Compositions and methods of activation of "stimulator of interferon genes"-dependent signali

专利摘要:

公开号:ES2754269T9
申请号:ES14800848T
申请日:2014-05-18
公开日:2020-08-19
发明作者:Thomas W Dubensky；David B Kanne；Meredith Lai Ling Leong；Laura Hix Glickman；Russell E Vance；Edward Emile Lemmens
申请人:University of California；Aduro Biotech Inc；
IPC主号:

专利说明:

[0002] Compositions and methods of activation of signaling dependent on the "stimulator of interferon genes"
[0003] Background of the invention
[0005] The human immune system can be broadly divided into two groups, which are called "innate immunity" and "adaptive immunity." The innate group of the immune system is predominantly responsible for an initial inflammatory response through a number of soluble factors, including the complement system and the chemokine / cytokine system; and various types of specialized cells, including mast cells, macrophages, dendritic cells (DC), and natural killer cells. In contrast, the adaptive immunity cluster involves a delayed and longer-lasting antibody response, along with CD8 + and c D4 + T cell responses that play a critical role in immunological memory against an antigen. A third group of the immune system can be identified involving T lymphocytes and T lymphocytes with limited repertoires of T lymphocyte receptors such as T n K lymphocytes and MAI T lymphocytes.
[0007] For an effective immune response to an antigen, antigen presenting cells (APC) must process and display the antigen in an appropriate MHC context to a T lymphocyte, which will then lead to stimulation of cytotoxic and helper T lymphocytes. Following antigen presentation, correct interaction of costimulatory molecules must occur on both APCs and T cells, or activation will be inhibited. GM-CSF and IL-12 serve as effective pro-inflammatory molecules in many tumor models. For example, GM-CSF induces myeloid precursor cells to proliferate and differentiate into dendritic cells (DCs), although additional signals are needed to activate their maturation to the efficient antigen-presenting cells necessary for the activation of T lymphocytes. Effective immune therapies include tolerance to the targeted antigen, which may limit the induction of CD8 cytotoxic T cells of appropriate magnitude and function, poor trafficking of generated T cells to malignant cell sites, and low persistence of the response of cells. induced T lymphocytes.
[0008] DCs that phagocytose tumor cell debris process material for the presentation of the major histocompatibility complex (MHC), up-regulate the expression of costimulatory molecules, and migrate to regional lymph nodes to stimulate tumor-specific lymphocytes. This pathway produces the proliferation and activation of CD4 + and CD8 + T lymphocytes that react to tumor-associated antigens. In fact, such cells can often be detected in the blood, lymphoid tissues, and malignant lesions of patients.
[0009] New insights into the mechanisms underlying immune evasion, along with combination treatment regimens that enhance the potency of therapeutic vaccination, either directly or indirectly, through combination with immune checkpoint inhibitors or other therapies , have served as the basis for the development of vaccines that induce effective antitumor immunity. The cyclic di-AMP (produced by Listeria monocytogenes) and its cyclic di-GMP analog (produced by Legionella pneumophila) are recognized by the host cell as a PAMP (pathogen-associated molecular pattern), which binds to the PRR (receptor for pathogen recognition) known as STING. STING is an adapter protein from the cytoplasm of mammalian host cells that activates the TANK-binding kinase (TBK1) -IRF3 signaling axis, leading to the induction of IFN-p and other IRF-3-dependent gene products. that greatly activate innate immunity. STING is now recognized as a component of the host cytosolic surveillance pathway, detecting infection with intracellular pathogens and, in response, inducing IFN-p production, leading to the development of an adaptive protective pathogen-specific immune response that It consists of antigen-specific CD4 and CD8 T lymphocytes, as well as pathogen-specific antibodies. Examples of purine cyclic dinucleotides are described in some detail in, for example, US Patent Nos. 7,709,458 and 7,592,326; WO2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18: 5631 (2008).
[0011] There remains a need for better compositions and methods for immunological strategies for treating diseases such as cancer that may be refractory to traditional therapeutic approaches.
[0013] Summary of the invention
[0015] It is an object of the present invention to provide compositions that modulate immune responses to disease.
[0017] In a first aspect, the present invention provides the purine cyclic dinucleotide of claim 1 that induces stimulator-dependent type I interferon production of interferon genes ("STING").
[0019] In their role as adjuvants, in certain embodiments, the present compositions can be used as adjuvants in a therapeutic or prophylactic strategy employing vaccine / s. Thus, the CDN of the present invention, or its pharmaceutically acceptable salts, can be used in conjunction with one or more selected vaccines to stimulate an immune response to one or more predetermined antigens. The CDN of the present invention, or its pharmaceutically acceptable salts, can be provided in conjunction with, or in addition to, such vaccines.
[0020] Said vaccine (s) may comprise inactivated or attenuated bacteria or viruses comprising the antigens of interest, purified antigens, live viral or bacterial delivery vectors designed recombinantly to express and / or secrete the antigens, vectors of presenting cells. antigen (APC) cells comprising cells that are loaded with the antigens or transfected with a composition comprising a nucleic acid encoding the antigens, liposomal antigen delivery vehicles, or naked nucleic acid vectors encoding the antigens. This list is not intended to be limiting. By way of example, said vaccine / s may also comprise an inactivated tumor cell that expresses and secretes one or more of GM-CSF, CCL20, CCL3, IL-12p70, FLT-3 ligand.
[0022] The CDN of the present invention, or its pharmaceutically acceptable salts, can be administered to individuals in need by various parenteral and non-parenteral routes in formulations containing pharmaceutically acceptable carriers, adjuvants, and vehicles. Preferred routes are parenteral and include, but are not limited to, one or more of subcutaneous, intravenous, intramuscular, intraarterial, intradermal, intrathecal, and epidural administration. Intratumoral routes are also preferred. In particular, subcutaneous administration is preferred. Preferred pharmaceutical compositions are formulated as aqueous or oil-in-water emulsions.
[0024] The compositions of the present invention may comprise, or be administered in conjunction with, one or more additional pharmaceutically active components such as adjuvants, lipids, interlayer cross-linked multilamellar vesicles, biodegradable nanoparticles or microparticles based on poly (D, L-lactic acid-co -glycolic) [PLGA] or based on poly-anhydride, and lipid bilayers supported by nanoporous particles, antagonists of the CTLA-4 and PD-1 pathway, PD-1 pathway blocking agents, inactivated bacteria that induce innate immunity (for For example, inactivated or attenuated Listeria monocytogenes ), compositions that mediate innate immune activation through Toll-like receptors (TLR), type I receptors (NOD) (NLR), retinoic acid-inducible gene-based type I receptors (RIG ) (RLR), C-type lectin receptors (CLR), pathogen-associated molecular patterns ("PAMPs"), chemotherapeutic agents, etc.
[0026] In a related aspect, the present invention relates to methods of inducing, stimulating or enhancing an immune response in an individual. These methods comprise administering the CDN of the present invention, or pharmaceutically acceptable salts thereof, to the individual. The preferred routes of administration are parenteral.
[0027] In certain embodiments, the method is a cancer treatment method. By way of example, the CDN of the present invention, or its pharmaceutically acceptable salts, can be provided alone, or in conjunction with or in addition to one or more cancer vaccine compositions that are known in the art. The patient receiving such treatment may be suffering from a cancer selected from the group consisting of a colorectal cancer cell, an aerodigestive squamous cancer, a lung cancer, a brain cancer, a liver cancer, a stomach cancer, a sarcoma , a leukemia, a lymphoma, a multiple myeloma, an ovarian cancer, a uterine cancer, a breast cancer, a melanoma, a prostate cancer, a pancreatic carcinoma and a renal carcinoma. In other embodiments, the method is a method of inducing, stimulating, or enhancing an immune response to a pathogen.
[0028] With respect to the treatment of a mammal suffering from cancer, the methods described herein may comprise administering to the mammal an effective amount of the CDN of the present invention, or pharmaceutically acceptable salts thereof, optionally before or after an administered primary therapy. to the mammal to kill or destroy cancer cells that express the cancer antigen. The compositions of the present invention can be provided as a neoadjuvant therapy; however, in preferred embodiments, the compositions of the present invention are administered after primary therapy. In various embodiments, the primary therapy comprises surgery to remove cancer cells from the mammal, radiation therapy to kill cancer cells from the mammal, or surgery and radiation therapy.
[0030] In other embodiments, the methods described herein may comprise administering to the mammal an effective amount of the CDN of the present invention for the treatment of disorders in which shifting Th1 to Th2 immunity confers clinical benefit. Cell-mediated immunity (CMI) is associated with CD4 + TH1 T cells that produce IL-2 cytokines, interferon (IFN) -y, and tumor necrosis factor (TNF) -a. In contrast, humoral immunity is associated with CD4 + TH2 T lymphocytes, which produce IL-4, IL-6, and IL-10. Immune drift toward TH1 responses normally results in activation of cytotoxic T lymphocytes (CTL), natural killer (NK) lymphocytes, macrophages, and monocytes. In general, Th1 responses are more effective against intracellular pathogens (viruses and bacteria that are within host cells) and tumors, while Th2 responses are more effective against extracellular bacteria, parasites, including helminths, and toxins. Additionally, activation of innate immunity is expected to normalize the balance of the T-helper immune system type 1 and 2 (Th1 / Th2) and suppress the overreaction of Th2-type responses that cause immunoglobulin (Ig) -dependent allergies. E and allergic asthma.
[0032] Brief description of the figures
[0034] Fig. 1 depicts purine cyclic dinucleotide ("CDN") mediated signaling. A CDN (e.g. cyclic di-AMP or cyclic di-GMP) induces IFN-p production by binding to the cytosolic STING receptor (stimulator of interferon genes) and inducing signaling through the TBK-1 / IRF-3 pathway, leading to both autocrine and paracrine activation of DCs through IFN receptor binding and subsequent signaling. Fig. 2A depicts a synthesis scheme for cyclic [G (2 ', 5') pG (3 ', 5') p] and dithium derivatives.
[0035] Fig. 2B depicts a synthesis scheme for cyclic [A (2 ', 5') pA (3 ', 5') p] and dithium derivatives.
[0036] Fig. 2C depicts the structures of compounds 10, 20, 21, 22 and 23.
[0037] Fig. 3A depicts the 1H NMR results for compound 9a.
[0038] Fig. 3B depicts the COZY results (3.5-6.0 ppm 1H axis) for compound 9a.
[0039] Fig. 3C depicts the HMBC results (1H axis 3.0-5.5 ppm) for compound 9a.
[0040] Fig. 3D depicts the 1H NMR results for compound 21.
[0041] Fig. 3E depicts the COZY results (1H axis of 3.5-6.0 ppm) for compound 21.
[0042] Fig. 3F depicts HMBC results (1H axis 0-9.5 ppm) for compound 21.
[0043] Fig. 3G depicts the HMBC results (3.5-5.5 ppm 1H axis) for compound 21.
[0044] Fig. 3H represents HPLC analytical results (2-20% ACN / 10mM-20 min TEAA buffer) for compound 19b.
[0045] Fig. 4 depicts [G (2 ', 5') pG (3 ', 5') p] cyclic and O-substituted dithio ribose derivatives.
[0046] Fig. 5 represents [A (2 ', 5') pA (3 ', 5') p] cyclic and O-substituted dithio ribose derivatives.
[0047] Fig. 6 represents cyclic [G (2 ', 5') pA (3 ', 5') p] and O-substituted dithio ribose derivatives.
[0048] FIG. 7 depicts the production of type 1 interferon in THP-1 lymphocytes after stimulation with various cyclic dinucleotide molecules.
[0049] Fig. 8 depicts the normalized RNA expression levels of type 1 interferons and interferon gamma in human PBMC from independent donors after stimulation with various cyclic dinucleotide molecules.
[0050] Fig. 9 (A-C) depicts the levels of type 1 interferon alpha and beta protein and interferon gamma protein in human PBMC from independent donors after stimulation with various cyclic dinucleotide molecules.
[0051] FIG. 10 depicts the induction of IFN-p in human cells as a hallmark of adjuvant potency after treatment with various cyclic dinucleotide molecules.
[0052] Fig. 11 (a) - (c) depict the up-regulation of surface CD69 expression in natural killer (NK) lymphocytes, CD4 + T lymphocytes, and CD8 + T lymphocytes, respectively, as a measure of immune activation after treatment with various cyclic dinucleotide molecules.
[0053] Fig. 12 depicts the resistance of various CDN derivatives to phosphodiesterase treatment.
[0054] Fig. 13 depicts several known STING variants.
[0055] Fig. 14 depicts stimulation of HEK293 cell lines encoding alleles of human STING variants by measuring the IFNp-LUC reporter induction factor.
[0056] Fig. 15A depicts the surface expression of MHC class I (HLA-ABC), CD80, CD83 and CD86 by stimulated human dendritic cells.
[0057] Fig. 15B are representative histograms of the expression of CD80, CD86, CD83 and MHC class I (HLA-ABC) in human DC after stimulation with LPS or CDN.
[0058] Figure 16 depicts OVA-specific CD8 T cell immunity in PBMC in C57BL / 6 mice at 7 days after vaccination with cyclic dinucleotide-adjuvanted OVA protein.
[0059] Figure 17 depicts OVA-specific CD8 T cell immunity in PBMC in C57BL / 6 or goldentickt (STING - / -) mice at 7 days after vaccination with cyclic dinucleotide-adjuvanted OVA protein.
[0060] Fig. 18 depicts tumor volume in a B 16 melanoma model after treatment with various cyclic dinucleotide molecules.
[0061] Fig. 19 depicts survival curves in a CT26 tumor model after treatment with various cyclic dinucleotide molecules.
[0062] Fig. 20A depicts tumor inhibition in wild-type C57BL / 6 mice after administration of ML RR-CDN compared to control mice receiving HBSS and CpG dinucleotide.
[0063] Fig. 20B represents the results obtained in STING deficient mice.
[0064] Fig. 21A depicts the rejection of established CT26 colon carcinomas after ML RR-CDN administration.
[0065] Fig. 21B depicts IFN-y induction of ML RR-CDA treated mice.
[0066] Fig. 22A depicts rejection of established 4T1 mammary carcinomas after ML RR-CDN administration.
[0067] Fig. 22B depicts protection against re-challenge with CT26 tumor cells.
[0068] Fig. 23 depicts the inhibition of the primary tumor treated in animals bearing both CT26 (A) and 4T1 (B) tumors after administration of ML RR-CDA, compared to the HBSS vehicle control. Fig. 24A represents the inhibition of the primary tumor treated in melanoma B 16 after administration of ML RR-CDA.
[0069] Figs. 24B and C represent the inhibition of the growth of distal lung tumor ganglia after the administration of m L RR-CDA, in comparison with the HBSS vehicle control in graphic form (B) and in the lung tissue itself in photographic form ( C).
[0070] Detailed description of the invention
[0071] The present invention relates to the use of a new highly active cyclic dinucleotide (CDN) immune stimulator that activates DCs through a recently discovered cytoplasmic receptor known as STING (stimulator of interferon genes). In particular, the 2 ', 5', 3 ', 5' CDN of the present invention induces STING-dependent type I interferon production.
