on activity, persistence and expansion of transplanted T cells⁷. Current approaches to increase T cell persistence and efficacy include combination with cytokine treatment⁸, T cell specific subset purification⁹ and genetic knock out of immune inhibitory molecules known as immune checkpoints¹⁰ amongst others⁷. There remains a need for new treatment regimens for improving T cell therapies.

BRIEF SUMMARY OF THE DISCLOSURE

Here the inventors aim to improve engineered TCR (eTCR) T cell and CAR T cell efficacy and persistence via targeting post-translational modification (PTM) and epigenetic regulation to overcome inhibitory mechanisms. The inventors employ hypomethylating drug 5-Aza-2’- deoxycytidine (5-Aza-2’) in their combination therapy. 5-Aza-2’ is a cytotoxic drug that is widely used against hematologic malignancies including acute myeloid leukaemia (AML) (Stomper et al., 2021). Interestingly, 5-Aza-2’ is suggested to also have immunotherapy enhancing potential. 5- Aza-2’ enhances transcription of granzyme B and perforin, driving CD8+ T cell towards a heightened activation state¹². Furthermore, hypomethylating agents overcome transcriptional repression induced by DNA methylation, enabling transcription of tumour suppressor genes and enabling tumour specific transcripts that encode for neoantigens or tumour associated antigens enhancing immunotherapy potential^{13 14}.

The second compound the inventors employ is the small molecule SUMO E1 inhibitor TAK-981¹⁵ because the PTM SUMO inhibits anti-tumour immune responses. Blocking of SUMOylation increases CD8+ T cell activation and augments anti-tumour responses predominantly via upregulation of type I interferon signaling^16-19. Furthermore, abolishment of SUMOylation enhances MHO class I antigen presentation and consequently efficacy of immunotherapy possibly as a result of increased interferon signaling²⁰.

The inventors have previously shown that combination of 5-Aza-2’ and SUMOylation inhibitor TAK-981 possesses synergistic anti-cancer potential. SUMOylation inhibition enhances 5-Aza-2’ induced entrapment of DNMT 1 via inhibition of SUMOylation signal degradation²¹. The inventors found recently that these drugs synergise to kill B cell lymphoma in vitro and in vivo²². The inventor’s previous study was conducted in immunodeficient mice and therefore only shows the combined cytotoxic potential of these drugs.

In this study the inventors combine sub-cytotoxic dosages of 5-Aza-2’ with TAK-981 to strengthen eTCR therapy and CAR therapy. The inventors investigate the potentiating effect of TAK-981 mediated SUMOylation inhibition and hypomethylation via 5-Aza-2’ on targeted eTCR T cell therapy against acute myeloid leukaemia (AML) and multiple myeloma (MM), and CAR T cell therapy against MM and Acute Lymphoblastic Leukemia (ALL). First line of treatment for AML is often effective, however relapses occur frequently and patients with relapsed or refractory AML have poor prognosis²³. Therefore, new therapies including eTCR therapy are in high demand and need to be further improved. The inventors employ the OCI-AML3 xenograft NSG mouse model with CD8+ NPM1-eTCR T cells that recognize a 4 base pair frameshift mutant of nucleophosmin 1 which is mutated in 30-35% of all AMLs⁴. High dosing of 5-Aza-2’ is employed to treat AML, however toxicity and therapy resistance are often observed for single compound use¹¹. Here, the inventors employ the immune modulatory potential of both drugs, potentially preventing drug resistance and overcoming major toxicity issues via use of sub-cytotoxic dosage and altered treatment frequency. The inventors found that the combination of drugs strongly synergizes to potentiate eTCR therapy and CAR therapy, by sustaining in vivo T cell expansion and T cell activation.

The inventors have therefore surprisingly found that combining T cell mediated therapies (such as eTCR T cell therapy or CAR T cell therapy) with the SUMO E1 inhibitor TAK-981 and the DNA methylation inhibitor 5-Aza-2’ resulted in strong anti-tumour activity against in vivo tumour models of established Acute Myeloid Leukemia, Multiple Myeloma and Acute Lymphoblastic Leukemia. The inventors have surprisingly found that the drug combination (SUMO E1 inhibitor TAK-981 and the DNA methylation inhibitor 5-Aza-2’) caused strong eTCR T cell and CAR T cell proliferation in vivo. Mechanistically, the drug combination increased cytokine signalling by T cells while simultaneously increasing the antigen presentation properties of the tumour. Accordingly, combining T cell mediated therapy with TAK-981 and 5-Aza-2’ is an important step towards improved clinical outcome. The advantageous effects observed herein for eTCR therapies and CAR therapies should apply to any T cell mediated therapy, irrespective of the background disease.

In one aspect, a DNA hypomethylating agent is provided for use in treating a patient undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy.

In one aspect, a SUMOylation inhibitor is provided for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

In one aspect, a T cell mediated therapy is provided for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

In one aspect, a DNA hypomethylating agent and a SUMOylation inhibitor is provided for use in treating a patient undergoing treatment with a T cell mediated therapy.

In one aspect, a DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy is provided for use in treating a patient.

In one aspect, a DNA hypomethylating agent is provided for use in treating a patient by inducing or enhancing a T cell response in the patient, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a SUMOylation inhibitor is provided for use in treating a patient by inducing or enhancing a T cell response in the patient, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a DNA hypomethylating agent and a SUMOylation inhibitor is provided for use in treating a patient by inducing or enhancing a T cell response in the patient.

In one aspect, a DNA hypomethylating agent is provided for use, or a SUMOylation inhibitor is provided for use, wherein the patient is undergoing treatment with a T cell mediated therapy.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a DNA hypomethylating agent, wherein the patient is undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a SUMOylation inhibitor, wherein the patient is undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a T cell mediated therapy, wherein the patient is undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a DNA hypomethylating agent and a SUMOylation inhibitor, wherein the patient is undergoing treatment with a T cell mediated therapy. In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a DNA hypomethylating agent, a SUMOylation inhibitor, and a T cell mediated therapy.

In one aspect, a method for treating a patient is provided by inducing or enhancing a T cell response, the method comprising: administering to the patient a DNA hypomethylating agent wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a method for treating a patient is provided by inducing or enhancing a T cell response, the method comprising: administering to the patient a SUMOylation inhibitor wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a method for treating a patient is provided by inducing or enhancing a T cell response, the method comprising: administering to the patient a SUMOylation inhibitor and a DNA hypomethylating agent.

In a specific embodiment, the patient is undergoing treatment with a T cell mediated therapy.

In a specific embodiment, the method further comprises administering to the patient a T cell mediated therapy.

In a specific embodiment, the induced or enhanced T cell response comprises induced or enhanced CD8+ T cell proliferation and/ or CD4+ T cell proliferation.

In a specific embodiment, the DNA hypomethylating agent for use, the SUMOylation inhibitor for use, the T cell mediated therapy for use, or method is for treating cancer.

In a specific embodiment, the cancer is a solid cancer.

In a specific embodiment, the cancer is a haematological malignancy.

In a specific embodiment, the haematological malignancy is a myeloid haematological malignancy or lymphoid haematological malignancy.

In a specific embodiment, the DNA hypomethylating agent is selected from the group consisting of: 5-Azacytidine, 5-Aza-2’-deoxycytidine, 5-Aza-4'-thio-2'-deoxycytidine (5-Aza-T-dCyd), 5- Fluro-2-deoxycytidine, SGI-110, Zebularine, CP-4200, RG108, Nanaomycin A, SW155246, GSK3735967, GSK-3484862, GSK-3685032, RX-3117 (TV-1360), DC-05, y-Oryzanol, DC_517, DNMT1-IN-3, Procainamide, lsofistularin-3, CM-272, and SGI-1027.

In a specific embodiment, the SUMOylation inhibitor is an E1 inhibitor.

In a specific embodiment, the E1 inhibitor is selected from the group consisting of: TAK-981 , Ginkgolic acid (15:1), Anacardic acid, Kerriamycin B, SUMO-AMSN, SUMO-AVSN, Compound 21 , Davidiin, Tannic acid, ML-792, COH-OOO, and ML-93.

In a specific embodiment, the T cell mediated therapy is TCR, CAR-T, virus-specific T cell, bispecific T-cell engagers, or a vaccine.

In a specific embodiment, the T cell mediated therapy is immune mobilising monoclonal T-cell receptors against cancer, or tumour infiltration lymphocyte (TIL).

The inventors have surprisingly found that the drug combination (SUMO E1 inhibitor TAK-981 and the DNA methylation inhibitor 5-Aza-2’) induces or increases cancer cell immunogenicity in vivo. Mechanistically, the drug combination caused downregulation of the key immune checkpoint inhibitor PD-L1. Accordingly, combining TAK-981 and 5-Aza-2’ for inducing or enhancing cancer cell immunogenicity is an important step towards improved clinical outcome.

In one aspect, a DNA hypomethylating agent is provided for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a SUMOylation inhibitor is provided for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a DNA hypomethylating agent and a SUMOylation inhibitor are provided for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression.

In one aspect, a method of inducing or enhancing cancer cell immunogenicity in a patient is provided by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a DNA hypomethylating agent wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a method of inducing or enhancing cancer cell immunogenicity in a patient is provided by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a SUMOylation inhibitor wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a method of inducing or enhancing cancer cell immunogenicity in a patient is provided by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a SUMOylation inhibitor and a DNA hypomethylating agent.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

DESCRIPTION OF THE FIGURES Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 TAK-981 and 5-Aza-2’ synergistically reduce OCI-AML3 viability. A The SUMOylation cycle. SUMO precursor protein is cleaved by SUMO specific proteases (SENPs) to produce mature SUMO. Target proteins are SUMOylated via an enzymatic cascade that consists of the E1 activating enzyme, the E2 conjugating enzyme and an E3 ligase. SUMO can be removed from a target protein by SENPs. Small molecule SUMOylation inhibitor TAK-981 inhibits the E1 enzyme. B Mode of action of hypomethylation drug 5-Aza-2’-deoxycytidine. 5-Aza-2’ incorporates into the DNA and entraps DNA methyl transferase 1 (DNMT1). Trapped DNTM1 is SUMOylated and degraded by the proteasome. C OCI-AML3 cell viability is shown after 4 days of 5-Aza-2’ treatment (0.025 - 20 pM) or control DMSO 0.01 % treatment. IC50 was calculated with GraphPad Prism 9.3.1 (n=3) D OCI-AML3 cell viability after 4 days of TAK-981 treatment (0.0001 - 0.1 pM) or control DMSO 0.01 % treatment. IC50 was calculated with GraphPad Prism 9.3.1 (n=3). E OCI- AML3 cell viability after 4 days of combination treatment with dose response range of 5-Aza-2’ combined with 10 nM of TAK-981 . Excess overbliss synergy calculations of single 5-Aza-2’ doses versus 5-Aza-2’ doses with 10 nM TAK-981.

Figure 2 SUMOylation inhibition in combination with hypomethylation activates the interferon pathway, cytokine production and cytolytic compound signalling in CD8+ T cells. A CD8+ T cells were isolated from three different healthy donors. CD8+ were treated 10 days post stimulation with 10 nM TAK-981 and/or 25 nM 5-Aza-2’ or DMSO 0.01% as control overnight. mRNA expression levels of IFNy, IFN(3, IFNa, STAT1 , IFNAR1, IFIT1 , IFITM3, ISG15, ISG56, IRF7, T- bet, TNF-a, GranzymeB, Perforin-1 , IL-2, IL-4, IL-5 and IL-10 were measured using qPCR. 18sRNA, SDHA and SRPR were used as housekeeping genes. Expression was plotted as ratio to DMSO 0.01% control, individual per donor (n=2), Donor 1 light grey dots, Donor 2 dark grey dots. The third donor did not give a response. * = P<0.05, ** P<0.01 , *** P <0.001 , two-way ANOVA compared to DMSO 0.01%, followed by Dunnett multiple comparison correction GraphPad Prism 9.3.1. B Experimental co-culture set up to measure production of IFNy by CD8+ T cell upon coculture with OCI-AML3 target cells. Either CD8+ T cells or OCI-AML3 cell were pre-treated with TAK-981 and/or 5-Aza-2’ pre- overnight co-culture. C OCI-AML3 target cells were pre-treated on day 4 and 1 with 10 nM TAK-981 and/or 250 nM 5-Aza-2’. Subsequently, OCI-AML3 cells were co-cultured overnight with CD8+ T cells. Supernatant was harvested and analysed by IFNy ELISA. P < 0.05 = *, two-way ANOVA compared to DMSO 0.01%, followed by Fisher’s LSD test, GraphPad Prism 9.3.1. Bars represent the following treatments (from left to right): DMSO 0.01 %, TAK-981 10nM, 5-Aza-2’ 250 nM, TAK-981 10 nM + 5-Aza-2’ 250 nM. D CD8+ T cells were pretreated on day 4 and 1 with 10 nM TAK-981 and/or 250 nM 5-Aza-2’. Subsequently, CD8+ T cells were co-cultured with OCI-AML3 cells overnight. Supernatant was harvested and analysed by IFNy ELISA. P < 0.05 = *, two-way ANOVA compared to DMSO 0.01 %, followed by Fisher’s LSD test, GraphPad Prism 9.3.1. Bars represent the following treatments (from left to right): DMSO 0.01 %, TAK-981 10nM, 5-Aza-2’ 250 nM, TAK-981 10 nM + 5-Aza-2’ 250 nM.

Figure 3 NPM1-eTCR CD8+ T cell anti tumour efficacy is enhanced by 5-Aza-2’ and TAK-981 in vivo. A Timeline of in vivo experiment. Luciferase expressing OCI-AML3 cells (1*10⁶) were injected intra-venously (i.v.) into the tail vain of NSG-mice and engrafted for 10 days. Tumour volume was measured by I VIS. At day 10 treatment was started. Two rounds of the drug treatment with TAK-981 (25 mg/kg) and/or 5-Aza-2’ (2.5 mg/kg) were carried out. Subsequently NPM1- eTCR or CM -eTCR CD8+ T cells (3*10⁶) were i.v. injected on day 15. Bi-weekly drug treatments were continued until day 50 post tumour injection. B OCI-AML3 tumour outgrowth average per group (n=6/7), control group consisted of HPBCD buffer (n=3) and CD8+ CMV-eTCR (n=3) . Relative bioluminescent signal (BLI photons/sec/cm2/r) per mouse at day 10 is shown. C Survival curves for each group from B. The spaced line at day 50 indicates the end of the drug treatment. D Average OCI-AML3-Luc tumour outgrowth per group (n=6) ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10. Graphs represent time point when all mice were present in the experiment. A selection of groups from B containing at least NPM1-eTCR as therapy are shown. One-way ANOVA analysis was performed for tumour signals at day 35, in GraphPad Prism 9.3.1.

Figure 4 Combination therapy of 5-Aza-2’ and TAK-981 potentiate CD8+ T cell proliferation in vivo. A Time line of in vivo experiment. Luciferase expressing NPM1-eTCR or CMV-eTCR CD8+ T cells (3*10⁶) were injected 18 days post OCI-AML3 (1*10⁶) engraftment. Two times dosing of compounds prior to T cell injection was performed and 3 times following T cell injection, matching the dosing time to Figure 3. Bioluminescence (photons/sec/cm2/r) was measured at indicated timepoints day 3, 6 and 9 post T cell injection. B Raw values of ventral BLI signal (photons/sec/cm2/r) for day 3, 6 and 9 are visualized per group. Each dot represents an individual mouse. Differences to CD8+ NPM1-eTCR Luc group were analysed per time point via two-way ANOVA (mixed model) followed by Tukey multiple comparisons. P < 0.05 = *, P < 0.01 = **, P < 0.001 = *** (Day 3: n=10 per group, Day 6: n=8 per group) C Visualization of a representative mouse per treatment imaged on day 3, 6 and 8. Scaling for Bioluminescence was kept the same for each timepoint (Living Image Software).

Figure 5 SUMOylation inhibition enhances NPM1-eTCR CD8+ T cell activation and in combination with 5-Aza-2’ increases proliferation in vivo. A Timeline of in vivo experiment; NSG- mice were engrafted with OCI-AML3 cells for 14 days followed by treatment with 25mg/kg TAK- 981 and/or 2.5mg/kg 5-Aza-2’ or with a buffer control on the indicated days. NPM1-eTCR CD8+ T cells were injected day 15 post OCI-AML3 engraftment. Harvesting of bone marrow occurred on days 2, 5 or 8 post inoculation with the NPM1-eTCR CD8+ Luc T cells and analysed with spectral flow cytometry, n = 4 per group for day 2, n = 3 per group for day 5 and 8. B Ratio of CD8+ cells per total cells in bone marrow. Samples were used for marker analysis (Figure 5C, D and E) of CD8+ NPM1-eTCR LucT cells and OCI-AML3 cells. C Bar-graph represents percentage of Ki67 positive NPM1-eTCR CD8+ T cells. Differences between groups per day were calculated via two-way ANOVA followed by Tukey multiple comparison P < 0.01 = **. MFI is depicted in Figure 11A. The bars above each time point (day 2, day 5, day 8, respectively) represent the following treatments (from left to right): NPM1-TCR, NPM1-TCR + 5-Aza-2’, NPM1TCR + TAK- 981 , NPM1-TCR +TAK-981 + 5-Aza-2’. D Bar-graph represents percentage of IRF1 positive NPM1-eTCR CD8+ T cells. Differences between groups per day were calculated via two-way ANOVA followed by Tukey multiple comparison P < 0.05 = *, P < 0.001 = ***, P < 0.0001 = ****. MFI is depicted in Figure 11C. The bars above each time point (day 2, day 5, day 8, respectively) represent the following treatments (from left to right): NPM1-TCR, NPM1-TCR + 5-Aza-2’, NPM1TCR + TAK-981 , NPM1-TCR +TAK-981 + 5-Aza-2’. E Bar-graphs represent percentage of ICOS, CD25, PD1 , CD137, HLA-DR and LAG3 positive NPM1-eTCR CD8+. Differences between days of treatment were calculated via two-way ANOVA followed by Tukey multiple comparison P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***, P < 0.0001 = ****. MFI is depicted in Figure 11 E. The first group of bars consisting of three bars (from left to right) represents treatment with NPM1- TCR, followed by the second group of bars consisting of three bars represents treatment with NPM1-TCR + 5-Aza-2’, followed by the third group of bars consisting of three bars represents treatment with NPM1TCR + TAK-981 , followed by the fourth group of bars consisting of three bars represents treatment with NPM1-TCR +TAK-981 + 5-Aza-2’.

Figure 6 SUMOylation inhibition and 5-Aza-2’ upregulate HLA class I. A Timeline of in vivo experiment; NSG-mice were engrafted with OCI-AML3 cells for 10 days followed by treatment with 25mg/kg TAK-981 and/or 2.5mg/kg 5-Aza-2’ or with a buffer control on the indicated days. Consequently, bone marrow was harvested on day 18 and analysed with spectral flow cytometry. The bars above each donor represent the following treatments (from left to right) DMSO 0.01%, TAK-981 100 nM, and 5-Aza-2’ 250 nM. B Histogram plots show marker expression of HLA-ABC, CD86, CD58, CD54, Ki67 and PD-L1 on OCI-AML3 cells. Plots include average MFI signal per group. Control (n=4), 5-Aza-2’(n=4), TAK-981 (n=4), TAK-981 and 5-Aza-2’ (n=2). Samples were removed from analysis if insufficient OCI-AML3 cells were present, as indicated in Figure 12B. Gating strategy is shown in Figure 12A. C Timeline of in vivo experiment; OCI-AML3 cells were engrafted for 10 days in NSG-mice. Mice were treated with 25mg/kg TAK-981 and/or 2.5mg/kg 5- Aza-2’ or with a buffer control on indicated days. NPM1-eTCR CD8+ T cells were injected day 15 post OCI-AML3 engraftment. OCI-AML3 cells were analysed from bone marrow harvested days 2, 5 or 8 post injection with the NPM1-eTCR CD8+ Luc T cells via spectral flow cytometry. D Histogram plots show marker expression of HLA-A2, Ki67 and PD-L1 on OCI-AML3 cells from mice also inoculated with NPM1-eTCR CD8+ T cells. Plots include average MFI signal per group. Samples were removed from analysis if insufficient OCI-AML3 cells were present in sample as indicated in Figure 12B. Gating strategy is shown in Figure 12A.

Figure 7 TAK-981 and 5-Aza-2’ synergistically reduce LI266 viability. A OCI-AML3 cells treated with TAK-981 (50 - 1000 nM) or DMSO 0.01% control for 4 hours. Cells were lysed and analysed by immunoblotting for SUMO2/3, SLIMO1 and ubiquitin. PonceauS staining was used as loading control. B LI266 cells treated with 50 - 1000 nM of TAK-981 or DMSO 0.01% control for 4 hours, were analysed for SUMO2/3, SLIMO1 and ubiquitin protein expression via Western Blotting. PonceauS staining was used as loading control. C LI266 cell viability is shown after 4 days of 5- Aza-2’ treatment (0.025 - 20 pM) or control DMSO 0.01 % treatment (n=3). D LI266 cell viability after 4 days of TAK-981 treatment (0.05 - 1 pM) or control DMSO 0.01% treatment. (n=3). E LI266 cell viability after 4 days of combination treatment with dose response range of 5-Aza-2’ with 25 nM of TAK-981. Excess overbliss synergy calculations of single 5-Aza-2’ doses versus 5-Aza-2’ doses with 25 nM TAK-981 are shown in the right y-axis per dose.

