New coupled nucleic acid molecules and their uses

Abstract

The present invention relates to novel nucleic acid molecules of therapeutic interest, in particular for the treatment of cancer.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to the field of oncology. Background Art

[0002] The DNA damage response (DDR) detects DNA lesions and promotes their repair. The wide diversity of DNA lesion types requires a variety of largely different DNA repair mechanisms such as mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), single-strand break repair (SSB) and double-strand break repair (DSB). For example, poly A ribose polymerase (PARP) is essentially involved in repairing SSBs, while repairing DSBs in DNA uses two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). In NHEJ, DSBs are recognized by the Ku protein, which then binds and activates the protein kinase DNA-PKc, leading to the recruitment and activation of end processing enzymes. The ability of cancer cells to repair treatment-induced DNA damage has been shown to affect the efficacy of treatment.

[0003] This has led to the targeting of DNA repair pathways and proteins to develop anticancer agents that increase sensitivity to traditional genotoxic therapies (chemotherapeutics, radiation therapy). Synthetic lethality approaches for cancer therapy offer new mechanisms to specifically target cancer cells while leaving non-cancerous cells unharmed, thereby reducing treatment-related toxicity.

[0004] In these synthetic lethal approaches, Dbait molecules are nucleic acid molecules that mimic double-stranded DNA lesions. They act as decoys for the DNA damage signaling enzymes PARP and DNA-PK, inducing "false" DNA damage signals and ultimately inhibiting the recruitment of many proteins involved in the DSB and SSB pathways at the damage site.

[0005] Dbait molecules have been extensively described in PCT patent applications WO2005 / 040378, WO2008 / 034866, WO2008 / 084087 and WO2017 / 013237. Dbait molecules can be defined by a number of features necessary for their therapeutic activity, such as their minimum length, which can vary as long as it is sufficient to allow appropriate binding of the Ku protein complex comprising Ku and DNA-PKc proteins. It has therefore been shown that the length of the Dbait molecule must be greater than 20 bp, preferably about 32 bp, to ensure binding to this Ku complex and to enable activation of DNA-PKc.

[0006] Potential predictive biomarkers for treatment using such Dbait molecules have been characterized. As described in PCT patent application WO2018 / 162439, sensitivity to Dbait molecules is in fact associated with a high spontaneous frequency of cells with micronuclei (MN). High basal levels of MN have been proposed as predictive biomarkers for treatment using Dbait molecules and subsequently validated in 43 solid tumor cell lines from a variety of different tissues and 16 cell and patient-derived xenograft models.

[0007] In addition, it has recently been proposed that micronuclei (MNs) provide a key platform as part of the DNA damage-induced immune response (Gekara J Cell Biol. 2017 Oct 2; 216 (10): 2999-3001). Recent studies have confirmed the role of MN formation in DNA damage-induced immune activation. Interestingly, in fact, the cytoplasmic DNA sensing pathway has been found to be the main link between DNA damage and innate immunity. DNA is usually present in the nucleus and mitochondria, so its presence in the cytoplasm acts as a danger-associated molecular pattern (DAMP) that triggers the immune response. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a sensor that detects DNA as a DAMP and induces type I IFN and other cytokines. DNA binds to cGAS in a sequence-independent manner; this binding induces a conformational change in the catalytic center of cGAS, allowing the enzyme to convert guanosine triphosphate (GTP) and ATP into the second messenger cyclic GMP-AMP (cGAMP). This cGAMP molecule is an endogenous high affinity ligand for the adaptor protein IFN gene stimulator STING. Thus, activation of the STING pathway can include, for example, stimulation of the inflammatory cytokines IP-10 (also known as CXCL10) and CCL5 or the receptors NGK2 and PD-L1.

[0008] Recent evidence suggests that the STING (stimulator of interferon genes) pathway is involved in the induction of anti-tumor immune responses. Therefore, STING agonists are now widely developed as a new class of cancer therapeutics. It has been shown that activation of STING-dependent pathways in cancer cells can lead to infiltration of tumors by immune cells and modulation of anti-cancer immune responses.

[0009] STING is an endoplasmic reticulum adapter that promotes innate immune signaling (a rapid nonspecific immune response against environmental insults, including but not limited to pathogens such as bacteria or viruses). STING is reported to be able to activate NF-kB, STAT6, and IRF3 transcriptional pathways to induce the expression of type I interferons (such as IFN-α and IFN-β) and exert an effective antiviral state after expression. However, the STING agonists developed so far are able to activate the STING pathway in all cell types and may trigger serious side effects associated with its activation in dendritic cells. Therefore, STING agonists are administered topically.

[0010] Therefore, there is a real need to find methods to specifically activate the STING pathway in tumor cells.

[0011] Therefore, there remains a need for therapies for cancer treatment, especially drugs that rely on several mechanisms, particularly DNA repair pathways and STING pathway activators, and for drugs that may help checkpoint inhibitors work in more patients and a wider range of cancers.

[0012] Cancer cells have a unique energy metabolism for maintaining rapid proliferation. The preference for anaerobic glycolysis under normoxic conditions is a unique feature of cancer metabolism and is known as the Warburg effect. Enhanced glycolysis also supports the generation of nucleotides, amino acids, lipids and folic acid as building blocks for cancer cell division. Nicotinamide adenine dinucleotide (NAD) is a coenzyme that mediates redox reactions in many metabolic pathways including glycolysis. Increased NAD levels enhance glycolysis and provide energy for cancer cells. In this case, NAD level depletion subsequently inhibits cancer cell proliferation by inhibiting energy production pathways such as glycolysis, tricarboxylic acid (TCA) cycle and oxidative phosphorylation. NAD also serves as a substrate for several enzymes, thereby regulating DNA repair, gene expression and stress response through these enzymes. Therefore, NAD metabolism is also involved in cancer pathogenesis in addition to energy metabolism, and is considered to be a promising therapeutic target for cancer treatment, particularly for cancer cells that exhibit NAD defects due to DNA repair gene defects (such as ERCC1 and ATM defects) or IDH (isocitrate dehydrogenase) mutations.

[0013] There remains a need for new therapeutic approaches that successfully target cancer cell populations without the emergence of cancer cells that are resistant to therapy. Summary of the invention

[0014] The present invention provides novel coupled nucleic acid molecules that specifically target DNA repair pathways and stimulate the STING pathway in cancer cells. More specifically, the nucleic acid molecules are capable of activating PARP without any activation of DNA-PK.

[0015] The present invention relates to a coupled nucleic acid molecule, comprising: a double-stranded nucleic acid component, wherein the 5' end of the first strand and the 3' end of the complementary strand are connected together through a loop; and an endocytosis-promoting molecule optionally connected to the loop,

[0016] in

[0017] - the length of the double-stranded nucleic acid component is 10 to 20 base pairs;

[0018] - the sequence of the double-stranded nucleic acid component has less than 80% sequence identity with any gene in the human genome;

[0019] - the double-stranded nucleic acid component comprises deoxyribonucleotides and up to 30% of ribonucleotides or modified deoxyribonucleotides relative to the total number of nucleotides in the nucleic acid molecule; and

[0020] - The ring has a structure selected from one of the following structural formulas:

[0021] -OP(X)OH-O-{[(CH2)2-O] g -P(X)OH-O} r -KOP(X)OH-O-{[(CH2)2-O] h -P(X)OH-O-} s (I)

[0022] wherein r and s are independently integers 0 or 1; g and h are independently integers from 1 to 7, and the sum of g+h is from 4 to 7;

[0023] where K is

[0024]

[0025] wherein i, j, k and l are independently integers from 0 to 6, preferably from 1 to 3;

[0026] or

[0027] -OP(X)OH-O-[(CH2) d -C(O)-NH] b -CHR-[C(O)-NH-(CH2) e ] c -OP(X)OH-O-(II)

[0028] wherein b and c are independently integers from 0 to 4, and the sum of b+c is from 3 to 7;

[0029] d and e are independently integers from 1 to 3, preferably from 1 to 2; and

[0030] Where R is -L f -J,

[0031] X is O or S, L is a linker, and f is an integer of 0 or 1, and J is a molecule that promotes endocytosis or is H.

[0032] The nucleic acid molecule may comprise one of the following sequences:

[0033] 5'CCCAGCAAACAAGCCT-∫(SEQ ID NO 1)

[0034] 3'GGGTCGTTTGTTCGGA-∫

[0035] and

[0036] 5'CAGCAACAAG-∫ (SEQ ID NO 2)

[0037] 3'GTCGTTGTTC-∫

[0038] Or a sequence in which 1 to 3 nucleotides are replaced by ribonucleotides or modified deoxyribonucleotides or ribonucleotides.

[0039] The molecule promoting endocytosis may be selected from cholesterol, single-chain or double-chain fatty acids, ligands targeting cell receptors capable of receptor-mediated endocytosis, or transferrin.

[0040] More specifically, the molecule that promotes endocytosis is cholesterol.

[0041] Alternatively, the molecule that promotes endocytosis is a ligand of a σ-2 receptor (σ2R). For example, the ligand of a σ-2 receptor (σ2R) comprises the following structural formula:

[0042]

[0043] wherein n is an integer from 1 to 20.

[0044] In one case, 1, 2 or 3 internucleotide bonds of the nucleotides at the free ends of the double-stranded component of the nucleic acid molecule may have a modified phosphodiester backbone such as phosphorothioate bonds, preferably on both strands. For example, 1 to 3 thymidines may be replaced by 2'-deoxy-2'-fluoroarabinouridine, or 1 to 3 guanosines may be replaced by 2'-deoxy-2'-fluoroarabinoguanosine, or 1 to 3 cytidines may be replaced by 2'-deoxy-2'-fluoroarabinocytidine.

[0045] The ring may have the structural formula (I), and K is

[0046]

[0047] Optionally, f is 1, and LJ is selected from -C(O)-(CH2) m -NH-[(CH2)2-O] n -(CH2) p -C(O)-J, -C(O)-(CH2) m -NH-C(O)-[(CH2)2-O] n -(CH2) p -J, C(O)-(CH2) m -NH-C(O)-CH2-O-[(CH2)2-O] n -(CH2) p -J, -C(O)-(CH2) m -NH-C(O)-[(CH2)2-O] n -(CH2) p -C(O)-J and -C(O)-(CH2) m -NH-C(O)-CH2-O-[(CH2)2-O] n -(CH2) p -C(O)-J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3.

[0048] Optionally, the ring has the structural formula (I)

[0049] -OP(X)OH-O-{[(CH2)2-O] g -P(X)OH-O} r -KOP(X)OH-O-{[(CH2)2-O] h -P(X)OH-O-} s (I)

[0050] Where X is S, r is 1, g is 6, s is 0, and K is

[0051]

[0052] wherein f is 1, and L is C(O)-(CH2)5-NH-[(CH2)2-O]3-(CH2)2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-[(CH2)2-O]3-(CH2)3-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]5-CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]9-CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]13 -CH2-C(O)-J or -C(O)-(CH2)5-NH-C(O)-J.

[0053] Optionally, f is 1, and LJ is -C(O)-(CH2) m -NH-[C(O)] t -[(CH2)2-O] n -(CH2) p -[C(O)] v -J or -C(O)-(CH2) m -NH-[C(O)-CH2-O] t -[(CH2)2-O] n -(CH2) p -[C(O)] v -J, wherein m is an integer from 0 to 10, n is an integer from 0 to 15; p is an integer from 0 to 4; t and v are integers 0 or 1, wherein at least one of t and v is 1.

[0054] In a particular case, L can be selected from -C(O)-(CH2) m -NH-[(CH2)2-O] n -(CH2) p -C(O)-J, -C(O)-(CH2) m -NH-C(O)-[(CH2)2-O] n -(CH2) p -J, C(O)-(CH2) m -NH-C(O)-CH2-O-[(CH2)2-O] n -(CH2) p -J, -C(O)-(CH2) m -NH-C(O)-[(CH2)2-O] n -(CH2) p -C(O)-J and -C(O)-(CH2) m -NH-C(O)-CH2-O-[(CH2)2-O] n -(CH2) p -C(O)-J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3.

[0055] In very specific cases, L can be selected from -C(O)-(CH2)5-NH-[(CH2)2-O]3-(CH2)2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-[(CH2)2-O]3-(CH2)3-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]5-CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]9-CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O] 13 -CH2-C(O)-J or -C(O)-(CH2)5-NH-C(O)-J.

[0056] In certain cases, the coupled nucleic acid molecule is selected from the following structural formula or a pharmaceutically acceptable salt thereof:

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; the italicized U is 2'-deoxy-2'-fluoroarabino uridine; the italicized G is 2'-deoxy-2'-fluoroarabino guanosine; and the italicized C is 2'-deoxy-2'-fluoroarabino cytidine.

[0064] The present invention further relates to a pharmaceutical composition comprising a coupled nucleic acid molecule according to the disclosure. Optionally, the pharmaceutical composition further comprises another therapeutic agent, which is preferably selected from immunomodulators such as immune checkpoint inhibitors (ICI), cancer immunotherapy based on T-cells such as adoptive cell transfer (ACT), genetically modified T-cells or engineered T-cells such as chimeric antigen receptor cells (CAR-T cells), or conventional chemotherapy, radiotherapy or anti-angiogenic agents, HDAC inhibitors (such as Belinostat) or targeted immunotoxins.

[0065] The present invention also relates to a coupled nucleic acid molecule or a pharmaceutical composition according to the disclosure, which is used as a medicament, in particular for treating cancer. It also relates to a method for treating cancer in a subject in need thereof, the method comprising repeated or long-term administration of a therapeutically effective amount of a coupled nucleic acid molecule or a pharmaceutical composition according to the present invention. Optionally, the method comprises administration of repeated treatment cycles, preferably at least two administration cycles, and even more preferably at least three or four administration cycles.

[0066] Repeated or long-term administration of the coupled nucleic acid molecules according to the present invention does not cause cancer cells to develop resistance to the therapy. It can be used in combination with immunomodulators such as immune checkpoint inhibitors (ICI), T-cell-based cancer immunotherapy including adoptive cell transfer (ACT), genetically modified T-cells or engineered T-cells such as chimeric antigen receptor cells (CAR-T cells).

[0067] Therefore, the coupled nucleic acid molecule or pharmaceutical composition is used in combination with another therapeutic agent for treating cancer, and the other therapeutic agent is preferably selected from immunomodulators such as immune checkpoint inhibitors (ICI), T-cell-based cancer immunotherapy such as adoptive cell transfer (ACT), genetically modified T-cells or engineered T-cells such as chimeric antigen receptor cells (CAR-T cells), or conventional chemotherapy, radiotherapy or anti-angiogenic agents, HDAC inhibitors (e.g., belinostat) or targeted immunotoxins.

[0068] In certain cases, the present invention also relates to a method for possible selection strategy or clinical stratification strategy to obtain + Patients with tumors that carry defects in synthesis. These patients may be better responders to drug treatment according to the invention, especially patients with tumors that carry defects in two DNA repair pathways (e.g. ERCC1 and ATM defects) or IDH mutations.

[0069] In certain cases, the coupled nucleic acid molecule or pharmaceutical composition is used to obtain a target for NAD in cancer treatment. + Targeted effect of tumor cells with defects in synthesis. More specifically, the tumor cells also have a DNA repair pathway defect selected from ERCC1 or ATM defect or IDH mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1: OX401-induced target effects. Cells were treated with increasing doses of OX401 or AsiDNA TMAfter 24 hours of treatment, (A) DNA-PK activation was assessed by H2AX phosphorylation (γH2AX) and (B) PARP hyperactivation was assessed by measuring cellular PARylation by detecting poly(ADP-ribose) (PAR) polymers. ***, p<0.001.