[0072] Recent insights into adjuvant design and development are based on a fundamental understanding that conserved microbial structures known as pathogen-associated molecular patterns (PAMPs) are detected by host cell pattern recognition receptors (PRRs), generating a downstream signaling cascade that leads to the induction of cytokines and chemokines, and the initiation of a specific adaptive immune response. The way in which the innate immune system is compromised by a microbe's complement PAMP shapes the development of an adaptive response that is appropriate to combat the invading disease-causing pathogen. One goal of adjuvant design is to select for defined PAMPs or specific synthetic molecules from designated PRRs to initiate a desired response. Adjuvants such as monophosphoryl lipid A (MPL) and CpG are PAMPs recognized by Toll-like receptors (TLRs), a class of transmembrane PRRs that signal through adapter molecules MyD88 and Trif, and mediate the induction of dependent pro-inflammatory cytokines. of NF-kB. MPL (TLR-4 agonist) and CpG (TLR-9 agonist) are clinically advanced adjuvants, and are components of FDA approved or pending approval vaccines. While TLRs present on the cell surface (e.g. TLR-4) and endosomes (e.g. CpG) detect extracellular and vacuolar pathogens, the productive growth cycle of multiple pathogens, including intracellular viruses and bacteria, occurs in the cytosol. The compartmentalisation of extracellular, vacuolar, and cytosolic PRRs has led to the hypothesis that the innate immune system distinguishes between pathogenic and nonpathogenic microbes by controlling the cytosol. It should be apparent to one of skill in the art that specific PRR agonists that comprise the cytosolic surveillance pathway initiate the development of protective immunity against intracellular pathogens, and this is relevant to vaccine design. These same targeting ligands will also be essential in the development of effective vaccines targeting malignant tumors, which are known to require tumor-specific CD4 + and CD8 + T cells.
[0073] Activation of the cytosolic surveillance pathway (CSP) is an integral part of the development of protective immunity towards intracellular pathogens. CSP detects bacteria, viruses, and protozoan pathogens, leading to activation of the TANK-binding kinase (TBK-1) / IRF-3 signaling axis and induction of IFN-p and others genes simultaneously regulated. Viral and bacterial nucleic acids activate this pathway, and IFN-p induction is independent of MyD88 and Trif. Although type I interferon is generally considered primarily as an antiviral host response, IFN-p induction is a hallmark of cytosolic growth in macrophages infected with the intracellular bacterium Listeria monocytogenes (Lm). A well-known dichotomy in the mouse listeriosis model is that, whereas wild-type Lm stimulates the potent immunity of CD4 and CD8 T cells that protects mice against bacterial challenge, Lm vaccination with removal of listeriolysin O (LLO) does not generate functional T lymphocytes or induce protective immunity. This difference is evidence of the need for host cell gene expression and cytosolic access by Lm to generate functional T-lymphocyte-mediated protective immunity. The level of IFN-p in infected host cells is regulated by Lm's multi-drug evacuation pumps (mDr), which secrete structurally unrelated small molecules, including antibiotics. IFN-p is not induced in host cells infected with Lm LLO mutants that are confined to the phagolysosome. Normal IFN-p levels are induced in infected MyD88- / 'T rif' macrophages deficient in all TLR-mediated signaling. These data demonstrate that although Lm involves TLRs, in response to infection with wild type Lm, host cell CSP is required for the development of protective immunity, correlated with IFN-p induction.
[0075] Cyclic dinucleotides (CDNs) activate the cytosolic surveillance pathway through direct binding to the cytosolic PRR, STING. The response of type I interferon to infection by Lm and other intracellular bacteria is due to the secretion of cyclic di-AMP or its related cyclic dinucleotide (CDN), cyclic di-GMP, and its direct binding to DDX41 and box helicase DEAD (aspartate-glutamate-alanine-aspartate) and STING (stimulator of interferon genes), a newly defined receptor of the cytosolic surveillance pathway. CDNs are second messengers expressed by most bacteria, and they regulate various processes, including motility and biofilm formation. In addition to activating the TBK-1 / IRF-3 signaling pathway, in response to binding to CDNs, STING also activates the IkB kinase, leading to the translocation of the transcription factor NF-kB to the nucleus, activating the expression of multiple pro-inflammatory genes.
[0077] Until recently, it was unknown how STING detects cytoplasmic DNA. Unlike AIM2, which binds directly to dsDNA, STING lacks obvious DNA-binding domains. It was unknown whether other candidate DNAs, such as DDX41, DNA-PK, and DAI kinase, were essential mediators of dsDNA signaling through STING. This puzzle was solved with the discovery of cyclic GMP-AMP synthase (cGAS), a host cell nucleotidyl transferase that, in response to dsDNA binding, synthesizes a second messenger, di-GMP-cyclic AMP, which binds directly to STINg and initiates a signaling cascade through the TBK-1 / IRF-3 axis, leading to IFN induction. In addition, the cGAS innate immune DNA detector produces a non-canonical cyclic dinucleotide that activates STING signaling. Unlike the cyclic dinucleotide second messenger produced by bacteria, in which the internucleotide phosphate bridge is linked by bis- (3 ', 5') bonds, the GMP-cyclic AMP phosphate internucleotide bridge synthesized by cGAS is bound by non-canonical 2 ', 5' and 3 ', 5' bonds, represented by c [G (2 ', 5') pA (3 ', 5') p] -. Thus, STING (stimulator of interferon genes) has become a central pathway to detect pathogenic cytosolic nucleic acids, either through the direct binding of cyclic dinucleotides (CDN) secreted by intracellular bacteria6 or through the binding of a second c-GMP-AMP messenger, synthesized by cyclic GMP-AMP synthase (cGAS) of the host cell in response to the binding of cytosolic pathogenic nucleic acids.
[0079] Native CDN molecules are sensitive to degradation by phosphodiesterases that are present in host cells, for example antigen presenting cells, taking vaccine formulations containing such native CDN molecules. The potency of a defined adjuvant may be diminished by such degradation, since the adjuvant could not bind and activate its defined PRR target. A lower adjuvant potency could be measured, for example, by a lower amount of induced expression of a characteristic molecule of innate immunity (for example, IFN-p), correlated with a weaker vaccine potency, as defined by the magnitude of a measured antigen-specific immune response.
[0081] In the present invention, a dithio diphosphate derivative of 2 ', 5', 3 ', 5'-c-di-AMP is provided. The synthesis process for said dithio-diphosphate derivative produces a mixture of diastereomers, including Rp, Rp, Sp, Sp, SpRp and Rp, Sp. These individual species can be separated and present substantial differences in their pharmaceutical characteristics. The compound of the present invention is Rp, Rp.
[0083] Definitions
[0085] "Administration", as used herein with respect to a human, mammal, mammalian subject, animal, veterinary subject, placebo subject, research subject, experimental subject, cell, tissue, organ or biological fluid, refers to, without limitation, upon contact of an exogenous ligand, reagent, placebo, small molecule, pharmaceutical agent, therapeutic agent, diagnostic agent, or composition with the biological subject, cell, tissue, organ, or fluid, and the like. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. Treatment of a cell encompasses the contact of a reagent with the cell, as well as the contact of a reagent with a fluid, where the fluid is in contact with the cell. "Administration" also encompasses in vitro and ex vivo treatments, for example, of a cell, by a reagent, a diagnostic, a binding composition or another cell. By "managed together" it is not meant that two or more agents are administered as a single composition. Although the present invention contemplates administration as a single single composition, said agents can be administered to a single subject as separate administrations, which can be at the same time or at different times, and which can be by the same route or different routes of administration. .
[0087] An "agonist" as regards a ligand and a receptor, comprises a molecule, a combination of molecules, a complex or a combination of reagents, that stimulates the receptor. For example, a granulocyte macrophage colony stimulating factor (GM-CSF) agonist can encompass GM-CSF, a mutein or derivative of GM-CSF, a GMCSF mimetic peptide, a small molecule that mimics the biological function of GM-CSF or an antibody that stimulates the GM-CSF receptor.
[0089] An "antagonist", as it relates to a ligand and a receptor, comprises a molecule, a combination of molecules or a complex, which inhibits, counteracts, down-regulates and / or desensitizes the receptor. "Antagonist" encompasses any reagent that inhibits a constitutive activity of the receptor. A constitutive activity is one that manifests itself in the absence of an interaction between ligand and receptor. "Antagonist" also encompasses any reagent that inhibits or prevents a stimulated (or regulated) activity of a receptor. By way of example, a GM-CSF receptor antagonist includes, without limitation, an antibody that binds to the ligand (GM-CSF) and prevents it from binding to the receptor, or an antibody that binds to the receptor and prevents where the ligand binds to the receptor, or where the antibody blocks the receptor in an inactive configuration.
[0091] By "substantially purified" with respect to the NDCs it is meant that a specific species represents at least 50%, more often represents at least 60%, usually represents at least 70%, more usually represents at least 75 %, most usually represents at least 80%, usually represents at least 85%, most generally represents at least 90%, most generally represents at least 95%, and conventionally represents at least 98% in weight, or more, of CDN activity present in a composition. The weights of water, buffers, salts, detergents, reducers, protease inhibitors, stabilizers (including an added protein such as albumin), and excipients, in general, are not used in determining purity.
[0092] Which "specifically" or "selectively" binds, referring to a ligand / receptor, nucleic acid / complementary nucleic acid, antibody / antigen, or other binding pair (eg, a cytokine to a cytokine receptor) (termed, in In general, each "target biomolecule" or "target") indicates a binding reaction related to the presence of the target in a heterogeneous population of proteins and other biological products. Specific binding may mean, for example, that the binding compound, nucleic acid ligand, antibody, or binding composition derived from the antigen-binding site of an antibody, of the contemplated method binds to its target with a affinity which is usually at least 25% higher, more usually at least 50% higher, most normally at least 100% (2 times) higher, usually at least ten times higher, more usually at least 20 times higher and more usually at least 100 times higher than the affinity for a non-target molecule.
[0094] "Ligand" refers to a small molecule, nucleic acid, peptide, polypeptide, saccharide, polysaccharide, glycan, glycoprotein, glycolipid, or combinations thereof that bind to a target biomolecule. While such ligands can be agonists or antagonists of a receptor, a ligand also encompasses a binding agent that is not an agonist or antagonist, and does not have agonist or antagonist properties. The specific binding of a ligand to its cognate target is often expressed in terms of an "affinity". In preferred embodiments, the ligands of the present invention bind with affinities between about 10 ⁴ M ^'1 and about 10 ⁸ M ^-1 . Affinity is calculated as Kd = koff / kon (koff is the dissociation rate constant, Kon is the association rate constant, and Kd is the equilibrium constant).
[0096] Affinity can be determined at equilibrium by measuring the bound fraction (r) of the labeled ligand at various concentrations (c). The data are represented using the Scatchard equation: r / c = K (nr): where r = moles of bound ligand / mole of receptor at equilibrium; c = concentration of free ligand at equilibrium; K = equilibrium association constant; and n = number of ligand binding sites per receptor molecule. By graphical analysis, r / c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. The measurement of affinity by Scatchard analysis is well known in the art. See, for example, van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988. In an alternative, affinity can be measured by isothermal titration calorimetry (ITC). In a typical ITC experiment, a solution of ligand is titrated in a solution of its cognate target. The heat released after their interaction (HA) is monitored over time. As successive amounts of the ligand are titrated in the ITC cell, the amount of heat absorbed or released is directly proportional to the amount of binding. As the system reaches saturation, the heat signal decreases until only heats of dilution are observed. A binding curve is then obtained from a plot of the heats of each injection versus the ratio of ligand and binding partner in the cell. The binding curve is analyzed with the appropriate binding model to determine Kb, n and HA. It should be noted that Kb = 1 / Kd.
[0098] The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both diseases human and veterinary. In certain embodiments, the subjects are "patients," that is, living human beings receiving medical care for a disease or condition. This includes people without defined disease who are being investigated for signs of pathology. Subjects having an existing diagnosis of a particular cancer to which the compositions and methods of the present invention are directed are preferred. Preferred cancers for treatment with the compositions described herein include, but are not limited to, prostate cancer, renal carcinoma, melanoma, pancreatic cancer, cervical cancer, ovarian cancer, colon cancer, head cancer, and neck, lung and breast cancer.
[0100] "Therapeutically effective amount" is defined as an amount of a reagent or a pharmaceutical composition that is sufficient to show a benefit to the patient, that is, to cause a decrease, prevention or amelioration of the symptoms of the condition being treated. . When the agent or pharmaceutical composition comprises a diagnostic agent, a "diagnostic effective amount" is defined as an amount that is sufficient to produce a diagnostic signal, image, or other parameter. The effective amounts of the pharmaceutical formulation will vary according to factors such as the degree of susceptibility of the individual, the age, gender and weight of the individual, and idiosyncratic responses of the individual. "Effective amount" encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, prevent or diagnose a symptom or sign of a medical condition or disorder, or a causal process thereof. Unless otherwise stated, explicitly or by context, an "effective amount" is not limited to a minimal amount sufficient to ameliorate a condition.
[0102] "Treatment" or "treat" (with respect to a condition or disease) is an approach to obtaining beneficial or desired results that preferably include clinical results. For the purposes of the present invention, beneficial or desired outcomes with respect to a disease include, but are not limited to, one or more of the following: prevention of a disease, amelioration of a condition associated with a disease, cure of a disease, decrease in the severity of a disease, delay the progression of a disease, alleviate one or more symptoms associated with a disease, increase the quality of life of a person suffering from a disease and / or prolong survival. Similarly, for the purposes of the present invention, beneficial or desired outcomes with respect to a condition include, but are not limited to, one or more of the following: prevention of a condition, amelioration of a condition, cure of a condition, decrease in the severity of a condition, delay the progression of a condition, alleviate one or more symptoms associated with a condition, increase the quality of life of a person suffering from a condition, and / or prolong survival. For example, in embodiments in which the compositions described herein are used for the treatment of cancer, beneficial or desired results include, but are not limited to, one or more of the following: reduction of proliferation (or destruction) of neoplastic or cancer cells, reduction of metastasis of neoplastic cells found in cancers, reduction in the size of a tumor, decreased symptoms resulting from cancer, increased quality of life for cancer sufferers, reduced dose from other medications necessary to treat disease, delay cancer progression and / or prolong survival of cancer patients. Depending on the context, the "treatment" of a subject may imply that the subject is in need of treatment, eg, in the situation where the subject comprises a disorder that is expected to be ameliorated by administration of a reagent.
[0104] "Vaccine" encompasses preventive vaccines. The vaccine also encompasses therapeutic vaccines, eg, a vaccine administered to a mammal comprising a condition or disorder associated with the antigen or epitope provided by the vaccine.
[0106] Cyclic purine dinucleotides
[0108] Prokaryotic cells and eukaryotic cells use different small molecules for cell signaling and intracellular and intercellular communication. Cyclic nucleotides such as cGMP, cAMP, etc. they are known to have regulatory and initiating activity in prokaryotic and eukaryotic cells. Unlike eukaryotic cells, prokaryotic cells also use cyclic purine dinucleotides as regulatory molecules. In prokaryotic cells, the condensation of two GTP molecules is catalyzed by the enzyme diguanylate cyclase (DGC), giving cyclic diGMP, which represents an important regulator in bacteria.