Figure 8 Higher dosing of TAK-981 and 5-Aza-2’ increasingly activate interferon signalling, also causing more cytotoxicity. A mRNA expression levels of IFNa, IFNp, IFNy, STAT1 , IFNAR1 , ISG15, ISG56, IFIT1 , IFITM3, IRF7, TNFa, Granzyme B and Perforin 1 were measured using qPCR, for CD8+ T cells isolated from three different healthy donors. CD8+ T cells were treated 10 days post stimulation with 100 nM TAK-981 , 250 nM 5-Aza-2’ or DMSO 0.01 % as control overnight. 18sRNA, SDHA and SRPR were used as housekeeping genes. Expression was plotted as ratio to DMSO 0.01 % control, individual per donor. B Experimental co-culture set up for CD8+ T cell survival upon TAK-981 and/or 5-Aza-2’ treatment, quantification by flow cytometry. C NPM1-eTCR CD8+ T cell counts are shown upon 5 days of co-culture with irradiated OCI-AML3 cells (to prevent overgrowth). CD8+ T cells were treated with TAK-981 at 10 nM and/or 5-Aza-2’ at 250 nM or DMSO 0.01 % control. Co-cultures were set up in T cell medium deficient of IL2. Data represent three different CD8+ T cell donor replicates. Each replicate is plotted individually as ratio to normalized DMSO control.

Figure 9 NPM1-eTCR CD8+ T cells and HA2 CD8+ T cells anti-tumour efficacy is enhanced by 5-Aza-2’ and TAK-981 in vivo. A OCI-AML3 tumour outgrowth average per group (n=6); ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10 is shown. Graphs represent time point when all mice were present in the experiment. Only compound groups from Figure 3B are shown. One-Way ANOVA analysis was performed for tumour signals at day 31 , in GraphPad Prism 9.3.1. B TAK-981 enhances tumour cell killing by NPM1-eTCR. Data from Figure 3B. OCI- AML3 tumour outgrowth average per group (n=6) ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10. Graphs represent time point when all mice were present in the experiment. C 5-Aza-2’ enhances tumour cell killing by NPM1-eTCR. Data from Figure 3B. OCI- AML3 tumour outgrowth average per group (n=6); ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10. Graphs represent time point when all mice were present in the experiment. D TAK-981 and 5-Aza-2’ enhance tumour cell killing by NPM1-eTCR. Data from Figure 3B. OCI-AML3 tumour outgrowth average per group (n=6) ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10. Graphs represent time point when all mice were present in the experiment. E Timeline of in vivo experiment shown in Figure 3F. Luciferase expressing OCI-AML3 cells (1*10⁶) were injected intra-venously (i.v.) into the tail vain of NSG- mice and engrafted for 14 days. Drug dosing was used as described in Figure 3A. HA2-eTCR CD8+ T cells (3*10⁶) were injected on day 18. F OCI-AML3 tumour outgrowth average per group (n=6); ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10. Graphs represent time point when all mice were present in the experiment. One-Way ANOVA analysis was performed for tumour signals at day 31 , in GraphPad Prism 9.3.1 .

Figure 10 MAGE-A1 and BOB1-eTCR CD8+ T cell anti-tumour efficacy is enhanced by 5-Aza-2’ and TAK-981 in vivo. A U266-Luc tumour outgrowth per group (n=6) NSG (male) mice. Data is presented as ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 21. Graphs represent time point when all mice were present in the experiment. Only groups treated with compound are shown. One-Way ANOVA analysis was performed for tumour signals at day 36, in GraphPad Prism 9.3.1. B U266-Luc tumour outgrowth average per group (n=3) NSG (female) mice data is presented as ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10 upon BOB1-eTCR with or without TAK-981 and/or 5-Aza-2’. One-Way ANOVA analysis was performed for tumour signals at day 28, in GraphPad Prism 9.3.1. C U266-Luc tumour outgrowth average per group (n=3) NSG (female) mice data is presented as ratio to bioluminescent (BLI photons/sec/cm2/r) signal per mouse at day 10 upon MAGE1-A2-eTCR with or without TAK-981 and/or 5-Aza-2’. One-Way ANOVA analysis was performed for tumour signals at day 28, in GraphPad Prism 9.3.1.

Figure 11 SUMOylation inhibition enhances NPM1-eTCR CD8+ T cell activation and in combination with 5-Aza-2’ increases CD8+ T cell proliferation in vivo. A Bar-graph represents mean fluorescent intensity (MFI) of Ki67 in NPM1-eTCR CD8+ T cells. Differences between groups per day were calculated via two-way ANOVA followed by Tukey multiple comparison. The bars above each time point (day 2, day 5, day 8, respectively) represent the following treatments (from left to right): NPM1-TCR, NPM1-TCR + 5-Aza-2’, NPM1TCR + TAK-981 , NPM1-TCR +TAK- 981 + 5-Aza-2’. B Bar graphs present data of A compared between days per treatment. The first group of bars consisting of three bars (from left to right) represents treatment with NPM1-TCR, followed by the second group of bars consisting of three bars represents treatment with NPM1- TCR + 5-Aza-2’, followed by the third group of bars consisting of three bars represents treatment with NPM1TCR + TAK-981 , followed by the fourth group of bars consisting of three bars represents treatment with NPM1-TCR +TAK-981 + 5-Aza-2’. C Bar-graph represents MFI of IRF1 in NPM1-eTCR CD8+ T cells. Differences between groups per day were calculated via two-way ANOVA followed by Tukey multiple comparison. The bars above each time point (day 2, day 5, day 8, respectively) represent the following treatments (from left to right): NPM1-TCR, NPM1- TCR + 5-Aza-2’, NPM1TCR + TAK-981 , NPM1-TCR +TAK-981 + 5-Aza-2’. D Bar graphs present data of C compared between days per treatment. The first group of bars consisting of three bars (from left to right) represents treatment with NPM1-TCR, followed by the second group of bars consisting of three bars represents treatment with NPM1-TCR + 5-Aza-2’, followed by the third group of bars consisting of three bars represents treatment with NPM1TCR + TAK-981 , followed by the fourth group of bars consisting of three bars represents treatment with NPM1-TCR +TAK- 981 + 5-Aza-2’. E Bar-graphs represent mean fluor intensity (MFI) of ICOS, CD25, PD1 , CD137, H LA-DR. Differences between days per group were calculated via two-way ANOVA followed by Tukey multiple comparison. The first group of bars consisting of three bars (from left to right) represents treatment with NPM1-TCR, followed by the second group of bars consisting of three bars represents treatment with NPM1-TCR + 5-Aza-2’, followed by the third group of bars consisting of three bars represents treatment with NPM1TCR + TAK-981 , followed by the fourth group of bars consisting of three bars represents treatment with NPM1-TCR +TAK-981 + 5-Aza- 2’.

Figure 12 Gating strategy for data presented in Figure 5 and 6 and Figure 11. ZombieRed staining was used to gate live cells. Human cells were separated via differential mouse versus human CD45 staining. Human cells were gated and CD8+ cells were separated from OCI-AML3 cells via HLA-A2/HLA-ABC versus CD8+. For selected markers, mean fluor intensity (MFI) and percentage positive cells were analysed for CD8+ population and HLA-A2 or HLA-ABC populations. Samples were measured with Cytek Aurora spectral flow cytometer 3L (Cytekbio) and analyzed with OMIQ.ai (Dotmatics). B Counts of OCI-AML3 tumour cells per femur harvested from NSG-mice of which data is presented in Figure 6A and B. Indicated samples were removed from analysis, depending on total cell count. C Counts of OCI-AML3 tumour cells per femur harvested from NSG-mice 2 days post T cell injection of which data is presented in Figure 6C and D. Indicated samples were removed from analysis, depending on total cell count. D Counts of OCI-AML3 tumour cells per femur harvested from NSG-mice 5 days post T cell injection of which data is presented in Figure 6C and D. Indicated samples were removed from analysis, depending on total cell count. E Counts of OCI-AML3 tumour cells per femur harvested from NSG-mice 8 days post T cell injection of which data is presented in Figure 6C and D. Indicated samples were removed from analysis, depending on total cell count. Figure 13 TAK981 and 5-Aza-2’ synergize to induce in vivo activity of CAR T-cell treatment. A Multiple Myeloma (MM) Model in which mice were treated with 5-Aza-2', TAK981 , and BCMA- CAR T cells; B-D Acute Lymphoblastic Leukemia (ALL) Model in which mice were treated with 5- Aza-2', TAK981 , and CD19-CAR T cells.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

Various aspects of the invention are described in further detail below.

DETAILED DESCRIPTION

Medicaments and methods for use in treating patients

Methods for treating a patient, DNA hypomethylating agents for use in treating a patient, SUMOylation inhibitors for use in treating a patient, and T cell mediated therapies for use in treating a patient are provided herein as outlined below.

In one aspect, a DNA hypomethylating agent is provided for use in treating a patient undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy.

In one aspect, a SUMOylation inhibitor is provided for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

In one aspect, a T cell mediated therapy is provided for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

In one aspect, a DNA hypomethylating agent and a SUMOylation inhibitor is provided for use in treating a patient undergoing treatment with a T cell mediated therapy.

In one aspect, a DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy is provided for use in treating a patient.

In one aspect, a DNA hypomethylating agent is provided for use in treating a patient by inducing or enhancing a T cell response in the patient, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a SUMOylation inhibitor is provided for use in treating a patient by inducing or enhancing a T cell response in the patient, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a DNA hypomethylating agent and a SUMOylation inhibitor is provided for use in treating a patient by inducing or enhancing a T cell response in the patient.

In one aspect, a DNA hypomethylating agent is provided for use, or a SUMOylation inhibitor is provided for use, wherein the patient is undergoing treatment with a T cell mediated therapy. In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a DNA hypomethylating agent, wherein the patient is undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a SUMOylation inhibitor, wherein the patient is undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a T cell mediated therapy, wherein the patient is undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a DNA hypomethylating agent and a SUMOylation inhibitor, wherein the patient is undergoing treatment with a T cell mediated therapy.

In one aspect, a method for treating a patient is provided, the method comprising: administering to the patient a DNA hypomethylating agent, a SUMOylation inhibitor, and a T cell mediated therapy.

In one aspect, a method for treating a patient is provided by inducing or enhancing a T cell response, the method comprising: administering to the patient a DNA hypomethylating agent wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a method for treating a patient is provided by inducing or enhancing a T cell response, the method comprising: administering to the patient a SUMOylation inhibitor wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a method for treating a patient is provided by inducing or enhancing a T cell response, the method comprising: administering to the patient a SUMOylation inhibitor and a DNA hypomethylating agent.

In one aspect, a DNA hypomethylating agent is provided for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

In one aspect, a SUMOylation inhibitor is provided for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a DNA hypomethylating agent and a SUMOylation inhibitor are provided for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression.

In one aspect, a method of inducing or enhancing cancer cell immunogenicity in a patient is provided by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a DNA hypomethylating agent wherein the patient is undergoing treatment with a SUMOylation inhibitor. In one aspect, a method of inducing or enhancing cancer cell immunogenicity in a patient is provided by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a SUMOylation inhibitor wherein the patient is undergoing treatment with a DNA hypomethylating agent.

In one aspect, a method of inducing or enhancing cancer cell immunogenicity in a patient is provided by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a SUMOylation inhibitor and a DNA hypomethylating agent.

The terms “DNA hypomethylating agent”, “SUMOylation inhibitor”, “T cell mediated therapy”, “patient”, “undergoing treatment”, “treat”, “treating”, “treatment”, “induced or enhanced immune response”, ““inducing or enhancing a T cell response”, “administer,” “administering”, “administration”, “cancer”, “cancer cell immunogenicity”, “down-regulating PD-L1 cancer cell surface expression” and similar or equivalent terms have the general definitions provided herein, which apply to all aspects of the invention.

DNA hypomethylatinq agent

As used herein, the term “DNA hypomethylating agent” or “hypomethylating agent” refers to a compound or agent that inhibits DNA methylation. DNA hypomethylating agents inhibit the activity of methyltransferase, the enzymes that mediate DNA methylation thereby causing hypomethylation of DNA. Consequently, regular DNA synthesis is prevented. Suitable DNA hypomethylating agents can be identified by using methods known in the art. For example, cells can be treated with a potential hypomethylating agent and compared to negative and positive controls. The DNA of the three tested cells is extracted and completely digested to the single base, followed by analysis using HPLC or mass spectrophotometry, thereby determining the global DNA methylation status.

Suitable DNA hypomethylating agents include, but are not limited to 5-Azacytidine, 5-Aza-2’- deoxycytidine, 5-Aza-4'-thio-2'-deoxycytidine (5-Aza-T-dCyd), 5-Fluro-2-deoxycytidine, SGI-110, Zebularine, CP-4200, RG108, Nanaomycin A, SW155246, GSK3735967, GSK-3484862, GSK- 3685032, RX-3117 (TV-1360), DC-05, y-Oryzanol, DC_517, DNMT1-IN-3, Procainamide, lsofistularin-3, CM-272, and SGI-1027. See for example⁴².

As used herein, “5-Aza-2’-deoxycytidine” is used interchangeably with decitabine.

In a specific embodiment, the DNA hypomethylating agent is selected from the group consisting of 5-Azacytidine, 5-Aza-2’-deoxycytidine, 5-Aza-4'-thio-2'-deoxycytidine (5-Aza-T-dCyd), 5-Fluro- 2-deoxycytidine, SGI-110, Zebularine, CP-4200, RG108, Nanaomycin A, SW155246, GSK3735967, GSK-3484862, GSK-3685032, RX-3117 (TV-1360), DC-05, y-Oryzanol, DC_517, DNMT1-IN-3, Procainamide, lsofistularin-3, CM-272, and SGI-1027. In a specific embodiment, the DNA hypomethylating agent is 5-Azacytidine, 5-Aza-4'-thio-2'- deoxycytidine (5-Aza-T-dCyd) or 5-Aza-2’-deoxycytidine.

In a specific embodiment, the DNA hypomethylating agent is 5-Azacytidine.

In a specific embodiment, the DNA hypomethylating agent is 5-Aza-4'-thio-2'-deoxycytidine (5- Aza-T-dCyd), an orally active DNA methyltransferase I (DNMT1) inhibitor which is a variant of decitabine.

The DNA hypomethylating agent may be administered using any suitable method and dosage form, as described in detail below.

The terms “administer,” “administering,” or “administration” of a DNA hypomethylating agent refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a DNA hypomethylating agent described herein (in or on a subject). The compounds described herein can be administered by any suitable route (e.g. any conventional route) including enteral (e.g., oral, for example in tablet form), parenteral, intravenous, intramuscular, intracerebral, intravascular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, intracavity, transdermal, intradermal, rectal, intravaginal, percutaneous, intratracheal, intralesional, epidural, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol, and/or as an injection, and/or by infusion, and/or by gradual infusion over time. Specifically, contemplated routes are intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, direct administration to an affected site, and/or oral administration. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

The DNA hypomethylating agent described herein may therefore be in a form suitable for the appropriate mode of administration. For example, suitable forms for oral administration include a tablet or capsule; suitable forms for nasal administration or administration by inhalation include a powder or solution; suitable forms for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion; suitable forms for topical administration include a patch, an ointment or cream; and suitable forms for rectal administration include a suppository. Alternatively, the route of administration may be by injection (e.g. i.v.). Preferably, the DNA hypomethylating agent described here is provided at an effective dose. The actual dose used will depend on a number of parameters.

In one specific embodiment, the DNA hypomethylating agent is administered at a sub-cytotoxic dose. As used herein, “sub-cytotoxic” refers to a dose that is not capable of inducing cell death. In a non-limiting example, the DNA hypomethylating agent in a sub-cytotoxic dose induces cytokine transcription during T cell mediated therapy. In a non-limiting example, the T cell mediated therapy is a TCR therapy. In a non-limiting example, the DNA hypomethylating agent in a sub-cytotoxic dose induces transcription of cytolytic molecules. In another non-limiting example, the induced cytolytic molecule is Granzyme B. The person skilled in the art can easily work out which dose is sub-cytotoxic and dose the DNA hypomethylating agent accordingly.

In one specific embodiment, the sub-cytotoxic dose of the DNA hypomethylating agent is roughly about 1 nM, roughly about 2.5 nM, roughly about 5 nM, roughly about 7.5 nM, roughly about 10 nM, roughly about 12.5 nM, roughly about 15 nM, roughly about 17.5 nM, roughly about 20 nM, roughly about 22.5 nM, roughly about 25 nM, roughly about 27.5 nM, roughly about 30 nM, roughly about 32.5 nM, roughly about 35 nM, roughly about 37.5 nM, roughly about 40 nM, roughly about 42.5 nM, roughly about 45 nM, roughly about 47.5 nM, roughly about 50 nM, roughly about 55 nM, roughly about 60 nM, roughly about 65 nM, roughly about 70 nM, roughly about 80 nM, roughly about 90 nM, roughly about 100 nM.

In one specific embodiment, the sub-cytotoxic dose of the DNA hypomethylating agent 5- Azacytidine is 25 nM.

In one specific embodiment, the sub-cytotoxic dose of the DNA hypomethylating agent is roughly

110 nM, roughly about 120 nM, roughly about 130 nM, roughly about 140 nM, roughly about 150 nM, roughly about 160 nM, roughly about 170 nM, roughly about 180 nM, roughly about 190 nM, roughly about 200 nM, roughly about 21 O nM, roughly about roughly about 220 nM, roughly about 230 nM, roughly about 240 nM, roughly about 250 nM, roughly about 260 nM, roughly about 270 nM, roughly about 280 nM, roughly about 290 nM, roughly about 300 nM, roughly about 310 nM, roughly about 320 nM, roughly about 330 nM, roughly about 340 nM, roughly about 350 nM, roughly about 400 nM, roughly about 450 nM, roughly about 500 nM, roughly about 550 nM, roughly about 600 nM, roughly about 650 nM, roughly about 700 nM, roughly about 750 nM, roughly about 800 nM, roughly about 850 nM, roughly about 900 nM, roughly about 950 nM, roughly about 1000 nM. In one specific embodiment, the sub-cytotoxic dose of the DNA hypomethylating agent 5- Azacytidine is 250 nM.

In one specific embodiment, the sub-cytotoxic dose of the DNA hypomethylating agent (e.g. 5- Azacytidine) is from about 1 nM to about 100 nM. For example, it may be from about 10 nM to about 100 nM, or from about 10 nM to about 50 nM.

In one specific embodiment, the sub-cytotoxic dose of the DNA hypomethylating agent (e.g. 5- Azacytidine) is from about 100 nM to about 500 nM. For example, it may be from about 150 nM to about 400 nM, or from about 200 nM to about 300 nM.

The DNA hypomethylating agent may advantageously be presented in unit dosage form. Dosage forms (also called unit doses) are pharmaceutical drug products in the form in which they are marketed for use, with a specific mixture of active ingredients and inactive components (excipients), in a particular configuration (such as a capsule shell, for example), and apportioned into a particular dose. Depending on the route of administration, dosage forms include liquid, solid, and semisolid dosage forms. Common dosage forms include pills, tablet, capsule, drinks or syrups. In a non-limiting example, the DNA hypomethylating agent is presented as a single injection volume, or volume for intravenous administration (i.e. volume applied on one location at a certain time point), comprising a total pharmaceutical dosage.

SUMOylation inhibitor

As used herein, an “inhibitor” refers to any agent that inhibits, reduces, slows, halts, blocks, suppresses, abolishes and/or prevents the activity and/or expression of a target protein in a cell. An inhibitor may reduce, slow, halt, block, suppress, abolish and/or prevent the activity and/or expression of a target protein in a cell relative to a cell subjected to a control (or a cell that is not subjected to the inhibitor). As would be clear to a person skilled in the art, an inhibitor may function at the level of the target gene, transcript or protein. The term “inhibitor” as used herein may be used to refer to an SUMOylation inhibitor.

In some examples, “inhibit”, “reduce”, “block”, “suppress”, “prevent” etc means that the activity being inhibited, blocked, reduced, suppressed, or prevented is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the activity of a control (e.g., activity in the absence of the inhibitor). In some examples, “inhibit”, “block”, “reduce”, “suppress”, “prevent” etc means that the expression of the target of the inhibitor (e.g. SUMOylation) is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% as compared to a control (e.g., the expression in the absence of the inhibitor). As used herein, the term “SUMOylation inhibitor” refers to any agent that inhibits, reduces, slows, halts, blocks, supresses, abolishes and/or prevents the activity and/or expression of the conjugation of SUMO proteins to substrate proteins. Accordingly, as would be clear to a person skilled in the art, a SUMOylation inhibitor may function at the level of the gene, transcript or protein. For example, a SUMOylation inhibitor can be an agent (e.g. an inhibitory nucleic acid, a binding molecule (e.g. a small molecule or an antibody), or peptide) that inhibits the dimeric SUMO- activating enzyme E1 (also known as SAE1/UBA2), the single conjugating E2 enzyme (also known as ubiquitin-conjugating enzyme 9 (UBC9), and E3 ligase. A person of skill in the art will be able to readily identify suitable SUMOylation inhibitors using known methods in the art (e.g. based on assaying the effect of potential inhibitors on known SUMO activity). For example, cells can be treated with a potential SUMOylation inhibitor and compared to negative and positive controls, followed by for example, protein extraction. Then, the expression status of the one or more SUMO subunits E1 , E2, and/or E3 can be determined, for example, by measuring the level of protein using methods known in the art, such as but not limited to, Western blot, ELISA or ELISPOT, antibodies microarrays, mass spectrometry, immunofluorescence or immunohistochemistry. In specific embodiments, SUMOylation inhibitors also encompass SUMO inhibitors such as SENP (SUMO/sentrin specific protease)modulators. SENPs are responsible for the maturation of SUMO and for the deconjugation of SUMO from substrate proteins. Without wishing to be bound to theory, activation of SENPs has the same effect as inhibiting the SUMO conjugation machinery. In a specific embodiment, the SENP modulator is a SENP activator. In a specific embodiment, the SENP modulator is a SENP inhibitor. In specific embodiments, SUMOylation inhibitors also encompass modulators of SUMO proteases such as the DeSI (deSUMOylating isopeptidase) family and USPL1 (ubiquitin-specific peptidase-like protein 1) as described in Nayak and Muller, Genome Biology, (15): 422 (2014). SUMO protease family members include but are not limited to DeSI-1 and DeSI-2 as well as USPL1 (Schulz et al EMBO Rep (10): 930 (2021).