[0071] Figure 2: OX401 exhibits tumor-specific cytotoxicity. (A) Tumor cells and (B) non-tumor cells were treated with OX401 or AsiDNA TM Treatment, and use XTT assay to assess cell viability. Cell viability was calculated as the ratio of live treated cells to live untreated cells. IC50 was calculated from dose response curves using GraphPad Prism software.

[0072] Figure 3 : OX401 triggers tumor immune response. Evaluation of OX401 or AsiDNA TM (A) % of micronucleus-positive cells, (B) amounts of secreted CCL5 and CXCL10 chemokines (using ELISA assays), and (C) total PD-L1 (by western blot) and (D) levels of surface-bound PD-L1 (by flow cytometry analysis) of chronically treated MDA-MB-231 cells. cGAMP, STING agonist; **, p<0.01.

[0073] Figure 4 : OX402 induces PARP activation. Cells were treated with increasing doses of OX402 for 24 hours and PARP hyperactivation was assessed by measuring cellular PARylation by detecting poly(ADP-ribose) (PAR) polymers.

[0074] Figure 5 OX401 induces PARylation and efficient NAD in tumor cells + Depletion. Cells were treated with OX401 (5 μM) for 48 h, 7 days, or 13 days, and PARP hyperactivation (by western blot analysis of PARylated proteins) (A, D), NAD+ intracellular levels (B, E), and cell viability (C, F) were assessed. + The % and viability are expressed as the ratio of treated cells to untreated cells (NT). (A, B, C) MDA-MB-231 tumor cells, (D, E, F) MRC5 lung fibroblasts.

[0075] Figure 6: OX401 abolishes the homologous recombination repair pathway. Cells were treated with OX401 (5 μM) for 48 hours, and DSB levels were assessed using (A) flow cytometry to detect phosphorylated forms of H2AX (γH2AX) or (B) immunofluorescence to detect γH2AX foci. (CD) After 48 hours of treatment with olaparib (5 μM) with or without OX401 (5 μM), the efficacy of the homologous recombination pathway was analyzed by (C) detection of the recruitment of Rad51 protein to DSB sites and (D) quantification of Rad51 foci. ***, p<0.001.

[0076] Figure 7 :Tumor cells treated with OX401 do not develop drug resistance.(A) Cells were treated with talazopanib (2 μM) or OX401 (1.5 μM) and counted after each treatment and amplification cycle.(B) Cell viability was estimated by dividing the number of treated cells by the mean number of untreated cells and determined after each treatment period.(C) After 4 days of treatment with talazopanib with increasing doses, resistance to talazopanib was confirmed in three isolated colonies (Tal1, Tal2 and Tal3) compared with U937 parental cells using the XTT assay. Survival percentages were normalized using untreated conditions.

[0077] Figure 8 : OX401 enhances antitumor immune responses. MDA-MB-231 cells were co-cultured with T lymphocytes (4:1 ratio of effector cells to target tumor cells) in the presence or absence of OX401 (5 μM) for 48 h, and (A) tumor cell proliferation, (B) the amount of secreted granzyme B (using ELISA assay), and (C, D) activation of the STING pathway (by western blot (C) or ELISA assay to quantify secreted CCL5 chemokine (D)) were assessed. LT a , activated T lymphocytes; MDA, MDA-MB-231 tumor cells.

[0078] Fig. 9 : Binding kinetics of OX401, OX402, OX406, OX407, OX408, OX410 and OX411 to PARP-1 (k on ) and interaction strength (K D ). DETAILED DESCRIPTION OF THE INVENTION

[0080] The present invention relates to novel nucleic acid molecules coupled to molecules that promote endocytosis, such as cholesterol-nucleic acid conjugates, which specifically target and activate PARP, induce a profound downregulation of cellular NAD, and are therefore particularly useful for cancer treatment, especially for cancer cells that exhibit NAD deficiency due to DNA repair gene defects (such as ERCC1 and ATM defects) or IDH (isocitrate dehydrogenase) mutations.

[0081] The present invention relates to novel nucleic acid molecules coupled to molecules promoting endocytosis, such as cholesterol-nucleic acid conjugates, which target the DDR machinery and are also STING agonists, allowing them to be used in combination with immune checkpoint therapy (ICT) for optimal treatment of cancer.

[0082] Therefore, the present inventors surprisingly found that:

[0083] 1) The coupled nucleic acid molecules of the present invention activate PARP but not DNA-PK, and compared with Dbait molecules, the use alone can lead to an increase in cancer cells with micronuclei, cytoplasmic chromatin fragmentation (CCF) and cytotoxicity.

[0084] 2) Specific increases in micronuclei (MN) and cytoplasmic chromatin fragments (CCF) in cancer cells lead to early increases in STING pathway activation, as shown by the release of inflammatory cytokines (CXCL10 and CCL5) and increased expression of PD-L1 and NKG2 on cancer cells. These effects are specific to cancer cells. This cancer cell specificity avoids general and widespread inflammation and subsequent possible harmful side effects.

[0085] 3) Activation of the STING pathway through inhibition of DNA repair pathways and generation of micronuclei and CCFs represents a very attractive way to specifically activate the STING pathway in tumor cells, especially through activation of innate immunity.

[0086] Based on these observations, the present invention relates to:

[0087] - a coupled nucleic acid molecule as described below;

[0088] - A pharmaceutical composition comprising a coupled nucleic acid molecule as described below and a pharmaceutically acceptable carrier, in particular for the treatment of cancer;

[0089] - a conjugated nucleic acid molecule as described below for use as a medicament, in particular for the treatment of cancer;

[0090] - a use of a coupled nucleic acid molecule as described below for the manufacture of a medicament, in particular for the treatment of cancer;

[0091] - A method of treating cancer in a patient in need thereof, the method comprising administering an effective amount of a coupled nucleic acid molecule disclosed herein;

[0092] - A pharmaceutical composition comprising a coupled nucleic acid molecule as described below, an additional therapeutic agent and a pharmaceutically acceptable carrier, in particular for the treatment of cancer;

[0093] - a product or kit containing (a) a coupled nucleic acid molecule as disclosed below and optionally (b) an additional therapeutic agent as a combined preparation for simultaneous, separate or sequential use, in particular for the treatment of cancer;

[0094] - A combined preparation comprising (a) a hairpin nucleic acid molecule as disclosed below, (b) an additional therapeutic agent as described below, for simultaneous, separate or sequential use, in particular for the treatment of cancer;

[0095] - A pharmaceutical composition comprising a coupled nucleic acid molecule as disclosed below, said pharmaceutical composition being used for treating cancer in combination with an additional therapeutic agent;

[0096] - Use of a pharmaceutical composition comprising a coupled nucleic acid molecule as disclosed below for the manufacture of a medicament for the treatment of cancer in combination with an additional therapeutic agent;

[0097] - A method of treating cancer in a patient in need thereof, said method comprising administering an effective amount of a) a coupled nucleic acid molecule as disclosed below and b) an effective amount of an additional therapeutic agent;

[0098] - A method of treating cancer in a patient in need thereof, the method comprising administering an effective amount of a pharmaceutical composition comprising a coupled nucleic acid molecule disclosed herein and an effective amount of an additional therapeutic agent;

[0099] - A method for improving the efficiency of treating cancer with a therapeutic anti-tumor agent or enhancing the sensitivity of a tumor to treatment with a therapeutic anti-tumor agent in a patient in need thereof, said method comprising administering an effective amount of a coupled nucleic acid molecule as disclosed below;

[0100] - A method for treating cancer, said method comprising repeated or chronic administration of a coupled nucleic acid molecule disclosed herein by repeated treatment cycles, preferably at least two administration cycles, even more preferably at least three or four administration cycles;

[0101] - A method of treating cancer in a patient having tumor cells with a defect in NAD+ synthesis and optionally with a defect in a DNA repair pathway selected from ERCC1 or ATM deficiency or an IDH mutation.

[0102] definition

[0103] Whenever "treating cancer" or the like is mentioned throughout this specification with respect to the pharmaceutical compositions, kits, products and combination preparations of the present invention, it means: a) a method of treating cancer comprising administering the pharmaceutical compositions, kits, products and combination preparations of the present invention to a patient in need of such treatment; b) the pharmaceutical compositions, kits, products and combination preparations of the present invention for treating cancer; c) the use of the pharmaceutical compositions, kits, products and combination preparations of the present invention in the preparation of a medicament for treating cancer; and / or d) the pharmaceutical compositions, kits, products and combination preparations of the present invention for treating cancer.

[0104] In the context of the present invention, the term "treatment" means curative, symptomatic and preventive treatment. The pharmaceutical composition, kit, product and combination preparation of the present invention can be used for humans who already have cancer or tumors, including those in the early or late stages of cancer progression. The pharmaceutical composition, kit, product and combination preparation of the present invention do not necessarily cure the patient who already has cancer, but will delay or slow the progression of the disease or prevent its further progression, thereby improving the patient's condition. Specifically, the pharmaceutical composition, kit, product and combination preparation of the present invention reduce the occurrence of tumors in mammalian hosts, reduce tumor burden, produce tumor regression, and / or prevent the occurrence of metastasis and cancer recurrence. In cancer treatment, the pharmaceutical composition, kit, product and combination preparation of the present invention are administered in a therapeutically effective amount.

[0105] As used herein, the terms "kit", "product" or "combination preparation" particularly define a "set of preparations" in the sense that the combination partners (a) and (b) as defined above can be administered independently or using different fixed combinations with different amounts of the combination partners (a) and (b), i.e., administered simultaneously or at different time points. The components of the set of preparations can then be administered simultaneously or staggered in time, i.e., at different time points and with equal or different time intervals for any part of the set of preparations. The ratio of the total amount of the combination partner (a) to be administered to the combination partner (b) in the combination preparation can vary. The combination partners (a) and (b) can be administered by the same route or by different routes.

[0106] "Effective amount" means the amount of the pharmaceutical composition, kit, product and combination preparation of the present invention, alone or in combination with other active ingredients of the pharmaceutical composition, kit, product or combination preparation, to prevent, eliminate or alleviate the harmful effects of cancer in mammals including humans. It should be understood that the dosage of administration can be changed by those skilled in the art according to the patient, condition, mode of administration, etc.

[0107] The term "STING" refers to the receptor for stimulator of interferon genes, also known as TMEM173, ERIS, MITA, MPYS, SAVI or NET23. When used herein, the terms "STING" and "STING receptor" are used interchangeably and include different isoforms and variants of STING. The mRNA and protein sequences of the longest isoform, human STING isoform 1, have NCBI reference sequences [NM_198282.3] and [NP_938023.1]. The mRNA and protein sequences of the shorter isoform, human STING isoform 2, have NCBI reference sequences [NM_001301738.1] and [NP_001288667.1].

[0108] As used herein, the term "STING activator" refers to a molecule that can activate the STING pathway. Activation of the STING pathway may include, for example, stimulation of inflammatory cytokines, including interferons, such as type 1 interferons including IFN-α, IFN-β, type 3 interferons such as IFN-λ, IP-10 (interferon-γ-induced protein, also known as CXCL10), PD-L1, TNF, IL-6, CXCL9, CCL4, CXCL11, NKG2D ligands (MICA / B), CCL5, CCL3, or CCL8. Activation of the STING pathway may also include stimulation of TANK binding kinase (TBK) 1 phosphorylation, interferon regulatory factor (IRF) activation (e.g., IRF3 activation), secretion of IP-10 or other inflammatory proteins and cytokines. Activation of the STING pathway can be determined, for example, by the ability of a compound to stimulate activation of the STING pathway, as detected using an interferon stimulation assay, a reporter gene assay (e.g., an hSTING wt assay or a THP1-Dual assay), a TBK1 activation assay, an IP-10 assay, or other assays known to those skilled in the art. Activation of the STING pathway can also be determined by the ability of a compound to increase the transcription level of a gene encoding a protein activated by STING or the STING pathway. Such activation can be detected, for example, using an RNAseq assay.

[0109] Activation of the STING pathway can be determined by one or more "STING assays" selected from the following: interferon stimulation assay, hSTING wt assay, THP1-Dual assay, TANK binding kinase 1 (TBK1) assay, interferon-γ-induced protein 10 (IP-10) secretion assay or PD-L1 assay.

[0110] More specifically, a molecule is a STING activator if it can stimulate the production of one or more STING-dependent cytokines in cells expressing STING by at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times or more compared to untreated STING-expressing cells. Preferably, the STING-dependent cytokine is selected from interferon, type 1 interferon, IFN-α, IFN-β, type 3 interferon, IFN-λ, CXCL10 (IP-10), PD-L1 TNF, IL-6, CXCL9, CCL4, CXCL11, NKG2D ligand (MICA / B), CCL5, CCL3 or CCL8, more preferably CCL5 or CXCL10.

[0111] Coupled nucleic acid molecules

[0112] Another advantage of certain coupled nucleic acid molecules according to the present invention is based on the fact that they can be synthesized as one molecule by using only oligonucleotide solid phase synthesis, thus allowing low cost and high manufacturing scale.

[0113] The coupled nucleic acid molecule of the present invention comprises: a double-stranded nucleic acid component, wherein the 5' end of the first strand and the 3' end of the complementary strand are connected together through a loop; and an optional molecule that promotes endocytosis connected to the loop. The other end of the double-stranded nucleic acid component is free.

[0114] The coupled nucleic acid molecules according to the invention can be defined by various features necessary for their therapeutic activity, such as their minimum and maximum length, the presence of at least one free end and the presence of a double-stranded portion, preferably a double-stranded DNA portion.

[0115] The coupled nucleic acid molecule is capable of activating PARP-1 protein. On the other hand, the coupled nucleic acid molecule does not activate DNA-PK.

[0116] The present invention also relates to pharmaceutically acceptable salts of the conjugated nucleic acid molecules of the present invention.

[0117] Nucleic acid molecules

[0118] The length of the coupling nucleic acid molecule can vary, as long as it is sufficient to allow proper binding or activation of the PARP (PARP-1) protein and insufficient to allow proper binding of the Ku protein complex comprising the Ku and DNA-PKc proteins. As has been shown, in order to ensure binding to this Ku complex and to allow DNA-PKc activation, the length of the coupling nucleic acid molecule must be greater than 20 bp, preferably about 32 bp, and thus the length is at most 20 bp. Furthermore, it has been shown that in order to allow proper binding and activation of PARP, the length of the coupling nucleic acid molecule must be greater than 8 bp.

[0119] The length of the double-stranded nucleic acid component is 10 to 20 base pairs. The length of up to 20bp prevents the molecule from activating DNA-PK. In specific cases, the length of the double-stranded nucleic acid component is 11 to 19 base pairs. For example, the length can be 11 to 19bp, 12 to 19bp, 13 to 19bp, 14 to 19bp, 15 to 19bp, 16 to 19bp, 12 to 16bp, 12 to 17bp, 12 to 18bp, 13 to 16bp, 13 to 17bp, 13 to 18bp, 14 to 16bp, 14 to 17bp, 14 to 18bp, 15 to 16bp, 15 to 17bp or 15 to 18bp. In very specific cases, the length of the double-stranded nucleic acid component is 16bp. "bp" means that the molecule comprises a double-stranded portion of the indicated length.