[0110] Recent work suggests that cyclic diGMP or its analogs may also stimulate or enhance the immune or inflammatory response in a patient, or may enhance the immune response to a vaccine by serving as an adjuvant in mammals. Cytosolic detection of pathogen-derived Dn requires signaling through the binding kinase TANK 1 (TBK1) and its downstream transcription factor, IFN regulatory factor 3 (IRF3). A transmembrane protein called STING (IFN gene stimulator; also known as MITA, ERIS, MPYS and TMEM173) functions as the signaling receptor for these purine cyclic dinucleotides, generating stimulation of the TBK1-IRF3 signaling axis and a response of STING-dependent type I interferon. See, for example, Fig. 1. Burdette et al., Nature 478: 515-18, 2011 demonstrated that STING binds directly to cyclic diguanylate monophosphate, but not to other unrelated nucleotides or nucleic acids.
[0112] Cyclic purine dinucleotides for use as precursors to obtain the CDN of the present invention are described in some detail in, for example, Gao et al., Cell (2013) 153: 1094-1107, doi: 10.1016 / j .cell.2013.04.046; US Patent Nos. 7,709,458 and 7,592,326; WO2007 / 054279; and Yan et al., Bioorg. Med. Chem Lett. 18: 5631 (2008). These CDNs can be modified using conventional organic chemistry techniques to produce the CDN of the present invention. The CDN of the present invention is a phosphorothioate. Phosphorothioates are a variant of normal nucleotides in which one of the free oxygen is replaced by sulfur. Sulfidation of the internucleotide bond dramatically reduces the action of endonucleases and exonucleases, including DNA exonuclease POL 1 5 'to 3' and 3 'to 5', S1 and PI nucleases, RNases, serum nucleases and phosphodiesterase of snake venom. In addition, it increases the potential to cross the lipid bilayer.
[0114] A phosphorothioate bond is inherently chiral. The person skilled in the art will recognize that phosphates of this structure may exist in R or S form. Thus, Rp, Rp, Sp, Sp, Sp, Rp and Rp, Sp forms are possible.
[0116] The CDN compositions described herein can be administered to a host, either alone or in combination with a pharmaceutically acceptable excipient, in an amount sufficient to induce, modify or stimulate an appropriate immune response. The immune response may comprise, without limitation, specific immune response, non-specific immune response, specific and non-specific response, innate response, primary immune response, adaptive immunity, secondary immune response, memory immune response, immune cell activation, proliferation of immune cells, immune cell differentiation, and cytokine expression. In certain embodiments, the CDN compositions are administered in conjunction with one or more additional compositions that include vaccines designed to stimulate an immune response to one or more predetermined antigens; adjuvants; CTLA-4 and PD-1 pathway antagonists, lipids, liposomes, chemotherapeutic agents, immunomodulatory cell lines, etc.
[0118] The CDN compositions can be administered before, after and / or in conjunction with an additional therapeutic or prophylactic composition or modality. These include, without limitation, costimulatory molecule B7, interleukin-2, interferon, and GM-CSF, CTLA-4 antagonists, OX-40 / OX-40 ligand, CD40 / CD40 ligand, sargramostim, levamisole, vaccinia, Bacillus Calmette-Guerin (BCG), liposomes, alum, Freund's complete or incomplete adjuvant, detoxified endotoxins, mineral oils, surfactants such as lipolecithin, pluronic polyols, polyanions, peptides, and oil or hydrocarbon emulsions. Vehicles for inducing a T cell immune response that preferentially stimulate a killer T cell response over an antibody response are preferred, although those that stimulate both types of response may also be used. In cases where the agent is a polypeptide, the polypeptide itself or a polynucleotide encoding the polypeptide can be administered. The vehicle can be a cell, such as an antigen presenting cell (APC) or a dendritic cell. Antigen-presenting cells include cell types such as macrophages, dendritic cells, and B lymphocytes. Other professional antigen-presenting cells include monocytes, marginal zone Kupffer cells, microglia, Langerhans cells, interdigitating dendritic cells, follicular dendritic cells, and lymphocytes. T. Facultative antigen-presenting cells can also be used. Examples of facultative antigen-presenting cells include astrocytes, follicular cells, endothelium, and fibroblasts. The vehicle can be a bacterial cell that is transformed to express the polypeptide or to deliver a polynucleotide that is subsequently expressed in cells of the vaccinated individual. Adjuvants, such as aluminum hydroxide or aluminum phosphate, can be added to increase the ability of the vaccine to generate, enhance, or prolong an immune response. Other additional materials, such as cytokines, chemokines, and bacterial nucleic acid sequences, such as CpG, a Toll-like receptor (TLR) 9 agonist, as well as additional agonists for TLR 2, TLR 4, TLR 5, TLR 7, TLR 8 , TLR9, including lipoprotein, LPS, monophosphoryl lipid A, lipoteichoic acid, imiquimod, resiquimod, and in addition, retinoic acid inducible gene I (RIG-I) agonists such as poly I: C, used alone or in combination with the Described compositions are also possible adjuvants. Other representative examples of adjuvants include synthetic adjuvant QS-21 which comprises a homogeneous saponin purified from the bark of Quillaja saponaria and Corynebacterium parvum (McCune et al., Cancer, 1979; 43: 1619). It will be understood that the adjuvant is subject to optimization. In other words, the person skilled in the art can participate in routine experiments to determine the best adjuvant to use.
[0120] Methods for co-administration with an additional therapeutic agent are well known in the art (Hardman, et al. (Eds.), (2001) "Goodman and Oilman's The Pharmacological Basis of Therapeutics", 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) "Pharmacotherapeutics for Advanced Practice: A Practical Approach", Lippincott, Williams & Wilkins, Philadelphia, PA; Chabner and Longo (eds.) (2001) "Cancer Chemotherapy and Biotherapy ", Lippincott, Williams & Wilkins, Philadelphia., PA).
[0122] Due to the adjuvant properties of the compound of the present invention, its use can also be combined with other therapeutic modalities, including other vaccines, adjuvants, antigen, antibodies, and immune modulators. Examples are provided below.
[0124] Adjuvants
[0126] In addition to the cyclic purine dinucleotide described above, the compositions of the present invention may further comprise one or more additional substances which, by their nature, may act to stimulate or otherwise utilize the immune system to respond to cancer antigens present in the tumor cell / s is / are inactivated. Such adjuvants include, but are not limited to, lipids, liposomes, inactivated bacteria that induce innate immunity (e.g., inactivated or attenuated Listeria monocytogenes ), compositions that mediate Innate immune activation through Toll-like receptors (TLR), type-I receptors (NOD) (NLR), retinoic acid-inducible gene-based (RIG) type I receptors (RLR) and / or lectin-like receptors C (CLR). Examples of PAMPs include lipoproteins, lipopolypeptides, peptidoglycans, zymosan, lipopolysaccharide, Neisseria porins , flagellin, profilin, galactoceramide, muramil dipeptide. Peptidoglycans, lipoproteins, and lipoteichoic acids are components of the cell wall of Gram-positive organisms. Lipopolysaccharides are expressed by most bacteria, MPL being an example. Flagellin refers to the structural component of bacterial flagella that is secreted by pathogenic and commensal bacteria. Α-Galactosylceramide (α-GalCer) is an activator of natural killer T lymphocytes (NKT). Muramil dipeptide is a bioactive peptidoglycan motif common to all bacteria. This list is not intended to be limiting. The preferred adjuvant compositions are described below.
[0127] CTLA-4 and PD-1 pathway antagonists
[0128] CTLA-4 is believed to be an important negative regulator of the adaptive immune response. Activated T lymphocytes positively regulate CTLA-4, which binds to CD80 and CD86 on antigen presenting cells with higher affinity than CD28, thus inhibiting T lymphocyte stimulation, IL-2 gene expression, and proliferation of T lymphocytes. Antitumor effects of CTLA4 blockade have been observed in murine models of colon carcinoma, metastatic prostate cancer, and metastatic melanoma.
[0129] Ipilimumab (Yervoy ™) and tremelimumab are humanized monoclonal antibodies that bind to human CTLA4 and prevent its interaction with CD80 and CD86. Phase I and II studies with ipilimumab and tremelimumab have shown clinical activity in cancer patients. Other negative immune regulators that can be targeted by a similar strategy include programmed cell death 1, B and T lymphocyte attenuator, transforming growth factor beta p, interleukin-10, and vascular endothelial growth factor.
[0130] PD-1 is another negative regulator of the adaptive immune response that is expressed on activated T lymphocytes. PD-1 binds to B7-H1 and B7-DC, and PD-1 binding suppresses T-cell activation. Anti-tumor effects have been demonstrated with blocking of the PD-1 pathway. BMS-936558, MK3475, CT-011, AMP-224 and MDX-1106 have been presented in the literature as examples of blockers of the PD-1 pathway that may find use in the present invention. TLR agonists
[0131] The term "Toll-like receptor" (or "TLR"), as used herein, refers to a member of the family of Toll-like receptor proteins or a fragment thereof that detects a microbial product and / or or initiates an adaptive immune response. In one embodiment, a TLR activates a dendritic cell (DC). Toll-like receptors (TLRs) are a family of pattern recognition receptors that were initially identified as detectors of the innate immune system that recognize microbial pathogens. TLRs comprise a family of molecules spanning conserved membranes that contain a leucine-rich repeat ectodomain, a transmembrane domain, and an intracellular TIR domain (Toll / IL-1R). TLRs recognize different structures in microbes, which are often referred to as "PAMPs" (pathogen-associated molecular patterns). The binding of the ligand to TLRs generates a cascade of intracellular signaling pathways that induce the production of factors involved in inflammation and immunity.
[0132] In humans, ten TLRs have been identified. TLRs that are expressed on the surface of cells include TLR-1, -2, -4, -5, and -6, while TLR-3, -7/8, and -9 are expressed with the ER compartment. Human dendritic cell subsets can be identified based on different TLR expression patterns. By way of example, the myeloid or "conventional" subset of DCs (mDCs) expresses TLR 1-8 when stimulated, and a cascade of activation markers occurs (eg, CD80, CD86, MHC class I and II , CCR7), proinflammatory cytokines and chemokines. One result of this stimulation and the resulting expression is the stimulation of antigen-specific CD4 + and CD8 + T lymphocytes. These DCs acquire a greater capacity to capture antigens and present them adequately to T lymphocytes. In contrast, the plasmacytoid subset of DC (pDC) only expresses TLR7 and TLR9 after activation, with a resulting activation of n K lymphocytes, thus as from T lymphocytes. As dying tumor cells may adversely affect DC function, it has been suggested that activation of DCs with TLR agonists may be beneficial in stimulating antitumor immunity in an immunotherapy approach to cancer treatment. . It has also been suggested that successful treatment of breast cancer using radiation and chemotherapy requires activation of TLR4.
[0133] TLR agonists known in the art and finding use in the present invention include, but are not limited to, the following:
[0134] Pam3Cys, a TLR-1/2 agonist;
[0135] CFA, a TLR-2 agonist;
[0136] MALP2, a TLR-2 agonist;
[0137] Pam2Cys, a TLR-2 agonist;
[0138] FSL-1, a TLR-2 agonist;
[0139] Hib-OMPC, a TLR-2 agonist;
[0140] Polyribosinic acid: polyribocytidic (Poly I: C), an agonist of TLR-3;
[0141] polyadenosine-polyuridyl acid (poly AU), an agonist of TLR-3;
[0142] poly-L-lysine and carboxymethylcellulose stabilized polycinosinic-polycytidylic acid (Hiltonol®), a TLR-3 agonist;
[0143] monophosphoryl lipid A (MPL), a TLR-4 agonist;
[0144] LPS, a TLR-4 agonist; bacterial flagellin, a TLR-5 agonist;
[0145] sialyl-Tn (STn), a carbohydrate associated with the MUC1 mucin in a series of human cancer cells and a TLR-4 agonist;
[0146] imiquimod, a TLR-7 agonist;
[0147] resiquimod, a TLR-7/8 agonist;
[0148] loxoribine, a TLR-7/8 agonist; and
[0149] Unmethylated CpG dinucleotide (CpG-ODN), an agonist of TLR-9.
[0150] Due to their adjuvant qualities, TLR agonists are preferably used in combinations with other vaccines, adjuvants, and / or immune modulators, and can be combined in different combinations. Thus, in certain embodiments, the cyclic purine dinucleotide that binds to STING and induces STING-dependent activation of TBK1 and an inactivated tumor cell that expresses and secretes one or more cytokines that stimulate induction, recruitment, and / or Dendritic cell maturation, as described herein, can be administered in conjunction with one or more TLR agonists for therapeutic purposes.
[0151] Antibody therapeutic agents
[0152] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a cell-mediated immune defense mechanism whereby an effector cell of the immune system actively clears a target cell, the membrane surface antigens of which have been bound by specific antibodies. It is one of the mechanisms through which antibodies, as part of the humoral immune response, can act to limit and contain the infection. Classic ADCC is mediated by natural killer (NK) lymphocytes; macrophages, neutrophils, and eosinophils can also mediate ADCC. ADCC is an important mechanism of action of therapeutic monoclonal antibodies, including trastuzumab and rituximab, against tumors. The compounds of the present invention can act to enhance ADCC.
[0153] The following is an illustrative list of antibodies that can be used in conjunction with the compound of the present invention. Muromonab-CD3 - used to prevent acute organ rejection, eg kidney, transplants. Humanized versions show promise for inhibiting autoimmune beta cell destruction in type 1 diabetes mellitus.
[0154] Infliximab (Remicade®) and adalimumab (Humira®): bind tumor necrosis factor alpha (TNF-a). They are used in some inflammatory diseases such as rheumatoid arthritis, psoriasis, Crohn's disease.
[0155] Omalizumab (Xolair®). It binds to IgE thus preventing IgE from binding to mast cells. It is used against allergic asthma. Daclizumab (Zenapax®). It binds to part of the IL-2 receptor exposed on the surface of activated T lymphocytes. It is used to prevent acute rejection of transplanted kidneys.
[0156] Rituximab (trade name = Rituxan®). It binds to the CD20 molecule found on most B lymphocytes and is used to treat B lymphocyte lymphomas.
[0157] Ibritumomab (trade name = Zevalin®). This is a monoclonal antibody against the CD20 molecule in B lymphocytes (and lymphomas) conjugated to isotopes. It is administered to the patient with lymphoma supplemented with Rituxan.
[0158] Tositumomab (Bexxar®). It is a conjugate of a monoclonal antibody against CD20 and the radioactive isotope iodine-131 (131I).
[0159] Cetuximab (Erbitux®). It blocks HER1, an epidermal growth factor (EGF) receptor found on some tumor cells (some breast cancers, lymphomas).
[0160] Trastuzumab (Herceptin®). It blocks HER2, an overexpressed growth factor receptor in approximately 20% of breast cancers.