In some examples, the SUMOylation inhibitor inhibits, reduces, slows, halts, blocks, supresses, abolishes and/or prevents the SUMO-activating enzyme E1.

In another example, the SUMOylation inhibitor inhibits, reduces, slows, halts, blocks, supresses, abolishes and/or prevents the conjugating activity of E2.

In a further example, the SUMOylation inhibitor inhibits, reduces, slows, halts, blocks, supresses, abolishes and/or prevents ligating activity of E3.

In some examples, an SUMOylation inhibitor is a molecule that reduces or prevents SUMOylation. In another particular example, the SUMOylation inhibitor is a direct inhibitor. A “direct inhibitor”, as used herein, refers to an inhibitor that directly targets the target gene, transcript or protein. In the context of SUMOylation, a direct inhibitor directly inhibits the E1 , and/or E2, and/or E3 protein, a E1 , and/or E2, and/or E3 transcript and/or the E1 , and/or E2, and/or E3 gene, thereby reducing SUMOylation.

In a non-limiting example, the SUMOylation inhibitor exerts its activity by the formation of an adduct between the SUMOylation inhibitor itself and the SUMO protein. The resulting SUMOylation inhibitor-SUMO conjugate binds to SAE2 (also known as UBA2), the catalytic subunit of E1 , thereby inhibiting its activity.

Accordingly, in some examples, the inhibitor directly targets the E1 gene, RNA transcript or protein. For example, the inhibitor may directly bind to the E1 gene, RNA transcript or protein. In some examples, the inhibitor directly targets the E2 gene, RNA transcript or protein. For example, the inhibitor may directly bind to the E2 gene, RNA transcript or protein. In some examples, the inhibitor directly targets the E3 gene, RNA transcript or protein. For example, the inhibitor may directly bind to the E3 gene, RNA transcript or protein.

Any suitable E1 inhibitor, and/or E2 inhibitor, and/or E3 inhibitor may be used. In a particular example, the E1 inhibitor, and/or E2 inhibitor, and/or E3 inhibitor may be selected from the group consisting of: an inhibitory nucleic acid, a binding molecule (e.g. a small molecule or an antibody (including functional fragments thereof)) and a peptide. Suitable examples of inhibitory nucleic acids, binding molecules (e.g. small molecules, or antibodies (including functional fragments thereof)) or peptides would be readily identifiable to a person of skill in the art. For example, an extensive list of suitable SUMO E1 inhibitors is provided in US20160009744A1 , which is incorporated herein in its entirety.

In some examples, the inhibitor may be an “inhibitory nucleic acid” whose presence in a cell causes the degradation of or inhibits the function, transcription, or translation of its target gene in a sequence -specific manner. Exemplary inhibitory nucleic acids include aptamers, siRNA, microRNA-adapted shRNA, shRNA, precursor microRNA (pre-miRNA), pri-miRNA, miRNA, amiRNA, interfering RNA or RNAi, dsRNA, ribozymes, antisense oligonucleotides (ASO), and DNA expression cassettes encoding said inhibitory nucleic acids.

In some examples, the inhibitory nucleic acid is an antisense oligonucleotide. Antisense oligonucleotides (AONs) generally inhibit their target by binding target mRNA and sterically blocking expression by obstructing the ribosome. AONs can also inhibit their target by binding target mRNA thus forming a DNA-RNA hybrid that can be a substance for Rnase H. AONs may also be produced as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, oligonucleotide mimetics, or regions or portions thereof. Such compounds have also been referred to in the art as hybrids or gapmers. AONs typically comprise between 12 to 80, preferably between 15 to 40, nucleobases. Preferably, the AONs comprise a stretch of at least s nucleobases having 100% complementarity with the target mRNA. The skilled person would recognize that antisense compounds can be unmodified or modified. Modified antisense compounds may comprise modified nucleobases, modified sugars, modified backbones, or any combination of the foregoing modifications. Examples of modifications include, but are not limited to 2’0-Me modifications, 2’-F modification, substitution of unlocked nucleobase analogs, and phosphorothioate backbone modification.

In another example, the inhibitory nucleic acid may be transcribed into a ribozyme or catalytic RNA, which affects expression of a target mRNA. See, U.S. Pat. No. 6,423,885. Ribozymes can be designed to specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.

In some examples, the inhibitory nucleic acid is a double-stranded RNAi molecule specific for mRNA encoded by E1 , E2, or E3 gene. Methods for using RNAi to inhibit the expression of a gene are known to those of skill in the art. See, e.g., U.S. Pat. Nos. 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753, 139; and 6,777,588. See also WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; and U.S. Patent Publications 20030175965, 20030175783, 20040214330 and 20030180945.

In some examples, the inhibitor is a binding molecule. Preferably, the binding molecule binds to E1 , E2, and/or E3 and inhibits its activity (for example it inhibits one or more of the activities for E1 , E2, and/or E3 described elsewhere herein). In a non-limiting example, the binding molecule may bind to E1 and thereby inhibit, reduce, slow, halt, block, supress, abolish and/or prevent E1- SUMO complex formation. In another non-limiting example, the binding molecule may bind to E2 and thereby inhibit, reduce, slow, halt, block, supress, abolish and/or prevent E2-SUMO complex formation. In another non-limiting example, the binding molecule may bind to E3 and thereby inhibit, reduce, slow, halt, block, supress, abolish and/or prevent E3-SUMO complex formation.

An example of a binding molecule is a small molecule. Binding molecules also include antibodies as well as non-immunoglobulin binding agents, such as phage display-derived peptide binders, and antibody mimics, e.g., affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains, aptamers and affilins. The term “antibody” includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab’, F(ab’)2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments. Antibodies which bind a particular epitope can be generated by methods known in the art. For example, polyclonal antibodies can be made by the conventional method of immunizing a mammal (e.g., rabbits, mice, rats, sheep, goats). Polyclonal antibodies are then contained in the sera of the immunized animals and can be isolated using standard procedures (e.g., affinity chromatography, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography). Monoclonal antibodies can be made by the conventional method of immunization of a mammal, followed by isolation of plasma B cells producing the monoclonal antibodies of interest and fusion with a myeloma cell (see, e.g., Mishell, et al., 1980). Screening for recognition of the epitope can be performed using standard immunoassay methods including ELISA techniques, radioimmunoassays, immunofluorescence, immunohistochemistry, and Western blotting (Ausubel, et al., 1992). In vitro methods of antibody selection, such as antibody phage display, may also be used to generate antibodies (see, e.g., Schirrmann et al. 2011). Preferably, a nuclear localization signal is added to the antibody in order to increase localization to the nucleus.

In some examples, the activity and/or expression of a target protein (e.g. E1 , E2 or E3) in a cell may be inhibited, reduced, slowed, halted, blocked, suppressed, abolished and/or prevented by introduction of a mutation that disrupts the target gene (e.g. by introduction of a mutation that disrupts the E1 , E2 or the E3 gene). As would be clear to a skilled person, such a mutation may decrease expression of the protein encoded by the target gene (e.g. E1 , E2 or E3), abrogate expression of the gene entirely, or render the gene product non-functional. Accordingly, in some examples, the mutation may be a loss of function mutation. In some examples, the mutation may be a point mutation, an insertion, a substitution or a deletion. In some examples, both alleles of the target gene are mutated. In some examples, the mutation may be located in a coding region (e.g., in an exon of E1 , E2 or E3) and/or in a non-coding region of the target gene (e.g. in the promoter region of E1 , E2 or E3).

Mutation of the E1 , E2, or E3 gene may be accomplished by methods well known in the art, including gene editing techniques (Doudna, 2020). For example, a mutation may be introduced into the E1 , E2 and/or E3 gene using a targeted genome editing technique (e.g. using targeted genome editing construct(s)). For example, a mutation may be introduced into the E1 gene, and/or E2 genes, and/or the E3 gene using zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) nucleases (e.g. RNA-guided DNA endonuclease Cas9 (or variants thereof)) or meganucleases. In some examples, meganucleases, ZFNs orTALENs may be used to introduce a mutation to the E1 , and/or E2, and or E3 gene since meganuclease, ZFN, and TALEN proteins can be engineered to recognize specific target DNA sequences (e.g. specific regions of the E1 , E2, or E3 gene). Meganuclease, ZFN, and TALEN specificity is achieved through protein-DNA interactions.

Accordingly, in some examples, the inhibitor may be a binding molecule that comprises a meganuclease, a zinc-finger nuclease, or a transcription activator-like effector nuclease. The inhibitor may also be a binding molecule that is part of a binding complex, where the binding complex comprises one or more (e.g. a pair of) meganucleases, zinc-finger nucleases, or transcription activator-like effector nucleases. Meganucleases integrate nuclease and DNA- binding domains whereas ZFN and TALEN proteins comprise individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively.

In some examples, the CRISPR/Cas system may be used to introduce a mutation to the E1 , and/or E2, and or E3 gene. Any suitable CRISPR/cas system may be used herein. The CRISPR/Cas system is based on RNA-guided Cas9 nuclease from the type II prokaryotic CRISPR adaptive immune system (see, e.g., Belahj et al., Plant Methods 9:39, 2013). This system comprises three components: Cas9 protein, crRNA, and tracrRNA. The crRNA and tracrRNA are usually provided as a single RNA molecule referred to as gRNA (guideRNA or signal guide RNA (sgRNA)). This system involves targeting Cas9 to a specific genomic locus (i.e. the E1 , E2, or E3 locus) via a gRNA. Accordingly, the gRNA is designed to bind to the target sequence, for example, the E1 gene, E2 gene, or E3 gene. The binding site is chosen such that the DNA targeted is followed by a PAM sequence (protospacer adjacent motif). The canonical length of the DNA-recognition sequence of the gRNA is 20bp. Accordingly, in some examples, where the CRISPR/Cas system is used to introduce a mutation to the E1 gene, and/or E2 gene, and/or the E3 gene, the inhibitor may be a binding molecule that is a gRNA.

Accordingly, as would be clear to a person skilled in the art, the term “inhibitor” encompasses binding molecules that are used in gene editing techniques to introduce a mutation that disrupts the target gene such that the activity and/or expression of the target protein (e.g. E1 , E2, or E3) in a cell is inhibited, reduced, slowed, halted, blocked, suppressed, abolished and/or prevented. Such binding molecules include, by way of example, meganucleases, zinc-finger nucleases, transcription activator-like effector nucleases and gRNA.

A person skilled in the art would readily be able to design suitable targeted genome editing construct(s) that inhibit, reduce, slow, halt, block, suppress, abolish and/or prevent the activity and/or expression of E1 , E2, or E3 in a cell (e.g. suitable targeted genome editing construct(s) that inhibit, reduce, slow, halt, block, suppress, abolish and/or prevent the activity and/or expression of E1 , E2, or E3 in a cell via introduction of a mutation to the E1 gene, and/or E2 gene, and/or the E3 gene. For example, a skilled person would readily be able to design meganuclease(s) specific for the E1 gene, the E2 gene or the E3 gene, a skilled person would readily be able to design zinc-finger nuclease(s) specific for the E1 gene, the E2 gene or the E3 gene, a skilled person would readily be able to design transcription activator-like effector nuclease(s) specific for the E1 gene, and/or E2 gene, and/or the E3 gene, a skilled person would readily be able to design a gRNA (for use with any suitable CRISPR nuclease) specific for the E1 gene, and/or E2 gene, and/or the E3 gene. In a specific embodiment, the SUMOylation inhibitor is TAK-981 , Ginkgolic acid (15:1), Anacardic acid, Kerriamycin B, SUMO-AMSN, SUMO-AVSN, Compound 21 , Davidiin, Tannic acid, ML-792, COH-OOO, ML-93, GSK145A, 2-D08, Spectomycin B, Compound 2, SLIBINs, Compound 38, Triptolide, Compound J5, GN6958, Compound 69 and 117, Compound 13m, Momordin Ic (Me), Compound 3, Streptonigrin, Ebselen, or Compound 6, 7, and 10. Further details of these compounds are provided in Kroonen and Vertegaal, Trends Cancer 7(6): 496-510 (2021), Table 1 and the structural formulae provided herein.

Table 1: SUMOylation Inhibitors

The structural formulae of Compound 21 , Compound 2, Compound 38, Compound J5, Compound 69, Compound 117, Compound 13m, Compound 3, Compound 6, Compound 7, and Compound 10 are displayed in Table 2 below: Table 2:Structural formulae

In a specific embodiment, the SUMOylation inhibitor is Compound 38, Triptolide, Compound J5, GN6958, Compound 69 and 117, Compound 13m, Momordin Ic (Me), Compound 3, Streptonigrin, Ebselen, or Compound 6, 7, and 10.

In a specific embodiment, the SUMOylation inhibitor is a E1 SUMOylation inhibitor.

In a specific embodiment, the E1 SUMOylation inhibitor is TAK-981 , Ginkgolic acid (15:1), Anacardic acid, Kerriamycin B, SUMO-AMSN, SUMO-AVSN, Compound 21 , Davidiin, Tannic acid, ML-792, COH-OOO, and ML-93. Further details of these compounds are provided in Kroonen and Vertegaal, Trends Cancer 7(6): 496-510 (2021) and Table 1 and the structural formulae provided herein.

In a specific embodiment, the SUMOylation inhibitor is TAK-981.

In a specific embodiment, the SUMOylation inhibitor is a E2 SUMOylation inhibitor.

In a specific embodiment, the E2 SUMOylation inhibitor is GSK145A, 2-D08, Spectomycin B, Compound 2, or SUBINs.

In a specific embodiment, the SUMOylation inhibitor is a E3 SUMOylation inhibitor.

In a specific embodiment, the SUMOylation inhibitor is a SENP modulator.

In a specific embodiment, the SUMOylation inhibitor is a SENP1 modulator.

In a specific embodiment, the SENP1 SUMOylation inhibitor is Compound 38, Triptolide, Compound J5, GN6958, Compound 13m, Momordin Ic (Me), Streptonigrin, or Compound 6, 7, and 10. Further details of these compounds are provided in Kroonen and Vertegaal, Trends Cancer 7(6): 496-510 (2021), Table 1 and the structural formulae provided herein.

In a specific embodiment, the SUMOylation inhibitor is a SENP2 modulator

In a specific embodiment, the SENP2 SUMOylation inhibitor is Compound 69 and 117 or Ebselen.

In a specific embodiment, the SUMOylation inhibitor is a SENP1/2 modulator.

In a specific embodiment, the SENP1/2 SUMOylation inhibitor is Compound 3.

In a specific embodiment, the SUMOylation inhibitor is a SENP3 modulator.

In a specific embodiment, the SUMOylation inhibitor is a SENP5 modulator.

In a specific embodiment, the SUMOylation inhibitor is a SENP6 modulator.

In a specific embodiment, the SUMOylation inhibitor is a SENP7 modulator.

In a specific embodiment, the SUMOylation inhibitor is a modulator of SUMO-specific isopeptidases or SUMO-specific proteases.

In a specific embodiment, the SUMOylation inhibitor is a DeSI-1 modulator.

In a specific embodiment, the SUMOylation inhibitor is a DeSI-2 modulator.

In a specific embodiment, the SUMOylation inhibitor is a USPL1 modulator.

The SUMOylation inhibitor may be administered using any suitable method and dosage form, as described in detail below.

The terms “administer,” “administering,” or “administration” of a SUMOylation inhibitor refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a the SUMOylation inhibitor described herein (in or on a subject). The compounds described herein can be administered by any suitable route (e.g. any conventional route) including enteral (e.g., oral, for example in tablet form), parenteral, intravenous, intramuscular, intracerebral, intravascular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, intracavity, transdermal, intradermal, rectal, intravaginal, percutaneous, intratracheal, intralesional, epidural, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, buccal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol, and/or as an injection, and/or by infusion, and/or by gradual infusion over time. Specifically, contemplated routes are intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, direct administration to an affected site, and/or oral administration. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration).

The SUMOylation inhibitor described herein may therefore be in a form suitable for the appropriate mode of administration. For example, suitable forms for oral administration include a tablet or capsule; suitable forms for nasal administration or administration by inhalation include a powder or solution; suitable forms for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion; suitable forms for topical administration include a patch, an ointment or cream; and suitable forms for rectal administration include a suppository. Alternatively, the route of administration may be by injection (e.g. i.v.).

Preferably, the SUMOylation inhibitor described herein is provided at an effective dose. The actual dose used will depend on a number of parameters.

In one specific embodiment, the SUMOylation inhibitor is administered at a sub-cytotoxic dose. As used herein, “sub-cytotoxic” refers to a dose that is not capable of inducing cell death. In a non-limiting example, the SUMOylation inhibitor in a sub-cytotoxic dose induces cytokine transcription of T cell mediated therapy. In a non-limiting example, the T cell mediated therapy is TOR therapy. In a non-limiting example, the SUMOylation inhibitor in a sub-cytotoxic dose induces transcription of IFNa, IFN/3, IFNy and interferon related genes such as ISG56 and IRF7. The person skilled in the art can easily work out which dose is sub-cytotoxic and dose the SUMOylation inhibitor accordingly. See for example¹⁷.

In one specific embodiment, the sub-cytotoxic dose of the SUMOylation inhibitor is roughly about

1 nM, roughly about 2.5 nM, roughly about 5 nM, roughly about 7.5 nM, roughly about 10 nM, roughly about 12.5 nM, roughly about 15 nM, roughly about 17.5 nM, roughly about 20 nM, roughly about 22.5 nM, roughly about 25 nM, roughly about 27.5 nM, roughly about 30 nM, roughly about 32.5 nM, roughly about 35 nM, roughly about 37.5 nM, roughly about 40 nM, roughly about 42.5 nM, roughly about 45 nM, roughly about 47.5 nM, roughly about 50 nM, roughly about 55 nM, roughly about 60 nM, roughly about 65 nM, roughly about 70 nM, roughly about 80 nM, roughly about 90 nM, roughly about 100 nM.

In one specific embodiment, the sub-cytotoxic dose of the SUMOylation inhibitor TAK-981 is 10 nM.

In one specific embodiment, the sub-cytotoxic dose of the SUMOylation inhibitor is roughly 110 nM, roughly about 120 nM, roughly about 130 nM, roughly about 140 nM, roughly about 150 nM, roughly about 160 nM, roughly about 170 nM, roughly about 180 nM, roughly about 190 nM, roughly about 200 nM, roughly about 21 O nM, roughly about roughly about 220 nM, roughly about

230 nM, roughly about 240 nM, roughly about 250 nM, roughly about 260 nM, roughly about 270 nM, roughly about 280 nM, roughly about 290 nM, roughly about 300 nM, roughly about 310 nM, roughly about 320 nM, roughly about 330 nM, roughly about 340 nM, roughly about 350 nM, roughly about 400 nM, roughly about 450 nM, roughly about 500 nM, roughly about 550 nM, roughly about 600 nM, roughly about 650 nM, roughly about 700 nM, roughly about 750 nM, roughly about 800 nM, roughly about 850 nM, roughly about 900 nM, roughly about 950 nM, roughly about 1000 nM.

In one specific embodiment, the sub-cytotoxic dose of the SUMOylation inhibitor TAK-981 is 100 nM.

In one specific embodiment, the sub-cytotoxic dose of the SUMOylation inhibitor (e.g. TAK-981) is from about 1 nM to about 100 nM. For example, it may be from about 5 nM to about 50 nM, or from about 10 nM to about 30 nM.

In one specific embodiment, the sub-cytotoxic dose of the SUMOylation inhibitor (e.g. TAK-981) is from about 100 nM to about 500 nM. For example, it may be from about 50 nM to about 300 nM, or from about 100 nM to about 300 nM.

The SUMOylation inhibitor may advantageously be presented in unit dosage form. Suitable dosage forms (also called unit doses) are described elsewhere herein and will depend on the route of administration.