[0120] The effect of the nucleic acid molecule is independent of its sequence. Therefore, the nucleic acid molecule can be defined as comprising the following formula

[0121] 5'NNNN(N) a N-∫

[0122] 3'NNNN(N) a N-∫

[0123] Wherein N is a nucleotide, "a" is an integer from 5 to 15, and the two chains are complementary to each other. "-∫" indicates that the nucleotide is connected to a ring. In a specific case, "a" is an integer from 6 to 14. In another specific case, "a" can be an integer from 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 11 to 14, 6 to 13, 7 to 13, 8 to 13, 9 to 13, 10 to 13, 11 to 13, 6 to 12, 7 to 12, 8 to 12, 9 to 12 or 10 to 12.

[0124] Preferably, the sequence of the nucleic acid molecule is of non-human origin (i.e., their nucleotide sequence and / or conformation are not present in human cells as is). The coupled nucleic acid molecule preferably has no significant degree of sequence homology or identity with known genes, promoters, enhancers, 5'- or 3'-upstream sequences, exons, introns, etc. In other words, the coupled nucleic acid molecule has a sequence identity lower than 80% or 70%, or even lower than 60% or 50% with any gene in the human genome. Methods for determining sequence identity are well known in the art, and include, for example, BLASTN 2.2.25. For example, percent identity can be determined using the 37th edition of the human genome (Human Genome Build 37) (see GRCh37.p2 and alternative assemblies). The coupled nucleic acid molecule does not hybridize with human genomic DNA under stringent conditions. Typical stringent conditions are conditions that allow fully complementary nucleic acids to be distinguished from partially complementary nucleic acids.

[0125] Furthermore, the sequence of the coupled nucleic acid molecule preferably does not contain 5'-CpG-3' to avoid the well-known toll-like receptor (TLR)-mediated immune response.

[0126] The coupled nucleic acid molecule must have a free end as a mimic of double-strand breaks. The free end can be a free blunt end or a 5'- / 3'-overhanging end. "Free end" in this article refers to a nucleic acid molecule having both a 5' end and a 3' end, particularly a double-stranded nucleic acid component.

[0127] For example, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises the following sequence (SEQ ID NO: 1) or consists of the sequence:

[0128] 5'CCCAGCAAACAAGCCT-∫

[0129] 3'GGGTCGTTTGTTCGGA-∫

[0130] In a specific embodiment, the coupled nucleic acid molecule has a double-stranded component, and the double-stranded component comprises the same nucleotide sequence as SEQ ID NO: 1. Optionally, the coupled nucleic acid molecule has the same nucleotide composition as SEQ ID NO: 1, but the nucleotide sequence is different. Therefore, the coupled nucleic acid molecule comprises a double-stranded component whose one strand has 6 A, 7 C, 2 G and 1 T. Preferably, the sequence of the coupled nucleic acid molecule does not contain any 5'-CpG-3' dinucleotide. Alternatively, the double-stranded component comprises at least 9, 10, 11, 12, 13, 14, 15 or 16 consecutive nucleotides of SEQ ID NO: 1. In a more specific embodiment, the double-stranded component consists of 9, 10, 11, 12, 13, 14, 15 or 16 consecutive nucleotides of SEQ ID NO: 1.

[0131] In another specific aspect, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises the following sequence (SEQ ID NO: 2) or consists of the sequence:

[0132] 5'CAGCAACAAG-∫

[0133] 3'GTCGTTGTTC-∫

[0134] In a specific embodiment, the coupled nucleic acid molecule has a double-stranded component, and the double-stranded component comprises the same nucleotide sequence as SEQ ID NO: 2. Optionally, the coupled nucleic acid molecule has the same nucleotide composition as SEQ ID NO: 2, but the nucleotide sequence is different. Therefore, the coupled nucleic acid molecule comprises a double-stranded component, one strand of which has 5 A, 3 C and 2 G, and preferably, the sequence of the coupled nucleic acid molecule does not contain any 5'-CpG-3' dinucleotide.

[0135] The double-stranded nucleic acid component may include nucleotides with a modified phosphodiester backbone, particularly to protect them from degradation. Preferably, the nucleotides with a modified phosphodiester backbone are located at the free ends of the double-stranded components of the nucleic acid molecule. In one case, 1, 2 or 3 internucleotide bonds of the nucleotides at the free ends of the double-stranded components of the nucleic acid molecule have a modified phosphodiester backbone, preferably on both chains. Alternatively, the preferred coupled nucleic acid molecule has a 3'-3' nucleotide bond at the end of the chain.

[0136] In a specific embodiment, the nucleic acid molecule can be defined as comprising the formula

[0137] NNNN (N) a N-∫

[0138] NNNN (N) a N-∫

[0139] The nucleotides are underlined N The internucleotide bonds have a modified phosphodiester backbone.

[0140] For example, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises the following sequence (SEQ ID NO: 1) or consists of the sequence:

[0141] 5' CCCA GCAAACAAGCCT-∫

[0142] 3' GGGT CGTTTGTTCGGA-∫

[0143] The nucleotides are underlined N The internucleotide bonds have a modified phosphodiester backbone.

[0144] In another specific aspect, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises the following sequence (SEQ ID NO: 2) or consists of the sequence:

[0145] 5' CAGC AACAAG-∫

[0146] 3' GTCG TTGTTC-∫

[0147] The nucleotides are underlined N The internucleotide bonds have a modified phosphodiester backbone.

[0148] The modified phosphodiester backbone may be a phosphorothioate backbone.

[0149] When the modified phosphodiester bond is a phosphorothioate bond, the molecule may be the following:

[0150] 5'CsCsCsAGCAAACAAGCCT-∫

[0151] 3'GsGsGsTCGTTTGTTCGGA-∫

[0152] or

[0153] 5'CsAsGsCAACAAG-∫

[0154] 3'GsTsCsGTTGTTC-∫

[0155] In alternative cases, the double-stranded nucleic acid component may comprise a modified phosphodiester bond, such as a phosphorothioate bond, on the two last nucleotides at the 3' end of the molecule, on the two last nucleotides at the 5' end of the molecule, or on the two last nucleotides at both the 3' end and the 5' end of the molecule.

[0156] For example, the double-stranded nucleic acid component comprises or consists of components selected from the group consisting of:

[0157] 5'CCCAGCAAACAAGCCT-∫

[0158] 3' GG GTCGTTTGTTCGGA-∫

[0159] 5'CAGCAACAAG-∫

[0160] 3' GT CGTTGTTC-∫

[0161] 5' CC CAGCAAACAAGCCT-∫

[0162] 3' GG GTCGTTTGTTCGGA-∫

[0163] and

[0164] 5' CA GCAACAAG-∫

[0165] 3' GT CGTTGTTC-∫.

[0166] When the modified phosphodiester bond is a phosphorothioate bond, the molecule may be the following:

[0167] 5'CCCAGCAAACAAGCCT-∫

[0168] 3'GsGGTCGTTTGTTCGGA-∫

[0169] 5'CAGCAACAAG-∫

[0170] 3'GsTCGTTGTTC-∫

[0171] 5'CsCCAGCAAACAAGCCT-∫

[0172] 3'GsGGTCGTTTGTTCGGA-∫

[0173] and

[0174] 5'CsAGCAACAAG-∫

[0175] 3′GsTCGTTGTTC-∫.

[0176] In another alternative, the double-stranded nucleic acid component may comprise three modified phosphodiester bonds, such as phosphorothioate bonds, on the last three nucleotides at the 3' end of the molecule or on the last four nucleotides at the 5' end of the molecule.

[0177] For example, the double-stranded nucleic acid component comprises or consists of components selected from the group consisting of:

[0178] 5' CCCA GCAAACAAGCCT-∫

[0179] 3'GGGTCGTTTGTTCGGA-∫

[0180] 5' CAGC AACAAG-∫

[0181] 3'GTCGTTGTTC-∫

[0182] 5'CCCAGCAAACAAGCCT-∫

[0183] 3' GGGT CGTTTGTTCGGA-∫

[0184] and

[0185] 5'CAGCAACAAG-∫

[0186] 3' GTCG TTGTTC-∫.

[0187] When the modified phosphodiester bond is a phosphorothioate bond, the molecule may be the following:

[0188] 5'CsCsCsAGCAAACAAGCCT-∫

[0189] 3'GGGTCGTTTGTTCGGA-∫

[0190] 5'CsAsGsCAACAAG-∫

[0191] 3'GTCGTTGTTC-∫

[0192] 5'CCCAGCAAACAAGCCT-∫

[0193] 3'GsGsGsTCGTTTGTTCGGA-∫

[0194] and

[0195] 5'CAGCAACAAG-∫

[0196] 3'GsTsCsGTTGTTC-∫.

[0197] The double-stranded nucleic acid component basically comprises deoxyribonucleotides. However, it may also include some ribonucleotides or modified deoxyribonucleotides or ribonucleotides. In one case, the double-stranded nucleic acid component comprises only deoxyribonucleotides. In another case, the double-stranded nucleic acid component comprises deoxyribonucleotides and up to 30, 20, 15 or 10% of ribonucleotides or modified deoxyribonucleotides relative to the total number of nucleotides of the nucleic acid molecule. In a specific case, the double-stranded nucleic acid component comprises a first chain containing only deoxyribonucleotides and a complementary chain with ribonucleotides or modified deoxyribonucleotides. According to one embodiment, the coupled nucleic acid molecule comprises a modification corresponding to position 2 of ribose. For example, the coupled nucleic acid molecule may comprise at least one 2'-modified nucleotide, for example with a 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA) modification or for example a 2'-deoxy-2'-fluoroarabinonucleotide (FANA). However, such a 2'-modified nucleotide is preferably not located at the 5' or 3' end of the chain.

[0198] In certain cases, the coupled nucleic acid molecule has at least 1, 2, 3 or more 2'-deoxy-2'-fluoroarabinonucleotides (FANA). FANA adopts a DNA-like structure, resulting in the recognition of the coupled nucleic acid molecule by the protein of interest being unchanged. FANA includes the following pyrimidine 2'-fluoroarabinonucleotides and purine 2'-fluoroarabinonucleotides:

[0199] 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine (2'-FANA-A);

[0200] 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)guanine (2'-FANA-G);

[0201] 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)cytosine (2'-FANA-C);

[0202] 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil (2'-FANA-U).

[0203] For example, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises the following sequence (SEQ ID NO: 1) or consists of the sequence:

[0204] 5'CCCAGCAAACAAGCCT-∫

[0205]

[0206] wherein U is 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)uracil (2'-F-ANA-U) or 2'-deoxy-2'-fluoroarabinouridine. Specifically, the double-stranded nucleic acid component comprises the following sequence (SEQ ID NO: 1) or consists of the sequence:

[0207] 5' CCCA GCAAACAAGCCT-∫

[0208]

[0209] More specifically

[0210] 5'CsCsCsAGCAAACAAGCCT-∫

[0211]

[0212] In another example, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises or consists of the following sequence (SEQ ID NO: 1):

[0213] 5'CCCAGCAAACAAGCCT-∫

[0214]

[0215] wherein G is 2'-deoxy-2'-fluoroarabinoguanosine. Specifically, the double-stranded nucleic acid component comprises the following sequence (SEQ ID NO: 1) or consists of the sequence:

[0216] 5' CCCA GCAAACAAGCCT-∫

[0217]

[0218] More specifically

[0219] 5'CsCsCsAGCAAACAAGCCT-∫

[0220]

[0221] In another example, the double-stranded nucleic acid component or nucleic acid of the molecule according to the present invention comprises or consists of the following sequence (SEQ ID NO: 1):

[0222]

[0223] 3'GGGTCGTTTGTTCGGA-∫

[0224] wherein C is 2'-deoxy-2'-fluoroarabinocytidine. Specifically, the double-stranded nucleic acid component comprises the following sequence (SEQ ID NO: 1) or consists of the sequence:

[0225]

[0226] 3' GGGT CGTTTGTTCGGA-∫

[0227] More specifically

[0228]

[0229] 3'GsGsGsTCGTTTGTTCGGA-∫

[0230] ring

[0231] The loop is linked to the 5' end of the first strand of the double-stranded component and to the 3' end of the complementary strand, and optionally to a molecule that promotes endocytosis.

[0232] The ring preferably comprises a chain of 10 to 100 atoms, preferably 15 to 25 atoms.

[0233] The ring may comprise 2 to 10 nucleotides, preferably 3, 4 or 5. Non-nucleotide rings include, non-exhaustively, abasic nucleotides, polyethers, polyamines, polyamides, peptides, sugars, lipids, polyhydrocarbons or other polymeric compounds (e.g., oligoethylene glycols, e.g., oligoethylene glycols having between 2 and 10 ethylene glycol units, preferably 4, 5, 6, 7 or 8 ethylene glycol units). In one embodiment, the ring can be selected from N-(5-hydroxymethyl-6-phosphohexyl)-11-(3-(6-phosphohexylthio)succinimidyl))undecylamide, 1,3-bis-[5-hydroxypentylamido]propyl-2-(6-phosphohexyl), hexaethylene glycol, tetradeoxythymidylic acid (T4), 1,19-bis(phospho)-8-hydrazino-2-hydroxy-4-oxa-9-keto-nonadecane and 2,19-bis(phospho)-8-hydrazino-1-hydroxy-4-oxa-9-keto-nonadecane.

[0234] The molecule that promotes endocytosis is optionally coupled to the ring via a connector. Any connector known in the art can be used to covalently attach the molecule that promotes endocytosis to the ring. For example, WO09 / 126933 provides a broad overview of convenient connectors on pages 38-45. Non-exhaustive, the connector can be an aliphatic chain, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon or other polymeric compound (e.g., oligoethylene glycol, such as oligoethylene glycol with 2 to 10 ethylene glycol units, preferably 3, 4, 5, 6, 7 or 8 ethylene glycol units, more preferably 6 ethylene glycol units), and any bond that can be broken by chemical or enzymatic means is incorporated, such as a disulfide bond, a protected disulfide bond, an acid-labile bond (e.g., a hydrazone bond), an ester bond, an orthoester bond, a phosphoramide bond, a bio-cleavable peptide bond, an azo bond or an aldehyde bond. Such cleavable linkers are described in detail on pages 12-14 of WO2007 / 040469, pages 22-28 of WO2008 / 022309.

[0235] The molecule promoting endocytosis is linked to the ring by any means known to those skilled in the art, optionally via an oligoethylene glycol spacer.

[0236] In certain embodiments, the linker between the molecule that promotes endocytosis and the ring comprises C(O)-NH-(CH2-CH2-O) n or NH-C(O)-(CH2-CH2-O) n , wherein n is an integer from 1 to 10, preferably n is selected from 3, 4, 5 and 6. In a very specific embodiment, the linker is CO-NH-(CH2-CH2-O)4 (formamidotriethylene glycol).

[0237] In another specific embodiment, the linker between the endocytosis-promoting molecule and the ring molecule is a dialkyl-disulfide {e.g. (CH2) p -SS-(CH2) q , wherein p and q are integers from 1 to 10, preferably from 3 to 8, for example 6}.

[0238] In another specific embodiment, the loop has been developed so as to be compatible with oligonucleotide solid phase synthesis. Thus, the loop can be incorporated during the synthesis of the nucleic acid molecule, thereby facilitating synthesis and reducing its cost.