[0161] Adcetris®. A conjugate of a monoclonal antibody that binds to CD30, a cell surface molecule expressed by the cells of some lymphomas, but not found in normal stem cells required to repopulate the bone marrow.
[0162] Alemtuzumab (Campath-1H®). It binds to CD52, a molecule found on lymphocytes and depletes T lymphocytes and B lymphocytes. It has produced a complete remission of chronic lymphocytic leukemia and shows promise in preventing the rejection of kidney transplants.
[0163] Lym-1 (Oncolym®). It binds to the HLA-DR encoded histocompatibility antigen that can be expressed at high levels on lymphoma cells.
[0164] Ipilimumab (Yervoy®) which works to boost the body's immune response to tumors.
[0165] Vitaxin. It binds to a vascular integrin (alpha-v / beta-3) that is found in the blood vessels of tumors, but not in the blood vessels that supply normal tissues. In phase II clinical trials, Vitaxin has shown promise in shrinking solid tumors without harmful side effects.
[0166] Bevacizumab (Avastin®). It binds to vascular endothelial growth factor (VEGF) preventing it from binding to its receptor. It is used to treat colorectal cancers.
[0167] Abciximab (ReoPro®). It inhibits the clumping of platelets by binding to receptors on their surface that are normally bound by fibrinogen. Useful in the prevention of re-obstruction of the coronary arteries in patients undergoing angioplasty.
[0168] Management agents
[0169] Liposomes are vesicles formed from one ("unilamellar") or more ("multilamellar") layers of phospholipids. Due to the amphipathic character of the phospholipid building blocks, liposomes typically comprise a hydrophilic layer having a hydrophilic outer face and enclosing a hydrophilic core. The versatility of liposomes in incorporating hydrophilic / hydrophobic components, their non-toxic nature, biodegradability, biocompatibility, adjuvance, induction of cellular immunity, property of sustained release and prompt absorption by macrophages, make them attractive candidates. for the administration of antigens.
[0170] WO2010 / 104833 describes suitable liposomal preparations. Said liposomal formulations, referred to herein as VesiVax® (Molecular Express, Inc.), with or without the aforementioned "immunogenic polypeptide (s) or carbohydrate (s)", may contain one or more additional components such as peptidoglycan, lipopeptide, lipopolysaccharide, monophosphoryl lipid A, lipoteichoic acid, resiquimod, imiquimod, flagellin, oligonucleotides containing unmethylated CpG motifs, beta-galactosylceramide, muramyl dipeptide, all-trans-retinoic acid, double-stranded viral RNA, heat shock proteins, bromide proteins of dioctadecyldimethylammonium, cationic surfactants, Toll-like receptor agonists, dimyristoyltrimethylammoniopropane, and Nod-like receptor agonists. Advantageously, these liposomal formulations can be used to deliver the CDN in accordance with the present invention.
[0171] Furthermore, while the liposomal formulations discussed above employ a "steroid derivative" as an anchor to attach an immunogenic carbohydrate or polypeptide to a liposome, the steroid can simply be provided as an unconjugated steroid such as cholesterol.
[0172] Suitable methods for preparing liposomes from lipid mixtures are well known in the art. See, for example, Basu and Basu, "Liposome Methods and Protocols (Methods in Molecular Biology)", Humana Press, 2002; Gregoriadis, "Liposome Technology", 3rd Edition, Informa Healthcare, 2006. Preferred methods include extrusion, homogenization and sonication methods described in those references. An illustrative method of preparing liposomes for use in the present invention, which comprises drying a mixture of lipids, followed by hydration in an aqueous vehicle and sonication to form liposomes, is described in WO2010 / 104833.
[0173] In certain embodiments, the liposomes are provided within a particular average size range. The size of the liposomes can be selected, for example, by extruding an aqueous vehicle comprising liposomes through membranes having a preselected pore size and collecting material that flows through the membrane. In preferred embodiments, the liposomes are selected to be essentially between 50 and 500 nm in diameter, more preferably essentially between 50 and 200 nm in diameter, and most preferably essentially between 50 and 150 nm in diameter. The term "essentially", as used herein in this context, means that at least 75%, more preferably 80%, and most preferably at least 90% of the liposomes are within the designated range. .
[0174] Other lipids and lipid-like adjuvants that may find use in the present invention include oil-in-water (o / w) emulsions (see, for example, Muderhwa et al., J. Pharmaceut. Sci. 88: 1332-9, 1999 )), TLR VesiVax® (Molecular Express, Inc.), digitonin (see, for example, U.S. Patent No. 5,698,432), and glucopyranosyl lipids (see, for example, U.S. Patent Application USA No. 20100310602).
[0175] Nanoparticles also represent drug delivery systems suitable for most routes of administration. Over the years, various natural and synthetic polymers have been explored for the preparation of nanoparticles, of which poly (lactic acid) (PLA), poly (glycolic acid) (p Ga) and their copolymers (PLGA) have been extensively investigated due to its biocompatibility and biodegradability. Nanoparticles and other nanocarriers act as potential carriers for various classes of drugs, such as anticancer agents, antihypertensive agents, immunomodulators, and hormones; and macromolecules such as nucleic acids, proteins, peptides, and antibodies. See, for example, Crit. Rev. Ther. Drug Carrier Syst. 21: 387-422, 2004; "Nanomedicine: Nanotechnology, Biology and Medicine" 1: 22-30, 2005.
[0177] Chemotherapeutic agents
[0179] In additional embodiments, the methods further involve administering to the subject an effective amount of one or more chemotherapeutic agents as additional treatment. In certain embodiments, the one or more chemotherapeutic agents are selected from abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro-W- (3-fluoro-sulfonamide -4-methoxyphenyl) benzene, bleomycin, W, W-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proli-1-L-proline-t-butylamide, cachectin, cemadotin, chlorambucil , cyclophosphamide, 3 ', 4'-didehydro-4'-deoxy-8'-norvin-caleucoblastine, docetaxol, docetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazin (DTIC), dactorhomycin decitabine dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechlorethamine nitrogen, ismechlorethamine, mivobelfine mustard, nitrogen mustardine sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, onapriston a, paclitaxel, prednimustine, procarbazine, RPR109881, estramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine.
[0181] Immunomodulatory cell lines
[0183] By "inactivated tumor cell" is meant a tumor cell (either "autologous" or "allogeneic" to the patient) that has been treated to prevent cell division. For the purposes of the present invention, said cells preserve their immunogenicity and metabolic activity. Said tumor cells are genetically modified to express a transgene that is expressed within a patient as part of cancer therapy. Thus, a composition or vaccine of the invention comprises neoplastic (eg, tumor) cells that are autologous or allogeneic to the patient undergoing treatment, most preferably being the same general type of tumor cell that affects the patient. For example, a patient suffering from melanoma will normally be given a genetically modified cell derived from a melanoma. Tumor cell inactivation methods for use in the present invention, such as the use of irradiation, are well known in the art.
[0185] The inactivated tumor cells of the present invention are administered to the patient in conjunction with one or more costimulatory agents or molecules. A preferred costimulatory agent comprises one or more cytokines that stimulate dendritic cell induction, recruitment and / or maturation. The methods of evaluating such costimulatory agents are well known in the literature. The induction and maturation of DC is normally assessed by increasing the expression of certain membrane molecules such as CD80 and CD86, and / or the secretion of pro-inflammatory cytokines, such as IL-12 and type I interferons upon stimulation. .
[0187] In preferred embodiments, the inactivated tumor cells are modified to express and secrete one or more cytokines that stimulate dendritic cell induction, recruitment, and / or maturation. The present invention is described in illustrative terms with respect to the use of GM-CSF. Thus, by way of example, the tumor cell may express a transgene encoding GM-CSF as described in US Patent Nos. 5,637,483, 5,904,920, 6,277,368, and 6,350,445. , as well as in US Patent Publication No. 20100150946. In US Patent Nos. 6,033,674 and 5,985,290, a form of genetically modified cancer cells expressing GM-CSF or a "cellular vaccine expressing cytokines" is described for the treatment of cancer of pancreas.
[0189] Other suitable cytokines that may be expressed by such inactivated tumor cells and / or control cells in place of, or in conjunction with, GM-CSF include, but are not limited to, one or more of CD40, IL-12, CCL3, CCL20, and ligand CCL21. This list is not intended to be limiting.
[0191] While it is preferred that the inactivated tumor cells administered to the subject express one or more cytokines of interest, the tumor cell line may be accompanied by an inactivated control cell line that expresses and secretes one or more cytokines that stimulate induction, recruitment, and / or or dendritic cell maturation. The control cell line can provide all the cytokines that stimulate the induction, recruitment and / or maturation of dendritic cells, or it can complement the cytokines that stimulate the induction, recruitment and / or maturation of dendritic cells expressed and secreted by the cells. inactivated tumor cells. By way of example, Control cell lines expressing immunomodulatory cytokines are described in US Patent Nos. 6,464,973 and 8,012,469, Dessureault et al., Ann. Surg. Oncol. 14: 869-84, 2007, and Eager and Nemunaitis, Mol. Ther. 12: 18-27, 2005.
[0193] By "granulocyte-macrophage colony-stimulating factor (GM-CSF) polypeptide" is meant a cytokine or fragment thereof that has immunomodulatory activity and has at least about 85% amino acid sequence identity with the n GenBank Registration No. AAA52122.1.
[0194] Vaccinations
[0196] In certain embodiments, the CDN compositions are administered in conjunction with one or more vaccines intended to stimulate an immune response to one or more predetermined antigens. Examples of target antigens that may find use in the invention are listed in the following table. The target antigen can also be a fragment or a fusion polypeptide that comprises an immunologically active part of the antigens listed in the table. This list is not intended to be limiting.
[0198] Table 1. Antí enos.
[0201] (continuation)
[0202]
[0203] (continuation)
[0204]
[0205] (continuation)
[0206]
[0207] (continuation)
[0208]
[0209] (continuation)
[0210]
[0211] (continuation)
[0212]
[0213] (continuation)
[0214]
[0215] (continuation)
[0220] Other organisms for which suitable antigens are known in the art include, but are not limited to, Chlamydia trachomatis, Streptococcus pyogenes (Group A Srep), Streptococcus agalactia (Group B Strep), Streptococcus pneumonia, Staphylococcus aureus, Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrheae, Vibrio cholerae, Salmonella species (including typhi, typhimurium), enterica ( including Helicobactor pylori Shigella flexneri and other group D shigella species), Burkholderia mallei, Burkholderia, Kseudomallei species Closesomallei (including C. difficile), Vibrio parahaemolyticus, and V. vulnificus. This list is not intended to be limiting.
[0222] Pharmaceutical compositions
[0224] The term "pharmaceutical", as used herein, refers to a chemical substance intended for use in the cure, treatment or prevention of disease, and which is subject to an approval process by the Administration of US Food and Drug Administration (or a non-US equivalent thereof) as a prescription or over-the-counter drug product. Details on techniques for the formulation and administration of such compositions can be found in Remington, "The Science and Practice of Pharmacy" 21st edition (Mack Publishing Co., Easton, PA), and Nielloud and Marti-Mestres, "Pharmaceutical Emulsions and Suspensions ": 2nd edition (Marcel Dekker, Inc, New York).
[0226] For the purposes of the present disclosure, the pharmaceutical compositions can be administered by various means, including orally, parenterally, by inhalation spray, topical, or rectally in formulations containing pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral, as used herein, includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarterial, intradermal, intrathecal, and epidural injection with various infusion techniques. Intra-arterial and intravenous injection, as used herein, includes administration through catheters. Administration through intracoronary stents and intracoronary deposits is also contemplated. Intratumor administration of the compounds of the present invention can directly activate locally infiltrating DCs, directly enhance apoptosis of tumor cells, or sensitize tumor cells to cytotoxic agents. The term oral as used herein includes, but is not limited to, oral ingestion, or administration by the sublingual or buccal route. Oral administration includes liquid drinks, energy bars, as well as pill formulations.
[0228] The pharmaceutical compositions can be in any form suitable for the intended method of administration. When used for oral use, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups and elixirs can be prepared. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents, including sweetening agents, flavoring agents, coloring agents, and preserving agents, for the purpose to provide a tasty preparation. Tablets containing a drug compound in combination with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets are acceptable. These excipients can be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as cornstarch or alginic acid; binding agents, such as starch, gelatin, or acacia; and lubricating agents; such as magnesium stearate, stearic acid, or talc. The tablets may be uncoated, or they may be coated by known techniques including enteric coating, colonic coating, or microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and / or provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed, alone or with a wax.
[0230] Formulations for oral use can also be presented in the form of hard gelatin capsules, where the drug compound is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules, in which the active ingredient It is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil.
[0232] The pharmaceutical compositions can be formulated in the form of aqueous suspensions mixed with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and dispersing or wetting agents such as naturally-occurring phosphatides (eg, lecithin), a condensation product of an alkylene oxide with a fatty acid (for example, polyoxyethylene stearate), a condensation product of ethylene oxide with a long-chain aliphatic alcohol (for example, heptadecaethylenexyketanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride ( eg, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
[0234] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. Oral suspensions can contain a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents can be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an antioxidant, such as ascorbic acid.
[0236] The dispersible powders and granules of the disclosure, suitable for the preparation of an aqueous suspension by the addition of water, provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening agents, flavoring and coloring agents, may also be present.
[0238] The pharmaceutical compositions of the disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil or peanut oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally occurring gums, such as acacia and gum tragacanth, naturally occurring phosphatides, such as soy lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion can also contain sweetening and flavoring agents.
[0240] Syrups and elixirs can be formulated with sweetening agents, such as glycerol, sorbitol, or sucrose. Such formulations may also contain an emollient, a preservative, a flavoring or a coloring agent.
[0242] The pharmaceutical compositions of the disclosure may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using the suitable dispersing or wetting agents and the suspending agents mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent such as a solution in 1,3-butanediol or prepared in the form of a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile non-volatile oils can be employed in conventional manner as a solvent or suspending medium. For this purpose, any mild non-volatile oil may be employed, including synthetic mono- or diglycerides. Furthermore, fatty acids, such as oleic acid, can also be used in the preparation of injectables.
[0244] The amount of active ingredient that can be combined with the carrier material, producing a unit dosage form will vary depending on the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain from about 20 to 500 mg of active material compounded with an appropriate and convenient amount of carrier material, which can range from about 5 to about 95%. of the total compositions. Preparation of the pharmaceutical composition that provides easily measurable amounts for administration is preferred. Generally, an effective amount to be administered systemically is from about 0.1 mg / kg to about 100 mg / kg, and depends on a number of factors including, for example, the age and weight of the subject (for example , a mammal such as a human), the exact condition requiring treatment and its severity, the route of administration, ultimately remaining at the discretion of the attending physician or veterinarian. It will be understood, however, that the specific dose level for any particular patient will depend on a number of factors including the activity of the specific compound employed, age, body weight, general health, sex, and diet of the patient. person treated; the time and route of administration; the rate of excretion; other drugs that have been previously administered; and the severity of the particular condition undergoing therapy, as is well understood by those of skill in the art.