T cell mediated therapy

As used herein, a “T cell mediated therapy” is any therapy that involves a T cell mediated approach, or T cell mediated mechanism. The skilled person is well aware that for example bispecific T cell engagers are designed bind to a T cell target such as CD3 and a target antigen on a target cell different than said T cell. Accordingly, the T cells are engaged and directed to the target cells of interest. Similarly, the recognition of fragments of antigens (peptides) is facilitated by T cells; a mechanism exploited by vaccines. Accordingly, therapeutics including but not limited to bi-specific T cell engagers, vaccines, immune mobilising monoclonal T-cell receptors against X diseases, and immune mobilising monoclonal T-cell receptors against cancer are encompassed. T cell therapies including but not limited to adoptive T cell therapy approaches are encompassed such as (engineered) T cell receptor (TCR) therapy, tumour-infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor (CAR) T cell therapy, virus specific T cell therapy. Accordingly, T cells that are present in a patient already (for example, due to a previous therapy including but not limited to T cell receptor (TCR) therapy, tumour-infiltrating lymphocyte (TIL) therapy, chimeric antigen receptor (CAR) T cell therapy, virus specific T cell therapy) are also encompassed by T cell mediated therapy. Bi-specific T cell engagers, vaccines, immune mobilising monoclonal T-cell receptors against X diseases, immune mobilising monoclonal T-cell receptors against cancer, (engineered) T cell receptor (TCR) therapy, tumour-infiltrating lymphocyte (TIL) Therapy, and chimeric antigen receptor (CAR) T cell therapy are further defined herein.

In a specific embodiment, the T cell mediated therapy is a TCR, CAR-T, virus-specific T cell, bi- specific T-cell engager, immune mobilising T-cell receptor against X disease, immune mobilising T-cell receptor against cancer, tumour infiltrating lymphocyte (TIL), or a vaccine.

In a specific embodiment, the T cell mediated therapy is a TCR, CAR-T, virus-specific T cell,, bi- specific T-cell engager, immune mobilising T cell receptor against X disease, or a vaccine.

In a specific embodiment, the T cell mediated therapy is a TCR, CAR-T, virus-specific T cell, bi- specific T-cell engagers, or a vaccine.

In a specific embodiment, the T cell mediated therapy is a immune mobilising T-cell receptor against cancer, or tumour infiltrating lymphocyte (TIL).

As used herein, “TCR” (T cell receptor) is a molecule found on the surface of T cells (T lymphocytes) that is responsible for recognising a peptide that is bound to (presented by) a major histocompatibility complex (MHC) molecule on a target cell. Preferably, the TCR is an engineered TCR. In a non-limiting example, the TCR is an antigen binding fragment of a TCR such as for example a single chain TCR (scTCR) or a chimeric dimer composed of the antigen binding fragments of the TCR a and TCR chain linked to transmembrane and intracellular domains of a dimeric complex so that the complex is a chimeric dimer TCR (cdTCR). Other appropriate binding proteins that comprise target antigen specific -TCR components are also encompassed. TCRs, in particular engineered T cells expressing TCRs that are antigen specific TCRs are one nonlimiting example of T cell mediated therapies. TCR therapies are well known in the art. For example, BOB1-TCRs are described in WO 2016/071758 (which describes the BOB1-TCR from clone 4G11 used in the example section below) and WO 2022/071795 (which describes the BOB1-TCR from clone 1C5.6 referred to below). As a further example, NPM1-TCRs are described in WO 2019/004831 , MAGEA1-TCRs are described in de Rooij et al, Mol Ther Oncolytics 28: 1-14 (2023) as a further example, and HA2-TCRs are described in Heemskerk et al, Blood 109 (1): 235-243 (2007). Appropriate TCR therapies are readily identifiable by the person skilled in the art.

In a specific embodiment, the TCR is a BOB1-TCR, a NPM-TCR, a MAGEA1-TCR, or a HA2- TCR.

In a specific embodiment, the TCR is a BOB1-TCR. Accordingly, said TCR is a BOB1 specific TCR.

In a non-limiting example, the BOB1 specific TCR is for use in the treatment of multiple myeloma.

In a specific embodiment, the BOB1 specific TCR is the TCR of clone 4G11 and HLA- B*07:02 restricted.

In a non-limiting example, the BOB1 specific TCR of clone 4G11 is for use in the treatment of multiple myeloma.

In a specific embodiment, the BOB1 specific TCR is the TCR of clone 1C5.6 and HLA- B*35:01 restricted.

In a specific embodiment, the TCR is a NPM-TCR. Accordingly, said TCR is a NPM specific TCR.

In a non-limiting example, the NPM specific TCR is for use in the treatment of acute myeloid leukaemia.

In a specific embodiment, the NPM specific TCR is the TCR of clone 1A2 and HLA-A*02:01 restricted.

In a non-limiting example, the NPM specific TCR of clone 1A2 is for use in the treatment of acute myeloid leukaemia.

In a specific embodiment, the TCR is a MAGEA1-TCR. Accordingly, said TCR is a MAGEA1 specific TCR. In a non-limiting example, the MAGEA1- specific TCR is for use in the treatment of multiple myeloma.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 4F7, 10C1 , 6G4, 3H4, 3B2, 3G2, or 2C2.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 4F7 and HLA-A*02:01 restricted.

In a non-limiting example, the MAGEA1 specific TCR of clone 4F7 is for use in the treatment of multiple myeloma.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 10C1 and HLA-C*07:02 restricted.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 6G4 and HLA-A*01 :01 restricted.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 3H4 and HLA-A*03:01 restricted.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 3B2 and HLA-A*03:01 restricted.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 3G2 and HLA- B*07:02 restricted.

In a specific embodiment, the MAGEA1 specific TCR is the TCR of clone 2C2 and HLA- B*07:02 restricted.

In a specific embodiment, the TCR is a HA2-TCR. Accordingly, said TCR is a HA2 specific TCR.

In a specific embodiment, the HA2 specific TCR is the clone of HA2.5 and therefore, HLA- A*02 restricted.

In a non-limiting example, the HA2 specific TCR is the TCR of clone HA2.5 is for use in the treatment of haematological malignancies. In a non-limiting example, the HA2 specific TCR is the TCR of clone HA2.5 is for use in the treatment of acute myeloid leukaemia.

In a specific embodiment, the HA2 specific TCR is the TCR of clone of HA2.6 and therefore, HLA- A*02 restricted.

In a non-limiting example, the HA2 specific TCR is the TCR of clone HA2.6 is for use in the treatment of haematological malignancies.

In a specific embodiment, the HA2 specific TCR is the TCR of clone of HA2.19 and therefore, HLA- A*02 restricted.

In a non-limiting example, the HA2 specific TCR is the TCR of clone HA2.19 is for use in the treatment of haematological malignancies.

In a specific embodiment, the HA2 specific TCR is the TCR of clone of HA.2.20 and therefore, HLA- A*02 restricted.

In a non-limiting example, the HA2 specific TCR is the TCR of clone HA2.20 is for use in the treatment of haematological malignancies.

As used herein, “CAR” (chimeric antigen receptor) refers to a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs described herein include an extracellular portion comprising an antigen binding domain (i.e. , obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as an scFv derived from an antibody or TCR specific for a cancer antigen, or an antigen binding domain derived or obtained from a killer immunoreceptor from an NK cell, or the extracellular part of an HLA class I or II molecule either linked with a specific peptide or not) linked to a transmembrane domain and one or more intracellular signalling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al, Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al, Cancer Immunol. Immunother., 63(11): 1163 (2014), Gille et al, HLA, (4): 436-448 (2023)).

As used herein, “CAR (chimeric antigen receptors)-T cells” are cells which have been engineered encoding a CAR transgene of interest to specifically recognise surface antigens on target cells. Accordingly, the CAR-T cells express a CAR protein which is a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. The target cells include but are not limited to cancer cells. CAR-T cells may be generated from any suitable source of T cells known in the art including, but not limited to, T cells collected from a subject. The collected T cells may be expanded ex vivo using methods commonly known in the art before transduction with a CAR to generate a CAR-T cell.

Methods for CAR design, delivery and expression in T cells, and the manufacturing of clinical- grade CAR-T cell populations are known in the art. See, for example, Lee et al., Clin. Cancer Res. 2012, 18(10): 2780-9, intracellular signalling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al, Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), and Stone et al, Cancer Immunol. Immunother., 63(11): 1163 (2014)).

In a non-limiting example, the CAR T cell therapy is a BCMA-CAR T cell therapy or a CD19-CAR T cell therapy. Several appropriate BCMA-CAR T cell therapies or CD19-CAR T cell therapies are known in the art; see for example⁴⁰ for a BCMA CAR and⁴¹ for a CD19 CAR.

As used herein, “virus-specific T cells” are T cells specific for a virus such as human gammaherpesvirus 4 (EBV), human cytomegalovirus (CMV), adenovirus, BK polyomavirus, JC polyomavirus, SARS-CoV2. In a non-limiting example, said virus-specific T cells are from seropositive donors. In a non-limiting example, said donor virus-specific T cells are further processed by virus-specific T cell selection and/or expansion. A method of producing virusspecific T cells and virus-specific T cells for use in treating patients is for example described in Houghtelin and Bollard, Front Immunol. 8: 1272 (2017).

As used herein, “bi-specific T-cell engagers” also known as BiTEs are bispecific monoclonal antibodies. They comprise two scFvs comprised in one single amino acid sequence. In a nonlimiting example, the two scFvs are from different antibodies. In a non-limiting example, the two scFvs comprise four different amino acid sequences from different genes that are linked via a peptide. The first scFv binds to a surface protein of T cells, including but not limited to CD3. The second scFv binds to a surface protein of a target cell, including but not limited to a cancer cell. Bi-specific T-cell engagers include but are not limited to Blinatumomab, Glofitamab, Mosunetuzumab, Solitomab, and Talquetamab.

As used herein, immune mobilising monoclonal T-cell receptors against X disease molecules also known as immTAC molecules are molecules comprising a TCR connected to an anti-CD3 antibody. ImmTACs are therefore bispecific, combining target recognizing TCR components with immune activating complexes. In a non-limiting example, immTAC molecules activate T cell responses to specifically kill for example, infected cells. In a non-limiting example, the immTAC molecules kill infected cells via an immune activating effector function. In a non-limiting example, the infected cells can be infected with Human Immunodeficiency Virus (HIV), or Hepatitis B Virus (HBV). Accordingly, in a non-limiting example, the immTAC molecule can be targeted to Gag (A02) in the example of HIV, or Envelope (A02) in the example of HBV.

As used herein, a “vaccine” is for example a peptide vaccine for treating or preventing a disease. The isolated peptide may be administered to induce or enhance an immune response to the presented peptide. These peptides are presented to the T cell repertoire of a subject in vivo and induce T cell activation.

In a specific embodiment, the T cell mediated therapy is a immune mobilising monoclonal T-cell receptors against cancer molecule, or tumour infiltrating lymphocytes (TIL).

As used herein, “immune mobilising monoclonal T-cell receptors against cancer” molecules also known as immTAC molecules are molecules comprising a TCR connected to an anti-CD3 antibody. ImmTACs are therefore bispecific, combining target recognizing TCR components with immune activating complexes. In a non-limiting example, the immTAC molecules activate T cell responses to specifically kill target cancer cells. In a non-limiting example, the immTAC molecules kill target cancer cells via an immune activating effector function. Accordingly, the immTAC molecule can be targeted to PRAME (A02), PRAME-HLE (A02), PRAME (A24), or PIWIL1 (A02).

In a specific embodiment, the immTAC molecule is Tebentafusp.

As used herein, “tumour infiltrating lymphocytes” (TIL) is a cell therapy leveraging the patient’s TIL from a tumour, harvesting, culturing, and amplifying said infiltrated lymphocytes in vitro, followed by infusing them back to the patient to be treated. In a non-limiting example, the harvested TIL are subjected to gene editing, for example a gene of interest is overexpressed or knocked out by for example TALEN or CRISPR-Cas. TIL can be administered for example, after a chemotherapy. In another non-limiting example, after administration of TIL, administration of immune system activating agents including but not limited to IL-2 can be administered. TIL is another non-limiting example of a T cell mediated therapy given that T cells and especially CD8+ cytolytic T cells (CTL) are regarded as major anti-tumour immune effector cells.

Cancer cell immunogenicity’ As used herein, “cancer cell immunogenicity” is the ability of a cancer to induce an immune response. Said immune response can, for example, control cancer growth by arresting cancer growth, or can shrink the cancer, or can even lead to the eradication of the cancer. Cancer cell immunogenicity is influenced by the cancer cells as well as the cancer microenvironment comprising various immune cells such as dendritic cells and T cells. In a non-limiting example, cancer cell immunogenicity is induced or enhanced. As used herein, the phrase “induced or enhanced tumour immunogenicity” refers to an increased immune response (e.g. a cell mediated immune response such as a T cell mediated immune response) of the subject during or after treatment compared to their immune response prior to treatment. “Induced or enhanced tumour immunogenicity” therefore encompasses any measurable increase in the immune response that is directly targeted to the tumour being treated. Accordingly, in a non-limiting example, a patient having a cancer is being treated.

“Down-regulating PD-L1 cancer cell surface expression”

As used herein “down-regulating PD-L1 cancer cell surface expression” refers to the reduction of protein PD-L1 levels on the cancer cell surface as measured for example, by FACS, immunohistochemistry, or immunofluorescence. This reduction of protein PD-L1 levels means that the PD-L1 expression is decreased in comparison to what is expected, or compared to baseline. “Compared to baseline” can be a comparison to another experiment but typically refers to a comparison prior to treatment.

Combination of DNA hypomethylating agent and SUMOylation inhibitor

In some examples, the DNA hypomethylating agent and the SUMOylation inhibitor may have an additive or synergistic effect on the T cell mediated therapy. In one example, the combination of DNA hypomethylating agent and SUMOylation inhibitor is defined as affording an “additive effect” “synergistic effect” or a “synergistic treatment” if the effect on the T cell mediated therapy is superior, as measured by, for example, increased transcription of type I and II interferon, interferon stimulated genes and transcription factors, transcription of interleukins, increased production of interferons, in particular IFNy, increased reactivity of T cells towards targeted cells, increase of CD8+ T cells, increase of CD4+ T cells, increase of HLA class I presentation, and/or PD-L1 downregulation to that achievable on dosing one or other of the components of the combination treatment at its conventional dose. For example, the effect of the combination treatment is additive if the effect is superior to the effect achievable with for example, DNA hypomethylating agent alone, or SUMOylation inhibitor alone. For example, the effect of the combination treatment may be synergistic if the effect of the combination treatment supersedes the effect of the individual treatments. Further, the effect of the combination is beneficial (e.g. additive or synergistic) if a beneficial effect is obtained in a T cell therapy that does not respond (or responds poorly) to any members of the combination alone. In addition, the effect of the combination treatment is defined as affording a benefit (e.g. additive or synergistic effect) if one of the components is dosed at its conventional dose and the other component is dosed at a reduced dose and the effect, as measured by, for example, increased transcription of type I and II interferon, interferon stimulated genes and transcription factors, transcription of interleukins, increased production of interferons, in particular IFNy, increased reactivity of T cells towards targeted cells, increase of CD8+ T cells, increase of CD4+ T cells, increase of HLA class I presentation, and/or PD-L1 downregulation, is equivalent to or better than that achievable on dosing conventional amounts of either one of the components of the combination treatment.

In an alternative example, the combination of DNA hypomethylating agent and SUMOylation inhibitor is defined as affording an “additive effect” “synergistic effect” or a “synergistic treatment” when used with T cell mediated therapy, if the effect is therapeutically superior, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, to that achievable on dosing one or other of the components of the combination treatment at its conventional dose. For example, the effect of the combination treatment is additive if the effect is therapeutically superior to the effect achievable with for example, DNA hypomethylating agent alone, or SUMOylation inhibitor alone. For example, the effect of the combination treatment may be synergistic if the effect of the combination treatment supersedes the effect of the individual treatments. Further, the effect of the combination is beneficial (e.g. additive or synergistic) if a beneficial effect is obtained in a group of subjects that does not respond (or responds poorly) to any members of the combination alone. In addition, the effect of the combination treatment is defined as affording a benefit (e.g. additive or synergistic effect) if one of the components is dosed at its conventional dose and the other component is dosed at a reduced dose and the therapeutic effect, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, is equivalent to or better than that achievable on dosing conventional amounts of either one of the components of the combination treatment.

Combination of a DNA hypomethylatinq agent, a SUMOylation inhibitor, and a T cell mediated therapy

In some examples, the DNA hypomethylating agent, the SUMOylation inhibitor and T cell mediated therapy may have an additive or synergistic effect on the treatment of a disease in a subject in need thereof. The combination treatment of DNA hypomethylating agent, SUMOylation inhibitor and T cell mediated therapy is defined as affording an “additive effect” “synergistic effect” or a “synergistic treatment” if the effect is superior, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, to that achievable on dosing one of the components of the combination treatment at its conventional dose. For example, the effect of the combination treatment comprising DNA hypomethylating agent, SUMOylation inhibitor and T cell mediated therapy is additive if the effect is therapeutically superior to the effect achievable with for example, DNA hypomethylating agent and T cell mediated therapy alone. For example, the effect of the combination treatment may be synergistic if the effect of the combination treatment supersedes the effect of the individual treatments added together. Further, the effect of the combination is beneficial (e.g. additive or synergistic) if a beneficial effect is obtained in a group of subjects that does not respond (or responds poorly) to any members of the combination alone. In addition, the effect of the combination treatment is defined as affording a benefit (e.g. additive or synergistic effect) if one of the components is dosed at its conventional dose and the other component is dosed at a reduced dose and the therapeutic effect, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, is equivalent to or better than that achievable on dosing conventional amounts of either one of the components of the combination treatment.

Inducing or enhancing an immune response (e.g. a T cell response)

The medicaments and methods for use in treating patients described herein may induce or enhance an immune response. In particular, the medicaments and methods for use in treating patients described herein may induce or enhance a T cell response in the patient. In particular, the medicaments and methods for use in treating patients described herein may induce or enhance cancer cell immunogenicity in the patient.

As used herein, the phrase “induced or enhanced immune response” refers to an increased immune response (e.g. a cell mediated immune response such as a T cell mediated immune response) of the subject during or after treatment compared to their immune response prior to treatment. Accordingly, “inducing or enhancing a T cell response” as well as “induced or enhanced T cell response” refers to an increased T cell mediated immune response of the subject during or after treatment compared to their T cell response prior to treatment. An “induced or enhanced immune response” therefore encompasses any measurable increase in the immune response that is directly or indirectly targeted to the disease being treated.

In a specific embodiment, the induced or enhanced T cell response comprises induced or enhanced T cell proliferation. As used herein, “induced T cell proliferation” means that the T cell response is elicited, or in another words started. As used herein, “enhanced T cell proliferation” means that the T cell proliferation is heightened, or increased in comparison to what is expected, or compared to baseline. “Compared to baseline” can be a comparison to another experiment but typically refers to a comparison prior to treatment.

The phrase “induced or enhanced T cell proliferation” therefore refers to increased T cell proliferation during or after treatment compared to T cell proliferation prior to treatment. “Induced or enhanced T cell proliferation” therefore encompasses any measurable increase in the T cell number.

In a specific embodiment, the induced or enhanced T cell response comprises induced or enhanced CD8+ T cell proliferation.

As used herein, “CD8+ T cells” are a subset of T cells that are positive for CD8. The skilled person knows appropriate methods and techniques to identify CD8+ T cells.

In a specific embodiment, the induced or enhanced T cell response comprises induced or enhanced CD4+ T cell proliferation.

As used herein, “CD4+ T cells” are a subset of T cells that are positive for CD4. The skilled person knows appropriate methods and techniques to identify CD4+ T cells.

In a specific embodiment, the induced or enhanced T cell response comprises induced or enhanced CD8+ and CD4+T cell proliferation.

In a specific embodiment, the induced or enhanced T cell response comprises increase of HLA class I presentation or PD-L1 downregulation in target cells, e.g. cancer cells.

In a specific embodiment, the induced or enhanced T cell response comprises increase of HLA class I presentation, and PD-L1 downregulation in target cells, e.g. cancer cells.

In a specific embodiment, cancer cell immunogenicity is induced or enhanced.

In a specific embodiment, cancer cell immunogenicity is induced or enhanced by down-regulating PD-L1.

Diseases

The medicaments and methods for use in treating patients described herein may be for use in treating cancer.

As used herein, "treatment of cancer," "treat cancer," and "treating cancer" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of e.g. cancer. “Treatment” therefore encompasses a reduction, slowing or inhibition of the amount or concentration of malignant cells, for example as measured in a sample obtained from the subject, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the amount or concentration of malignant cells before treatment. Methods of measuring the amount or concentration of malignant cells include, for example, qRT-PCR, and quantification of hyperproliferative specific biomarkers in a sample obtained from the subject. Methods of measuring the amount or concentration of malignant cells also include, for example, histo-pathologic examination of tumour (e.g. malignant tumour) material and quantification of tumour biomarkers (not limited to hyperproliferative biomarkers).

In a specific embodiment, the cancer is a solid tumour.

Examples of cancers that are solid tumours include, but are not limited to, lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, a.k.a. Wilms' tumour, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangio sarcoma, lymphangioendotheliosarcoma, hemangio sarcoma); appendix cancer; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumour; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endothelio sarcoma (e.g., Kaposi' s sarcoma, multiple idiopathic haemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); oesophageal cancer (e.g., adenocarcinoma of the oesophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumour (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumours; immunocytic amyloidosis; liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; mesothelioma;; neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumour (GEP-NET), carcinoid tumour); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumours); penile cancer (e.g., Paget' s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumour (PNT); paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g. , prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumour (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; uveal melanoma; vaginal cancer; and vulvar cancer (e.g., Paget' s disease of the vulva).