[0239] The ring may have a structure selected from one of the following structural formulas:

[0240] -OP(X)OH-O-{[(CH2)2-O] g -P(X)OH-O} r -KOP(X)OH-O-{[(CH2)2-O]h -P(X)OH-O-} s (I)

[0241] wherein r and s are independently integers 0 or 1; g and h are independently integers from 1 to 7, and the sum of g+h is from 4 to 7;

[0242] where K is

[0243]

[0244] wherein i, j, k and l are independently integers from 0 to 6, preferably from 1 to 3;

[0245] or

[0246] -OP(X)OH-O-[(CH2) d -C(O)-NH] b -CHR-[C(O)-NH-(CH2) e ] c -OP(X)OH-O-(II)

[0247] wherein b and c are independently integers from 0 to 4, and the sum of b+c is from 3 to 7;

[0248] d and e are independently integers from 1 to 3, preferably from 1 to 2;

[0249] Where R is -L f -J,

[0250] wherein X is O or S, L is a linker, preferably a linear alkylene and / or oligoethylene glycol optionally interrupted by one or more groups selected from amino, amide and keto groups, f is an integer of 0 or 1, and J is a molecule promoting endocytosis or H.

[0251] When J is H, the molecule can be used as a synthon to prepare the molecule coupled to the molecule that promotes endocytosis. Alternatively, the molecule can also be used as a drug without being coupled to any molecule that promotes endocytosis.

[0252] In certain embodiments, the molecule may be

[0253]

[0254] In the first case, the ring has a structure according to formula (I):

[0255] -OP(X)OH-O-{[(CH2)2-O] g -P(X)OH-O} r -KOP(X)OH-O-{[(CH2)2-O] h-P(X)OH-O-} s (I)

[0256] X is O or S. In each occurrence of -OP(X)OH-O- in formula (I), X may vary between O and S. Preferably, X is S.

[0257] The sum of g+h is preferably from 5 to 7, in particular 6. Thus, if r is 0, h may be from 5 to 7 (wherein s is 1); if g is 1, h may be from 4 to 6 (wherein r and s are 1); if g is 2, h may be from 3 to 5 (wherein r and s are 1); if g is 3, h may be from 2 to 4 (wherein r and s are 1); if g is 4, h may be from 1 to 3 (wherein r and s are 1); if g is 5, h may be from 1 to 2 (wherein r is 1 and s is 0 or 1); or if g is 6 or 7, s is 0 (wherein r is 1).

[0258] Preferably, i and j may be the same integer or may be different. i and j may be selected from integers 1, 2, 3, 4, 5 or 6, preferably 1, 2 or 3, more particularly 1 or 2, particularly 1.

[0259] Preferably, k and l are the same integer. In one embodiment, k and l are integers selected from 1, 2 or 3, preferably 1 or 2, more preferably 2.

[0260] Therefore, K can be

[0261]

[0262] In a preferred embodiment, K is

[0263]

[0264] In a particular instance, the ring has the structural formula (I)

[0265] -OP(X)OH-O-{[(CH2)2-O] g -P(X)OH-O} r -KOP(X)OH-O-{[(CH2)2-O] h -P(X)OH-O-} s (I)

[0266] Where X is S, r is 1, g is 6, s is 0, and K is

[0267]

[0268] In a specific case, f is 1, and LJ is -C(O)-(CH2) m -NH-[C(O)] t-[(CH2)2-O] n -(CH2) p -[C(O)] v -J or -C(O)-(CH2) m -NH-[C(O)-CH2-O] t -[(CH2)2-O] n -(CH2) p -[C(O)] v -J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; p is an integer from 0 to 4; t and v are integers 0 or 1, wherein at least one of t and v is 1.

[0269] More specifically, f is 1, and LJ is selected from -C(O)-(CH2) m -NH-[(CH2)2-O] n -(CH2) p -C(O)-J, -C(O)-(CH2) m -NH-C(O)-[(CH2)2-O] n -(CH2) p -J, C(O)-(CH2) m -NH-C(O)-CH2-O-[(CH2)2-O] n -(CH2) p -J, -C(O)-(CH2) m -NH-C(O)-[(CH2)2-O] n -(CH2) p -C(O)-J and -C(O)-(CH2) m -NH-C(O)-CH2-O-[(CH2)2-O] n -(CH2) p -C(O)-J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3.

[0270] Optionally, f is 1, and LJ is selected from -C(O)-(CH2)5-NH-[(CH2)2-O] 3-13 -CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-[(CH2)2-O] 3-13 -CH2-J、C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O] 3-13 -CH2-J, -C(O)-(CH2)5-NH-C(O)-[(CH2)2-O] 3-13-CH2-C(O)-J and -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O] 3-13 -CH2-C(O)-J or -C(O)-(CH2)5-NH-C(O)-J.

[0271] For example, f can be 1, and LJ is selected from -C(O)-(CH2)5-NH-[(CH2)2-O]3-(CH2)2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-[(CH2)2-O]3-(CH2)3-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]5-CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O]9-CH2-C(O)-J, -C(O)-(CH2)5-NH-C(O)-CH2-O-[(CH2)2-O] 13 -CH2-C(O)-J or -C(O)-(CH2)5-NH-C(O)-J.

[0272] In very specific cases, f is 1 and LJ is -C(O)-(CH2) m -NH-[(CH2)2-O] n -(CH2) p -C(O)-J, wherein m is an integer from 0 to 10, preferably from 4 to 6, in particular from 5; n is an integer from 0 to 6; and p is an integer from 0 to 2. In a specific case, m is 5 and n and p are 0. In another specific case, m is 5, n is 3 and p is 2.

[0273] In the second aspect of the present disclosure, the ring has a structure according to formula (II):

[0274] -OP(X)OH-O-[(CH2) d -C(O)-NH] b -CHR-[C(O)-NH-(CH2) e ] c -OP(X)OH-O-(II)

[0275] Where X is O or S;

[0276] b and c are independently integers from 0 to 4, and the sum of b+c is from 3 to 7;

[0277] d and e are independently integers from 1 to 3, preferably from 1 to 2;

[0278] Where R is –(CH2) 1-5-C(O)-NH-L f -J or –(CH2) 1-5 -NH-C(O)-L f -J, and

[0279] wherein L is a linker, preferably a linear alkylene group or an oligoethylene glycol, f is an integer of 0 or 1, and J is a molecule that promotes endocytosis.

[0280] If b and / or c is 2 or more, then at each occurrence of [(CH2) d -C(O)-NH] or -[C(O)-NH-(CH2) e ] when d and e can be different.

[0281] In one embodiment, when d and e are 2, the sum of b+c is 3 to 5, particularly 4. For example, b may be 0 and c is 3 to 5; b may be 1 and c is 2 to 4; b may be 2 and c is 1 to 3; or b may be 3 to 5 and c is 0.

[0282] In one embodiment, when d and e are 1, the sum of b+c is 4 to 7, in particular 5 or 6. For example, b may be 0 and c is 3 to 6; b may be 1 and c is 2 to 5; b may be 2 and c is 1 to 4; or b may be 3 to 6 and c is 0.

[0283] In one case, b, c, d and e are selected such that the ring comprises a chain of 10 to 100 atoms, preferably 15 to 25 atoms.

[0284] In a non-exhaustive list of examples, the ring may be one of the following structures:

[0285] -OP(X)OH-O-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-CHR-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-OP(X)OH-O-

[0286] -OP(X)OH-O-(CH2)2-C(O)-NH-CHR-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-OP(X)OH-O-

[0287] -OP(X)OH-O-CHR-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-OP(X)OH-O-

[0288] -OP(X)OH-O-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-CHR-C(O)-NH-(CH2)2-OP(X)OH-O-

[0289] -OP(X)OH-O-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-CHR-OP(X)OH-O-

[0290] -OP(X)OH-O-(CH2)2-C(O)-NH-(CH2)-C(O)-NH-CHR-C(O)-NH-(CH2)-C(O)-NH-(CH2)2-OP(X)OH-O-

[0291] -OP(X)OH-O-(CH2)-C(O)-NH-(CH2)2-C(O)-NH-CHR-C(O)-NH-(CH2)2-C(O)-NH-(CH2)-OP(X)OH-O- or

[0292] -OP(X)OH-O-(CH2)-C(O)-NH-(CH2)-C(O)-NH-CHR-C(O)-NH-(CH2)-C(O)-NH-(CH2)-OP(X)OH-O-

[0293] In certain cases, the ring may be the following structure:

[0294] -OP(X)OH-O-(CH2)2-C(O)-NH-(CH2)2-C(O)-NH-CHR-C(O)-NH-(CH2)2-C(O)-NH-(CH2)2-OP(X)OH-O-

[0295] Where R is -L f -J; and

[0296] wherein L is a linker, preferably a linear alkylene group and / or an oligoethylene glycol optionally interrupted by one or more groups selected from amino, amide and keto groups, and f is an integer of 0 or 1.

[0297] Preferably, X is S.

[0298] L can be –(CH2) 1-5 -C(O)-J, preferably -CH2-C(O)-J or -(CH2)2-C(O)-J.

[0299] Alternatively, LJ can be –(CH2)4-NH-[(CH2)2-O] n -(CH2) p -C(O)-J, wherein n is an integer from 0 to 6; and p is an integer from 0 to 2. In a specific case, n is 3 and p is 2.

[0300] Molecules that promote endocytosis

[0301] The nucleic acid molecules of the present invention are optionally coupled to a molecule that promotes endocytosis, which is referred to as J in the above formula. Thus, in the first case, J is a molecule that promotes endocytosis. In an alternative case, J is hydrogen.

[0302] The molecule that promotes endocytosis can be a lipophilic molecule such as cholesterol, a single-chain or double-chain fatty acid, or a ligand that targets a cell receptor capable of receptor-mediated endocytosis, such as folic acid and folic acid derivatives or transferrin (Goldstein et al., Ann. Rev. Cell Biol. 19851: 1-39; Leamon & Lowe, Proc Natl Acad Sci USA. 1991, 88: 5572-5576). The fatty acid can be saturated or unsaturated and can be C4-C 28 , preferably C 14 -C 22 More preferably C 18 Fatty acids, such as oleic acid or stearic acid. Specifically, fatty acids can have octadecyl or dioleoyl. Fatty acids can exist as double-stranded forms connected with suitable connectors such as glycerol, phosphatidylcholine or ethanolamine or connected together by connectors for attachment to the coupled nucleic acid molecules. When used in this article, the term "folic acid" means folic acid and folic acid derivatives, including pteroic acid derivatives and analogs. Analogs and derivatives suitable for folic acid of the present invention include but are not limited to folic acid antagonists, dihydrofolate, tetrahydrofolate, folinic acid, pteroyl polyglutamic acid, 1-deaza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10-dideaza, 8,10-dideaza and 5,8-dideaza folic acid, folic acid antagonists and pteroic acid derivatives. Other folic acid analogs are described in US2004 / 242582.

[0303] Therefore, the molecule that promotes endocytosis can be selected from single-chain or double-chain fatty acids, folic acid and cholesterol. More preferably, the molecule that promotes endocytosis is selected from dioleoyl, octadecyl, folic acid and cholesterol. In the most preferred embodiment, the molecule that promotes endocytosis is cholesterol.

[0304] Therefore, in a preferred embodiment, the conjugated nucleic acid molecule (also referred to as OX401) has the following structural formula:

[0305]

[0306] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0307] In another specific embodiment, the conjugate nucleic acid molecule (also referred to as OX402) has the following structural formula:

[0308]

[0309] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0310] In another preferred embodiment, the coupled nucleic acid molecule has the following structural formula:

[0311]

[0312] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0313] In other preferred embodiments, the coupled nucleic acid molecule has any one of the following structural formulas:

[0314] -OX-406:

[0315]

[0316] OX407:

[0317]

[0318] OX408:

[0319]

[0320] OX410:

[0321]

[0322] and

[0323] OX411:

[0324]

[0325] Wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond; the italicized U is 2'-deoxy-2'-fluoroarabino uridine, the italicized G is 2'-deoxy-2'-fluoroarabino guanosine; and the italicized C is 2'-deoxy-2'-fluoroarabino cytidine.

[0326] Alternatively, the molecule promoting endocytosis may also be tocopherol, sugars such as galactose and mannose and oligosaccharides thereof, peptides such as RGD and bombesin, and proteins such as integrins.

[0327] σ -2 receptor ligand

[0328] In certain cases, the molecule promoting endocytosis is selected so as to target cancer cells. Thus, it is selected to be a ligand for a receptor that is specifically expressed in cancer cells or that is overexpressed in cancer cells compared to normal cells.

[0329] In this case, the molecule that promotes endocytosis may be a ligand for the sigma-2 receptor (σ2R).

[0330] The term "σ-2 receptor (σ2R)" refers to a σ receptor subtype that has been found to be highly expressed in malignant cancer cells (e.g., breast cancer, ovarian cancer, lung cancer, brain cancer, bladder cancer, colon cancer, and melanoma). The σ-2 receptor is a cytochrome-related protein located in lipid rafts, most commonly associated with P450 proteins, and coupled to the PGRMC1 complex, EGFR, mTOR, caspases, and various ion channels.

[0331] The term "sigma-2 receptor (σ2R) ligand" refers to a synthetic or non-synthetic agonist compound that binds to σ2R with high selectivity and affinity and then is internalized by endocytosis. σ2R agonists inhibit tumor cell proliferation and induce apoptosis in cancer cells.

[0332] In a preferred embodiment, the σ-2 receptor (σ2R) ligand is an azabicyclononane analog, more particularly an N-substituted-9-azabicyclo[3.3.1]nonan-3α-ylcarbamate analog described in Vangveravong et al., Bioorg. Med. Chem (2006), comprising the following structural formula:

[0333]

[0334] in particular

[0335]

[0336] wherein n is an integer from 1 to 20. Optionally, n is an integer from 1 to 10, 2 to 9, 3 to 8, 4 to 7 or 5 to 6.

[0337] In a first specific case, the σ2R ligand has the following structural formula

[0338]

[0339] wherein n is an integer from 1 to 20. Optionally, n is an integer from 1 to 10, 2 to 9, 3 to 8, 4 to 7 or 5 to 6.

[0340] In a specific embodiment, the σ2R ligand is referred to as SV119 (n=6) and has the following structural formula:

[0341]

[0342] In yet another specific embodiment, the σ2R ligand is referred to as SW43 (n=10) and has the following structural formula:

[0343]

[0344] In another embodiment, the σ2R ligand is an N-substituted-9-azabicyclo[3.3.1]nonan-3α-ylcarbamate analog and has the following structural formula:

[0345]

[0346] wherein n is an integer from 1 to 20, and m is an integer from 0 to 10.

[0347] In certain embodiments, the σ2R ligand has the following structural formula:

[0348]

[0349] The σ2R ligand is coupled to the nucleic acid molecule via the ring, through the carboxyl or amino group, optionally via a linker.

[0350] Therefore, in a preferred embodiment, the coupled nucleic acid molecule has the following structural formula:

[0351]

[0352] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0353] Therefore, in another preferred embodiment, the coupled nucleic acid molecule has the following structural formula:

[0354]

[0355] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0356] Therefore, in another preferred embodiment, the conjugated nucleic acid molecule (also referred to as OX405) has the following structural formula:

[0357]

[0358] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0359] In another preferred embodiment, the conjugated nucleic acid molecule (also referred to as OX407) has the following structural formula:

[0360]

[0361] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0362] In other preferred embodiments, the coupled nucleic acid molecule has any one of the following structural formulas:

[0363]

[0364]

[0365] The above compound is also known as OX403

[0366]

[0367] and

[0368]

[0369] The above compound is also known as OX404

[0370] wherein the internucleotide bond "s" represents a phosphorothioate internucleotide bond.