[0246] As indicated above, the formulations of the disclosure suitable for oral administration can be presented as discrete units such as capsules, sachets or tablets, each containing a predetermined quantity of the active principle, in the form of a powder or granules; in the form of a solution or suspension in an aqueous or non-aqueous liquid, or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The pharmaceutical compositions can also be administered in the form of a bolus, electuary, or paste.
[0248] A tablet can be made by compression or molding, optionally, with one or more auxiliary ingredients.
[0249] Tablets prepared by compression can be prepared by pressing in a suitable machine the active ingredient in fluid form such as a powder or granules, optionally mixed with a binder (for example, povidone, gelatin, hydroxypropylethylcellulose), lubricant, inert diluent, preservative, disintegrant (eg, sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surfactant or dispersant. Moldable tablets can be made on a suitable machine using a mixture of the powdered compound moistened with an inert liquid diluent. Optionally, the tablets can be coated or scored and can be formulated to provide a slow or controlled release of the active ingredient therefrom using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. Tablets can optionally be provided with an enteric or colonic coating to provide delivery in parts of the intestine other than the stomach. This is particularly advantageous with compounds of formula 1 when said compounds are susceptible to acid hydrolysis.
[0251] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; lozenges comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid vehicle.
[0253] Formulations for rectal administration may be in the form of a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
[0255] Formulations suitable for vaginal administration can be presented in the form of vaginal ovules, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the active principle, the vehicles considered appropriate in the art.
[0257] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and sterile aqueous and non-aqueous suspensions which may include suspending agents and thickening agents. The formulations can be presented in sealed unit dose or multidose containers, for example ampoules and vials, and can be stored in a freeze-dried state (lyophilized), requiring only the addition of the sterile liquid vehicle, for example water for injections. , immediately before use. Injection solutions and suspensions can be prepared from powders, granules and tablets of the type described above.
[0259] As used herein, pharmaceutically acceptable salts include, but are not limited to: acetate, pyridine, ammonium, piperazine, diethylamine, nicotinamide, formic acid, urea, sodium, potassium, calcium, magnesium, zinc, lithium, cinnamic acid, methylamino, methanesulfonic acid, picric acid, tartaric acid, triethylamino, dimethylamino, and tris (hydroxymethyl) aminomethane. Additional pharmaceutically acceptable salts are known to those skilled in the art.
[0260] An effective amount for a particular patient may vary depending on factors such as the condition to be treated, the general health of the patient, the route and dose of administration, and the severity of the side effects. Guidance is available for treatment and diagnostic methods (see, for example, Maynard, et al. (1996) "A Handbook of SOPs for Good Clinical Practice," Interpharm Press, Boca Raton, FL; Dent (2001) " Good Laboratory and Good Clinical Practice ", Urch Publ., London, UK).
[0262] An effective amount can be administered in one dose, but is not restricted to one dose. Thus, the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of pharmaceutical composition. When there is more than one administration of a pharmaceutical composition in the present methods, the administrations may be separated by time intervals of one minute, two minutes, three, four, five, six, seven, eight, nine, ten or more minutes, at intervals of approximately one hour, two hours, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. In the context of hours, the term "approximately" means plus or minus any time interval plus / minus 30 minutes. The administrations can also be separated by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days and their combinations. The invention is not limited to dosage intervals that are equally spaced in time, but encompasses doses at unequal intervals.
[0264] A dosing schedule of, for example, once / week, twice / week, three times / week, four times / week, five times / week, six times / week, seven times / week, once every two weeks, once every three weeks, once every four weeks, once every five weeks and the like is available for the invention. Dosing guidelines cover dosing over a total time period of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months , seven months, eight months, nine months, ten months, eleven months and twelve months.
[0265] Cycles of the above dosing guidelines are provided. The cycle can be repeated approximately, for example, every seven days; every 14 days; every 21 days; every 28 days; every 35 days; 42 days; every 49 days; every 56 days; every 63 days; every 70 days; and the like. Between each cycle, a no-dosing interval can be given, where the interval can be approximately, for example, seven days; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63 days; 70 days; and the like. In this context, the term "approximately" means plus or minus a day, plus or minus two days, plus or minus three days, plus or minus four days, plus or minus five days, plus or minus six days, or plus or minus seven days.
[0266] Methods for co-administration with an additional therapeutic agent are well known in the art (Hardman, et al. (Eds.), (2001) "Goodman and Gilman's The Pharmacological Basis of Therapeutics", 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) "Pharmacotherapeutics for Advanced Practice: A Practical Approach", Lippincott, Williams & Wilkins, Philadelphia, PA; Chabner and Longo (eds.) (2001) "Cancer Chemotherapy and Biotherapy ", Lippincott, Williams & Wilkins, Philadelphia., PA).
[0267] As indicated, The compositions of the present invention are preferably formulated as pharmaceutical compositions for parenteral or enteral administration. A typical pharmaceutical composition for administration to an animal comprises a pharmaceutically acceptable carrier such as aqueous solutions, non-toxic excipients, including salts, preservatives, buffers, and the like. See, for example, "Remington's Pharmaceutical Sciences", 15th Ed., Easton ed., Mack Publishing Co., p. 1405-1412 and 1461-1487 (1975); "The National Formulary XIV", 14th Ed., American Pharmaceutical Association, Washington, DC (1975). Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's solution with dextrose, etc. Intravenous vehicles include liquid and nutrient boosters. Preservatives include antimicrobial agents, antioxidants, chelating agents, and inert gases. The pH and exact concentration of the different components of the pharmaceutical composition are adjusted in accordance with ordinary skill in the art.
[0268] Repeated administrations of a given vaccine (homologous booster) have been shown to increase humoral responses. Such an approach may not be effective in increasing cellular immunity, because pre-vector immunity tends to impair robust antigen presentation and the generation of appropriate inflammatory signals. One approach to get around this problem has been the sequential administration of vaccines using different antigen delivery systems (heterologous boost). In a heterologous booster regimen, at least one primary or booster administration comprises administration of the cyclic purine dinucleotide / inactivated tumor cell compositions described herein. The heterologous group of the regimen may comprise antigen administration using one or more of the following strategies: inactivated or attenuated bacteria or viruses comprising the antigen of interest, which are particles that have been treated with some denaturing condition to render them ineffective or inefficient in the generation of a pathogenic invasion;
[0269] purified antigens, which are typically naturally produced antigens purified from a cell culture of the pathogen or a tissue sample containing the pathogen, or a recombinant version thereof; Live bacterial or viral delivery vectors recombinantly designed to express and / or secrete antigens in host cells of the subject. These strategies are based on attenuating (for example, by genetic engineering) the viral or bacterial vectors so that they are not pathogenic or toxic;
[0270] antigen presenting cell (APC) vectors, such as a dendritic cell (DC) vector, comprising cells that are loaded with an antigen or transfected with a composition comprising a nucleic acid encoding the antigen (e.g., Provenge® (Dendreon Corporation) for the treatment of castration-resistant metastatic prostate cancer);
[0271] liposomal antigen delivery vehicles; and
[0272] naked DNA vectors and naked RNA vectors that can be delivered by gene gun, electroporation, bacterial ghosts, microspheres, microparticles, liposomes, polycationic nanoparticles, and the like.
[0273] A prime vaccine and a booster vaccine can be administered by any one of the following routes or a combination thereof. In one aspect, the prime vaccine and the booster vaccine are administered by the same route. In another aspect, the prime vaccine and the booster vaccine are administered by different routes. The term "different pathways" encompasses, but is not limited to, different sites in the body, for example, a site that is oral, non-oral, enteric, parenteral, rectal, intranodal (lymph node), intravenous, arterial, subcutaneous, intramuscular, intratumoral. , peritumoral, intratumoral, infusion, mucosal, nasal, in the cerebrospinal space or cerebrospinal fluid, etc., as well as by different modes, for example, oral, intravenous and intramuscular.
[0274] An effective amount of a prime or booster vaccine can be given in one dose, but is not restricted to one dose. Thus, the administration can be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more, administrations of the vaccine. When there is more than one administration of a vaccine, the administrations may be separated by time intervals of one minute, two minutes, three, four, five, six, seven, eight, nine, ten or more minutes, at intervals of approximately one minute. hour, two hours, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and so on. In the context of hours, the term "approximately" means plus or minus any time interval plus / minus 30 minutes. The administrations can also be separated by time intervals of one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days and their combinations. The invention is not limited to dosage intervals that are evenly spaced in time, but encompasses doses at unequal intervals, such as a sensitization regimen consisting of administration at 1 day, 4 days, 7 days and 25 days, just to provide a non-limiting example.
[0275] Examples
[0277] The following examples serve to illustrate the present invention. These examples are not intended to limit the scope of the invention in any way.
[0279] Example 1. General methods
[0281] Suitable anhydrous solvents and reagents for solution phase oligonucleotide synthesis were purchased and handled under dry argon or nitrogen using anhydrous technique. Amidite coupling reactions and cyclizations were carried out in anhydrous acetonitrile or pyridine under dry argon or nitrogen. The starting materials for all dry pyridine reactions were dried by concentration (three times) in pyridine. Silica gel preparative flash chromatography was carried out using high purity Fluka 60A silica or Merck Grade 9385 silica using gradients of methanol in dichloromethane. Analytical HPLC was carried out on a Varian ProStar 210 HPLC system with a ProStar 330 photodiode array detector at 254 nm using a Microsorb Varian 10 micron C18 250 x 4.6 mm column or a Varian 3 micron column. 100 x 4.6 mm C18 and gradients of 10 mM TEAA and acetonitrile. Preparative HPLC was carried out on a Shimadzu LC20-AP preparative HPLC system, equipped with a SPD-20A UV / Vis detector operating at 254 nm on a 41.6 x Microsorb 60-8 Varian C-18 column. 250 mm using gradients of 10 mM TEAA and acetonitrile at a flow rate of 50 ml / min. Solid phase extractions using Sep-Pak C-18 (Waters) were carried out at 3% (w / w) loading. LC / MS (ESI / APCI) was obtained on a single Shimadzu 2010EV quadrupole instrument with PDA, Ms and ELSD detection using a Shimadzu LC20D analytical HPLC. High resolution FT-ICR mass spectrometry was obtained from the WM Keck Foundation Biotechnology Resources Laboratory at Yale University in New Haven, CT and the QB3 / Chemistry Mass Spect Lab at UC Berkeley.
[0283] Spectra of 1H, 31P, 1H-1H COZY (2D NMR correlation spectroscopy), 1H-31P HMBC (heteronuclear multiple bond correlation spectroscopy) were acquired in d6-DMSO with 10 ul D2O (delay of 16 hours after the addition of D2O) at 45 ° C in a Varian INOVA-500 NMR spectrometer operating at 500 MHz for 1H and at 202 MHz for 31P. The resulting FIDs were transferred to a PC and processed using Acorn NMR Inc.'s NUTS NMR processing software. Chemical shifts were referenced with respect to DMSO solvent, 2.50 ppm for 1H. According to IUPAC recommendations for NMR spectrum referencing, 31P chemical shifts were referenced using the "unified scale" at the 1H absolute frequency of 0 ppm. Some of the 1H and 31P spectra were acquired on a JEOL ECX-400 NMR spectrometer operating at 400 MHz for 1H and 162 MHz for 31P.
[0285] The gradient COZY spectrum was acquired in absolute value mode using 2,048 data points in the direct dimension and 256 time points in the indirect dimension. Both dimensions were apodized using sinusoidal bell squared functions. The indirect dimension was filled to zero, giving a final matrix size of 2048 x 2048 points and a resolution of 3.91 Hz / data point in both dimensions.
[0286] Regiochemistry assignment at phosphodiester bond: 1H-1H COZY experiments were used in combination with 1H-31P HMBC (and in some cases phosphor uncoupling) to provide direct evidence that the regiochemistry of phosphodiester bonds are 2 ', 5'-3', 5 '(see discussion in experimental section for 9a and Fig. 3A-G). Similar 1H-31P HMBC experiments confirmed the non-canonical (2 ', 5'-3', 5 ') regiochemistry at the phosphodiester bond of all synthesized cyclic dinucleotides after final silyl deprotection or ion exchange.
[0287] The assignment of the RR and RS diastereomers (main CDN products of the synthetic sequence) followed the methods in the literature (Zhao et al. "Nucleosides, Nucleotides and Nucleic Acids" 289: 352-378, 2009J.
[0288] All CDN products (Fig. 2A-2C) were> 95% pure as indicated by C18 reverse phase HPLC analysis (UV detection at 254 nm)
[0289] Abbreviations and acronyms: Guanine = G. isobutyryl-guanine = Gib. 4,4-dimethoxytrityl = DMT. OCH2CH2CN = CEO. tert- butyldimethylsilyl = TBS. adenine = A. benzoyl-adenine = ABz [A (2 ', 5') pA (3 ', 5') p] cyclic = ML-CDA = 19a (TEA salt). dithio- [RP, RP] - [A (2 ', 5') pA (3 ', 5') p] cyclic = ML-RR-CDA = 19b (TeA salt); 21 (sodium salt); 22 (ammonium salt). dithio- [Rp, SP] - [A (2 ', 5') pA (3 ', 5') p] cyclic = ML-RS-CDA = 19c (TEA salt). [G (2 ', 5') pG (3 ', 5') p] cyclic = ML-CDG = 9a (salt of TEA). dithio- [Rp, Rp] - [G (2 ', 5') pG (3 ', 5') p] cyclic = ML-RR-CDG = 9b (TEA salt). dithio- [Rp, Sp] - [G (2 ', 5') pG (3 ', 5') p] cyclic = ML-RS-CDG = 9c (TEA salt). [G (2 ', 5') pA (3 ', 5') p] cyclic = ML-cGAMP. dithio - ["P," P] - [G (2 ', 5') pA (3 ', 5') p] cyclic = ML-RR-cGAMP = 20 (TEA salt). monothio- [A (2 ', 5') pA (3 ', 5') Rp] cyclic = ML-3 ', 5'-R-CDA = 19d (TEA salt).
[0290] 2'-O-myristoyl- [G (2 ', 5') pG (3 ', 5') p] cyclic = C14-ML-CDG = 10 (TEA salt). ML-cGAMP = 2 ', 3'-cGAMP = [G (2', 5 ') pA (3', 5 ') p] cyclic = 23 (TEA salt)
[0291] ML-cGAMP (structure 23 in Fig. 2c) was prepared enzymatically from cellular cGAS and purified by prep HpLC.
[0293] Example 2. General experimental section for the ML-CDG series (Fig. 2a): synthesis of [G (2 ', 5') pG (3 ', 5') p] cyclic 9a. (not part of the invention)
[0295] 1) Preparation of 3. To a solution of 4.87 g (5.0 mmol) of N ² -isobutyryl-5'-O- (4,4'-dimethoxytrityl) -2'-O-fercbutyldimethylsilyl-3'- O - [(2-cyanoethyl) -W, W-diisopropylaminophenyl] guanosine (1) in 25 ml of acetonitrile, 0.18 ml (10 mmol) of water and 1.23 g (6 mmol) of pyridinium trifluoroacetate were added . After 5 minutes of stirring at room temperature, 25 ml of f-butylamine was added, and the reaction was stirred for 15 minutes at room temperature. The solvents were removed under reduced pressure, giving 2 as a foam which was then evaporated together with acetonitrile (2 x 50 ml). To a solution of 2 in 60 ml of dichloromethane, 0.9 ml (50 mmol) of water and 60 ml of 6% (v / v) dichloroacetic acid in dichloromethane (44 mmol) were added. After 10 minutes at room temperature, the reaction was quenched by the addition of pyridine (7.0 mL, 87 mmol). The reaction mixture was concentrated to an oil which was dried by three joint evaporations with 40 ml of anhydrous acetonitrile, the last time leaving 3 in a volume of 12 ml.