As used herein, haematological malignancies are a group of neoplastic diseases of the blood, bone marrow, lymph and lymphatic system. Also myeloproliferative diseases which tend to be benign at time of diagnosis are encompassed by the expression “haematological malignancies”, in line with the WHO Guidelines. The skilled person knows that there may be an overlap between different classifications, or classification systems. The skilled person is also aware that one disease can progress to another disease. For example, it is commonly known that patients having myelodysplastic syndrome (MDS) have a high risk for developing acute myeloid leukaemia (AML). The skilled person is also aware that haematological malignancies can present simultaneously, such as the concurrent presentation of multiple myeloma and Non-Hodgkin’s Lymphoma as reported for example by Lee et al, Korean J Intern Med 9(2): 113-115 (1994). For example, chronic myeloid leukaemia (CML) can be a myeloid haematological malignancy but also a leukaemia and a myeloproliferative disorder (MPD), depending on the type of classification.

In a specific embodiment, the cancer is a haematological malignancy.

Examples of haematological malignancies include, but are not limited to, acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML); chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), including hairy cell leukaemia, chronic neutrophilic leukaemia (CNL), benign monoclonal gammopathy; heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), myelodysplastic syndrome (MDS); myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, hypereosinophilic syndrome (HES)), multiple myeloma, plasmacytoma, Hodgkin’ lymphoma (e.g., nodular sclerosis Hodgkin lymphoma (NSCHL), mixed cellularity Hodgkin lymphoma (MCCHL), lymphocyte rich Hodgkin lymphoma (LRCHL), lymphocyte depleted Hodgkin lymphoma (LDCHL) nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), Non-Hodgkin lymphoma (e.g., mature 13- cell lymphomas such as diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), Lymphoblastic lymphoma, Burkitt lymphoma (BL), Primary mediastinal (thymic) large B-cell lymphoma (PMBCL), Transformed follicular and transformed mucosa-associated lymphoid tissue (MALT) lymphomas, High-grade B-cell lymphoma with double or triple hits (HBL), Primary cutaneous DLBCL, leg type, Primary DLBCL of the central nervous system, Primary central nervous system (CNS) lymphoma, Acquired immunodeficiency syndrome (AIDS)-associated lymphoma, Follicular lymphoma (FL), Marginal zone lymphoma (MZL), Chronic lymphocytic leukaemia/small-cell lymphocytic lymphoma (CLL/SLL), Gastric mucosa-associated lymphoid tissue (MALT) lymphoma, Lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia (WM), Nodal marginal zone lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), mature T-cell and natural killer (NK)-cell lymphomas, such as Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), Systemic anaplastic large-cell lymphoma (ALCL), Lymphoblastic lymphoma, Hepatosplenic T-cell lymphoma, Enteropathy-associated intestinal T- cell lymphoma, Monomorphic epitheliotropic intestinal T-cell lymphoma, Angioimmunoblastic T- cell lymphoma (AITL), Adult T-cell leukaemia/lymphoma, Extranodal natural killer (NK)/T-cell lymphoma (ENK/TCL), nasal type, Primary cutaneous lymphomas, e.g. Cutaneous T-cell lymphoma (CTCL) such as Mycosis fungoides (MF), Sezary syndrome (SS), Primary cutaneous anaplastic large-cell lymphoma (pcALCL), Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) such as Primary cutaneous gamma delta T-cell lymphoma.

In a specific embodiment, the haematological malignancy is a myeloid haematological malignancy or lymphoid haematological malignancy.

As used herein, myeloid haematological malignancies are disorders that concern haematopoietic stem cells and myeloid progenitor cells. Accordingly, examples of myeloid haematological malignancy include but are not limited to acute myeloid leukaemia (AML); chronic myeloid leukaemia (CML), chronic neutrophilic leukaemia (CNL), myelodysplastic syndrome (MDS), myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, hypereosinophilic syndrome (HES)).

As used herein, lymphoid haematological malignancies are disorders that arise from malignant transformation of normal lymphoid cells at various stages of differentiation. Accordingly, examples of lymphoid haematological malignancy include but are not limited to multiple myeloma, plasmacytoma, Hodgkin’ lymphoma (e.g., nodular sclerosis Hodgkin lymphoma (NSCHL), mixed cellularity Hodgkin lymphoma (MCCHL), lymphocyte rich Hodgkin lymphoma (LRCHL), lymphocyte depleted Hodgkin lymphoma (LDCHL) nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), Non-Hodgkin lymphoma (e.g., mature B-cell lymphomas such as diffuse large B-cell lymphoma (DLBCL), Mantle cell lymphoma (MCL), Lymphoblastic lymphoma, Burkitt lymphoma (BL), Primary mediastinal (thymic) large B-cell lymphoma (PMBCL), Transformed follicular and transformed mucosa-associated lymphoid tissue (MALT) lymphomas, High-grade B-cell lymphoma with double or triple hits (HBL), Primary cutaneous DLBCL, leg type, Primary DLBCL of the central nervous system, Primary central nervous system (CNS) lymphoma, Acquired immunodeficiency syndrome (AIDS)-associated lymphoma, Follicular lymphoma (FL), Marginal zone lymphoma (MZL), Chronic lymphocytic leukaemia/small-cell lymphocytic lymphoma (CLL/SLL), Gastric mucosa-associated lymphoid tissue (MALT) lymphoma, Lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia (WM), Nodal marginal zone lymphoma (NMZL), Splenic marginal zone lymphoma (SMZL), mature T-cell and natural killer (NK)-cell lymphomas, such as Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), Systemic anaplastic large-cell lymphoma (ALCL), Lymphoblastic lymphoma, Hepatosplenic T-cell lymphoma, Enteropathy-associated intestinal T-cell lymphoma, Monomorphic epitheliotropic intestinal T-cell lymphoma, Angioimmunoblastic T-cell lymphoma (AITL), Adult T-cell leukaemia/lymphoma, Extranodal natural killer (NK)/T-cell lymphoma (ENK/TCL), nasal type, Primary cutaneous lymphomas, e.g. Cutaneous T-cell lymphoma (CTCL) such as Mycosis fungoides (MF), Sezary syndrome (SS), Primary cutaneous anaplastic large-cell lymphoma (pcALCL), Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) such as Primary cutaneous gamma delta T-cell lymphoma.

In a specific embodiment, the haematological malignancy is a chronic myeloproliferative disorder.

Examples of chronic myeloproliferative disorders include, but are not limited to, chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), including hairy cell leukaemia, chronic neutrophilic leukaemia (CNL), benign monoclonal gammopathy; heavy chain disease (e.g. , alpha chain disease, gamma chain disease, mu chain disease), myelodysplastic syndrome (MDS); myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, hypereosinophilic syndrome (HES)).

In a specific embodiment, the haematological malignancy is a myeloid haematological malignancy.

In a specific embodiment, the haematological malignancy is a lymphoid haematological malignancy.

In a specific embodiment, the haematological malignancy is plasmacytoma, multiple myeloma or acute myeloid leukaemia (AML).

In a specific embodiment, the haematological malignancy is multiple myeloma or acute myeloid leukaemia (AML). In a specific embodiment, the haematological malignancy is multiple myeloma.

In a specific embodiment, the haematological malignancy is acute myeloid leukaemia (AML).

In a specific embodiment, the haematological malignancy is acute Lymphoblastic leukaemia (ALL).

In some examples, the patient is undergoing treatment with, has been treated with, or has been prescribed treatment with, one or more anti-cancer therapy.

In some examples, the methods or uses further comprise administering one or more anti-cancer therapy to the subject.

In some examples, the patient is undergoing treatment with, has been treated with, or has been prescribed treatment with, one or more anti-cancer therapy; and/or the method further comprises administering one or more anti-cancer therapy to the subject.

As used herein, "anti-cancer therapy" refers to any agent, composition or medical technique (e.g., surgery, radiation treatment, etc.) useful for the treatment of cancer. For example, an anti-cancer agent can be a small molecule, antibody, peptide or antisense compound. Examples of antisense compounds include, but are not limited to interfering RNAs (e.g., dsRNA, siRNA, shRNA, miRNA, and amiRNA) and antisense oligonucleotides (ASO).

In some examples, the anti-cancer therapy is selected from the group consisting of surgery, radiation therapy, chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, adjuvant therapy, and immunotherapy.

As used herein, the phrase “chemotherapeutic agent” refers to (but is not limited to) compounds that are used in chemotherapy for the treatment of proliferative disorders such as cancer. For the avoidance of doubt, reference to a chemotherapeutic agent herein refers to an additional agent to the DNA hypomethylating agent and/or SUMOylation inhibitor and/or T cell mediated therapy. Accordingly, where the claims or description refers to a DNA hypomethylating agent and/or SUMOylation inhibitor and a chemotherapeutic agent, an additional chemotherapeutic agent is intended, in addition to the DNA hypomethylating agent and/or SUMOylation inhibitor.

Several chemotherapeutic agents are known, some of which are clinically approved or awaiting approval as cancer therapies. Suitable examples include nucleoside analogues, topoisomerase inhibitors, platinum complexes, microtubule-targeting drugs and combinations thereof. In some examples, the chemotherapy is a cytotoxic agent. As used herein a “cytotoxic agent” refers to any substance that kills cells, including cancer cells e.g. the cytotoxic agent may stop cancer cells from dividing and growing and may cause tumours to shrink in size. In some examples, the chemotherapy comprises administering to the subject a cytotoxic agent in an amount effective to treat the cancer.

In some examples, the cytotoxic agent is selected from the group consisting of a platinum agent, mitomycin C, a poly (ADP-ribose) polymerase (PARP) inhibitor, a poltheta inhibitor, a radioisotope, a vinca alkaloid, an antitumor alkylating agent, a monoclonal antibody and an antimetabolite.

Examples of platinum agents include, but are not limited to cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, and Lipoplatin. Examples of antitumor alkylating agents include, but are not limited to nitrogen mustards, cyclophosphamide, mechlorethamine or mustine (HN2), uramustine or uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, nitrosoureas, carmustine, lomustine, streptozocin, alkyl sulfonates, busulfan, thiotepa, procarbazine, altretamine, triazenes, dacarbazine, mitozolomide, and temozolomide.

Examples of anti-cancer monoclonal antibodies include, but are not limited to necitumumab, dinutuximab, nivolumab, blinatumomab, pembrolizumab, ramucirumab, obinutuzumab, adotrastuzumab emtansine, pertuzumab, brentuximab, ipilimumab, ofatumumab, catumaxomab, bevacizumab, cetuximab, tositumomab-1131 , ibritumomab tiuxetan, alemtuzumab, gemtuzumab ozogamicin, trastuzumab, and rituximab. Examples of vinca alkaloids include, but are not limited to vinblastine, vincristine, vindesine, vinorelbine, desoxyvincaminol, vincaminol, vinburnine, vincamajine, vineridine, vinburnine, and vinpocetine. Examples of antimetabolites include, but are not limited to fluorouracil, cladribine, capecitabine, mercaptopurine, pemetrexed, fludarabine, gemcitabine, hydroxyurea, methotrexate, nelarbine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, and thioguanine. In some embodiments, the anti-cancer therapy is an immunotherapy, such as, but not limited to, cellular immunotherapy, antibody therapy or cytokine therapy. Examples of cellular immunotherapy include, but is not limited to, dendritic cell therapy and Sipuleucel-T. Examples of antibody therapy include, but is not limited to Alemtuzumab, Ipilimumab, Nivolumab, Ofatumumab, Pembrolizumab, and Rituximab. Examples of cytokine therapy include, but is not limited to, interferons (for example, IFNa, lENp, lENy, I ENA) and interleukins. In some embodiments, the immunotherapy comprises one or more immune checkpoint inhibitors. Examples of immune checkpoint proteins include, but are not limited to, CTLA-4 and its ligands CD80 and CD86, PD-1 with its ligands PD-L1 and PD-L2, and 4- IBB. Additional examples of anti-cancer therapies include, but are not limited to, abiraterone acetate (e.g., ZYTIGA), ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, ado- trastuzumab emtansine (e.g., KADCYLA), afatinib dimaleate (e.g., GILOTRIF), aldesleukin (e.g., PROLEUKIN), alemtuzumab (e.g., CAMPATH), anastrozole (e.g., ARIMIDEX), arsenic trioxide (e.g., TRISENOX), asparaginase erwinia chrysanthemi (e.g., ERWINAZE), axitinib (e.g., INLYTA), azacitidine (e.g., MYLOSAR, VIDAZA), BEACOPP, belinostat (e.g., BELEODAQ), bendamustine hydrochloride (e.g., TREANDA), BEP, bevacizumab (e.g., AVASTIN), bicalutamide (e.g., CASODEX), bleomycin (e.g., BLENOXANE), blinatumomab (e.g., BLINCYTO), bortezomib (e.g., VELCADE), bosutinib (e.g., BOSULIF), brentuximab vedotin (e.g., ADCETRIS), busulfan (e.g., BUSULFEX, MYLERAN), cabazitaxel (e.g., JEVTANA), cabozantinib- s-malate (e.g., COMETRIQ), CAF, capecitabine (e.g., XELODA), CAPOX, carboplatin (e.g., PARAPLAT, PARAPLATIN), carboplatin-taxol, carfilzomib (e.g., KYPROLIS), carmustine (e.g., BECENUM, BICNU, CARMUBRIS), carmustine implant (e.g., GLIADEL WAFER, GLIADEL), ceritinib (e.g., ZYKADIA), cetuximab (e.g., ERBITUX), chlorambucil (e.g., AMBOCHLORIN, AMBOCLORIN, LEUKERAN, LINFOLIZIN), chlorambucilprednisone, CHOP, cisplatin (e.g., PLATINOL, PLATINOL-AQ), clofarabine (e.g., CLOFAREX, CLOLAR), CMF, COPP, COPP- ABV, crizotinib (e.g., XALKORI), CVP, cyclophosphamide (e.g., CLAFEN, CYTOXAN, NEOSAR), cytarabine (e.g., CYTOSAR-U, TARABINE PFS), dabrafenib (e.g., TAFINLAR), dacarbazine (e.g., DTIC-DOME), dactinomycin (e.g., COSMEGEN), dasatinib (e.g., SPRYCEL), daunorubicin hydrochloride (e.g., CERUBIDINE), decitabine (e.g., DACOGEN), degarelix, denileukin diftitox (e.g., ONTAK), denosumab (e.g., PROLIA, XGEVA), Dinutuximab (e.g., UNITUXIN), docetaxel (e.g., TAXOTERE), doxorubicin hydrochloride (e.g., ADRIAMYCIN PFS, ADRIAMYCIN RDF), doxorubicin hydrochloride liposome (e.g., DOXIL, DOX-SL, EVACET, LIPODOX), enzalutamide (e.g., XTANDI), epirubicin hydrochloride (e.g., ELLENCE), EPOCH, erlotinib hydrochloride (e.g., TARCEVA), etoposide (e.g., TOPOSAR, VEPESID), etoposide phosphate (e.g., ETOPOPHOS), everolimus (e.g., AFINITOR DISPERZ, AFINITOR), exemestane (e.g., AROMASIN), FEC, fludarabine phosphate (e.g., FLUDARA), fluorouracil (e.g., ADRUCIL, EFUDEX, FLUOROPLEX), FOLFIRI , FOLFIRI-BEVACIZUMAB, FOLFIRI- CETUXIMAB, FOLFIRINOX, FOLFOX, FU-LV, fulvestrant (e.g., FASLODEX), gefitinib (e.g., IRESSA), gemcitabine hydrochloride (e.g., GEMZAR), gemcitabine-cisplatin, gemcitabineoxaliplatin, goserelin acetate (e.g., ZOLADEX), Hyper-CVAD, ibritumomab tiuxetan (e.g., ZEVALIN), ibrutinib (e.g., IMBRUVICA), ICE, idelalisib (e.g., ZYDELIG), ifosfamide (e.g., CYFOS, IFEX, IFOSFAMIDUM), imatinib mesylate (e.g., GLEEVEC), imiquimod (e.g., ALDARA), ipilimumab (e.g., YERVOY), irinotecan hydrochloride (e.g., CAMPTOSAR), ixabepilone (e.g., IXEMPRA), lanreotide acetate (e.g., SOMATULINE DEPOT), lapatinib ditosylate (e.g., TYKERB), lenalidomide (e.g., REVLIMID), lenvatinib (e.g., LENVIMA), letrozole (e.g., FEMARA), leucovorin calcium (e.g., WELLCOVORIN), leuprolide acetate (e.g., LUPRON DEPOT, LUPRON DEPOT-3 MONTH, LUPRON DEPOT-4 MONTH, LUPRON DEPOT-PED, LUPRON, VIADUR), liposomal cytarabine (e.g., DEPOCYT), lomustine (e.g., CEENU), mechlorethamine hydrochloride (e.g., MUSTARGEN), megestrol acetate (e.g., MEGACE), mercaptopurine (e.g., PURINETHOL, PURIXAN), methotrexate (e.g., ABITREXATE, FOLEX PFS, FOLEX, METHOTREXATE LPF, MEXATE, MEXATE-AQ), mitomycin c (e.g., MITOZYTREX, MUTAMYCIN), mitoxantrone hydrochloride, MOPP, nelarabine (e.g., ARRANON), nilotinib (e.g., TASIGNA), nivolumab (e.g., OPDIVO), obinutuzumab (e.g., GAZYVA), OEPA, ofatumumab (e.g., ARZERRA), OFF, olaparib (e.g., LYNPARZA), omacetaxine mepesuccinate (e.g., SYNRIBO), OPPA, oxaliplatin (e.g., ELOXATIN), paclitaxel (e.g., TAXOL), paclitaxel albumin-stabilized nanoparticle formulation (e.g., ABRAXANE), PAD, palbociclib (e.g., IBRANCE), pamidronate disodium (e.g., AREDIA), panitumumab (e.g., VECTIBIX), panobinostat (e.g., FARYDAK), pazopanib hydrochloride (e.g., VOTRIENT), pegaspargase (e.g., ONCASPAR), peginterferon alfa-2b (e.g., PEG-INTRON), peginterferon alfa-2b (e.g., SYLATRON), pembrolizumab (e.g., KEYTRUDA), pemetrexed disodium (e.g., ALIMTA), pertuzumab (e.g., PERJETA), plerixafor (e.g., MOZOBIL), pomalidomide (e.g., POMALYST), ponatinib hydrochloride (e.g., ICLUSIG), pralatrexate (e.g., FOLOTYN), prednisone, procarbazine hydrochloride (e.g., MATULANE), radium 223 dichloride (e.g., XOFIGO), raloxifene hydrochloride (e.g., EVISTA, KEOXIFENE), ramucirumab (e.g., CYRAMZA), R-CHOP, recombinant HPV bivalent vaccine (e.g., CERVARIX), recombinant human papillomavirus (e.g., HPV) nonavalent vaccine (e.g., GARDASIL 9), recombinant human papillomavirus (e.g., HPV) quadrivalent vaccine (e.g., GARDASIL), recombinant interferon alpha 2a (e.g., VELDONA), recombinant interferon alpha-2b (e.g., INTRON A), regorafenib (e.g., STIVARGA), rituximab (e.g., RITUXAN), romidepsin (e.g., ISTODAX), ropeginterferon alpha 2b, also known as peg-proline-interferon alpha-2b (e.g., BESREMI), ruxolitinib phosphate (e.g., JAKAFI), siltuximab (e.g., SYLVANT), sipuleucel-t (e.g., PROVENGE), sorafenib tosylate (e.g., NEXAVAR), STANFORD V, sunitinib malate (e.g., SUTENT), TAC, tamoxifen citrate (e.g., NOLVADEX, NOVALDEX), temozolomide (e.g., METHAZOLASTONE, TEMODAR), temsirolimus (e.g., TORISEL), thalidomide (e.g., SYNOVIR, THALOMID), thiotepa, topotecan hydrochloride (e.g., HYCAMTIN), toremifene (e.g., FARESTON), tositumomab and iodine 1 131 tositumomab (e.g., BEXXAR), TPF, trametinib (e.g., MEKINIST), trastuzumab (e.g., HERCEPTIN), VAMP, vandetanib (e.g., CAPRELSA), VEIP, vemurafenib (e.g., ZELBORAF), vinblastine sulfate (e.g., VELBAN, VELSAR), vincristine sulfate (e.g., VINCASAR PFS), vincristine sulfate liposome (e.g., MARQIBO), vinorelbine tartrate (e.g., NAVELBINE), vismodegib (e.g., ERIVEDGE), vorinostat (e.g., ZOLINZA), XELIRI, XELOX, ziv-aflibercept (e.g., ZALTRAP), zoledronic acid (e.g., ZOMETA), or a combination thereof.

In certain examples, the anti-cancer therapy is selected from the group consisting of epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., oestrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors, modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acids, and other agents that promote differentiation.

In some examples, the cancer is resistant to treatment with one or more cytotoxic agents alone. Cytotoxic agents are described elsewhere herein. As used herein, a cancer that is resistant to one or more cytotoxic agents means that the cancer does not respond to one or more cytotoxic agents, for example as evidenced by continued proliferation and/or increasing tumour growth and burden. In some examples, the cancer may have initially responded to treatment with such one or more cytotoxic agents (referred to herein as a previously administered therapy) but may have grown resistant after a time. In some examples, the cancer may have never responded to treatment with one or more cytotoxic agents at all.