[0371] Therapeutic uses of the nucleic acid molecules

[0372] The coupled nucleic acid molecules according to the invention are capable of activating PARP. They lead to an increase in micronuclei and cytotoxicity in cancer cells. They show specificity for cancer cells, which can exclude or limit side effects. In addition, the specific increase in micronuclei in cancer cells leads to an early activation of the STING pathway.

[0373] Therefore, the coupled nucleic acid molecules according to the present invention can be used as drugs, in particular for the treatment of cancer.

[0374] Therefore, the present invention relates to a coupled nucleic acid molecule according to the present invention for use as a medicament. It also relates to a pharmaceutical composition comprising a coupled nucleic acid molecule according to the present invention, in particular for use in the treatment of cancer.

[0375] The pharmaceutical compositions contemplated herein may also include a pharmaceutically acceptable carrier in addition to the active ingredient. The term "pharmaceutically acceptable carrier" is intended to encompass any carrier (e.g., support, substance, solvent, etc.) that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered. For example, for parenteral administration, the active compound can be formulated into a unit dosage form for injection in a medium such as saline, dextrose solution, serum albumin, and Ringer's solution.

[0376] The pharmaceutical composition can be formulated in a manner known in the art as a solution in a pharmaceutically compatible solvent or an emulsion, suspension or dispersion in a suitable pharmaceutical solvent or medium or a pill, tablet or capsule containing a solid medium. The dosage form of the present invention suitable for oral administration can take the form of discrete units as capsules, sachets, tablets or lozenges each containing a predetermined amount of active ingredients; in the form of powders or granules; in the form of solutions or suspensions in aqueous liquids or non-aqueous liquids; or in the form of oil-in-water emulsions or water-in-oil emulsions. Dosage forms suitable for parenteral administration conveniently contain a sterile oily or aqueous preparation of the active ingredient, which is preferably isotonic with the blood of the recipient. Each such dosage form may also contain other pharmaceutically compatible and non-toxic adjuvants, such as stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances. Therefore, the dosage form of the present invention comprises an active ingredient and optionally other therapeutic ingredients associated with a pharmaceutically acceptable carrier. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the dosage form and not harmful to its recipient. Advantageously, the pharmaceutical composition is administered by injection or intravenous infusion of a suitable sterile solution or as an oral dosage form through the digestive tract. Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature.

[0377] The pharmaceutical compositions and products, kits or combined preparations described in the present invention can be used to treat cancer in a subject.

[0378] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, solid tumors and hematological cancers, including carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, neuroblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumors, and head and neck cancer. Other cancer indications are disclosed herein.

[0379] In certain embodiments, "cancer" refers to a cancer with a defect in NAD, such as one selected from ERCC1 or ATM. + Exhausted tumor cells or cancer cells with IDH mutations.

[0380] In a very specific embodiment, for a patient having a NAD + Patients with tumors that are deficient in NAD synthesis, particularly those with + For patients with depleted tumors, clinical stratification or selection of better responders is possible.

[0381] The determination of the optimal dose generally involves a balance between the therapeutic benefit level of the treatment of the present invention and any risk or harmful side effects. The selected dose level depends on various factors, including but not limited to the activity of the coupled nucleic acid molecule, the route of administration, the time of administration, the excretion rate of the compound, the duration of treatment, other drugs, compounds and / or materials used in combination, and the age, sex, weight, condition, overall health and previous medical history of the patient. The amount of the coupled nucleic acid molecule and the route of administration are ultimately at the discretion of the physician, although the dosage should generally reach a local concentration at the site of action that achieves the desired effect.

[0382] The administration route of the conjugated nucleic acid molecules disclosed herein can be oral, parenteral, intravenous, intratumoral, subcutaneous, intracranial, intraarterial, topical, rectal, transdermal, intradermal, nasal, intramuscular, intraperitoneal, intraosseous, etc. In a preferred embodiment, the conjugated nucleic acid molecules are administered or injected near the tumor site to be treated.

[0383] For example, the effective amount of the coupled nucleic acid molecule is 0.01 to 1000 mg, for example, preferably 0.1 to 100 mg. Of course, the dosage and regimen can be changed by professionals according to chemotherapy and / or radiotherapy regimens.

[0384] The coupled nucleic acid molecules according to the present invention can be used in combination with other therapeutic agents. The other therapeutic agent can be, for example, an immunomodulator such as an immune checkpoint inhibitor, a cancer immunotherapy based on T- cells including adoptive cell transfer (ACT), genetically modified T- cells or engineered T- cells such as chimeric antigen receptor cells (CAR-T cells), conventional chemotherapy, radiotherapy or anti-angiogenic agents, HDAC inhibitors (such as Belisstat) or targeted immunotoxins.

[0385] Combination with immunomodulators / immune checkpoint inhibitors (ICIs)

[0386] As indicated by the activation of the STING pathway and the increase in PD-L1 expression, the inventors have confirmed the high anti-tumor therapeutic efficiency of the combination of coupled nucleic acid molecules and immunomodulators, such as immune checkpoint inhibitors (ICI), preferably inhibitors of the PD-1 / PD-L1 pathway. Therefore, the present invention provides a combination therapy in which the coupled nucleic acid molecules of the present invention are administered to the patient simultaneously, before or after an immunomodulator such as an immune checkpoint inhibitor (ICI).

[0387] Therefore, the present invention relates to a pharmaceutical composition comprising a coupled nucleic acid molecule of the present invention and an immunomodulator, more specifically for the treatment of cancer. The present invention also relates to a product comprising a coupled nucleic acid molecule of the present invention and an immunomodulator as a combined preparation for simultaneous, separate or sequential use, more specifically for the treatment of cancer. In a preferred embodiment, the immunomodulator is an inhibitor of the PD-1 / PD-L1 pathway.

[0388] The present invention also provides a method for treating cancer by administering to a patient in need thereof a coupled nucleic acid molecule of the present invention in combination with one or more immunomodulators (e.g., one or more activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules). In a preferred embodiment, the immunomodulator is an inhibitor of the PD-1 / PD-L1 pathway. Activators of co-stimulatory molecules:

[0389] In certain embodiments, the immunomodulator is an activator of a co-stimulatory molecule. In one embodiment, the agonist of the co-stimulatory molecule is selected from OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, an agonist of B7-H3 or CD83 ligand (e.g., agonist antibodies or their antigen binding fragments or soluble fusions).

[0390] Inhibitors of immune checkpoint molecules:

[0391] In certain embodiments, the immunomodulator is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulator is an inhibitor of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, NKG2D, NKG2L, KIR, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGFRβ. In one embodiment, the inhibitor of the immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3 or CTLA-4 or any combination thereof. The term "inhibition" or "inhibitor" includes a reduction in certain parameters of a given molecule such as an immune checkpoint inhibitor, such as activity. For example, the term includes at least 5%, 10%, 20%, 30%, 40%, 50% or more inhibition of activity such as PD-1 or PD-L1 activity. Therefore, inhibition does not have to be 100%.

[0392] The inhibition of inhibitory molecules can be carried out at the DNA, RNA or protein level. In certain embodiments, inhibitory nucleic acids (e.g., dsRNA, siRNA or shRNA) can be used to inhibit the expression of inhibitory molecules. In other embodiments, the inhibitor of the inhibitory signal is a polypeptide bound to the inhibitory molecule, such as a soluble ligand (e.g., PD-1Ig or CTLA-4Ig) or an antibody or its antigen-binding fragment; for example, an antibody or its fragment (also referred to herein as "antibody molecule") bound to PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, NKG2D, NKG2L, KIR VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and / or TGFRβ, or a combination thereof.

[0393] In one embodiment, the antibody molecule is a complete antibody or a fragment thereof (e.g., Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In other embodiments, the heavy chain constant region (Fc) of the antibody molecule is selected from, for example, the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly selected from, for example, the heavy chain constant region of IgG1, IgG2, IgG3, and IgG4, more particularly the heavy chain constant region of IgG1 or IgG4 (e.g., human IgG1 or IgG4). In one embodiment, the heavy chain constant region is the heavy chain constant region of human IgG1 or human IgG4. In one embodiment, the constant region is changed, for example, by mutation, to improve the performance of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). In certain embodiments, the antibody molecule takes the form of a bispecific or multispecific antibody molecule.

[0394] PD-1 inhibitors

[0395] In certain embodiments, the coupled nucleic acid molecules of the present invention are administered in combination with a PD-1 inhibitor. In certain embodiments, the PD-1 inhibitor is selected from PDR001 (Novartis), nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co), pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte) or AMP-224 (Amplimmune).

[0396] Exemplary PD-1 Inhibitors

[0397] In certain embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Alternative names of nivolumab include MDX-1106, MDX-1106-04, ONO-4538, BMS-936558 or Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in U.S. Patent No. 8,008,449 and PCT Publication No. WO2006 / 121168, which are incorporated herein by reference in their entireties.

[0398] In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (trade name KEYTRUDA, formerly Lambrolizumab, also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab is disclosed in, for example, Hamid, O. et al., (2013) New England Journal of Medicine 369 (2): 134-44, PCT Publication No. WO 2009 / 114335, and U.S. Pat. No. 8,354,509, which are incorporated herein by reference in their entirety.

[0399] In certain embodiments, the anti-PD-1 antibody is pidilizumab. pidilizumab (CT-011; CureTech) is a humanized IgG1 k monoclonal antibody that binds to PD1. pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in PCT Publication No. WO 2009 / 101611, which is incorporated herein by reference in its entirety.

[0400] Other anti-PD1 antibodies are disclosed in US Patent No. 8,609,089, US Publication No. 2010028330, and / or US Publication No. 20120114649, which are incorporated herein by reference in their entireties. Other anti-PD1 antibodies include AMP514 (Amplimmune).

[0401] In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO2012 / 145493, which are incorporated herein by reference in their entirety.

[0402] In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (Regeneron).

[0403] In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer).

[0404] In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 or BGB-108 (Beigene).

[0405] In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210.

[0406] In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011.

[0407] Other known anti-PD-1 antibodies include those described in, e.g., WO 2015 / 112800, WO 2016 / 092419, WO 2015 / 085847, WO 2014 / 179664, WO 2014 / 194302, WO 2014 / 209804, WO 2015 / 200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731, and US 9,102,727, which are incorporated herein by reference in their entirety.

[0408] In one embodiment, the anti-PD-1 antibody is an antibody that competes for binding and / or binds to the same epitope on PD-1 as one of the anti-PD-1 antibodies described herein.

[0409] In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, such as described in US 8,907,053, which is incorporated herein by reference in its entirety. In certain embodiments, the PD-1 inhibitor is an immunoadhesin {e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)}. In certain embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg (Amplimmune)), such as disclosed in WO 2010 / 027827 and WO 2011 / 066342, which are incorporated herein by reference in their entirety.

[0410] PD-L1 inhibitors

[0411] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-L1. In some embodiments, the coupled nucleic acid molecule of the present invention is administered in combination with a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from FAZ053 (Novartis), atezolizumab (Genentech / Roche), avelumab (Merck Serono and Pfizer), durvalumab (Medlmmune / AstraZeneca) or BMS-936559 (Bristol-MyersSquibb).

[0412] Exemplary PD-L1 Inhibitors

[0413] In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the anti-PD-L1 antibody molecule is Avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO 2013 / 079174, which is incorporated herein by reference in its entirety.

[0414] In one embodiment, the anti-PD-L1 antibody molecule is durvalumab (MedImmune / AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108, which is incorporated herein by reference in its entirety.

[0415] In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015 / 081158, which are incorporated herein by reference in their entirety.

[0416] Other known anti-PD-L1 antibodies include, for example, those described in WO 2015 / 181342, WO 2014 / 100079, WO 2016 / 000619, WO 2014 / 022758, WO 2014 / 055897, WO 2015 / 061668, WO 2013 / 079174, WO 2012 / 145493, WO 2015 / 112805, WO 2015 / 109124, WO 2015 / 195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082, which are incorporated herein by reference in their entireties.

[0417] In one embodiment, the anti-PD-L1 antibody is an antibody that competes for binding and / or binds to the same epitope on PD-L1 as one of the anti-PD-L1 antibodies described herein.

[0418] LAG-3 inhibitors

[0419] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of LAG-3. In some embodiments, the coupled nucleic acid molecule of the present invention is administered in combination with a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb) or TSR-033 (Tesaro).

[0420] Exemplary LAG-3 Inhibitors

[0421] In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986016 (Bristol-Myers Squibb), also known as BMS986016. BMS-986016 and other anti-LAG-3 antibodies are disclosed in WO 2015 / 116539 and US 9,505,839, which are incorporated herein by reference in their entirety.

[0422] In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tesaro).

[0423] In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and PrimaBioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO2008 / 132601 and US9,244,059, which are incorporated herein by reference in their entirety.

[0424] Other known anti-LAG-3 antibodies include, for example, those described in WO 2008 / 132601, WO 2010 / 019570, WO 2014 / 140180, WO 2015 / 116539, WO 2015 / 200119, WO 2016 / 028672, US 9,244,059, US 9,505,839, which are incorporated herein by reference in their entireties.

[0425] TIM-3 inhibitors

[0426] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of TIM-3. In some embodiments, the coupled nucleic acid molecule of the present invention is administered in combination with a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is MGB453 (Novartis) or TSR-022 (Tesaro).

[0427] Exemplary TIM-3 Inhibitors

[0428] In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio / Tesaro).

[0429] In one embodiment, the anti-TIM-3 antibody is APE5137 or APE5121. APE5137, APE512 and other anti-TIM-3 antibodies are disclosed in WO 2016 / 161270, which is incorporated herein by reference in its entirety.

[0430] Other known anti-TIM-3 antibodies include, for example, those described in WO 2016 / 111947, WO 2016 / 071448, WO 2016 / 144803, US 8,552,156, US 8,841,418, and US 9,163,087, which are incorporated herein by reference in their entireties.

[0431] NKG2D inhibitors

[0432] In some embodiments, the inhibitor of the NKG2D / NKG2DL pathway is an inhibitor of NKG2D. In some embodiments, the coupled nucleic acid molecules of the present invention are administered in combination with a NKG2D inhibitor. In some embodiments, the NKG2D inhibitor is an anti-NKG2D antibody molecule such as anti-NKG2D antibody NNC0142-0002 (also known as NN 8555, IPH2301 or JNJ-4500).

[0433] Exemplary NKG2D Inhibitors

[0434] In one embodiment, the anti-NKG2D antibody molecule is NNC0142-0002 (Novo Nordisk), as disclosed in WO 2009 / 077483 and US 7,879,985, which are incorporated herein by reference in their entireties.

[0435] In another embodiment, the anti-NKG2D antibody molecule is JNJ-64304500 (Janssen), as disclosed in WO 2018 / 035330, which is incorporated herein by reference in its entirety.

[0436] In certain embodiments, the anti-NKG2D antibody is human monoclonal antibodies 16F16, 16F31, MS and 21F2, the production, isolation and structural and functional characterization of which are described in US 7,879,985. Other known anti-NKG2D antibodies include, for example, those described in WO 2009 / 077483, WO 2010 / 017103, WO 2017 / 081190, WO 2018 / 035330 and WO 2018 / 148447, which are incorporated herein by reference in their entirety.

[0437] In certain other embodiments, the NKG2D inhibitor is an immunoadhesin {e.g., an immunoadhesin comprising an extracellular or NKG2D binding portion of NKG2DL fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)}, as described in WO 2010 / 080124, WO 2017 / 083545, and WO 2017 / 083612, which are incorporated herein by reference in their entireties.