[0297] 2) Preparation of a dry solution of 4. N ² -isobutyryl-5'-O- 4,4'-dimethoxytrityl) -3'-O-tert-butyldimethylsilyl-2'-O - [(2-cyanoethyl) was dissolved -W, W-diisopropylaminovinyl] guanosine (4.33 g, 6.5 mmol) in 40 ml of anhydrous acetonitrile and dried by three joint evaporations with 40 ml of anhydrous acetonitrile, the last time leaving 20 ml. Ten 3A molecular sieves were added and the solution was stored under argon until use.
[0299] 3) Coupling of 3 and 4, giving, after oxidation and detritylation, the linear dimer 2 ', 5' 6a. Azeotropically dried 4 (6.5 mmol) in 20 ml acetonitrile was added via syringe to 3 (5.0 mmol). After 5 minutes stirring at room temperature, 2.37 ml (15 mmol) of 5.5M f-butyl hydroperoxide in decane was added and the reaction was stirred for 30 minutes at room temperature. The reaction was then cooled to 0 ° C and 1.25 g of NaHSO3 in 2.5 ml of water was added, the ice bath was removed and the reaction was stirred for 5 minutes. The reaction was concentrated to a foam, which was then taken up in 80 ml of dichloromethane. 0.9 ml of water and 80 ml of 6% (v / v) dichloroacetic acid in dichloromethane were added, and the reaction was stirred for 10 minutes at room temperature. 50 ml of pyridine was added to inactivate the dichloroacetic acid. Solvents were removed under reduced pressure, yielding crude 6a as a solid.
[0301] 4) Cycle of 6th, giving 7th. 6a was dissolved in 50 ml of dry pyridine and 5 ml (1/10 of the total reaction, approximately 0.5 mmol) was transferred via syringe to 150 ml of dry pyridine. This was concentrated to a volume of approximately 100 ml. Then 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorin-2-oxide (DMOCP, 0.35 g, 1.8 mmol) was added and the reaction was stirred for 30 minutes at room temperature. 0.32 ml of water was added immediately, followed by the addition of 0.16 g of iodine, and the reaction was stirred for 5 minutes at room temperature. Then the reaction mixture was poured into 350 ml of water containing 0.1 g of NaHSO3 and stirred for 5 minutes at room temperature. 2 g of NaHCO3 were slowly added with stirring, then poured into a separatory funnel and extracted with 400 ml of 1: 1 ethyl acetate: diethyl ether. The aqueous layer was re-extracted with 400 ml of 1: 1 ethyl acetate: diethyl ether, and the organic layers were combined, dried over sodium sulfate, and concentrated under reduced pressure, yielding 0.75 g of a mixture containing 7a, the fully protected cyclic [G (2 ', 5') pG (3 ', 5') p].
[0303] 5) Deprotection of crude 7a with methylamine, yielding crude 8a. To 750 mg of 7a, 18 ml of methylamine in anhydrous ethanol (33% by weight) was added and the mixture was stirred for 90 minutes, at which time HPLC analysis indicated that the reaction was complete. The reaction mixture was concentrated to give an oil which, after treatment with 10 ml of hexane / ethyl acetate (50:50), produced an off-white solid. The trituration / wash solvent was decanted, and the residual solvent was removed under reduced pressure, yielding 240 mg of an off-white solid.
[0305] 6) Preparative HPLC of crude 8a. A 120 mg portion of crude 8a was taken up in 5 ml CH3CN / 10 mM aqueous triethylammonium acetate (20/80). After filtration through 0.45 micron PTFE, the injection sample was applied to a Dynamax C-18 column (40 x 250 mm). Elution was performed with a gradient of acetonitrile and 10 mM aqueous triethylammonium acetate (20% to 50% CH3CN for 20 minutes at 50 ml / min flow). The HPLC fractions from the two HPLC series containing pure 8a were pooled, evaporated to remove CH3CN, and lyophilized to remove most of the remaining water and volatile buffer, giving, after azeotropic drying with acetonitrile (3 x 4 ml), 42 mg of pure 8a in the form of the bis-triethylammonium salt. (It is also possible to defer prep HPLC purification until after the last step). HRMS (FT-ICR) m / z:
[0306] [MH] - calculated for C32H51N1üO14P2Si2917.2606; observed 917.2622. ¹ H NMR (DMSO-d ⁶ + trace D2O) 45 ° C 6 8.22 (1H, s), 7.85 (1H, s), 5.76-5.79 (2H, dd), 5, 21 (1H, m), 4.85 (1H, m), 4.58 (1H, t), 4.49 (1H, d), 4.31 (1H, m), 4.21 (1H, m), 3.97 (1H, d), 3.83 (3H, m), 2.94 (12H, m), 1.12 (18H, t), 0.90 (9H , s), 0.72 (9H, s), 0.14 (6H, d), 0.09 (3H, s), -0.02 (3H, s). ³¹ P NMR (DMSO-d ⁶ + trace D2O) 45 ° C. 6 -1.26, -2.02 (Fig. 3a-3c).
[0308] 7) Deprotection of the TBS groups of 8a with triethylamine trihydrofluoride, neutralization with TEAB and solid phase extraction with a Sep-Pak C-18, yielding pure 9a as the bis-triethylammonium salt. To 40 mg of 8a, 1.0 ml of triethylamine trihydrofluoride was added. The mixture was stirred at room temperature for 30 h. After confirming completion of the reaction by analytical HPLC, the sample was neutralized by dropwise addition to 12 ml of cooled 1M triethylammonium bicarbonate. The neutralized solution was desalted on a Waters C-18 Sep-Pak and the product was eluted with CHaCN / 10 mM aqueous triethylammonium acetate (1: 1). CH3CN was evaporated under reduced pressure, and the remaining aqueous solution was frozen and lyophilized overnight. Multiple evaporations in methanol (3 x 3 ml) and a final evaporation of 50% acetonitrile in methanol (1 x 3 ml) gave 29.3 mg of [G (2 ', 5') pG (3 ', 5' ) p] cyclic (9a) in the form of the bis-triethylammonium salt. HRMS (FT-ICR) m / z: [MH] 'calculated for C20H23N10O14P2689.0876; observed 689.0874. ¹ H NMR (DMSO-d ⁶ + trace D2O) 45 ° C 67.92 (1H, s), 7.90 (1H, s), 5.82 (1H, d), 5.80 (1H, d ), 4.97 (1H, m), 4.85 (1H, m), 4.68 (1H, m), 4.31 (1H, d), 4.21 (1H, t), 4.10 (2H, m), 3.79 (3H, m), 2.91 (14H, m), 1.13 (22H, t). ³¹ P NMR (DMSO-d ⁶ ) 45 ° C. 61.80, -1.05.
[0310] HPLC retention time of 9a is 7.25 min compared to 9.3 min for cyclic di-GMP using a gradient of 2 to 20% CH3CN in 10 mM triethylammonium acetate over 20 L min on a column C-18 (3 microns, 100 x 4.6 mm, 0.6 ml / min.) HRMS (FT-ICR) confirmed the expected elemental formula: [MH] 'calculated for C20H23N10O14P2689.0876; observed 689.0874. The ³¹ P NMR of 9a showed two maxima (integrating 1: 1) at 2.03 and -0.95 ppm coinciding with a 2 ', 5' / 3 ', 5' mixed bond (both c [G (3 ' , 5 ') pG (3', 5 ') p] as c [G (2', 5 ') pG (2', 5 ') p], for example, would only give a ³¹ P NMR signal due to symmetry). Direct evidence for the regiochemistry of phosphodiester bonds was obtained by ¹ H- ¹ H COZY in combination with phosphorus uncoupling experiments, and by 2-D NMR of ¹ H- ³¹ P HMBC (Fig. 3b and 3c). Anomeric protons (H-1) appear as an overlapping doublet of doublets (or triplet) at 5.82 ppm. The designation "A" was given to the downfield anomeric proton (H-1) and the designation "B" to the slightly upfield anomeric proton. Starting with the anomeric proton on ribose "A" and "B", a COZY experiment of ¹ H- ¹ H (Fig. 3b) allowed the assignment of H-2A (4.96 ppm), H-3A ( 4.31 ppm), as well as H-2B (4.67 ppm) and H-3B (4.84 ppm). The irradiation of the phosphorus downfield (2.03 ppm) converted the multiplet H-3B into a doublet, while the irradiation of the phosphorous upfield (-0.95 ppm) resulted in a simplification of the complex multiplet of H-2A . In both uncoupling experiments, the simplification of the 5 'ribose methylene multiplet was also observed. The bidimensional HMBC ¹ H- ³¹ P confirmed the result of decoupling experiments. The results of ¹ H- ¹ H COZY in combination with phosphor uncoupling and ¹ H- ³¹ P HMBC experiments provide direct evidence that the regiochemistry of phosphodiester bonds is 2 ', 5' / 3 ', 5' and that 9a is [G (2 ', 5') pG (3 ', 5') p] .cyclic.
[0312] Example 3. General experimental section for the ML-CDA series (Fig. 2b): synthesis of the Na salt of [A (2 ', 5') pA (3 ', 5') p] cyclic 21 (see compound Fig. 2c).
[0314] 1) Preparation of 13.
[0316] To a solution of 5 g (5.15 mmol) of N ⁶ -benzoyl-5'-O- (4,4'-dimethoxytrityl) -2'-O-tert-butyldimethylsilyl-3'-O - [(2- cyanoethyl) -W, W-diisopropylamino-phenyl] adenosine (11) in 25 ml of acetonitrile, 0.18 ml (10 mmol) of water and 1.20 g (6.2 mmol) of pyridinium trifluoroacetate were added. After 5 minutes of stirring at room temperature, 25 ml of tert-butylamine were added and the reaction was stirred for 15 minutes at room temperature. The solvents were removed under reduced pressure, giving 12 as a foam which was then evaporated together with acetonitrile (2 x 50 ml), then dissolved in 60 ml of dichloromethane. To this solution, water (0.9 ml, 50 mmol) and 60 ml of 6% (v / v) dichloroacetic acid (44 mmol) in dichloromethane were added. After 10 minutes at room temperature, the reaction was quenched by adding pyridine (7.0 ml, 87 mmol) and concentrated to an oil which was dried by three joint evaporations with 40 ml of anhydrous acetonitrile, the last time leaving 13 in a volume of 12 ml.
[0318] 2) Preparation of a dry solution of 14.
[0320] N ⁶ -benzoyl-5'-O- (4,4'-dimethoxytrityl) -3'-O-tert-butyldimethylsilyl-2'-O - [(2-cyanoethyl) -W, W-diisopropylaminophenyl] adenosine ( 14, 6.4 g, 6.6 mmol) in 40 ml of anhydrous acetonitrile and dried by three joint evaporations with 40 ml of anhydrous acetonitrile, the last time leaving 20 ml. Ten 3A molecular sieves were added and the solution was stored under argon until use.
[0322] 3) Preparation of the 2 ', 5'-monothio-linear dimer 16.
[0324] Azeotropically dried 14 (6.4 g, 6.6 mmol) in 20 ml of acetonitrile was added via syringe to a solution of 13 (5.15 mmol) in 12 ml of anhydrous acetonitrile. After 5 minutes stirring at room temperature, 1.14 g (5.6 mmol) of 3 - ((W, W-dimethylaminomethylidene) amino) -3H-1,2,4-dithiazole-5-thione (DDTT) were added , and the reaction was stirred for 30 minutes at room temperature. The reaction was concentrated and the residual oil was dissolved in 80 ml of dichloromethane. Water (0.9 ml, 50 mmol) and 80 ml of 6% (v / v) dichloroacetic acid (58 mmol) were added in dichloromethane, and the reaction was stirred for 10 minutes at room temperature. 50 ml of pyridine was added to inactivate the dichloroacetic acid. Solvents were removed under reduced pressure, yielding crude 16b as a solid.
[0325] 4) Cyclization and sulfidation of 16b, yielding the protected cyclic dithium diastereoisomers 17b and 17c.
[0326] 16b was dissolved in 150 ml of dry pyridine which was concentrated to a volume of approximately 100 ml. Then, 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorin-2-oxide (DMOCP, 3.44 g, 18 mmol) was added and the reaction was stirred for 5 minutes at room temperature. 3.2 ml of water was added immediately followed by the addition of 3-H-1,2-benzodithiol-3-one (1.3 g, 7.7 mmol), and the reaction was stirred for 5 minutes at room temperature . Then, the reaction mixture was poured into 700 ml of water containing 20 g of NaHCO3 and stirred for 5 minutes at room temperature, then poured into a separatory funnel and extracted with 800 ml of ethyl acetate: diethyl ether 1: 1. The aqueous layer was re-extracted with 600 ml of 1: 1 ethyl acetate: diethyl ether. The organic layers were combined and concentrated under reduced pressure, yielding approximately 11 g of an oil containing diastereoisomers 17b and 17c.
[0327] 5) Silica gel column chromatography of the crude mixture containing 17b and 17c.
[0328] The above crude mixture was dissolved in dichloromethane and applied to a 250 g silica column. The desired diastereoisomers were eluted from the column using a gradient of methanol in dichloromethane (0-10%). Fractions containing the desired diastereoisomers 17b and 17c were combined and concentrated, yielding 2.26 g of approximately 50% 17b and 50% 17c.
[0329] 6) Deprotection of fully protected cyclic diastereoisomers 17b and 17c to crude 18b and 18c.
[0330] 2.26 g of crude 17b and 17c were transferred from the silica gel column to a thick-walled glass pressure tube. 60 ml of methanol and 60 ml of concentrated aqueous ammonia were added, and the tube was heated with shaking in an oil bath at 50 ° C for 16 h (the last series were carried out 12 h after the material of starting right now). The reaction mixture was cooled to near room temperature, sparged with a stream of nitrogen gas for 30 minutes, and then transferred to a large round bottom flask. Most volatile substances were removed under reduced pressure with caution to avoid foaming and shock. When water was still present, the residue was frozen and lyophilized to dryness.
[0331] 7) Purification by preparative HPLC of crude 18b and 18c, yielding pure 18b.
[0332] The lyophilized crude mixture containing 18b and 18c was taken up in approximately 50 ml CHaCN / 10 mM aqueous triethylammonium acetate (60/40). After filtration through 0.45 micron PTFE, 4-5 ml sample portions were applied to a Dynamax C-18 column (40 x 250 mm). Elution was performed with a gradient of acetonitrile and 10 mM aqueous triethylammonium acetate (30% to 50% CH3CN for 20 minutes at 50 ml / min flow). The preparative HPLC series fractions containing pure 18b were combined, evaporated to remove CH3CN and lyophilized, yielding 360 mg of pure 18b (the RpRp diastereoisomer) as the bis-triethylammonium salt.