In some examples, the cancer is resistant to conventional first line therapy, including, for example, first line chemotherapy (such as nucleoside analogues, topoisomerase inhibitors, platinum-based drugs, microtubule-targeting drugs, anthracyclines, and combinations thereof) and/or irradiation.

Patient and treatment

As used herein, the term “patient” refers to an individual, e.g., a human, having a specified condition, disorder or symptom. The patient is in need of treatment in accordance with the invention. The patient may have received treatment for the condition, disorder or symptom in the past. Alternatively, the patient has not been treated prior to treatment in accordance with the present invention. Typically, the patient is a mammal, more typically a human.

As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (e.g. a hyperproliferative disease or condition, such as cancer). Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. In other words, terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of e.g. cancer. “Treatment” therefore encompasses a reduction, slowing or inhibition of the amount or concentration of malignant cells, for example as measured in a sample obtained from the subject, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the amount or concentration of malignant cells before treatment. Methods of measuring the amount or concentration of malignant cells include, for example, qRT-PCR, and quantification of hyperproliferative specific biomarkers in a sample obtained from the subject. Methods of measuring the amount or concentration of malignant cells also include, for example, histo-pathologic examination of tumour (e.g. malignant tumour) material and quantification of tumour biomarkers (not limited to hyperproliferative biomarkers). In some examples, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other examples, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms). Treatment may also be continued after symptoms have resolved, for example, to delay and/or prevent recurrence.

In some examples, the patient may be undergoing treatment with one or more of a SUMOylation inhibitor, a T cell mediated therapy, and/or a DNA hypomethylating agent (as appropriate). In other words, the patient may already have been prescribed (and typically may already be being treated, or may have ongoing treatment) with one or more of a SUMOylation inhibitor, a T cell mediated therapy, and/or a DNA hypomethylating agent (as appropriate) at the time of treatment according to the invention.

As a non-limiting example, one aspect of the invention provides a DNA hypomethylating agent for use in treating a patient undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy. In this context, the patient may already have been prescribed (and typically may already be being treated, or may have ongoing treatment) with a SUMOylation inhibitor and a T cell mediated therapy, and is now also starting treatment with a DNA hypomethylating agent in accordance with the invention. The treatment with a SUMOylation inhibitor and a T cell mediated therapy can be concurrent, or non-concurrent. For example, the treatment with a SUMOylation inhibitor and T cell mediated therapy may take place at identical timepoints, for example on the same day at the same time. Alternatively, the treatment with a SUMOylation inhibitor and T cell mediated therapy may take place at dispersed timepoints, for example on the same day at different times, or on different days in the same week, or in different weeks, or in different months. Accordingly, for example, the patient could first undergo treatment with a T cell mediated therapy, followed by treatment with a SUMOylation inhibitor. Nonetheless, the patient undergoes treatment with a SUMOylation inhibitor and a T cell mediated therapy.

In another non-limiting example, one aspect of the invention provides a SUMOylation inhibitor for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy. In this context, the patient may already have been prescribed (and typically may already be being treated, or may have ongoing treatment) with a DNA hypomethylating agent and a T cell mediated therapy, and is now also starting treatment with a SUMOylation inhibitor agent in accordance with the invention. The treatment with a DNA hypomethylating agent and a T cell mediated therapy can be concurrent, or non-concurrent. For example, the treatment with a DNA hypomethylating agent inhibitor and T cell mediated therapy may take place at identical timepoints, for example on the same day at the same time. Alternatively, the treatment with a DNA hypomethylating agent and T cell mediated therapy may take place at dispersed timepoints, for example on the same day at different times, or on different days in the same week, or in different weeks, or in different months. Accordingly, for example, the patient could first undergo treatment with a T cell mediated therapy, followed by treatment with a DNA hypomethylating agent. Nonetheless, the patient undergoes treatment with a DNA hypomethylating agent and a T cell mediated therapy.

In a further non-limiting example, the invention provides a T cell mediated therapy for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor. In this context, the patient may already have been prescribed (and typically may already be being treated, or may have ongoing treatment) with a DNA hypomethylating agent and a SUMOylation inhibitor, and is now also starting treatment with a T cell mediated therapy in accordance with the invention. The treatment with a SUMOylation inhibitor and a DNA hypomethylating agent can be concurrent, or non-concurrent. For example, the treatment with a SUMOylation inhibitor and DNA hypomethylating agent may take place at identical timepoints, for example on the same day at the same time. Alternatively, the treatment with a SUMOylation inhibitor and DNA hypomethylating agent may take place at dispersed timepoints, for example on the same day at different times, or on different days in the same week, or in different weeks, or in different months. Accordingly, for example, the patient could first undergo treatment with a DNA hypomethylating agent, followed by treatment with a SUMOylation inhibitor. Nonetheless, the patient undergoes treatment with a SUMOylation inhibitor and a DNA hypomethylating agent.

In a further non-limiting example, the invention provides a DNA hypomethylating agent and a SUMOylation inhibitor for use in treating a patient undergoing treatment with a T cell mediated therapy. In this context, the patient may already have been prescribed (and typically may already be being treated, or may have ongoing treatment) with a T cell mediated therapy, and is now also starting treatment with a DNA hypomethylating agent and a SUMOylation inhibitor in accordance with the invention.

In a further non-limiting example, the invention provides a DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy for use in treating a patient. In this context, the patient is now starting treatment with DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy.

In a further non-limiting example, the invention provides a DNA hypomethylating agent and a SUMOylation inhibitor for use in inducing or enhancing cancer cell immunogenicity in a patient. In this context, the patient may already have been prescribed (and typically may already be being treated, or may have ongoing treatment) with a T cell mediated therapy, and is now also starting treatment with a DNA hypomethylating agent and a SUMOylation inhibitor in accordance with the invention. The treatment with a SUMOylation inhibitor and a DNA hypomethylating agent can be concurrent, or non-concurrent. For example, the treatment with a SUMOylation inhibitor and DNA hypomethylating agent may take place at identical timepoints, for example on the same day at the same time. Alternatively, the treatment with a SUMOylation inhibitor and DNA hypomethylating agent may take place at dispersed timepoints, for example on the same day at different times, or on different days in the same week, or in different weeks, or in different months. Accordingly, for example, the patient could first undergo treatment with a DNA hypomethylating agent, followed by treatment with a SUMOylation inhibitor. Nonetheless, the patient undergoes treatment with a SUMOylation inhibitor and a DNA hypomethylating agent.

The person of skill in the art would understand that these principles apply to all of the aspects of the invention.

Order of administration

In a specific embodiment, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor prior to treatment with a T cell mediated therapy.

As used herein, “prior to treatment” means that patient receives the DNA hypomethylating agent and the SUMOylation inhibitor hours, days, or weeks before the T cell mediated therapy in a nonlimiting example. Accordingly, in a non-limiting example, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor one week, two weeks, or three weeks before T cell mediated therapy. In the alternative, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or 13 days before T cell mediated therapy. In another example, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours before T cell mediated therapy.

In a specific embodiment, the patient receives two rounds of DNA hypomethylating agent and the SUMOylation inhibitor prior to treatment with a T cell mediated therapy.

As used herein “round”, “round of”, “rounds”, or “rounds of” refers to the amount of times a given treatment such as the DNA hypomethylating agent and/or the SUMOylation inhibitor has been administered to the patient. Accordingly, in a non-limiting example, two rounds of DNA hypomethylating agent means that said DNA hypomethylating agent has been administered twice, or in other words, two times to the patient.

In a specific embodiment, the patient receives one round of DNA hypomethylating agent and SUMOylation inhibitor on day 0 and another round of DNA hypomethylating agent and the SUMOylation inhibitor on day 14 prior to treatment with a T cell mediated therapy.

In a specific embodiment, the patient receives one round of DNA hypomethylating agent and the SUMOylation inhibitor on day 0, another round of DNA hypomethylating agent and the SUMOylation inhibitor on day 14 prior to treatment with a T cell mediated therapy, followed by T cell mediated therapy on day 15.

In a specific embodiment, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor after treatment with a T cell mediated therapy.

In a specific embodiment, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor one week after treatment with a T cell mediated therapy.

As used herein, “after treatment” means that patient receives the DNA hypomethylating agent and the SUMOylation inhibitor hours, days, or weeks after the T cell mediated therapy in a non-limiting example. Accordingly, in a non-limiting example, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor one week, two weeks, or three weeks after T cell mediated therapy. In the alternative, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days ,9 days ,10 days, 11 days ,12 days, or 13 days after T cell mediated therapy. In another example, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours ,9 hours ,10 hours, 11 hours ,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, or 23 hours after T cell mediated therapy.

In a specific embodiment, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor two weeks after treatment with a T cell mediated therapy, followed by biweekly treatment with DNA hypomethylating agent and the SUMOylation inhibitor.

As used herein, “biweekly treatment" refers to a treatment that is administered twice weekly, or in other words, twice in on one week. In a non-limiting example, the patient receives treatment in week 1 on any two given days, such as for example, day 3 and day 6, or day 2 and day 4, or day 1 and 2 etc. Accordingly, any possible permutation, or in other words, combination of two days in one week is encompassed.

In an alternative embodiment, the treatment is administered once every two weeks. In a nonlimiting example, the patient receives treatment in week 1 but does not receive treatment in week 2. The patient receives treatment again in week 3 etc.

In a specific embodiment, the patient receives the DNA hypomethylating agent and the SUMOylation inhibitor prior to treatment and after treatment with a T cell mediated therapy.

In a specific embodiment, the T cell mediated therapy is repeated. In a non-limiting example, the T cell mediated therapy is repeated after weeks, months, or years.

In a specific embodiment, the T cell mediated therapy is not repeated.

In a specific embodiment, the type of T cell mediated therapy is exchanged for another T cell mediated therapy. In a non-limiting example, the patient starts therapy with a bi-specific T cell engager, followed by TCR. It is understood that these therapies do not have to follow immediately, the patient could undergo chemotherapy as defined elsewhere herein between the therapy with a bi-specific T cell engager and the TCR, or have a drug holiday.

In a specific embodiment, the patient receives one round of DNA hypomethylating agent and the SUMOylation inhibitor on day 0, another round of DNA hypomethylating agent and the SUMOylation inhibitor on day 14 prior to treatment with a T cell mediated therapy, and bi-weekly treatment with DNA hypomethylating agent and the SUMOylation inhibitor after T cell mediated therapy.

For example, the treatment with the DNA hypomethylating agent and the SUMOylation inhibitor may be interrupted in the context of a structured treatment interruption also known as drug holiday. Accordingly, the treatment with the DNA hypomethylating agent and the SUMOylation inhibitor is interrupted for a defined period of time, for example for days, weeks, or months. After the defined period of time, treatment with the DNA hypomethylating agent and the SUMOylation inhibitor is resumed.

In a non-limiting example, the treatment effect is preserved for roughly 10, 20, 30, 40, 50, 60, 70 days after cessation of rounds of DNA hypomethylating agent and the SUMOylation inhibitor. As used herein, the treatment effect is “preserved” is the treated disease does not progress, or the condition of the patient worsens due to the treated disease. In a non-limiting example, the treated disease is a solid tumour, i.e. cancer. The disease does not progress, if the tumour is stable (i.e., does not grow), or even shrinks.

In one example, chemotherapy is used as a bridging therapy to control disease progression prior to treatment with the DNA hypomethylating agent and the SUMOylation inhibitor. In another example, chemotherapy is used as a conditioning regimen prior to treatment with the DNA hypomethylating agent and the SUMOylation inhibitor. The goal of chemotherapy as a conditioning regimen is to enhance the expansion, engraftment and anti-tumour efficacy of adoptive T cell transfer by lymphodepletion. In a non-limiting example, cyclophosphamide and fludarabine are used as a conditioning regimen prior to treatment with the DNA hypomethylating agent and the SUMOylation inhibitor.

Accordingly, in a specific embodiment, the patient has received chemotherapy prior to treatment with the DNA hypomethylating agent and the SUMOylation inhibitor.

In a specific embodiment, the chemotherapy comprises cyclophosphamide and fludarabine.

In a specific embodiment, the treatment effect is reduced tumour growth.

In a specific embodiment, the DNA hypomethylating agent and the SUMOylation inhibitor prolong efficacy of the T cell mediated therapy.

As used herein, efficacy of the T cell mediated therapy is “prolonged”” by the DNA hypomethylating agent and the SUMOylation inhibitor, if the treated disease does not progress, or the condition of the patient worsens due to the treated disease for a longer time period compared to a time period, if treatment with a T cell mediated therapy alone takes place. In a nonlimiting example, the treated disease is a solid tumour, i.e. cancer. The efficacy of the T cell mediated therapy, if the tumour is stable (i.e., does not grow), or even shrinks.

In a specific embodiment, the DNA hypomethylating agent and the SUMOylation inhibitor heighten efficacy of the T cell mediated therapy.

As used herein, efficacy of the T cell mediated therapy is “heightened”” by the DNA hypomethylating agent and the SUMOylation inhibitor, if the efficacy is higher than compared to the efficacy of a treatment with a T cell mediated therapy alone. In a specific embodiment, the DNA hypomethylating agent and the SUMOylation inhibitor heighten efficacy of the T cell mediated therapy and prolong efficacy of the T cell mediated therapy.

In a specific embodiment, the DNA hypomethylating agent and the SUMOylation inhibitor yield T cells producing increased levels of interferon.

As used herein “increased levels of interferon” refers to the level of interferon that is produced by T cells in combination with the DNA hypomethylating agent and the SUMOylation inhibitor compared to T cells without DNA hypomethylating agent and the SUMOylation inhibitor. In a nonlimiting example, said T cells are T cells that are produced within the body of a patient.

In a specific embodiment, the DNA hypomethylating agent and the SUMOylation inhibitor yield a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher count of T cells.

As used herein “higher count of T cells” refers to the count of T cells that is yielded in combination with the DNA hypomethylating agent and the SUMOylation inhibitor compared to T cells without DNA hypomethylating agent and the SUMOylation inhibitor. In a non-limiting example, said T cells are T cells that are produced within the body of a patient.

In a specific embodiment, the higher count of T cells are CD8+ T cells or CD4+ T cells.

In a specific embodiment, the higher count of T cells are CD8+ T cells.

Exemplary embodiments

Methods for treating a patient, DNA hypomethylating agents for use in treating a patient, SUMOylation inhibitors for use in treating a patient, and T cell mediated therapies for use in treating a patient are described in detail above.

The following embodiments apply to all aspects of the invention described herein.

In a specific embodiment of any of the aspects of the invention, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is a E1 SUMOylation inhibitor. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any of the aspects of the invention, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is TAK-981. In a specific example of this embodiment, the T cell mediated therapy is a TOR. In a specific embodiment of any of the aspects of the invention, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is TAK-981.

In a specific embodiment of any aspects of the invention, the method or use is for treating cancer, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is a E1 SUMOylation inhibitor. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any of the aspects of the invention, the method or use is for treating cancer, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is TAK-981. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any of the aspects of the invention, the method or use is in enhancing cancer cell immunogenicity, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’- deoxycytidine and the SUMOylation inhibitor is TAK-981.

In a specific embodiment of any aspects of the invention, the method or use is for treating haematological malignancies, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’- deoxycytidine and the SUMOylation inhibitor is a E1 SUMOylation inhibitor. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any of the aspects of the invention, the method or use is for treating haematological malignancies, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’- deoxycytidine and the SUMOylation inhibitor is TAK-981 . In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any aspects of the invention, the method or use is for treating acute myeloid leukaemia, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is a E1 SUMOylation inhibitor. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any of the aspects of the invention, the method or use is for treating acute myeloid leukaemia, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’- deoxycytidine and the SUMOylation inhibitor is TAK-981 . In a specific example of this embodiment, the T cell mediated therapy is a TOR. In a specific embodiment of any of the aspects of the invention, when the method or use enhancing cancer cell immunogenicity is in an acute myeloid leukaemia patient, the DNA hypomethylating agent may be 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor may be TAK-981.

In a specific embodiment of any aspects of the invention, the method or use is for treating multiple myeloma, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is a E1 SUMOylation inhibitor. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

In a specific embodiment of any of the aspects of the invention, the method or use is for treating multiple myeloma, the DNA hypomethylating agent is 5-Azacytidine, or 5-Aza-2’-deoxycytidine and the SUMOylation inhibitor is TAK-981. In a specific example of this embodiment, the T cell mediated therapy is a TOR.

Pharmaceutical compositions

The DNA hypomethylating agent, and/or the SUMOylation inhibitor, and/or the T cell mediated therapy described herein may be part of a pharmaceutical composition.

As used herein, “pharmaceutical composition” refers to a composition comprising one or more compounds that is formulated for administration to a subject. Pharmaceutical compositions typically comprise one or more active ingredients (e.g. in this case a DNA hypomethylating agent and/or a SUMOylation inhibitor and/or a T cell mediated therapy) and one or more pharmaceutically acceptable materials. The pharmaceutical compositions described herein may therefore comprise a DNA hypomethylating agent and/or a SUMOylation inhibitor and/or a T cell mediated therapy and one or more other components. For example, the pharmaceutical composition may comprise a DNA hypomethylating agent and/or a SUMOylation inhibitor and/or a T cell mediated therapy and a pharmaceutically acceptable excipient, diluent and/or carrier. Pharmaceutical compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.

As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound (e.g. DNA hypomethylating agent and/or a SUMOylation inhibitor and/or a T cell mediated therapy) without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. In some examples, the pharmaceutical composition comprises a pharmaceutically acceptable diluent. Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

In some examples, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. the vaccine, cell cycle inhibitor, modulator of an immune suppression mechanism, or immune check point inhibitor (as appropriate)), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

In some examples, the pharmaceutical composition may comprise a DNA hypomethylating agent and/or a SUMOylation inhibitor and/or a T cell mediated therapy, and a pharmaceutically acceptable carrier. Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

Preferably, the pharmaceutical composition comprises or consists of an amount of DNA hypomethylating agent, and/or SUMOylation inhibitor, and/or T cell mediated therapy that constitutes a pharmaceutical dosage unit. A pharmaceutical dosage unit is defined herein as the amount of active ingredients (i.e. DNA hypomethylating agent, and/or SUMOylation inhibitor, and/or T cell mediated therapy) that is applied to a subject at a given time point. A pharmaceutical dosage unit may be applied to a subject in a single volume, i.e. a single intravenous administration, or may be applied in 2, 3, 4, 5 or more separate volumes or intravenous administrations that are applied preferably at different locations of the body, for instance in the right and the left limb. It is to be understood herein that the separate volumes of a pharmaceutical dosage may differ in composition, i.e. may comprise different kinds or composition of active ingredients and/or adjuvants. A single injection volume, or volume for intravenous administration (i.e. volume applied on one location at a certain time point), comprising a total pharmaceutical dosage, or part thereof in case multiple volumes applied at substantially the same time point, may between 100 mL and 2000 mL, or between 100 and 1000 mL. The single injection volume may be 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, 1000 mL, 1100 mL, 1200 mL, 1300 mL, 1400 mL, 1500 mL, 1600 mL, 1700 mL, 1800 mL, 1900 mL, 2000 mL, or any value in between, depending on for example, the patient’s body surface area. In a non-limiting example, if a given patient is exceptionally tall and/or heavy, the single injection volume may be higher. In another non-limiting example, if the patient is exceptionally short and/or light, or a child, the single injection volume may lower.

The pharmaceutical dosage unit applied to a subject at a given time point, either in a single volume or in multiple volumes at a certain time point, comprises an effective amount of DNA hypomethylating agent, and/or SUMOylation inhibitor, and/or T cell based therapy. The skilled person is aware that effective dosage of DNA hypomethylating agents and SUMOylation inhibitors are provided in mg/m² of the body surface, or milligrams (mg), respectively. Similarly, the skilled person is well aware that the effective dosage of T cell mediated therapy depends on the nature of the T cell mediated therapy. In a non-limiting example, the T cell mediated therapy is a CAR-T or TOR and the dosage is provided in viable CAR-T cells or engineered T cells having the desired TCR, such as for example 0.1 to 5 x 10⁶ viable T cells, or 0.1 to 6 x 10⁸ viable T cells. In a nonlimiting example, the T cell mediated therapy is virus-specific T cells and the dosage is provided in viable virus-specific T cells or engineered T cells having the desired virus specific TCR, such as for example 0.1 to 5 x 10⁶ viable T cells, or 0.1 to 6 x 10⁸ viable T cells.

DEFINITIONS

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1 94); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

Aspects of the invention are demonstrated by the following non-limiting examples.

EXAMPLES

Example 1

Materials and Methods

Compounds

5-Aza-2’-deoxycytidine (5-Aza-2’, Merck) was dissolved in DMSO for in vitro usage and in 20% (2-Hydroxypropyl)-p-268 cyclodextrin (HPBCD, Merck) for in vivo purposes. TAK-981 (Chemietek) was dissolved in DMSO for in vitro usage and in 20% HPBCD for in vivo purposes.

Cell culture

OCI-AML3 cells were obtained from DSMZ (Braunschweig, Germany) and U266 cell line was obtained from Prof. Dr T. Mutis (Department of Hematology, VUMC, NL). Cell lines were cultured in IMDM (Lonza), supplemented with 1.5% glutamine (Lonza), 10% fetal bovine serum (Gibco, Life Technologies) and 1% Penicillin-Streptomycin (Lonza). Cells were cultured at 37 °C and 5% CO2 in a humidified incubator.