[0438] NKG2DL inhibitors

[0439] In certain embodiments, the inhibitor of the NKG2D / NKG2DL pathway is an inhibitor of NKG2DL, such as a member of the MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, or RAET1 family. In certain embodiments, the conjugated nucleic acid molecules of the invention are administered in combination with a NKG2DL inhibitor. In certain embodiments, the NKG2DL inhibitor is an anti-NKG2DL antibody molecule such as an anti-MICA / B antibody. Exemplary MICA / MICB inhibitors

[0440] In one embodiment, the anti-MICA / B antibody molecule is IPH4301 (Innate Pharma), as disclosed in WO 2017 / 157895, which is incorporated herein by reference in its entirety.

[0441] Other known anti-MICA / B antibodies include, for example, those described in WO 2014 / 140904 and WO 2018 / 073648, which are incorporated herein by reference in their entireties.

[0442] KIR inhibitors

[0443] In some embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of KIR. In some embodiments, the coupled nucleic acid molecule of the present invention is administered in combination with a KIR inhibitor. In some embodiments, the KIR inhibitor is liruilumab (formerly also known as BMS-986015 or IPH2102).

[0444] Exemplary KIR Inhibitors

[0445] In one embodiment, the anti-KIR antibody molecule is lirelumab (Innate Pharma / AstraZeneca), as disclosed in WO 2008 / 084106 and WO 2014 / 055648, which are incorporated herein by reference in their entireties.

[0446] Other known anti-KIR antibodies include, for example, those disclosed in WO 2005 / 003168, WO 2005 / 009465, WO 2006 / 072625, WO 2006 / 072626, WO 2007 / 042573, WO 2008 / 084106, WO 2010 / 065939, WO 2012 / 071411, and WO / 2012 / 160448, which are incorporated herein by reference in their entireties.

[0447] Combination with conventional chemotherapy, radiotherapy, anti-angiogenic agents or histone deacetylase inhibitors (HDACi)

[0448] The present invention also provides combination therapies, wherein the conjugated nucleic acid molecules of the present invention are used simultaneously with, before or after surgery or radiation therapy, or are administered to a patient simultaneously with, before or after conventional chemotherapy, radiation therapy or anti-angiogenic agents, HDAC inhibitors (e.g., belinostat) or targeted immunotoxins.

[0449] The present invention also provides a method for treating cancer by the following means: administering the coupled nucleic acid molecules of the present invention in combination with conventional chemotherapy, radiotherapy or anti-angiogenic agents or HDACi or targeted immunotoxins to patients in need. The present invention also relates to a pharmaceutical composition comprising the coupled nucleic acid molecules of the present invention and conventional chemotherapy, radiotherapy or anti-angiogenic agents or HDACi or targeted immunotoxins, more particularly for the treatment of cancer. The present invention also relates to a product comprising the coupled nucleic acid molecules of the present invention and conventional chemotherapy, radiotherapy or anti-angiogenic agents, or HDACi, or targeted immunotoxins as a combined preparation for simultaneous, separate or sequential use, more particularly for the treatment of cancer.

[0450] Other aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative rather than limiting the scope of the present application.Many references are cited in this specification, each of which is incorporated herein by reference. Example

[0451] Example 1: Synthesis of exemplary nucleic acid molecules

[0452] Example 1-1: Synthesis of OX401

[0453] The synthesis of OX401 was based on standard solid phase DNA synthesis using solid phase phosphoramidite chemistry (dA(Bz); dC(Bz); dG(Ibu); dT(-)), HEG and Chol6 phosphoramidite.

[0454] The detritylation step was performed using 3% DCA in toluene, oxidation was performed using 50 mM iodine in pyridine / water 9 / 1, and sulfidation was performed using 50 mM DDTT in pyridine / ACN 1 / 1. Endcapping was performed using 20% ​​NMI in ACN together with 20% Ac2O in 2,6-lutidine / ACN (40 / 60). Cleavage and deprotection were performed using 20% ​​diethylamine in ACN for 25 min to remove the cyanoethyl protecting group on the phosphate / phosphorothioate and concentrated ammonia at 45°C for 18 h, respectively.

[0455] The crude solution was loaded onto a preparative AEX-HPLC column (TSK gel SuperQ5PW20). Purification was then performed using a sodium bromide salt gradient containing 20% ​​by volume acetonitrile at pH 12. After the fractions were combined, desalting was performed by TFF on regenerated cellulose.

[0456] Purity of OX401: 91.8% as measured by AEX-HPLC; molecular weight as measured by ESI-MS: 11046.5 Da.

[0457] HEG phosphoramidite (hexaethylene glycol phosphoramidite)

[0458]

[0459] (CLP-9765, ChemGenes Corp)

[0460] Chol6 phosphoramidite

[0461]

[0462] (No. 51230, AM Chemicals)

[0463] Example 1-2: Synthesis of OX402

[0464] The synthesis, cleavage and deprotection steps were the same as for OX401.

[0465] The crude solution was loaded onto a preparative AEX-HPLC column. Purification was then performed using a sodium bromide gradient containing 20% ​​by volume acetonitrile at pH 8. After pooling the fractions, desalting was performed by SEC on stabilized cellulose.

[0466] Purity of OX402: 92.2% as measured by AEX-HPLC; Molecular weight as measured by ESI-MS: 7340.7 Da.

[0467] Example 1-3: Synthesis of backbone = OX499

[0468] The synthesis of OX499 was based on the same protocol as OX401, except that dC(Ac) was used instead of dC(Bz) and NH2-C6 phosphoramidite was used.

[0469] Cleavage and deprotection were performed using 20% ​​diethylamine in ACN and AMA (NH3, methylamine), respectively.

[0470] The crude solution was first purified on a preparative AEX-HPLC column at pH 12 and then by RP-HPLC at pH 7. After pooling the fractions, desalting was performed by SEC on stabilized cellulose.

[0471] Purity of OX499: 95.7% as measured by AEX-HPLC; Molecular weight as measured by ESI-MS: 10637.0 Da.

[0472] Example 1-4: Synthesis of OX403

[0473] SV119 (0.123 mmol) was first coupled with 9 units of activated PEG (1.2 eq.) and then coupled with OX499. The final coupled compound OX403 was purified using a RP column.

[0474] Example 1-5: Synthesis of OX404

[0475] The synthesis followed the same synthetic route as OX403.

[0476] Example 1-6: Synthesis of OX406

[0477] The synthesis, cleavage, deprotection and purification (AEX-HPLC column) steps were identical to those of OX401.

[0478] Purity of OX406: 96.5% as measured by AEX-HPLC; molecular weight as measured by ESI-MS: 11054.3 Da.

[0479] Example 1-7: Synthesis of OX407

[0480] The synthesis, cleavage, deprotection and purification (AEX-HPLC column) steps were identical to those of OX401.

[0481] Purity of OX407: 95.7% as measured by AEX-HPLC; Molecular weight as measured by ESI-MS: 10966.2 Da.

[0482] Example 1-8: Synthesis of OX408

[0483] The synthesis, cleavage, deprotection and purification (AEX-HPLC column) steps were identical to those of OX401.

[0484] Purity of OX408: 88.4% as measured by AEX-HPLC; Molecular weight as measured by ESI-MS: 10982.2 Da.

[0485] Example 1-9: Synthesis of (OX410)

[0486] The synthesis, cleavage, deprotection and purification steps were consistent with OX401.

[0487] The crude solution was purified first on a preparative AEX-HPLC column and then by a RP-HPLC column.

[0488] Purity of OX410: 83.6% as measured by AEX-HPLC; molecular weight as measured by ESI-MS: 11051.3 Da.

[0489] Example 1-10: Synthesis of (OX411)

[0490] The synthesis, cleavage, deprotection and purification steps were consistent with OX401.

[0491] The crude solution was purified first on a preparative AEX-HPLC column and then by a RP-HPLC column.

[0492] Purification of OX411: 83.1% by AEX-HPLC; molecular weight by ESI-MS: 11051.3 Da.

[0493] Example 2: OX401 hyperactivates PARP but not DNA-PK

[0494] Materials and methods

[0495] Cell culture

[0496] The triple negative breast cancer cell line MDA-MB-231 was purchased from ATCC and grown according to the supplier's instructions. Briefly, MDA-MB-231 cells were grown in L15 Leibovitz medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere with 0% CO2.

[0497] ELISA anti-PAR

[0498] Poly (ADP-ribose) (PAR) polymers were detected using sandwich ELISA. Cells were boiled in tissue protein extraction (T-PER) buffer (Thermo Scientific) supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma). Cell extracts were then diluted in Superblock buffer (Thermo Scientific) before ELISA assay. 96-well polystyrene plates (Thermo Scientific Pierce, white opaque) were coated overnight at 4°C with 100 μl of carbonate buffer (1.5 g / l sodium carbonate Na2CO3, 3 g / l NaHCO3) containing capture antibody (mouse anti-PAR antibody, 4 μg / ml, Trevigen 4335) per well, and then washed with PBST solution. The wells were then coated with Superblock at 37°C for 1 h. 10 μl of cell extract was then added to 65 μL Superblock and added to each well in triplicate, incubated overnight at 4°C, and then washed with PBST solution. Then add the detection antibody (rabbit anti-PAR antibody, Trevigen 4336, diluted 1 / 1000 in PBS / 2% milk powder / 1% mouse serum) and incubate at room temperature for 1 hour. After washing, apply the secondary antibody, i.e., HRP-coupled anti-rabbit antibody (Abcam, ab97085, diluted 1 / 5000 in PBS / 2% milk powder / 1% mouse serum) to each well and incubate for 1 hour. For reading, add 75 μl enzyme substrate (Supersignal Pico, Pierce) to each well. Determine the chemiluminescence reading immediately.

[0499] ELISA anti-γH2AX

[0500] The phosphorylated form of histone H2AX (γH2AX) was detected using sandwich ELISA. The cells were boiled in a tissue protein extraction (T-PER) buffer (Thermo Scientific) supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride, SIGMA). The cell extract was then diluted in a Superblock buffer (Thermo Scientific) before the ELISA assay. A 96-well polystyrene plate (Thermo Scientific Pierce, white opaque) was coated overnight at 4°C with 100 μl of carbonate buffer (1.5 g / l sodium carbonate Na2CO3, 3 g / l NaHCO3) containing a capture antibody (mouse anti-γH2AX antibody, 4 μg / ml, Millipore 05-636) per well, and then washed with a PBST solution. The wells were then coated with Superblock at 37°C for 1 h. 50 μl of cell extract was then applied to each well in triplicate and incubated at 25°C for 2 h, then washed with a PBST solution. Then add detection antibody (rabbit anti-H2AX antibody, Abcam ab11175, diluted 1 / 500 in PBS / 2% milk powder) and incubate at 25°C for 1 h. After washing, apply HRP-conjugated anti-rabbit secondary antibody (Abcam, ab97085, diluted 1 / 20000 in PBS / 2% milk powder) to each well and incubate at 25°C for 1 h. For reading, add 75 μl enzyme substrate (SupersignalPico, Pierce) to each well. Chemiluminescent readings are measured immediately.

[0501] Statistical analysis

[0502] All statistical analyses were performed using two-tailed Student's t test.

[0503] result

[0504] First, the inventors analyzed the activity of OX401 in MDA-MB-231 cells by monitoring the activation of DNA-dependent protein kinase (DNA-PK) and poly (ADP-ribose) polymerase (PARP). TM After interaction of DNA components, both enzymes are activated to modify their targets. MDA-MB-231 cells treated with AsiDNA showed dose-dependent phosphorylation of histone H2AX (γH2AX) and accumulation of poly(ADP-ribose) (PAR) polymers (PARylation) after treatment, which were caused by DNA-PK and PARP activation, respectively ( Figure 1A , B). With AsiDNA TM In contrast, cells treated with OX401 did not interact and activate DNA-PK enzymes ( Figure 1A However, OX401 hyperactivated PARP and induced dose-dependent PARylation, which provided a promising target for AsiDNA TM Twice as high ( Figure 1B ). Thus, they observed a target effect in MDA-MB-231 cells, indicated by OX401-induced pseudo DNA damage signaling (PARylation).

[0505] Example 3: OX401 exhibits specific anti-tumor activity

[0506] Materials and methods

[0507] Cell culture

[0508] Cell culture was performed using triple negative breast cancer cell line MDA-MB-231, histiocytic lymphoma cell line U937, and non-tumor breast cell line MCF-10A. Cells were grown according to the instructions of the supplier. Cell lines were maintained at 37°C and in a humidified atmosphere of 5% CO2, with the exception of the MDA-MB-231 cell line, which was maintained at 0% CO2.

[0509] Drug treatment and measurement of cell viability

[0510] MDA-MB-231(5x10 3 cells / well), MCF-10A (5x10 3 cells / well) and U937 (2x10 4 Cells / well) were seeded in 96-well plates and incubated at +37°C for 24 hours, followed by the addition of gradually increasing concentrations of drugs for 4 to 7 days. After drug exposure, cell viability was measured using the XTT assay (Sigma Aldrich). Briefly, XTT solution was added directly to each well containing the cell culture, and the cells were incubated at 37°C for 5 hours, and then the absorbance at 490nm and 690nm was read using a microplate reader (BMG Fluostar, Galaxy). Cell viability was calculated as the ratio of live treated cells to live mock-treated cells. Using GraphPad Prism software (version 5.04), IC was calculated by a nonlinear regression model by plotting the survival percentage of each cell line against the logarithm of the drug concentration. 50 (It represents the dose at which 50% of the cells survive).

[0511] result

[0512] Since OX401 and AsiDNA TMCompared to inducing PARP targets without inducing DNA-PK, we wanted to ensure that it exhibited interesting anti-tumor activity. Tumor (MDA-MB-231, U937) and non-tumor (MCF-10A) cells were treated with AsiDNA (black) or OX401 (dark gray) and survival was measured using the XTT assay 4 days (U937) or 7 days (MDA-MB-231 and MCF-10A) after treatment (Figure 2). OX401 exhibited a significantly higher survival rate than AsiDNA. TM Higher antitumor activity, as indicated by the IC 50 AsiDNA TM 3 times lower than that shown ( Figure 2A MCF10A non-tumor cells were insensitive to OX401, highlighting the tumor specificity of OX401 ( Figure 2B The absence of any effect in non-tumor cells predicts that OX401 treatment will be non-toxic and highly safe in normal tissues.

[0513] Example 4: OX401 induces tumor immune response

[0514] Materials and methods

[0515] Cell culture

[0516] Cell culture was performed using the triple negative breast cancer cell line MDA-MB-231 and the non-tumor breast cell line MCF-10A. The cells were grown according to the instructions of the supplier. The cell lines were maintained at 37°C and in a humidified atmosphere of 5% CO2, except for the MDA-MB-231 cell line, which was maintained at 0% CO2.

[0517] Use of OX401 or AsiDNA TM Long-term treatment

[0518] The cells were seeded at appropriate density in 6-well culture plates and incubated at 37°C for 24 h, and then OX401 or AsiDNA was added at a concentration of 5 μM. TM Cells were harvested on day 7 after treatment, washed to remove drug, and seeded again in 6-well plates for 7 days of recovery. One week treatment / one week release period constituted one treatment cycle. Further analysis (micronucleus quantification; western blot; ELISA; flow cytometry) was performed after each treatment cycle.