[0333] 8) Deprotection of the two 18b TBS groups with triethylamine trihydrofluoride, neutralization with TEAB, solid phase extraction with a Sep-Pak C-18 and lyophilization, yielding pure 19b as the bis-triethylammonium salt. 8a) To 270 mg (0.24 mmol) of 18b, 5.0 ml of neat triethylamine trihydrofluoride was added. The mixture was stirred at room temperature for approximately 40 h. After confirming completion of the reaction by analytical HPLC, the sample was neutralized by dropwise addition to 45 ml of chilled and stirred 1M triethylammonium bicarbonate. The neutralized solution was desalted on a Waters Sep-Pak C-18 and the product was eluted with CHsCN / 10 mM aqueous triethylammonium acetate (5: 1). CH3CN was evaporated under reduced pressure, and the remaining aqueous solution was frozen and lyophilized. Multiple series of lyophilization in water gave 122 mg (57%) of dithium- (Rp, Rp) - [A (2 ', 5') pA (3 ', 5') cyclic p] (19b) as the bis salt -triethylammonium.
[0334] 8b) 90 mg (0.08 mmol) of 18b was co-evaporated three times with 10 ml of dry acetonitrile. The dry residue was taken up in 0.4 ml of anhydrous pyridine. The flask with a vent needle was placed in a 50 ° C oil bath, and 0.62 ml of triethylamine trihydrofluoride and 1.0 ml of triethylamine were added simultaneously to the stirring mixture. The mixture was stirred at 50 ° C for two hours. After confirming completion of the reaction by analytical HPLC, the sample was neutralized by dropwise addition to 25 ml of chilled and stirred 1M triethylammonium bicarbonate. The neutralized solution was desalted on a Waters Sep-Pak C-18 and the product was eluted with CHsCN / 10 mM aqueous triethylammonium acetate (1: 4). CH3CN was evaporated under reduced pressure, and the remaining aqueous solution was frozen and lyophilized. Multiple series of lyophilization in water gave 54 mg (76%) of dithium- (Rp, Rp) - [A (2 ', 5') pA (3 ', 5') cyclic p] (19b) as the bis salt -triethylammonium.
[0335] 8c) A variant of TEA-HF deprotection by heating in neat TEA-HF at 45 ° C followed by neutralization of TEAB, desalting with Sep-Pak and lyophilization.
[0336] TEA ^ 3HF (1 ml, 6.1 mmol) was added to 18b (41 mg, 0.04 mmol) to a flask fitted with a vent needle, and the mixture was stirred at 45 ° C. The progress of the reaction was monitored by LC and after consumption of the starting material and mono-TBS analogs (~ 2 h), the mixture was cooled to room temperature. The mixture was slowly pipetted into a solution of 1 M TEAB (4.9 ml) and TEA (1.6 ml) at 0 ° C and a slightly basic pH was confirmed by pH paper. The neutralized solution was desalted on a Waters Sep-Pak C-18 (10 g) and the product was eluted with 15% CH3CN / 10 mM aqueous triethylammonium acetate. Lyophilization gave 21 mg (64%) of 19b (bistriethylammonium salt) as a white solid. Analysis by analytical HPLC (2-20% acetonitrile / 10 nM-20 min TEAA buffer) showed> 95% purity (Fig. 3h). ¹ H NMR (500 MHz, 45 ° C, (C ³ ) ² SO-15 pl of D2O) or 8.58 (s, 1H), 8.41 (s, 1H), 8.18 (s, 1H ), 8.15 (s, 1H), 6.12 (d, J = 8.0, 1H), 5.92 (d, J = 7.0, 1H), 5.30 (td, J = 8 , 5, 4.0, 1H), 5.24 5.21 (m, 1H), 5.03 (dd, J = 7.5, 4.5, 1H), 4.39 (d, J = 4 , 1H), 4.23 (dd, J = 10.5, 4.0, 1H), 4.18 (s, 1H), 4.14-4.08 (m, 2H), 3.85-3 , 83 (m, 1H), 3.73 (d, J = 12.0, 1H), 3.06 (c, J = 7.5, 12H), 1.15 (t, J = 7.5, 1 HOUR); ³¹ P NMR (200 MHz, 45 ° C, (CDa) ² SO-15 µl of D2O) or 58.81; 52.54; HRMS (FT-ICR) m / z calculated for C20H24O10N10P2S2 (MH) - 689.0521, found 689.0514.
[0337] 8d) Treatment of the TEA-HF reaction by acetone precipitation is also possible as described in Gaffney et al. 2010, but a somewhat cleaner product has been obtained using the modifications described in sections 8a-8c above.
[0338] 10) Conversion to sodium salt
[0339] The bis-TEA ML-RR-CDA salt (19b) is easily converted to the pharmaceutically acceptable sodium salt (21) by ion exchange as described below.
[0340] ML-RR-CDA ^ 2Na + (21). 100-200 mesh of BT AG ® 50W-X2 resin, The hydrogen form (100 mg) in suspension was packed with DI water on a Bio-spin® column. Excess DI water was drained by gravity. 3 bed volumes of 1 M NaOH (1 ml) were passed through the column by gravity, followed by 5 bed volumes of DI water (2 ml). After draining excess DI water by gravity, a solution of ML-RR-CDA ^ 2TEA (19b, 10 mg) in DI water (1 ml) was loaded onto the column. The column was eluted with 5 bed volumes of DI water (2 ml), the fractions were collected and the UV activity was verified by means of a TLC plate and a UV lamp. The fractions of interest were pooled, frozen and lyophilized overnight, yielding ML-RR-CDA ^ 2Na + quantitatively. ¹ H NMR (500 MHz, 45 ° C, (CDa) ² SO-30 µl D2O) or 8.54 (s, 1H), 8.40 (s, 1H), 8.17 (s, 1H) , 8.167 (s, 1H), 6.09 (d, J = 8.0, 1H), 5.92 (d, J = 8.0, 1H), 5.26 (td, J = 8.5; 4.5, 1H), 5.21-5.19 (m, 1H), 5.01 (dd, J = 7.5; 4.5, 1H), 4.42 (d, J = 4.1H ), 4.23 (dd, J = 10.5, 5.0, 1H), 4.17 (s, 1H), 4.15-4.00 (m, 2H), 3.90-3.82 (m, 1H), 3.73-3.70 (m, 1H); ³¹ P NMR (200 MHz, 45 ° C, (CDa) ² SO-30 µl D2O) or 58.85; 51.53 (Fig. 3d-3g); HRMS (FT-ICR) m / z calculated for C20H23O10N10P2S2 (MH) '689.0521, found 689.0503.
[0341] The direct test for the regiochemistry of phosphodiester bonds was obtained by ¹ H- ¹ H COZY in combination with 2-dimensional NMR of ¹ H- ³¹ P HMBC (Fig. 3e-3g) in a manner analogous to the experimental section of the series of ML-CDG (as described above).
[0342] ML-RR-CDG (9b). Compound 9b was synthesized analogously to ML-CDG following the procedures of the experimental section of the ML-CDG series (as described above) with the following modifications (Fig. 2a): e) DDTT; h) 3-H-1,2-benzodithiol-3-one; n) obtained as the TEA salt, no ion exchange was needed. ¹ H NMR (500 MHz, 45 ° C, (CDa) ² SO-15 pl of D2O) or 7.98 (s, 1H), 7.94 (s, 1H), 5.85 (d, J = 9.0, 1H), 5.80 (d, J = 7.5, 1H), 5.25-5.23 (m, 1H), 5.12 (dd, J = 8.5, 4.5 , 1H), 4.73 (dd, J = 8.0, 4.5, 1H), 4.42 (d, J = 4.0, 1H), 4.22 (t, J = 7.5, 1H), 4.14-4.10 (m, 2H), 3.94-3.90 (m, 2H), 3.77-3.73 (m, 1H), 3.05 (c, J = 7.0, 12H), 1.160 (t, J = 7.0, 1H); ³¹ P NMR (200 MHz, 45 ° C, (CDa) ² SO-15 µl of D2O) or 59.09; 50.37; HRMS (FT-ICR) m / z calculated for C20H23O12N10P2S2 (M -H) - 721.0419, found 721.0410.
[0343] ML-RS-CDG (9c). Compound 9c was synthesized analogously to ML-CDG following the procedures of the experimental section of the ML-CDG series (as described above) with the following modifications (Fig. 2a): e) DDTT; h) 3-H-1,2-benzodithiol-3-one; k) the diastereomer [Rp, Sp] 8c was collected; n) obtained as the TEA salt, no ion exchange was needed.
[0344] ¹ H NMR (500 MHz, 45 ° C, (CDa ^ SO-15 pl of D2O) or 8.01 (s, 1H), 7.98 (s, 1H), 5.86 (d, J = 8 , 5.1H), 5.79 (d, J = 8.0, 1H), 5.29 (dd, J = 8.5; 4.0, 1H), 5.20-5.19 (m, 1H), 4 , 68 (dd, J = 8.5, 4.0, 1H), 4.21-4.18 (m, 2H), 4.10-4.05 (m, 3H), 3.71-3, 68 (m, 2H), 2.96 (c, J = 7.0, 12H), 1.13 (t, J = 7.0, 18H); ³¹ P NMR (200 MHz, 45 ° C, ( CD ³ ) ² SO-15 pl D2O) or 59.89, 57.17, HRMS (FT-ICR) m / z calculated for C20H24O12N10P2S2 (MH) - 721.041904, observed 721.04143.
[0345] C14-ML-CDG (10): Compound 10 (Fig. 2c) was synthesized analogously to ML-CDG following the procedures of the experimental section of the ML-CDG series (as described above) with the following modifications (Fig. 2a): n) myristic anhydride, DMF.
[0346] To the bis-triethylamine salt of 9a (0.260 g, 0.291 mmol), 3.7 ml of DMF, 0.3 ml of pyridine and 128 mg (0.292 mmol) of myristic anhydride were added. The reaction mixture was heated for a total of 5 h at 60 ° C, cooled to room temperature and quenched with 100 ul MeOH. The LC trace indicated a 25% conversion to a major new product with the remainder of the mass appearing in the retention time range of the starting material. The mass of the main product was confirmed as the C14 acylated product by LC / MS in negative mode, with m / z (M-1) of 899 (calculated for C34H49N10O15P2 ": 889.3). After evaporation, the residue was collected in 2 ml of CH3CN, 3 ml of 0.1 M TEAA and enough MeOH to bring most of the material into solution.After a brief centrifugation to remove a small amount of particulate material, the solution was purified by prep HPLC of C18 using a CH3CN gradient of 25% -> 50% in 10 mM TEAA over 20 min Fractions containing the desired product were combined and lyophilized to dryness, yielding 36 mg of C14-ML-CDG 10 (triethylammonium salt ) as a white solid.
[0347] ¹ H NMR (500 MHz, 45 ° C, (CD ³ ) ² SO-15 µl of D2O) or 8.00 (s, 1H), 7.90 (s, 1H), 5.98 (d, J = 7.5, 1H), 5.83 (d, J = 8.5, 1H), 5.76 (dd, J = 7.5; 4.5, 1H), 5.15-5.10 (m, 1H) , 4.90-4.85 (m, 1H), 4.36 (d, J = 4.5, 1H), 4.30-4.27 (m, 1H), 4.07 (s, 1H) , 3.94-3.90 (m, 3H), 3.82-3.78 (m, 1H), 3.04 (c, J = 7.0, 12H), 2.37-2.23 ( m, 2H), 1.51-1.43 (m, 2H), 1.28-1.14 (m, 38H). 0.85 (t, J = 7.0, 3H); ³¹ P NMR (200 MHz, 45 ° C, (CDa ^ SO-15 pl of D2O) or -1.36, -2.12; HRMS (FT-ICR) m / z calculated for C34H49O15N10P2 (M - H) '899.2860, found 899.2834.
[0348] ML-CDA (19a). Compound 19a was synthesized analogously to ML-RR-CDA following the procedures of the experimental section of the ML-CDA series (as described above) with the following modifications (Fig. 2b): e) t-BuOOH; h) I2 / H2O; n) obtained as the TEA salt, no ion exchange was needed.
[0349] ¹ H NMR (500 MHz, 45 ° C, (CD ³ ) ² SO-15 pl of D2O) or 8.44 (s, 1H), 8.37 (s, 1H), 8.16 (s, 1H ), 8.14 (s, 1H), 6.08 (d, J = 8.0, 1H), 5.90 (d, J = 7.5, 1H), 5.10-5.0 (m , 3H), 4.30 (d, J = 4.5, 1H), 4.3-4.19 (m, 1H), 4.14 (d, J = 1.5, 1H), 4.05 (c, J = 11.5, 2H), 3.78-3.75 (m, 2H), 2.90 (c, J = 7.5, 18H), 1.08 (t, J = 7, 0.27H); ³¹ P NMR (200 MHz, 45 ° C, (CD ³ ) ² SO-15 µl of D2O) or 1.67, -0.47; HRMS (FT-ICR) m / z calculated for C20H24O12N10P2 (MH) - 657.097763, found 657.09680.
[0350] ML-RS-CDA (19c). Compound 19c was synthesized analogously to ML-RR-CDA following the procedures of the experimental section of the ML-CDA series (as described above) with the following modifications (Fig. 2b): k) the diastereomer was collected [Rp, Sp] 18c; n) obtained as the TEA salt, no ion exchange was needed.
[0351] ¹ H NMR (500 MHz, 45 ° C, (CD ³ ) ² SO-15 pl of D2O) or 8.52 (s, 1H), 8.37 (s, 1H), 8.16 (s, 1H ), 8.15 (s, 1H), 6.10 (d, J = 8.5, 1H), 5.90 (d, J = 7.5, 1H), 5.45 (dd, J = 8 , 5, 4.5, 1H), 5.31-5.26 (m, 1H), 5.00 (dd, J = 8.5, 4.5, 1H), 4.41-4.36 ( m, 1H), 4.22 (d, J = 5.0, 1H), 4.14-4.07 (m, 3H), 3.70-3.67 (m, 3H), 2.84 ( c, J = 7.0, 19H), 1.08 (t, J = 7.5, 29H); ³¹ P NMR (200 MHz, 45 ° C, (CD ³ ) ² SO-15 µl of D2O) or 59.98; 57.35; HRMS (FT-ICR) m / z calculated for C20H24O10N10P2S2 (M -2 H + Na) - 711.0340, found 711.0316.
[0352] ML-3'-5'-R-CDA (19e). Compound 19e was synthesized analogously to ML-RR-CDA following the procedures of the experimental section of the ML-CDA series (as described above) with the following modifications (Fig. 2b): e) t-BuOOH; h) 3-H-1,2-benzodithiol-3-one; n) obtained as the TEA salt, no ion exchange was needed.