T cells were cultured in IMDM (Lonza) supplemented with 5% fetal bovine serum (FBS; Gibco, Life Technologies), 5% human serum 1.5% glutamine (Lonza) and 1 % penicillin/streptomycin (Lonza) and 1001 U/ml IL2 (Proleukin; Novartis Pharma). CD8+ T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) by MACS using anti-CD8 MicroBeads (Miltenyi Biotec). CD8+ T cells were subsequently activated with irradiated autologous PBMCs (35 Gy) and 0.8 mg/mL phytohemagglutinin (PHA; Oxoid Microbiology Products, Thermo Fisher Scientific). PBMCs were obtained from the Leiden University Medical Center Biobank for Hematological Diseases (approval number B16.039). Samples were collected after written informed consent. eTCR transfer to healthy donor T cells

On day 2 post activation, CD8+ T cells were retrovirally transduced with NPM1-eTCR^(CLAA2), HA2- eTCR*™³^, BOB1/4G11-eTCR^{(APA B7)}, MageA1-eTCR^{(KVL A2)} or CMV-eTCR^{(NLV A2)}, using 24-well non-tissue culture plate coated with retronectin (30 mg/mL) (Takara) overnight at 4°C. Wells were blocked with 2% human serum albumin (HSA) (Sanquin) for 30 min. Viral supernatant was thawed and added to the 24-wells plate and spun for 20 min, 2,000g at 4°C. Virus supernatant was removed and 0.3*10⁶ activated CD8+ T cells were transferred to each well. After overnight incubation, T cells were transferred to a tissue culture plate. On day 7 post activation, eTCR transduced T cells were indirectly MACS enriched for eTCR expression on anti-mouse TCR-Cp APC antibody (mTCR APC; BD Pharmingen) followed by anti-APC MicroBeads (Milenyi Biotec). Purified T cells were used in experiments between day 10-14 after activation. eTCR expression was assessed by HLA tetramer binding; cells were stained for anti-mTCR APC antibody and PE labeled pHLA-tetramers. Cells were measured on the LSR II (BD Bioscience), and data were analyzed with FlowJo Version 10 software.

Viability assay

OCI-AML3 and LI266 cells were seeded in 96-well flat bottom plate format in a density of 1*10⁵ cells/mL. OCI-AML3 cells were treated for 4 days with increasing concentrations of TAK-981 (0.0001 - 0.1 pM) or 5-Aza-2’ (0.025 - 20 pM) as indicated in the figures; 0.01 % DMSO was used as control. For synergy analysis, a dose range of 5-Aza-2’ (0.025 - 0.10 pM) with or without 0.01 pM of TAK-981 was used. For synergy response of LI266, cells were treated with a dose range of 5-Aza-2’ (1.5 - 20 pM) with or without 0.25 pM of TAK-981. Presto Blue viability reagent (A13261 , Merck) was added 1 :10 into cell culture medium for 1 hour at 37°C and 5% CO2. Fluorescence was measured with a plate reader (Victor X3, Perkin Elmer) at 544/591 nm. Three technical replicates were used within each of three biological replicates performed for the viability assays performed. The excess overbliss model³⁸ was used to calculate the synergistic score, using the following formula with Fa as the fractional activity: Excess overbliss = (Fa1+2 - [(Fa1 + Fa2) - (Fa1 x Fa2)]) x 100.

Western Blot

Total cell lysates of OCI-AML3 and LI266 cells treated with TAK-981 (0.05 to 1 pM) or DMSO 0.01% were analyzed by western blotting for SUMO2/3, SLIMO1 and conjugation. CD8+ T cells treated with 100 nM TAK-981 and/or 250 nM 5-Aza-2’ overnight were analyzed by western blotting for p-STAT Tyr701 and SUMO2/3 and p-actin for loading control. Total lysates were prepared on ice in SNTBS buffer (2% SDS, 1% NP40, 50mM Tris pH 7.5, 150 mM NaCI) followed by 10 min at 100°C. Proteins were size separated with precast 4-12% Bis-Tris gradient gels (Thermo Fisher Scientific). Size-separated proteins were transferred to nitrocellulose membranes (0.45 pm, Amersham Protran Premium (Merck)). Membranes were incubated with primary antibodies against SUMO2/3 (1 :500, mouse monoclonal 8A2, University of Iowa), SUMO1 (1 :1000, 4930P, Cell Signaling Technology), ubiquitin (1 :5000, sc8017, Santa Cruz), and p-actin in 5% milk powder in PBS - 0.05% Tween20. Membranes were incubated with p-STAT1 Tyr701 (1 :1000, 58D6, Cell Signaling Technology) antibody in TBS - 0.05% Tween20 - 3% bovine serum albumin. Goat antimouse IgG- HRP (1 :2500) and Donkey anti- rabbit IgG- HRP (1 :10 000) were used as secondary antibodies in 5% milk in TBS - 0.05% Tween20 - 3% bovine serum albumin. ECL signal was detected using Pierce ECL2 (Life Technologies) and imaged using the iBright CL1500 (Invitrogen iBright Imaging Systems). qPCR

CD8+ T cells from three healthy donors were treated with 100 nM TAK-981 and/or 250 nM 5-Aza- 2’ or control DMSO 0.01% overnight, 10 days post stimulation. Total RNA of CD8+ T cells was isolated with use of SV total RNA isolation system (Promega). 0.5 - 1 pg of RNA was used for cDNA synthesis using random primers (Invitrogen) and reverse transcriptase ImProm-ll (Promega) following manufacturer’s protocol. Real-time quantitative PCR was performed with SYBR Green PCR Mastermix (Applied Biosystems) on a CFX384 real-time PCR detection system (Bio-Rad) according to the following protocol. 95 °C for 7 minutes, followed by 39 cycles of; 95 °C 15 seconds, 60 °C 15 seconds, 60 °C 35 seconds + measurement, the protocol was finalized with 95 °C 10 seconds, 65 °C 5 seconds + measurement and 95 °C 50 seconds. CT values of genes were normalized against the geometric mean of housekeeping genes (SRPR, 18S-RNA and SDHA). Primer sequences are listed in the reagent table.

Co-culture IFNy ELISA

5000 CD8+ T cells (NPM1-eTCR, CMV-eTCR) were co-cultured with OCI-AML3 as target cells in a E:T ratio of 1 :6. CD8+ T cells or OCI-AML3 target cells were pre-treated with 10 nM TAK-981 and/or 250 nM 5-Aza-2’ or 0.01 % DMSO control, on day 10 and 14 post stimulation of CD8+ T cells. Subsequently OCI-AML3 and CD8+ T cells were washed and co-cultured overnight in 60 uL of T cell medium. Supernatant was harvested and diluted 5x and 125x, IFNy was measured by ELISA (Diaclone).

CD8+ survival assay

Activated CD8+ T cells (NPM1-eTCR, CMV-eTCR, HA2.5-eTCR) were co-cultured with irradiated (50Gy) target cells (OCI-AML3, OCI-AML2) for 5 and 7 days in 96-well round bottom plates in an E:T ratio of 1 :5 (5,000 CD8+ T cells). During co-culture, cells were treated on day 1 and 4 with 10 nM TAK-981 and/or 250 nM 5-Aza-2’ or DMSO 0.01 % as control. CD8+ T cell counts were measured with help of flow cytometry (LSR-II). Cells were spun down in plates and CD8+-FITC conjugated ab (BD Pharmingen) was added for 30 min. Subsequently, cells were washed with PBS and resuspended in SytoxBIue (Thermofisher) dead marker (1 :1000). Target cells have an internal label of tdTomato, which was used for identification. Each sample was run for a standardized time of 23 seconds. CD8+ T cells were gated out as presented in Figure 9 and total counts were used for analysis. in vivo tumor therapy

OCI-AML3-Luc and U266-Luc cells were transduced with Luciferase-tdTomato. Cell lines were bulk enriched on tdTomato using an Aria III cell sorter (BD Biosciences) to reach >98% purity. Male and female NOD scid gamma (NSG) mice (NOD.Cg-Prkdc(scid) H2rg(tm1Wjl)/SzJ) originated from the Jackson Laboratory and were bred in house. Male NSG mice were inoculated with 1*10⁶ OCI-AML3-Luc cells intravenously (i.v.). Male and female NSG mice were inoculated with 2*10⁶ U266-Luc (multiple myeloma) cells i.v.

Tumor growth was measured bi-weekly with use of the In Vivo Imaging System (IVIS-spectrum, Perkin Elmer). Mice were subcutaneously injected with 150 pL of 7.5 mM D-luciferin potassium salt (Synchem) and bioluminescence (photons/sec/cm2/r) of U266-LUC and OCI-AML3 cells was measured.

Treatment with TAK-981 (25mg/kg) and/or 5-Aza-2’ (2.5mg/kg) or HPBCD-buffer as control was started on day 10 or day 14 post inoculation of OCI-AML3 and U266 respectively. On day 15 (0CI-AML3) or day 19 (U266) post inoculation, NPM1-eTCR (3x10⁶) and HA2-eTCR for the OCI- AML3 model and 4G11-eTCR (1x10⁶) and MAGEA1-eTCR (1x10⁶) for the U266 model respectively, or CMV-eTCR control were i.v. injected. Drug treatment was continued bi-weekly until day 50 post tumor inoculation. This study was approved by the national Ethical Committee for Animal Research (AVD116002017891).

In vivo NPM1-eTCR CD8+ T cell-LUC tracking

Male NSG-mice (n=6/group) were inoculated with 1*10⁶ OCI-AML3 cells via i.v. injection. Treatment with TAK-981 (25mg/kg) and/or 5-Aza-2’ (2.5mg/kg) or HPBCD-buffer control was started on day 14 post inoculation of OCI-AML3 cells and continued bi-weekly. NPM1 and CMV- eTCR CD8+ T cells were transduced with Luciferase-tdTomato. CD8+ T cells were bulk enriched on tdTomato using an Aria III cell sorter (BD Biosciences) to reach >98% purity. On day 18 post OCI-AML3 injection 3*10⁶ NPM1 or CMV-eTCR Luc CD8+ T cells were i.v. injected. Mice were monitored on day 3, 6 and 9 post injection, using s.c. injection with 150 pL of 7.5 mM D-luciferin potassium salt and bioluminescence of T cells was measured using the IVIS. To analyze CD8+ T cell activation in vivo, a similar experiment was carried out and bone marrow was harvested from mice on day 2 (n=4/group), day 5 (n=3/group) and day 8 (n=3/group). This study was approved by the national Ethical Committee for Animal Research (AVD116002017891).

Isolation of bone marrow and ex-vivo CD8+ T cell analysis

Bone marrow was harvested from euthanized mice. Femur bones were cleaned of all surrounding tissue and cut open on the knee-side. Subsequently the content of the femur was spun down at 2500g at room temperature into Eppendorf tubes containing 100 pL of T cell medium. Bone marrow suspension was filtered through a cellstraining (70pM) (sysmex) into a sterile tube. Subsequently, bone marrow was spun down (500g) and resuspended in 500 pL red blood cell lysis buffer (pharmacy LLIMC) for 10 minutes on ice. Lysed samples were spun and supernatant was removed. Samples were transferred to round bottom 96-well plates for staining. 20 pL of Zombie-red staining (Thermofisher) (1 :1000 in PBS) was added to each well for 25 min. Plates were washed with PBS and 100 pL of paraformaldehyde (1%) (pharmacy LUMC) was added and incubated for 8 min at room temperature for fixation. Plates were spun and PFA was removed from samples; subsequently 100 pL saponin-buffer (500 mL PBS, 2 mL 200g/L albumin, 1 % P/S, 0.1 % saponin (Quillaja)) was added and plates were incubated for 30 min at 4°C. After removal of the saponin-buffer, 20 pL of antibody mix (reagent table) including BV staining buffer (BD Pharmingen) plus (1/20) and 5% normal mouse serum (Thermofisher) was added and incubated for 30 min at room temperature. Finally, plates were washed with saponin-buffer and the samples were resuspended in 50 pL saponin-buffer and measurement on Cytek Aurora spectral flow cytometer 3L (Cytekbio) was performed. Bone marrow of mice with only tumor was used as unstained samples.

Table 3: Reagents and resources

Example 2: Synergistic cytotoxic potential of SUMOylation inhibition and 5-Aza-2’ to inhibit

Acute Myeloid Leukemia and Multiple Myeloma in vitro

First, the inventors addressed the synergistic cytotoxic capacity of TAK-981 and 5-Aza-2’. TAK- 981 is a SUMOylation inhibitor, acting via blocking the SUMO E1 enzyme and consequently abrogating SUMOylation of target proteins (Figure 1A). 5-Aza-2’ entraps DNMT1 to DNA, resulting in DNA-protein crosslinks (DPCs) that block replication, leading to cytotoxic stress (Figure 1 B). Trapped DNMT1 needs to be SUMOylated to be degraded, providing the molecular basis for the synergistic cytotoxic potential of these drugs^{21 22}

TAK-981 inhibited conjugation of SLIMOs to target proteins in AML and MM cell lines as expected and did not interfere with ubiquitylation, demonstrating specificity (Figure 7 A and B). TAK-981 and 5-Aza-2’ individually inhibited AML and MM cell growth in vitro, in a dose dependent manner (Figure 1C and D, Figure 7C and D). Combining low nanomolar dosage of TAK-981 and 5-Aza-2’ synergistically reduced tumor cell viability in AML in vitro (Figure 1 E ) consistent with current literature²². To reduce viability in MM, a 10-fold higher dosage of TAK-981 and 5-Aza-2’ was required (Figure 7E).

Example 3: Immunomodulatory effect of TAK-981 and 5-Aza-2’ on CD8+ T cells

Next, the inventors investigated the immunomodulatory properties of both drugs on T cells. Activated CD8+ T cells were treated overnight with low concentrations of TAK-981 and/or 5-Aza- 2’, after which expression of different cytokine signaling and cytolytic pathways were measured. The inventors measured interferon, interleukin and cytolytic molecule signaling at the transcriptional level via qPCR analysis. Combining low doses of TAK-981 (10nM) and 5-Aza-2’ (25nM) increased transcription of type I and II interferon, interferon stimulated genes and transcription factors, and transcription of interleukins (Figure 2A). 10-fold higher dosage of TAK- 981 single treatment induces transcription of interferon-related genes as expected (Figure 8A)¹⁷’¹⁸. The inventors observed that 100 nM TAK-981 (Figure 8A) resulted in substantially higher expression of interferon and interferon related genes compared to 10 nM TAK-981 (Figure 8A). Furthermore, 10-fold higher dosage of 5-Aza-2’ (250nM) induces transcription of cytolytic molecule Granzyme B as expected¹². Combination of higher dosage of TAK-981 and 5-Aza-2’ does not lead to a proper read out due to cytotoxic effects (Figure 8A). Therefore, the inventors used sub-cytotoxic dosage of both drugs for the combination treatment to induce cytokine transcription (Figure 2A).

Subsequently, the inventors investigated whether TAK-981 and 5-Aza-2’ enhance reactivity of NPM1-eTCR CD8+ T cells towards OCI-AML3 cells, since NPM1-eTCR CD8+ T cells recognize the mutated neoantigen NPM1 in the context of HLA-A*02 on OCI-AML3 cells⁴. OCI-AML3 or NPM1-eTCR CD8+ T cells were pre-treated for 4 days with 10 nM TAK-981 and/or 250nM 5-Aza- 2’ or DMSO 0.01% as control. Drug-treated OCI-AML3 or NPM1-eTCR CD8+ T cells were cocultured with untreated NPM1-eTCR CD8+ T cells or OCI-AML3 cells overnight (Figure 2B). Pretreatment of OCI-AML3 target cells did not lead to significant increase of IFNy production (Figure 2C), whereas pre-treatment of NPM1-eTCR CD8+ T cells with TAK-981 potentiated reactivity of T cells towards OCI-AML3 target cells, IFNy production doubled compared to DMSO control (Figure 2D). The large variation shown for the double combination could be assigned to a too high dosage of 5-Aza-2’ used. CD8+ survival assays indicate that 5-Aza-2’ at 250 nM is harmful for CD8+ T cells ( Figure 8B and C). Taken together, the inventors’ data show that sub-cytotoxic dosage of TAK-981 and 5-Aza-2’ have immunomodulatory capacity towards CD8+ T cells.

Example 4: TAK-981 and 5-Aza-2’ synergize to potentiate NPM1-eTCR activity in vivo

After establishing an immunomodulatory effect of 5-Aza-2’and TAK-981 in vitro, the inventors continued to apply this strategy in vivo. For in vivo validation of 5-Aza-2’, TAK-981 , and eTCR combination therapy, the inventors used an OCI-AML3 xenograft model. NSG mice were engrafted with luciferase transduced OCI-AML3 cells for 10 days, followed by two rounds of compound treatment and intravenous inoculation of NPM1-eTCR or CMV-eTCR CD8+ T cells on day 15 (Figure 3A). Compound treatment was continued bi-weekly. NPM1-eTCR therapy combined with 5-Aza-2’ and TAK-981 (“triple” therapy) demonstrated a striking better anti-tumor effect in the OCI-AML3 xenograft model compared to single and double treatments (Figure 3 B- D, Figure 9D). The reduction in tumor outgrowth obtained with the triple combination therapy was preserved for over 40 days in half of the mice after cessation of the drug treatment (Figure 3C). In contrast, single compound treatments did not significantly reduce OCI-AML3 tumor outgrowth (Figure 3B, Figure 9A). TAK-981 significantly potentiated N PM 1 -eTCR therapy, whereas TAK-981 treatment had no benefit over control therapy (Figure 3D, Figure 9B). The combination of 5-Aza- 2’ with NPM1-eTCR therapy was additive (Figure 3D, Figure 9C). The prolonged tumor reduction with combination therapies suggests prolonged and/or heightened effectivity of the eTCR T cells in combination with 5-Aza-2’ and TAK-981.

To investigate whether the efficacy of the triple therapy was not restricted to the NPM1 specificity of the eTCR, the inventors repeated the experiment with a similar dosing regimen but with another antigen specificity of the TCR modified CD8+ T cells (Figure 9E). OCI-AML3 engrafted NSG mice were treated with a suboptimal dose of HA2-eTCR CD8+ T cells specific for the HA2 minor histocompatibility antigen (MiHA) in the context of HLA-A*02. Treatment of OCI-AML3 engrafted NSG mice with the HA2-eTCR CD8+ T cells alone did not reduce OCI-AML3 outgrowth (Figure 9F). TAK-981 alone could not potentiate the HA2-eTCR T cells and 5-Aza-2’ gave some reduction of the tumor outgrowth. In contrast, combining HA2-eTCR therapy with both drugs showed major reduction in OCI-AML3 outgrowth, underlining the efficacy of the triple therapy (Figure 9F).

To further strengthen their findings, the inventors investigated whether the efficacy of the triple therapy was not restricted to AML but could be extended to other hematological malignancies. For this purpose, the inventors employed a multiple myeloma (MM) xenograft model with luciferase transduced LI266 cells targeted by BOB1-eTCR²⁷ or MAGE-A1-eTCR⁶. Single compound treatment had no effect on LI266 outgrowth (Figure 10A), comparable to the poor effect on OCI-AML3 (Figure 3B and Figure 9A). The inventors then treated MM engrafted NSG mice with suboptimal doses of MAGE-A1-eTCR CD8+ T cells to investigate increased efficacy with TAK-981 or 5-Aza-2’, or both. Treatment with low numbers of BOB1-eTCR T cells alone did not reduce LI266 outgrowth. In addition, TAK-981 could not potentiate the eTCR efficacy as single compound. However, combination of both drugs with eTCR therapy caused clear tumor reduction. Treatment with MAG E-A1 -eTCR T cells reduced LI266 outgrowth in vivo. Additional treatment with TAK-981 or 5-Aza-2’ did potentiate the T cell therapy. Once more, the triple combination gave an even faster and larger reduction in tumor outgrowth compared to the dual combinations. These results demonstrate the strength of the triple therapy. Boosting TCR therapy with single compounds partially depends on the potential of eTCR T cells towards the tumor. TAK-981 can potentiate eTCR therapy only when CD8+ T cells show initial effectivity.

Example 5: TAK-981 and 5-Aza-2’ synergize to induce CD8+ T cell proliferation in vivo

Based on the observation that combination treatment prolonged the in vivo responses induced by eTCR T cells, the inventors hypothesized that combination treatment might affect eTCR T cell persistence and/or heightened activation. To gain insight on the effect of TAK-981 and 5-Aza-2’ on NPM1-eTCR CD8+ T cell persistence and proliferation in vivo, the inventors generated luciferase expressing CD8+ T cells. This approach allowed the inventors to locate and quantify NPM1-eTCR CD8+ T cells in mice, using a similar experimental setting as presented in Figure 3. NSG-mice were transplanted with OCI-AML3 (non-luciferase) cells, treated at day 14 and 17 with the two drugs and subsequently inoculated with NPM1-eTCR CD8+ Luc T cells at day 18, and subsequently treated with drugs as indicated. Bioluminescence of N PM 1 -eTCR CD8+ T cells was measured on day 3, 6 and 9 post T cell injection (Figure 4A). Strikingly, combining 5-Aza-2’ and TAK-981 treatment led to a 10-fold higher count of NPM1-eTCR CD8+ T cells at day 6 compared to the single eTCR T cell treatment or double combinations (Figure 4B and C). TAK-981 treatment induced a modest increase in N PM 1 -eTCR CD8+ T cells on day 6 and 9. 5-Aza-2’ treatment boosted NPM1-eTCR CD8+ T cells at an early stage, however this boost was reduced on day 6.