[0519] Western blot analysis

[0520] Harvest with OX401 or AsiDNA TM(5 μ M) for one cycle of cells, seeded at a suitable density, and then treated again for 48 hours. The cells were then lysed in RIPA buffer (150 mM NaCl, 50 mM Tris base, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) containing protease and phosphatase inhibitors (Roche Applied Science, Germany). Protein concentration was measured using BCA protein assay (Thermo Fisher Scientific, USA). Equal amounts (15 μ g) of protein were electrophoresed with SDS-PAGE (12% gel), transferred to nitrocellulose membranes, blocked with TBS Tween 1% containing 5% skim milk powder at room temperature for 1 hour, and then incubated with the first antibody at 4 ° C overnight. After washing with TBS / Tween 1%, the membrane was incubated with the second antibody at room temperature for 1 hour. The bound antibody was detected using an enhanced chemiluminescent western blotting substrate kit (Ozyme, USA). Western blotting was performed using the following antibodies: rabbit anti-Sting monoclonal primary antibody (1 / 1,000 dilution; CST-13647), mouse anti-PD-L1 monoclonal primary antibody (1 / 1,000 dilution; abcam ab238697), mouse anti-β-actin monoclonal primary antibody (1 / 10,000 dilution, SigmaA1978), HRP-conjugated goat anti-rabbit IgG secondary antibody (1 / 2,000 dilution, Millipore 12-348), and HRP-conjugated goat anti-mouse IgG secondary antibody (1 / 2,000 dilution, Millipore 12-349).

[0521] Flow cytometry for detection of cell surface PD-L1

[0522] Harvest with OX401 or AsiDNA TM Cells treated with 5 μM for one cycle were seeded in 6-well plates at appropriate density and then treated again for 48 hours. The cells were then washed with PBS and incubated with Alexa Fluor 488-conjugated anti-PD-L1 monoclonal antibody (CST-14772) at 4°C for 1 hour. The cells were then washed with PBS and the fluorescence intensity was measured using Guava easyCyte (Merck). The data were analyzed using FlowJo software (Tree Star, California).

[0523] Micronucleus quantification

[0524] Micronuclei are caused by chromosome breakage or spindle damage. They appear in the nuclei of daughter cells after cell division and form single or multiple micronuclei in the cytoplasm.TM Cells treated with 5 μM for one cycle were grown on coverslips in petri dishes. The cells were then fixed with PFA (4%), permeabilized with Triton (0.5%), and stained with DAPI (0.5 mg / mL). The frequency of micronuclei was estimated as the percentage of cells with micronuclei to the total number of cells. At least 1,000 cells were analyzed for each condition.

[0525] ELISA for detection of CCL5 chemokine

[0526] Harvest with OX401 or AsiDNA TM (5 μM) for one cycle, seeded in a 6-well plate at an appropriate density, and then treated again for 48 hours. The cell culture supernatant was then centrifuged at 2,000 x g for 10 minutes to remove debris. The 96-well strips provided with the kit (Human SimpleStep ELISA Kit – Abcam – ab174446) were supplied ready for use. 50 μl of each supernatant was added to each well in duplicate with 50 μl of the antibody mixture, then incubated at room temperature for 1 h on a plate shaker set to 400 rpm, and then washed with 1X wash buffer PT. 100 μl of TMB substrate was then added to each well and incubated in the dark on a plate shaker set to 400 rpm for 10 min. 100 μl of stop solution was then added to each well, on a plate shaker for 1 minute, and the absorbance was measured at 450 nm.

[0527] ELISA for the detection of CXCL10 (IP-10) chemokine

[0528] Harvest with OX401 or AsiDNA TM (5 μM) for one cycle, seeded in 6-well plates at appropriate density, and then treated again for 48 hours. The cell culture supernatant was then centrifuged at 1,000 x g for 10 minutes to remove debris. The 96-well strips provided with the kit (IP-10 (CXCL10) Human ELISA Kit – Abcam – ab83700) were supplied ready for use. 100 μl of each supernatant was added to each well in duplicate, and then incubated at room temperature for 2 hours, and then washed with 1X Wash Buffer PT. 50 μl of biotinylated anti-IP-10 antibody was then added to each well and incubated for 1 hour, and then washed with 1X Wash Buffer PT. 100 μl of 1X Streptavidin-HRP solution was then added to each well, incubated for 30 minutes, and washed with 1X Wash Buffer PT. 100 μl of chromogen TMB substrate solution was then added to each well and incubated in the dark for 10-20 minutes. 100 μl of stop reagent was added to each well and the absorbance at 450 nm was immediately measured.

[0529] Statistical analysis

[0530] All statistical analyses were performed using two-tailed Student's t test.

[0531] result

[0532] Since OX401 is double-stranded DNA, we wondered whether it could be recognized by innate immune pathways. Stimulator of interferon genes (STING) is a cytoplasmic receptor that senses both exogenous and endogenous cytoplasmic DNA and triggers type I interferon and proinflammatory cytokine responses. Therefore, the inventors evaluated the activation of the STING pathway in cells treated with OX401. Interestingly, OX401 was not recognized as exogenous DNA by the STING pathway and did not trigger direct induction of chemokines and interferon cytokines (data not shown).

[0533] Since short-term treatment with OX401 does not directly induce anti-tumor immune responses, the inventors hypothesized that long-term treatment may indirectly trigger STING-dependent immune responses through the accumulation of unrepaired DNA structures. TM Cells treated with OX401 chronically (one cycle of one week treatment / one week release) showed a significant two-fold increase in the % of cells with micronuclei compared to cells treated with OX401. Figure 3 A). To verify the link between increased micronuclei and STING pathway activation, the inventors analyzed the release of CCL5 and CXCL10 target chemokines. Interestingly, cells chronically treated with OX401 secreted twice as much CCL5 and 1.5 times as much CXCL10 compared to untreated cells ( Figure 3 B). AsiDNA-treated cells did not show increased secretion of CCL5 or CXCL10 ( Figure 3 B). The results of STING pathway activation in tumor cells include upregulation of PD-L1 (programmed death ligand 1), which may be a response to provide protection against the immune system. The inventors analyzed the levels of total PD-L1 or cell surface-bound PD-L1 in cells treated for a long time. TM Compared with parental cells, OX401-treated cells showed total levels of ( Figure 3 C) and membrane-bound PD-L1 ( Figure 3 D) a significant improvement.

[0534] Taken together, these results confirm that OX401 triggers indirect STING pathway activation via micronucleus-induced accumulation and pave the way for combination treatment with anti-PD-L1 therapy.

[0535] Example 6: Pharmaceutical properties / PK / PD experiments

[0536] Injection of OX401 into mice at a dose of 2 mg by the intravenous (iv) route resulted in a maximum plasma concentration (CMAX) of 8 μM measured by the HPLC method. Surprisingly, this Cmax was 40-fold higher than the Cmax obtained using AsiDNA under the same experimental conditions.

[0537] Example 7: OX402 hyperactivates PARP - smaller but equally active as OX401

[0538] Materials and methods

[0539] Cell culture

[0540] The triple negative breast cancer cell line MDA-MB-231 was purchased from ATCC and grown according to the supplier's instructions. Briefly, MDA-MB-231 cells were grown in L15 Leibovitz medium supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere with 0% CO2.

[0541] ELISA anti-PAR

[0542] Poly (ADP-ribose) (PAR) polymers were detected using sandwich ELISA. Cells were boiled in tissue protein extraction (T-PER) buffer (Thermo Scientific) supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma). Cell extracts were then diluted in Superblock buffer (Thermo Scientific) before ELISA assay. 96-well polystyrene plates (Thermo Scientific Pierce, white opaque) were coated overnight at 4°C with 100 μl of carbonate buffer (1.5 g / l sodium carbonate Na2CO3, 3 g / l NaHCO3) containing capture antibody (mouse anti-PAR antibody, 4 μg / ml, Trevigen 4335) per well, and then washed with PBST solution. The wells were then coated with Superblock at 37°C for 1 h. 10 μl of cell extract was then added to 65 μL Superblock and added to each well in triplicate, incubated overnight at 4°C, and then washed with PBST solution. Then add the detection antibody (rabbit anti-PAR antibody, Trevigen 4336, diluted 1 / 1000 in PBS / 2% milk powder / 1% mouse serum) and incubate at room temperature for 1 hour. After washing, apply the secondary antibody, i.e., HRP-coupled anti-rabbit antibody (Abcam, ab97085, diluted 1 / 5000 in PBS / 2% milk powder / 1% mouse serum) to each well and incubate for 1 hour. For reading, add 75 μl enzyme substrate (Supersignal Pico, Pierce) to each well. Determine the chemiluminescence reading immediately.

[0543] result

[0544] The inventors also analyzed the minimum sequence length required to activate PARP and induce pseudo-injury signaling (PARylation). MDA-MB-231 cells were treated with OX402, a 10 base pair (bp) molecule, for 24 h and PARP activation was monitored using an anti-PARylation ELISA assay. MDA-MB-231 cells treated with OX402 showed dose-dependent PARylation (P) caused by PARP action and activation. Figure 4 ). Therefore, a 10 bp molecule is sufficient to hijack and activate PARP.

[0545] Example 8: OX401 induces intracellular NAD + Exhaustion

[0546] The PARP protein binds to DSBs with high affinity. Upon binding, PARP is auto-"PARylated" and activates other target proteins through the addition of poly(ADP-ribose) (PAR) polymers, a process known as PARylation. The kinetics of PARP activation were studied in MDA-MB-231 and MRC5 cells treated with OX401 by monitoring protein PARylation.

[0547] Materials and methods

[0548] Cell culture

[0549] Cell culture was performed using triple negative breast cancer cell line MDA-MB-231 and non-tumor MRC5 primary lung fibroblasts. All cell lines were purchased from ATCC and grown in a humidified atmosphere at 37°C and 5% CO2 according to the supplier's instructions, except for MDA-MB-231 (37°C and 0% CO2).

[0550] Treatment of cells with OX401 and assessment of viability

[0551] MDA-MB-231 or MRC5 cells were seeded at appropriate density in 60 mm diameter culture plates and incubated overnight at 37° C. Cells were then treated with 5 μM OX401 for 48 hours, 7 days, and 13 days, then washed, harvested, and counted using the Trypan Blue (4%) cell staining assay and Eve automated cell counter (VWR) for further analysis.

[0552] NAD + Measurement of intracellular levels of

[0553] NAD content was determined using the NAD / NADH-Glo assay kit (Promega, G9071) according to the manufacturer's instructions. The principle of the assay is a continuous conversion: first, NAD cycle enzymes convert NAD + is modified into NADH, which is used by the reductase to convert the substrate into luciferin. Luciferase then uses luciferin to produce light. Therefore, the luminescence produced is proportional to the NAD present in the cell. + is proportional to the amount.

[0554] Briefly, MDA-MB-231 or MRC5 cells treated with OX401 (5 μM) for 48 h, 7 days, or 13 days were harvested and seeded in 96-well plates (5 × 10 4cells / well). The cells were then lysed using 1% DTAB buffer, 25 μL of 0.4 M HCl was added to each well, incubated at 60°C for 15 min and at room temperature for 10 min. 25 μL of detection reagent Trizma and 100 μL of NAD detection reagent were added to each well. The plate was read on a microplate reader (Enspire TM The generated luminescent signal was measured on a Perkin-Almer microscope.

[0555] Western blot analysis

[0556] MDA-MB-231 or MRC5 cells treated with OX401 (5 μM) for 48 hours, 7 days or 13 days were harvested and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris base, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) containing protease and phosphatase inhibitors (Roche Applied Science, Germany). Protein concentration was measured using BCA protein assay (Thermo Fisher Scientific, USA). Equal amounts (15 μg) of protein were electrophoresed by SDS-PAGE (12% gel), transferred to nitrocellulose membranes, blocked with TBS Tween 1% containing 5% skim milk powder at room temperature for 1 hour, and then incubated with the first antibody at 4°C overnight. After washing with TBS / Tween 1%, the membrane was incubated with the second antibody at room temperature for 1 hour. The bound antibodies were detected using an enhanced chemiluminescence western blotting substrate kit (Ozyme, USA). Western blotting was performed using the following antibodies: anti-pan-ADP ribose binding reagent (1 / 1,500 dilution; Millipore MABE1016), mouse anti-β-actin monoclonal primary antibody (1 / 10,000 dilution, Sigma A1978), HRP-conjugated goat anti-rabbit IgG secondary antibody (1 / 2,000 dilution, Millipore 12-348) and HRP-conjugated goat anti-mouse IgG secondary antibody (1 / 2,000 dilution, Millipore 12-348).

[0557] result

[0558] MDA-MB-231 cells treated with OX401 (5 μM) showed accumulation of PARylated proteins after treatment, as shown in the photographs 7 days after treatment ( Figure 5 A). Due to the nicotinamide adenine dinucleotide (NAD + ) is used as a substrate by PARP to PARylate its target protein, so the intracellular NAD after OX401 treatment was analyzed +OX401 induces NAD levels in MDA-MB-231 cells + The maximum value was 55% of NAD after 7 days of treatment compared with untreated cells. + level, which was maintained until 13 days after treatment ( Figure 5 B) Due to this huge OX401-induced NAD + We hypothesized that tumor cells could not regulate and maintain NAD under OX401 treatment. + To test this hypothesis, we analyzed the cell survival of MDA-MB-231 cells under OX401 treatment. No effect on cell survival was observed 48 h after OX401 treatment, which is consistent with the fact that NAD + Significant effects on cell viability were observed 7 and 13 days after treatment (57% and 32%, respectively, compared to untreated cells), confirming that NAD + Importance of levels to cell viability ( Figure 5 C) All of these effects were specific to tumor cells, as no NAD + No cell death was observed after depletion ( Figure 5 DF).

[0559] Taken together, these results suggest that chronic treatment with OX401 induces PARP hyperactivation and NAD + Depletion of both. OX401 induces NAD + Levels rapidly drop below a threshold compatible with cell survival, which would exceed the cellular NAD + Replenish capacity and trigger massive tumor cell death.

[0560] Example 10

[0561] OX401 disrupts the homologous recombination (HR) repair pathway

[0562] Since OX401 induces PARP and induces pseudo-DNA damage PARylation signaling, the inventors tested whether OX401 could trigger the rapid accumulation of DNA damage.

[0563] The homologous recombination (HR) repair pathway is an error-free repair pathway that is essential for maintaining genetic stability and intact DNA information. HR is a well-organized multistep mechanism that consumes a lot of cellular energy. Since OX401 triggers high NAD +Depleting and thus inducing metabolic imbalance in tumor cells (Example 9), the inventors hypothesized that OX401 might disrupt the HR repair mechanism that is very energy-dependent. To test this hypothesis, the efficiency of HR repair after OX401 treatment was analyzed (by detecting the recruitment of Rad51 protein to DSB sites).

[0564] Materials and methods

[0565] Cell culture

[0566] Cell culture was performed using the triple negative breast cancer cell line MDA-MB-231. Cells were grown in complete L15 Leibovitz medium and maintained at 37°C in a humidified atmosphere with 0% CO2.

[0567] Homologous recombination pathway activity analysis

[0568] For immunostaining, cells were plated at 5 × 10 5 Cells were seeded on coverslips (Menzel, Braunschweig, Germany) at a concentration of 10 cells and incubated at 37°C for 1 day. Cells were then treated with olaparib (5 μM) + / - OX401 (5 μM). 48 h after treatment, cells were fixed in 4% paraformaldehyde / phosphate-buffered saline (PBS 1x) for 20 min, permeabilized in 0.5% Triton X-100 for 10 min, blocked with 2% bovine serum albumin / PBS 1x, and incubated with primary antibodies for 1 h at 4°C. All secondary antibodies were used at a dilution of 1 / 200 for 45 min at room temperature (RT), and DNA was stained with 4',6-diamidino-2-phenylindole (DAPI). The following antibodies were used: mouse anti-phospho-H2AX monoclonal primary antibody (Millipore, Guillancourt, France), rabbit anti-Rad51 antibody (Merk Millipore, Darmstadt, Germany), Alexa-633-conjugated goat anti-mouse IgG secondary antibody (Molecular Probes, Eugene, OR, USA), and Alexa-488-conjugated goat anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR, USA).

[0569] Analysis of drug-induced DNA damage by flow cytometry

[0570] Cells were treated with OX401 (5 μM) or olaparib (5 μM) for 48 hours, then fixed and permeabilized with cold (-20°C) 70% ethanol for at least 2 hours. After washing with PBS, cells were further permeabilized with 0.5% Triton in PBS for 20 minutes at RT, washed in PBS, and incubated with anti-γ-H2AX antibody (05-636 Millipore) in PBS containing 2% BSA. After washing with PBS, cells were incubated with a secondary antibody coupled to Alexa Fluor 488. Fluorescence intensity was measured using a Guava EasyCyte cytometer (Luminex). Data were analyzed using FlowJo software (Tree Star, California).

[0571] result

[0572] As expected, as measured by flow cytometry ( Figure 6 A) or γH2AX foci detected by immunofluorescence ( Figure 6 B) Olaparib induces accumulation of double-strand breaks (DSBs) in MDA-MB-231 cells 48 h after treatment, as measured by elevated histone H2AX phosphorylation (γH2AX). In comparison, OX401 does not induce an increase in the γH2AX DSB biomarker and therefore does not trigger direct DSB accumulation ( Figure 6 A, B).

[0573] MDA-MB-231 cells treated with olaparib (5 μM) for 48 h showed accumulation of γH2AX foci colocalized with Rad51 foci, indicating that olaparib-induced DSBs were repaired by the HR repair pathway ( Figure 6 C). Addition of OX401 (5 μM) significantly reduced the formation of Rad51 foci induced by olaparib ( Figure 6 C, D), demonstrating that OX401 may effectively perturb the HR pathway through energy depletion associated with metabolic imbalance.

[0574] Embodiment 11

[0575] Tumor cells do not acquire resistance to OX401

[0576] It is now recognized that cancer undergoes an evolutionary process proposed by Charles Darwin in his concept of natural selection. Natural selection is the process by which nature selects certain physical attributes or phenotypes to pass on to future generations so that organisms can better "adapt" to the environment. Under the selective pressure of targeted therapies, resistant populations of cancer cells continue to evolve, resulting in "resistant clones" that adapt to the new environment caused by the treatment. It has also been established that tumor cells require a lot of energy to develop resistance to anticancer treatments. Since OX401 induces NAD+ depletion and metabolic imbalance (Example 8), the inventors tested whether cells develop resistance to OX401.

[0577] Materials and methods

[0578] Cell culture

[0579] Cell culture was performed using the lymphoma cell line U937. Cells were grown in complete RPMI medium supplemented with 10% FBS and 1% penicillin / streptomycin and maintained at 37°C in a humidified atmosphere of 5% CO2. This cell line was selected based on its high sensitivity to both OX401 and talazopanib.

[0580] Selection for acquired resistance

[0581] For repeated treatment cycles to select for drug resistance, U937 cells were plated at an appropriate density (2×10 5 Cells / mL) were inoculated and incubated at 37°C for 24 h, and drugs were added at doses corresponding to 10-20% viability relative to untreated cells. Resistance was selected at 2 μM talazopanib or 1.5 μM OX401. Cells were harvested on day 4 after treatment, washed, and counted after staining with 0.4% trypan blue (Eve TM Counting slides, NanoEnTek). After counting, cells were plated in suitable culture plates and allowed to recover (drug-free period) for 3 to 7 days. Another treatment / recovery cycle was then started, up to 4 cycles.

[0582] Irreversibility of acquired drug resistance—a measure of cell survival

[0583] To evaluate the irreversibility of acquired resistance, U937 parental or resistant cells were seeded in 96-well plates (2 × 10 4Cells / well) and treated with talazopanib at gradually increasing concentrations for 4 days. After drug exposure, cell viability was measured using the XTT assay (Sigma Aldrich). Briefly, XTT solution was added directly to each well containing cell culture, and the cells were incubated at 37°C for 5 hours, and then the absorbance was read at 490nm and 690nm using a microplate reader (BMG Fluostar, Galaxy). Cell viability was calculated as the ratio of live treated cells to live mock-treated cells. Using GraphPad Prism software (version 5.04), IC50 (which represents the dose at which 50% of cells survive) was calculated by a nonlinear regression model by plotting the percentage of viability of each cell line against the logarithm of drug concentration.

[0584] result

[0585] U937 cells were subjected to treatment cycles with either OX401 or talazopanib. Cells treated with talazopanib recovered during the expansion period, whereas cells treated with OX401 did not grow during the drug-free expansion period ( Figure 7 A). Cells treated with talazopanib developed acquired resistance during the treatment cycles, with cell survival evolving from 10% after the first cycle to more than 50% after the fourth treatment cycle (33 days after the start of treatment) (p<0.01) ( Figure 7 B). To assess the irreversible resistance status to tazopanib, resistant cells were subjected to increasing doses of tazopanib to analyze their sensitivity compared to parental cells. Parental cells sensitive to tazopanib showed a low IC50 of 2 μM. Tal1, Tal2, and Tal3 resistant populations showed higher IC50s of more than 4 μM ( Figure 7 C).

[0586] Example 12

[0587] OX401 amplifies anti-tumor immune responses

[0588] In previous experiments ( Figure 3 ), the inventors showed that long-term treatment with OX401 exhibited micronucleus-induced STING pathway activation and increased secretion of CCL5 and CXCL10 chemokines. To test the effectiveness of these anti-tumor immune effects, co-culture of tumor cells with freshly isolated T cells was performed and T cell-induced cytotoxic effects were evaluated.

[0589] Materials and methods

[0590] Cell culture

[0591] Cell culture was performed using triple negative breast cancer cell line MDA-MB-231 and cervical tumor cell line HeLa. Cells were purchased from ATCC and grown according to the supplier's instructions. Cells were maintained at 37°C and in a humidified atmosphere of 5% CO2.

[0592] Isolation of PBMCs

[0593] Buffy coats from healthy donors were purchased from EFS Blood Center (Paris, France). PBMCs were isolated using the EasySep Direct Human PBMC Isolation Kit (19654, Stemcell, France) according to the manufacturer's protocol. The isolated PBMCs were adjusted to 5 x 10 in freezing medium (10% DMSO and 90% FBS). 7 The concentration of cells / ml was determined from which 1 ml aliquots were dispensed into cryovials and stored in liquid nitrogen at -196°C until use.

[0594] Isolation of T lymphocytes from PBMCs

[0595] T lymphocytes were isolated from PBMCs using the EasySep Human T Cell Isolation Kit (17951, Stemcell, France) according to the manufacturer's protocol. 6 Cells were suspended at a concentration of 10 cells / ml in ImmunoCult-XF T Cell Expansion Medium (10981, Stemcell, France) and activated for 24 h using ImmunoCult Human CD3 / CD28 / CD2 T Cell Activator (10970, Stemcell, France) before further experiments.

[0596] Co-culture of tumor and T lymphocytes

[0597] MDA-MB-231 cells were seeded in 12-well cell culture plates (5 x 10 4 cells / well) or 60 mm diameter cell culture plates (10 6 Cells / plate) and incubated at 37°C for 24 hours. Activated T cells were added to tumor cells at a ratio of 4:1 effector cells to target cells with or without OX401 (5 μM). The co-cultures were incubated at 37°C for 48 hours. At the end of the incubation, each cell type (adherent tumor cells or suspension T cells) was counted and the supernatant was harvested for cytokine release analysis.

[0598] Western blot analysis

[0599] Cells treated with OX401 (5 μM) containing or not T lymphocytes were harvested and then lysed in RIPA buffer (150 mM NaCl, 50 mM Tris base, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) containing protease and phosphatase inhibitors (Roche Applied Science, Germany). Protein concentration was measured using the BCA protein assay (Thermo Fisher Scientific, USA). Equal amounts (15 μg) of protein were electrophoresed by SDS-PAGE (12% gel), transferred to nitrocellulose membranes, blocked with TBS Tween 1% containing 5% skim milk powder at room temperature for 1 hour, and then incubated with the first antibody at 4°C overnight. After washing with TBS / Tween 1%, the membrane was incubated with the second antibody at room temperature for 1 hour. The bound antibody was detected using an enhanced chemiluminescent western blotting substrate kit (Ozyme, USA). Western blotting was performed using the following antibodies: rabbit anti-Sting monoclonal primary antibody (1 / 1,000 dilution; CST-13647), mouse anti-PD-L1 monoclonal primary antibody (1 / 1,000 dilution; abcam ab238697), mouse anti-β-actin monoclonal primary antibody (1 / 10,000 dilution, Sigma A1978), HRP-conjugated goat anti-rabbit IgG secondary antibody (1 / 2,000 dilution, Millipore 12-348), and HRP-conjugated goat anti-mouse IgG secondary antibody (1 / 2,000 dilution, Millipore 12-349).

[0600] ELISA for detecting CCL5 chemokine

[0601] The cells were treated with OX401 (5 μM) for 48 hours in the presence or absence of T lymphocytes. The cell culture supernatant was then centrifuged at 2,000 x g for 10 minutes to remove debris. The 96-well strips provided with the kit (Human SimpleStep ELISA Kit – Abcam – ab174446) were supplied ready for use. 50 μl of each supernatant was added to each well in duplicate with 50 μl of the antibody mixture, then incubated at RT for 1 h on a plate shaker set to 400 rpm, and then washed with 1X wash buffer PT. 100 μl of TMB substrate was then added to each well and incubated in the dark on a plate shaker set to 400 rpm for 10 min. 100 μl of stop solution was then added to each well, on a plate shaker for 1 minute, and the absorbance was measured at 450 nm.

[0602] ELISA for detection of granzyme B

[0603] Cells were treated with OX401 (5 μM) for 48 hours in the presence or absence of T lymphocytes. The cell culture supernatant was then centrifuged at 2,000 x g for 10 minutes to remove debris. The 96-well strips provided with the kit (Human SimpleStep Granzyme B ELISA Kit – Abcam – ab235635) were supplied ready for use. 50 μl of each supernatant was added to each well in duplicate with 50 μl of the antibody mixture and then incubated at RT for 1 h on a plate shaker set to 400 rpm, which was then washed with 1X wash buffer PT. 100 μl of TMB substrate was then added to each well and incubated in the dark on a plate shaker set to 400 rpm for 10 min. 100 μl of stop solution was then added to each well, on a plate shaker for 1 minute, and the absorbance was measured at 450 nm.

[0604] result

[0605] As revealed by the decreased survival of MDA-MB-231 tumor cells (50% survival compared to MDA-MB-231 cells without T cells) Figure 8 A), Freshly activated T cells triggered antitumor cytotoxic effects 48 and 72 hours after the start of co-culture. Addition of OX401 to co-culture further enhanced T cell-induced antitumor cytotoxicity (20% survival compared to MDA-MB-231 cells without OX401 treatment and without T cells) ( Figure 8 A). Interestingly, in the presence of MDA-MB-231 tumor cells treated with OX401, cytotoxic T cells secreted greater amounts of granzyme B ( Figure 8 B), with higher cytotoxic potency ( Figure 8 A). Given the importance of STING pathway activation in triggering higher immune cell recruitment and anti-tumor cytotoxicity, we analyzed this pathway in tumor / immune cell co-cultures in the presence or absence of OX401. After 48 h of co-culture, we observed a higher increase in STING protein levels in tumor cells treated with OX401 ( Figure 8 C). This was accompanied by higher IRF3 protein phosphorylation ( Figure 8 C) and the increase in secreted CCL5 chemokine ( Figure 8 D), indicating sustained STING pathway activation.

[0606] Taken together, these findings demonstrate that anti-tumor cytotoxic T cells are highly enhanced by OX401 through higher STING pathway activation, which may stimulate better recruitment of T cells to the vicinity of tumor cells.

[0607] Example 13: Binding Kinetics (kon ) and the interaction strength (K D )

[0608] Materials and methods

[0609] The interaction of different molecules according to the invention with human poly [ADP-ribose] polymerase 1 protein (PARP-1) (115 kDa) was characterized by SPR technology using a Biacore T100 instrument from GE Healthcare Life Sciences. PARP1-His was captured on an anti-His antibody immobilized on a carboxymethylated chip surface.

[0610] result

[0611] Binding kinetics (k on ) and the interaction strength (K D ) reported in Fig. 9 middle.

[0612] OX401, OX410 and OX411, which have modified phosphodiester backbones such as phosphorothioate bonds (OX401) or both phosphorothioate bonds and FANA modifications (OX410, OX411), on the first three nucleotides on the 3' and / or 5' strands, have similar affinities for PARP-1 (K D ) and binding kinetics (k on OX402, which has phosphorothioate bonds on the first three nucleotides on the 3' and / or 5' strands, has similar binding affinity to PARP-1 as the above mentioned molecules, but has lower binding kinetics to PARP-1.

[0613] The binding strength appeared to be higher using OX406 with two FANA modifications in the 3'-strand.

[0614] Reducing the number of phosphorothioate modifications to a single nucleotide (OX407 and OX408) significantly increased the interaction strength (K D values ​​decreased) and binding kinetics (k on ).

[0615] From this set of experiments it is clear that chemical modification of the first three nucleotides on the 3' and / or 5' strand modulates the interaction strength (K D ) and binding kinetics (k on ), have a strong influence on the interaction of coupled nucleic acid molecules with PARP, demonstrating that they are very interesting inhibitors of DNA repair pathways and therefore potential candidates for a role in cancer therapy. Sequence Listing <110> ONXEO <120> New coupled nucleic acid molecules and their uses <130> B2921PC <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> DNA <213> Artificial <220> <223> Nucleic acid molecules <400> 1 cccagcaaac aagcct 16 <210> 2 <211> 10 <212> DNA <213> Artificial <220> <223> Nucleic acid molecules <400> 2 cagcaacaag 10

Claims

1. A coupled nucleic acid molecule, wherein the coupled nucleic acid molecule is selected from the following structural formula or a pharmaceutically acceptable salt thereof: Wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; the italicized U is 2'-deoxy-2'-fluoroarabino-uridine, the italicized G is 2'-deoxy-2'-fluoroarabino-guanosine; and the italicized C is 2'-deoxy-2'-fluoroarabino-cytidine.

2. A pharmaceutical composition comprising the coupled nucleic acid molecule according to claim 1.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition further comprises an additional therapeutic agent selected from the group consisting of immunomodulators, T-cell based cancer immunotherapeutics, conventional chemotherapeutics, radiotherapeutics, anti-angiogenic agents, HDAC inhibitors and targeted immunotoxins.

4. Use of the coupled nucleic acid molecule according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 3 in the preparation of a drug for treating cancer.

5. Use of the pharmaceutical composition according to claim 2 in combination with another therapeutic agent in the preparation of a medicament for treating cancer, wherein the other therapeutic agent is selected from immunomodulators, T-cell-based cancer immunotherapeutics, conventional chemotherapeutics, radiotherapeutics, anti-angiogenic agents, HDAC inhibitors, targeted immunotoxins, immune checkpoint inhibitors, belinostat and chimeric antigen receptor cells.

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