Example 6: TAK-981 and 5-Aza-2’ synergize to induce in vivo activity of NPM1-eTCR CD8+ T cells

To gain more insight into the mechanisms underlying the efficacy of the triple therapy, the inventors conducted spectral flow cytometry analysis on the bone marrow of OCI-AML3 engrafted NSG-mice at several days after NPM1-eTCR T cell injection and compound treatment (Figure 5A). On days 2, 5 and 8 post NPM1-eTCR CD8+ T cell injection, bone marrow was harvested and spectral flow cytometry analysis was performed both on the N PM 1 -eTCR CD8+ T cells (Figure 5) and OCI-AML3 cells (Figure 6). The frequency of T cells expressing the proliferation marker Ki67 as well as the level of expression was upregulated in NPM1-eTCR T cells in mice treated with 5-Aza-2’ at day 5 and was even more pronounced in eTCR T cells of mice treated with combination of 5-Aza-2’ and TAK-981 (Figure 5C and Figure 11 A). In addition, NPM1-eTCR T cell counts were significantly higher in these experimental groups (Figure 5B), which was also consistent with the bioluminescence data in Figure 4B. Together these findings demonstrate that the combination of 5-Aza-2’ and TAK-981 leads to strong in vivo proliferation of transferred tumor targeting eTCR T cells.

Furthermore, the frequency and level of Interferon Transcription Factor 1 (I RF1) positive eTCR T cells in mice treated with 5-Aza-2’ and TAK-981 was dramatically increased and sustained compared to control or single drug treatment. Furthermore, 5-Aza-2’ single-treatment equally increased IRF1 frequency and level at day 2, however this effect did not persist to later timepoints (Figure 5D and Figure 11 B). The inventors’ data indicate that the tumor specific eTCR T cells in their triple therapy persistently produce high levels of interferon in vivo.

Interestingly, although the NPM1-eTCR T cells in triple treated mice proliferated much more pronounced, the T cells showed lower levels of early activation markers such as CD137, ICOS, CD25, and PD1 compared to no or single drug treated mice (Figure 5E and Figure 11 E). CD8+ NPM1-eTCR T cells in 5-Aza-2’ or TAK-981 treatment conditions showed increased expression of activation markers ICOS, PD1 , LAG3 and CD137. For 5-Aza-2’ treatment, the expression peaked on day 5, which corresponded with the peak in CD8+ NPM1-eTCR bioluminescence signal (Figure 4B) and CD8+ T cell counts at day 5 (Figure 5B). For TAK-981 the activation markers peaked on day 8, which corresponds with the hypothesis that TAK-981 facilitates prolonged T cell activation and persistence, and therefore prolonged repression of tumor outgrowth as presented in Figure 3. In contrast the inventors show that HLA-DR was typically upregulated for prolonged time in the CD8+ T cells of all treatment groups. In the triple therapy group, HLA-DR was the earliest and highest upregulated, correlating with the largest activation response. Taken together, in vivo T cell marker expression data, upregulation of I RF1 , Ki67 and HLA-DR, provide some mechanistic understanding of the increased efficacy of the NPM1-eTCR, 5-Aza-2’, TAK-981 triple therapy.

Example 7: TAK-981 and 5-Aza-2’ regulate HLA class I and other tumor cell surface markers Antigen presentation by HLA class I molecules is vital for CD8+ T cell recognition of tumor cells. To investigate whether 5-Aza-2’, TAK-981 or combination of treatment potentiates the T cell reactivity via changes in HLA class I expression as well as adhesion and co-inhibitory/ -stimulatory molecules, the inventors analysed OCI-AML3 tumor cells from the bone marrow of NSG mice by spectral flow cytometry. Bone marrow was harvested 18 days post engraftment after three rounds of treatment (Figure 6A). Tumor cell surface molecules involved in antigen presentation HLA class I, CD86, CD54 and CD58, and PD-L1 inhibitory ligand were investigated upon in vivo tumor treatment with 5-Aza-2’ and/or TAK-981 (Figure 6B). TAK-981 and 5-Aza-2’ single or combination treatments all induced upregulation of HLA class I and CD86 cell surface expression, whereas the levels of CD54 were slightly downregulated upon all treatments. Interestingly, PD-L1 , a key immune checkpoint facilitating immune escape, was downregulated upon 5-Aza-2’ single treatment and in combination with TAK-981 , indicating that the combination of 5-Aza-2 and TAK- 981 increases the immunogenicity of the tumor cells leading to better eradication of tumor cells by the tumor specific T cells (Figure 6B).

Subsequently, the inventors investigated whether the presence of CD8+ T cells influenced HLA class I molecule and PD-L1 expression on OCI-AML3 cells. Mice were drug treated two times prior to T cell injection, followed by 3 treatment rounds before the harvest of bone marrow to isolate the OCI-AML3 cells (Figure 6C). OCI-AML3 tumor cells were harvested from the bone marrow of NSG mice on day 2, 5 and 8 post CD8+ NPM1-eTCR T cell injection. HLA class I cell surface expression was further upregulated in the presence of NPM1-eTCR CD8+ T cells treated in vivo with 5-Aza-2’ and/or TAK-981 (Figure 6D). TAK-981 treatment supported persistent upregulation of HLA class I over time. Due to treatment efficiency, no OCI-AML3 cells were left for analysis in the triple combination group. The inventors hypothesise that the induced interferon production by the CD8+ T cells upon treatment with TAK-981 led to the increase in HLA class I presentation. 5-Aza-2’ is expected to upregulate HLA cell surface expression in a direct manner. PD-L1 downregulation by 5-Aza-2’ treatment was also found in the presence of CD8+ T cells (Figure 6B and D). Treatment with 5-Aza-2’ resulted in increased proliferation of the remaining tumor cells in the presence and absence of CD8+ T cells (Figure 6B and D).

Taken together, 5-Aza-2’ and TAK-981 regulate multiple tumor cell surface markers facilitating antigen presentation and while simultaneously restricting the expression of the PD-L1 immune checkpoint. Combined, these results provide insight into the potential molecular mechanism underlying the stimulation of eTCR therapy by 5-Aza-2’ and TAK-981.

Example 8: Discussion

In this study, the inventors evaluated a novel drug combination to augment eTCR T cell therapy. Excitingly, combining eTCR T cell therapy with the SUMO E1 inhibitor TAK-981 and the DNA methylation inhibitor 5-Aza-2’ resulted in strong anti-tumor activity against two in vivo tumor models of hematological malignancies using four different engineered TCR T cells. The inventors uncovered that the drug combination caused strong eTCR T cell proliferation in vivo. Mechanistically, the drug combination increased cytokine signaling in T cells and MHC class I cell surface expression on tumor cells.

Proliferating T cells that demonstrate active cytokine signaling is a unique signature of this therapy. Proliferation of T cells and active interferon signaling are thought to be mutually exclusive because of the anti-proliferative effect of interferon²⁸. Numbers of T cells drop drastically after day 6, which corresponds with the day where the lowest tumor volume is reached, indicating that most if not all tumor cells are cleared and T cells lose their targets and therefore decline in numbers.

Both compounds contribute distinctively to therapy efficacy. SUMOylation is known to block interferon transcription²⁹’³⁰; consequently, SUMOylation inhibition by TAK-981 enhances cytokine production in T cells. This is consistent with recent findings that TAK-981 enhances T cell interferon transcription and production^17-19, providing a mechanistic explanation for the positive effect of TAK-981 on eTCR T cell activation in vivo. TAK-981 efficacy is fully dependent on the presence of T cells, since single compound TAK-981 treatment did not block tumor growth. Ex vivo evaluation of T cell makers showed increased upregulation of activation markers over time. Hypomethylation via 5-Aza-2’ treatment results in additional removal of transcriptional blockage resulting in increased cytolytic compound production and in combination with TAK-981 overall increase cytokine signaling.

Furthermore, induction of MHC I cell surface expression is observed upon either 5-Aza-2’ or TAK- 981 treatment of OCI-AML3 in the absence of T cells. In contrast, in the presence of T cells a large increase in MHC I cell surface expression was observed in an early response to 5-Aza-2’ 2 days post T cells, whereas MHC I cell surface expression peaked 5 days after T cell injection in response to TAK-981 treatment. This is compatible with a slower but prolonged activation of TAK- 981 treatment compared to 5-Aza-2’ and suggests that interferon production by T cells strongly contributes to MHC I upregulation on tumor cells. The inventors’ data on MHC I regulation corresponds with recent literature²⁰, where it has been shown that active SUMO has repressive effects on MHC I cell surface expression. In addition, it was found previously that hypomethylation of antigen presentation complex (APC) related genes by 5-Aza-2’ enhanced antigen presentation on tumor cells³¹’³². Increased expression of APC machinery is not restricted to the MHC molecules but extends to CD86 co-stimulatory ligand. It has to be noted that CD54 was downregulated in our ex vivo analysis, in contrast to literature (Krishnadas et al., 2014). Interestingly, PD-L1 inhibitory ligand was also downregulated in response to 5-Aza-2’ treatment of tumor cells, potentially reducing immune checkpoint signaling. This is in contrast with literature where 5-Aza-2’ treatment upregulated PD-L1 expression³³³⁴, which potentially could be explained by differences in dosage.

These individual effects of both drugs potentially explain the striking efficacy of the triple therapy. The inventors observed that on the one hand 5-Aza-2’ is responsible for the gain in proliferative capacity of the CD8+ T cells in vivo and that TAK-981 increased their persistence and activation via cytokine signaling. Combined, these drugs mutually support the increased tumor targeting potential of eTCR therapy. Whereas normally high proliferation and tumour interaction of CD8 T cells is followed by ‘exhaustion’, the inventors’ data suggest that inhibition of SUMOylation might support the maintenance of an activated T cells state. Furthermore, it has been shown that SUMO plays a role in inducing exhaustion via aryl hydrocarbon receptor stability, whereas inhibition of SUMO leads to degradation of the aryl hydrocarbon receptor and consequently a decrease in exhaustion via this protein³⁵.

The inventors provide evidence for a novel strategy to enhance eTCR therapy with sub-cytotoxic dosing of TAK-981 and 5-Aza-2’. In summary, SUMOylation inhibition via TAK-981 in combination with 5-Aza-2’ synergizes to enhance cellular immunotherapy by altering transcriptional regulation of CD8+ T cells, increasing cytokine production including interferons indicating increased activation. Moreover, antigen presentation on the tumor cells is increased as well as co- stimulation whereas the PD-L1 immune checkpoint is reduced. Combining eTCR T cell therapy with TAK-981 and 5-Aza-2’ may be an important step towards improved clinical outcome.

Example 9: TAK981 and 5-Aza-2’ synerqize to induce in vivo activity of CAR T-cell treatment

The triple combination of a DNA hypomethylating agent, a SUMOylation inhibitor, and T-cell- mediated therapy is also effective in CAR T-cell treatment, as demonstrated in two in vivo models: Figure 13A shows data from a Multiple Myeloma Model in which NSG mice were intravenously inoculated with LI266 multiple myeloma cells expressing Luc2. Tumor cells were allowed to engraft for 10 days. Subsequently, some mice were treated bi-weekly with 5-Aza-2' (2.5 mg/kg) and TAK981 (25mg/kg). On day 14, BCMA-CAR T cells were infused either into mice without treatment (BCMA-CAR T cells) or those treated with the two compounds (BCMA-CAR + TAK981 + 5-Aza-2'). Additional control mice received only CD19 CAR T cells. Tumor progression was monitored using I VIS imaging after subcutaneous luciferin injection. As shown in figure 13A, by day 18 (four days after T-cell infusion), tumor eradication was more rapid in mice treated with the triple therapy compared to those that received only BCMA-CAR T cells.

Figure 13B-D show data from a Acute Lymphoblastic Leukemia (ALL) Model in which NSG mice were intravenously injected with the NALM-6 ALL cell line expressing Luc2. Starting on day 1 after tumor injection, two groups of mice received bi-weekly treatments with TAK-981 (25mg/kg) and 5-Aza-2’ (2.5mg/kg). One group continued receiving only these two compounds (TAK981 + 5-Aza-2'). On day 4, CD19-CAR T cells were infused into mice that either had not been treated with the compounds (CD19-CAR T cells group) or had received both compounds (CD19-CAR + TAK981 + 5-Aza-2' group). Control mice were given only control T cells (BCMA-CAR group). Tumor growth was monitored regularly using I VIS imaging. Figure 13B shows the average ventral radiance (p/s/cm²/sr) for each group. Figure 13D displays the individual ventral radiance (p/s/cm²/sr) for each mouse. In Figure 13C, the mean and standard deviation of tumor growth (average radiance measured by bioluminescence imaging) are shown for day 17 after tumor injection. Statistics depict one-way ANOVA with Turkey multiple comparisons post-hoc test on log-transformed data comparing bioluminiscence of all groups on day 17 after tumor injection. The results clearly demonstrate that the combination of a DNA hypomethylating agent, a SUMOylation inhibitor, and CAR T cells more effectively eradicated ALL tumor cells compared to CAR T cells or the two compounds used alone.

Conclusion

A novel strategy to enhance T cell mediated therapy has been demonstrated. This enhancement is facilitated by the combination of a DNA hypomethylating agent and a SUMOylation inhibitor leading to altered transcriptional regulation of T cells as well as increased activation of T cells. Statements that relate to embodiments of the invention are provided below as a numbered paragraphs (paras):

Para 1 . A DNA hypomethylating agent for use in treating a patient undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy.

Para 2. A SUMOylation inhibitor for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

Para 3. A T cell mediated therapy for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

Para 4. A DNA hypomethylating agent and a SUMOylation inhibitor for use in treating a patient undergoing treatment with a T cell mediated therapy.

Para 5. A DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy for use in treating a patient.

Para 6. A DNA hypomethylating agent for use in treating a patient by inducing or enhancing a T cell response in the patient, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

Para 7. A SUMOylation inhibitor for use in treating a patient by inducing or enhancing a T cell response in the patient, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

Para 8. A DNA hypomethylating agent and a SUMOylation inhibitor for use in treating a patient by inducing or enhancing a T cell response in the patient.

Para 9. The DNA hypomethylating agent for use according to para 6 or 8, or the SUMOylation inhibitor for use according to para 7, wherein the patient is undergoing treatment with a T cell mediated therapy.

Para 10. A method for treating a patient, the method comprising: administering to the patient a DNA hypomethylating agent, wherein the patient is undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy. Para 11. A method for treating a patient, the method comprising: administering to the patient a SUMOylation inhibitor, wherein the patient is undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

Para 12. A method for treating a patient, the method comprising: administering to the patient a T cell mediated therapy, wherein the patient is undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

Para 13. A method for treating a patient, the method comprising: administering to the patient a DNA hypomethylating agent and a SUMOylation inhibitor, wherein the patient is undergoing treatment with a T cell mediated therapy.

Para 14. A method for treating a patient, the method comprising: administering to the patient a DNA hypomethylating agent, a SUMOylation inhibitor, and a T cell mediated therapy.

Para 15. A method of treating a patient by inducing or enhancing a T cell response, the method comprising: administering to the patient a DNA hypomethylating agent wherein the patient is undergoing treatment with a SUMOylation inhibitor.

Para 16. A method of treating a patient by inducing or enhancing a T cell response, the method comprising: administering to the patient a SUMOylation inhibitor wherein the patient is undergoing treatment with a DNA hypomethylating agent.

Para 17. A method of treating a patient by inducing or enhancing a T cell response, the method comprising: administering to the patient a SUMOylation inhibitor and a DNA hypomethylating agent.

Para 18. The method of any one of paras 15 to 17, wherein the patient is undergoing treatment with a T cell mediated therapy.

Para 19. The method of any one of paras 15 to 18, wherein the method further comprises administering to the patient a T cell mediated therapy.

Para 20. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of any one of paras 6 to 9, or any one of paras 15 to 19, wherein the induced or enhanced T cell response comprises induced or enhanced CD8+ T cell proliferation and/ or CD4+ T cell proliferation. Para 21 . The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of any one of the preceding paras, wherein the use or method is for treating cancer.

Para 22. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of para 21 , wherein the cancer is a solid cancer.

Para 23. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of para 21 , wherein the cancer is a haematological malignancy.

Para 24. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of para 23, wherein the haematological malignancy is a myeloid haematological malignancy or lymphoid haematological malignancy.

Para 25. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of any one of the preceding paras, wherein the DNA hypomethylating agent is selected from the group consisting of: 5-Azacytidine, 5-Aza-2’-deoxycytidine, 5-Aza-4'- thio-2'-deoxycytidine (5-Aza-T-dCyd), 5-Fluro-2-deoxycytidine, SGI-110, Zebularine, CP-4200, RG108, Nanaomycin A, SW155246, GSK3735967, GSK-3484862, GSK-3685032, RX-3117 (TV- 1360), DC-05, y-Oryzanol, DC_517, DNMT1-IN-3, Procainamide, lsofistularin-3, CM-272, and SGI-1027.

Para 26. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of any one of the preceding paras, wherein the SUMOylation inhibitor is an E1 inhibitor, optionally wherein the E1 inhibitor is selected from the group consisting of: TAK981 , Ginkgolic acid (15:1), Anacardic acid, Kerriamycin B, SUMO-AMSN, SUMO-AVSN, Compound 21 , Davidiin, Tannic acid, ML-792, COH-OOO, and ML-93.

Para 27. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of any one of the preceding paras, wherein the T cell mediated therapy is a TCR, CAR-T, virus-specific T cells, immune mobilising T cell receptor against X disease, bispecific T-cell engagers, or a vaccine.

Para 28. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, or method of any one of paras 21 to 26, wherein the T cell mediated therapy is immune mobilising monoclonal T-cell receptors against cancer, or tumour infiltrating lymphocyte (TIL) . Para 29. A DNA hypomethylating agent for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

Para 30. A SUMOylation inhibitor for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

Para 31 .A DNA hypomethylating agent and a SUMOylation inhibitor for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 tumour cell surface expression.

Para 32. A method of inducing or enhancing tumor immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a DNA hypomethylating agent wherein the patient is undergoing treatment with a SUMOylation inhibitor.

Para 33.A method of inducing or enhancing cancer cell immunogenicity in a patient by downregulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a SUMOylation inhibitor wherein the patient is undergoing treatment with a DNA hypomethylating agent.

Para 34. A method of inducing or enhancing tumor immunogenicity in a patient by downregulating PD-L1 cancer cell surface expression, the method comprising: administering to the patient a SUMOylation inhibitor and a DNA hypomethylating agent.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

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Claims

1 . A DNA hypomethylating agent for use in treating a patient undergoing treatment with a SUMOylation inhibitor and a T cell mediated therapy.

2. A SUMOylation inhibitor for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a T cell mediated therapy.

3. A T cell mediated therapy for use in treating a patient undergoing treatment with a DNA hypomethylating agent and a SUMOylation inhibitor.

4. A DNA hypomethylating agent and a SUMOylation inhibitor for use in treating a patient undergoing treatment with a T cell mediated therapy.

5. A DNA hypomethylating agent, a SUMOylation inhibitor and a T cell mediated therapy for use in treating a patient.

6. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, according to any one of the preceding claims, wherein the use or method is for treating cancer.

7. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, of claim 6, wherein the cancer is a solid cancer.

8. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, of claim 6, wherein the cancer is a haematological malignancy.

9. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, of claim 8, wherein the haematological malignancy is a myeloid haematological malignancy or lymphoid haematological malignancy.

10. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, according to any one of the preceding claims, wherein the DNA hypomethylating agent is selected from the group consisting of: 5-Azacytidine, 5-Aza-2’-deoxycytidine, 5-Aza-4'- thio-2'-deoxycytidine, 5-Fluro-2-deoxycytidine, SGI-110, Zebularine, CP-4200, RG108, Nanaomycin A, SW155246, GSK3735967, GSK-3484862, GSK-3685032, RX-3117 (TV-1360), DC-05, y-Oryzanol, DC_517, DNMT1-IN-3, Procainamide, lsofistularin-3, CM-272, and SGI-1027.

11. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, according to any one of the preceding claims, wherein the SUMOylation inhibitor is an E1 inhibitor, optionally wherein the E1 inhibitor is selected from the group consisting of: TAK981 , Ginkgolic acid (15:1), Anacardic acid, Kerriamycin B, SUMO-AMSN, SUMO-AVSN, Compound 21 , Davidiin, Tannic acid, ML-792, COH-OOO, and ML-93.

12. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, according to any one of the preceding claims, wherein the T cell mediated therapy is a TOR, CAR-T, virus-specific T cells, immune mobilising T cell receptor against X disease, bispecific T-cell engagers, or a vaccine.

13. The DNA hypomethylating agent for use, SUMOylation inhibitor for use, T cell mediated therapy for use, of any one of claims 6 to 11 , wherein the T cell mediated therapy is immune mobilising monoclonal T-cell receptors against cancer, or tumour infiltrating lymphocyte (TIL) .

14. A DNA hypomethylating agent for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a SUMOylation inhibitor.

15. A SUMOylation inhibitor for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 cancer cell surface expression, wherein the patient is undergoing treatment with a DNA hypomethylating agent.

16. A DNA hypomethylating agent and a SUMOylation inhibitor for use in inducing or enhancing cancer cell immunogenicity in a patient by down-regulating PD-L1 tumour cell surface expression.