[0353] ¹ H NMR (500 MHz, 45 ° C, (CD ³ ) ² SO-15 pl of D2O) or 8.49 (s, 1H), 8.38 (s, 1H), 8.17 (s, 1H ), 8.14 (s, 1H), 6.09 (d, J = 8.5, 1H), 5.90 (d, J = 7.5, 1H), 5.23 (dd, J = 8 , 0, 5.0, 1H), 5.12-5.04 (m, 2H), 4.31 (d, J = 4.5, 1H), 4.21-4.14 (m, 3H) , 4.10 ( c, J = 11.0, 1H), 3.80-3.71 (m, 2H), 2.85 (c, J = 7.0, 18H), 1.08 (t, J = 7.5, 27H); ³¹ P NMR (200 MHz, 45 ° C, (CD ³ ) ² SO-15 µl of D2O) or 59.32; -0.37; HRMS (FT-ICR) m / z calculated for C2 ⁰ H2 ³ O11N1üP2S (MH) - 673.0749, found 673.0729.
[0354] ML-RR-CDA (22) in the form of an ammonia salt. Compound 22 was synthesized analogously to ML-RR-CDA following the procedures of the experimental section of the ML-CDA series (as described above) with the following modifications (Fig. 2b): n) 100-200 mesh BT resin AG ® 5UW-X2, hydrogen form, 1 M NH4OH ^1H NMR (500 MHz, 45 ° C, (CD ^{3) 2} sO-3U pl D2O) or 8.80 (s, 1H) , 8.44 (s, 1H), 8.39 (s, 2H), 6.45 (d, J = 10.0, 1H), 6.34 (s, 1H), 5.50 (td, J = 10.5, 4.5, 1H), 5.21-5.15 (m, 1H), 5.02 (d, J = 4.0, 1H), 4.92 (d, J = 4, 5.1H), 4.61-4.49 (m, 2H), 4.30-4.27 (m, 2H); ¹ HRMS (FT-ICR) m / z calculated for C2oH2 ³ O1oN-ioP2S2 (MH) - 689.0521, found 689.0504.
[0355] ML-RR-cGAMP (20). Compound 20 (Fig. 2c) was synthesized analogously to ML-RR-CDA following the procedures of the experimental section of the ML-CDA series (as described above) with the following modifications (Fig. 2b): d ) pir, 4; n) obtained as the TEA salt, no ion exchange was needed. ¹ H NMR (500 MHz, 45 ° C, (CD ³ ) ² SO-3U pl of D2O) or 8.34 (s, 1H), 8.15 (s, 1H), 8.01 (s, 1H ), 5.91 (d, J = 7.5, 1H), 5.86 (d, J = 8.5, 1H), 5.29-5.23 (m, 1H), 5.17-5 , 14 (m, 1H), 5.02 (dd, J = 7.5, 4.0, 1H), 4.41 (d, J = 4.5, 1H), 4.25 (dd, J = 5.0, 10.5, 1H), 4.13-4.03 (m, 3H), 3.95-3.85 (m, 1H), 3.78-3.74 (m, 1H), 2.84 (q, J = 7.5, 18H), 1.08 (t, J = 7.5, 28H); ³¹ P NMR (200 MHz, 45 ° C, (CD ³ ) ² SO-30 µl of D2O) or 58.81; 50.91; HRMS (FT-ICR) m / z calculated for C2üH2 ³ O ¹¹ N1oP2S2 (MH) - 705.0470, found 705.0451.
[0356] Example 4. Derivatives substituted at ribose 2 'and 3
[0358] For reference purposes, examples of derivatives are depicted in Figs. 4-6.
[0360] Example 5. Expression of type I interferon induced by CDN
[0362] To determine the relative level of type I interferon induced in human cells by each of the native and derived molecules as a hallmark of adjuvant potency, 4 x 10 ⁵ THP1-Blue ™ ISG lymphocytes (a monocyte cell line) were incubated. human transfected with an IRF-inducible secreted embryonic alkaline phosphatase reporter gene (Invivogen) expressing alkaline phosphatase under the control of a promoter composed of five IFN-stimulated response elements) with 100 pM of [G (3 ', 5') pG (3 ', 5') p] (CDG) cyclic, [G (2 ', 5') pG (3 ', 5') p] cyclic (mixed bond or ML-CDG) or HBSS for 30 min at 37 ° C with 5% CO2. After 30 minutes, cells were washed and seeded in 96-well plates in RPMI medium containing 10% FBS, and incubated at 37 ° C with 5% CO2. Cell culture supernatants from each sample were collected after overnight incubation, and 20 µl of cell culture supernatants were added to 180 µl of QUANTI-Blue reagent (Invivogen) and incubated for 45 minutes to assess levels of type I interferon protein. Absorbance readings were taken at 655 nm every 3 minutes using a Versa Max kinetic spectrophotometer (Molecular Diagnostics).
[0363] As shown in Fig. 7, [G (2 ', 5') pG (3 ', 5') p] (ML-CDG) cyclic induced significantly higher levels of IFN-p than [G (3 ', 5 ') pG (3', 5 ') p] cyclic at a wide selection of time points. These results demonstrate that a purified preparation of cyclic [G (2 ', 5') pG (3 ', 5') p] activates the innate immune response more profoundly than [G (3 ', 5') pG (3 ', 5 ') p] cyclic in a human monocyte cell line.
[0365] To determine the levels of IFN-a, -py -y induced by [G (2 ', 5') pG (3 ', 5') p] cyclic (ML-CDG) compared to [G (3 ', 5 ') pG (3', 5 ') p] cyclic as a hallmark of potency to activate innate immunity, 1 x 10 ⁶ primary human PBMC isolated from four independent human donors were incubated in a 96-well U-bottom plate for 30 minutes at 37 ° C, 5% CO2 with 5 or 0.5 pM of [G (3 ', 5') pG (3 ', 5') p] cyclic (CDG) or [G (2 ', 5 ') pG (3', 5 ') p] cyclic (ML-CDG), 1 pg / ml of interferon stimulatory DNA (iSd) or 4 pg / ml of Poly (I: C) using the Effectene transfection reagent ( Qiagen) to transfer the molecules to PBMC. ISD (interferon stimulatory DNA) is independent of TLR (Stetston, DB et al., Immunity 24, 93-103, January 2006) and signals through cGAS, and therefore depends on STING, whereas Poli ( I: C) can signal through the TLR3 and RIG-I pathways and are therefore independent of STING. After 30 minutes, cells were washed and replaced with RPMI media containing 10% FBS and incubated at 37 ° C, 5% CO2. After 6 hours of incubation, a part of the cells were harvested and evaluated by quantitative real-time RT-PCR to determine the gene expression of the type I cytokines interferon alpha 2 (IFNA2) and interferon beta 1 (IFNB1), and the type II cytokine gene interferon gamma (IFNG). Gene expression was determined by quantitative real-time RT-PCR using the PrimePCR RNA purification and cDNA analysis system, and was run on the CFX96 gene cycler (all from BioRad). The normalized expression for each was determined, which explains the different efficiencies of PCR amplification for the target (Ediana) and the reference (Ereference), and transforms the Cycle Threshold (UC) from raw data unit to logarithmic scale in the linear unit of normalized expression. The reference genes used were GUSB and PGK1, and it was confirmed that the genes had a coefficient variable (VC) below 0.5 and an M value below 1, so they did not vary with the different conditions of treatment. To evaluate the correlative levels of secreted proteins of these cytokines, the supernatants of the remaining cells were collected after 24 hours of incubation and the levels of IFN-a and IFN-y were determined using the Cytometric bead matrix (CBA, BD Biosciences). , while IFN-p levels were determined by ELISA (PBL).
[0367] As shown in Fig. 8, Interferon alpha 2 (IFNA2) gene expression was significantly higher for [G (2 ', 5') pG (3 ', 5') p] cyclic at 5 pM than for [G (3 ', 5') pG (3 ', 5') p] cyclic at 5 pM in all four donors. Similarly, the gene expression of interferon beta 1 (IFNB1) was significantly higher for [G (2 ', 5') pG (3 ', 5') p] cyclic at 5 pM than for [G (3 ', 5 ') pG (3', 5 ') p] cyclic at 5 pM in all four donors. Gene expression for interferon gamma (IFNG) was induced at a significantly higher level for [G (2 ', 5') pG (3 ', 5') p] cyclic at 5 pM than for [G (3 ', 5 ') pG (3', 5 ') p] cyclic in all four donors. These data demonstrate the increased potency of cyclic [G (2 ', 5') pG (3 ', 5') p] compared to [G (3 ', 5') pG (3 ', 5') p ] cyclic induces gene expression of critical innate immune cytokines in various human donors.
[0369] As shown in Fig. 9 (a), the levels of secreted IFN-α induced in primary human PBMC by cyclic [G (2 ', 5') pG (3 ', 5') p] at 5 pM are higher than those of cyclic [G (3 ', 5') pG (3 ', 5') p] at the same or lower dose in all four donors. In Fig. 9 (b), the levels of FN-p, as assessed by ELISA, for [G (2 ', 5') pG (3 ', 5') p] cyclic at 5 pM were also greater than with the levels induced by [G (3 ', 5') pG (3 ', 5') p] cyclic, as well as for the ISD and Poly (I: C) controls in the four donors. Fig. 9 (c) demonstrates a similar finding for IFN-y secretion, as assessed by CBA. At both 5 pM and 0.5 pM, cyclic [G (2 ', 5') pG (3 ', 5') p] induced higher levels of IFN-y than [G (3 ', 5' ) pG (3 ', 5') p] cyclic at the same doses and higher levels as the ISD and Poly (I: C) controls in all four donors. These data demonstrate the increased potency of cyclic [G (2 ', 5') pG (3 ', 5') p] compared to [G (3 ', 5') pG (3 ', 5') p ] cyclic to stimulate the production of IFNs of type I and II, critical for the induction of innate immunity through a large sample of human donors.
[0370] To determine the relative level of IFN-p induced in human cells by each of the native and derived molecules as a hallmark of adjuvant potency, 4 x 10 ⁵ THP1-Blue cells, a human monocyte cell line transfected with an IRF-inducible secreted embryonic alkaline phosphatase reporter gene (Invivogen), with 50 pM of [G (3 ', 5') pG (3 ', 5') p] (CDG) cyclic, [G (2 ', 5 ') pG (3', 5 ') p] cyclic (mixed bond or ML-CDG), Rp, Rp-dithio- [G (2', 5 ') pG (3', 5 ') p] cyclic (ML RR-Cd G), compared to [A (3 ', 5') pA (3 ', 5') p] (CDA), [A (2 ', 5') pA (3 ', 5') p ] cyclic (mixed bond or Ml-CDA), Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (Ml r R-CDA) or media control for 30 minutes at 37 ° C with 5% CO2. After 30 minutes, cells were washed and seeded in 96-well plates in RPMI medium containing 10% FBS, and incubated at 37 ° C with 5% CO2. Cell culture supernatants from each sample were collected after overnight incubation, and 20 µl of cell culture supernatants were added to 180 µl QUANTI-Blue reagent (Invivogen) and incubated for 45 minutes. Absorbance readings were taken at 655 nm at 15 minutes using a SpectraMax spectrophotometer (Molecular Diagnostics).
[0372] As shown in Fig. 10, the derivative Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) induced significantly higher levels of IFN -p than unmodified cyclic c-di-GMP (CDG) or modified CDG molecules. Similarly, the Rp molecule, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) induced significantly higher IFN-p levels than the unmodified CDA or ML CDA molecules. These results demonstrate that the purified preparations of ML RR-CDN derivatives activate the innate immune response more profoundly than parental CDN molecules in a human monocyte cell line.
[0374] To determine the relative ability of the derived molecules to activate immune responses, CDN compounds were administered to 6-8 week-old female BALB / c mice (in a total volume of 100 μl in HBSS) at doses of 50.5 and 0.5 pM by subcutaneous injection at the base of the tail. Mice were evaluated 24 hours later for activation of lymphocytic immune cells by fluorescent activated cell sorting (FACS) for upregulation of surface CD69 expression on natural killer (NK) lymphocytes, CD4 + T lymphocytes, and lymphocytes. CD8 + T, compared to IgG1 isotype controls.
[0375] As shown in Fig. 11 (ac), the Rp molecule, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) induced a potent immune activation of NK and T lymphocytes in a dose-dependent manner. The Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic molecule (ML RR-CDA) also induced the activation of NK and T lymphocytes, although to a lesser extent than the ML RR-CDG molecule. Both ML RR-CDN molecules induced greater activation of immune cells than ML CDN molecules at all doses. These data demonstrate the increased immune-activating properties of ML RR-CDN molecules compared to ML CDN molecules, and specifically, highlight the ability of the ML RR-CDG molecule to induce potent activation of cells. immune.
[0377] Example 6. Enhanced resistance of Rp, Rp-dithio-CDN to phosphodiesterases
[0379] Induction of type I interferon in human cells was measured to assess the potency of untreated and phosphodiesterase-treated oxo, Rp-monothium derivatives and Rp, Rp-dithium. Five compounds ([A (3 ', 5') pA (3 ', 5') p] cyclic (CDA), [A (2 ', 5') pA (3 ', 5') p] cyclic (ML- CdA), Rp-monothio (Rp, monothio- [A (2 ', 5') pA (3 ', 5') p] cyclic (m L R-CDa), Rp, Rp-dithio (Rp, Rp-dithio - [A (3 ', 5') pA (3 ', 5') p] cyclic (Rr -CDA) and Rp, Rp-dithio [A (2 ', 5') pA (3 ', 5') p ] cyclic (ML RR-CdA) were treated either with 160 pg of snake venom phosphodiesterase (SVPD) from Crotalus adamanteus (Sigma), 2.5 mU of Nuclease PI (NP1) from Penicillium citrinum (Sigma) or with sham treatment 7 pg of each compound were diluted in SVPD buffer (1 x PBS and 0.6 mM MgCh), NP1 buffer (30 mM Na acetate, pH 5.3, 2 mM ZnCh) or left untreated and then incubated for 2 hours at 37 ° C, followed by boiling for 10 minutes to inactivate nucleases. 4 x 10 ⁵ THP1-Blue ™ ISG lymphocytes (a human monocyte cell line transfected with an inducible secreted embryonic alkaline phosphatase reporter gene) were incubated by IRF (Invivogen) expressing alkaline phosphatase under the control of ap romotor composed of five response elements stimulated by IFN) with 50 pM of molecules with sham treatment, treated with SVPD or treated with NP1. After 30 minutes, cells were washed and seeded in a 96-well plate in RPMI medium containing 10% FBS, and incubated at 37 ° C with 5% CO2. Cell culture supernatants were collected from each sample after incubation for 16 h, and 20 µl of cell culture supernatants were added to 180 µl of QUANTI-Blue reagent (Invivogen) and incubated for 25 minutes to assess levels of type I interferon protein. Readings at 655 nm absorbance were measured with a Versa Max spectrophotometer (Molecular Diagnostics).
[0381] As shown in Figure 12, the untreated Rp, Rp-dithio compounds, Rp, Rp-dithio- [A (3 ', 5') pA (3 ', 5') p] cyclic (RR-CDA) and Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) are more potent inducers of type I interferon than oxo ([A (3 ', 5') cyclic pA (3 ', 5') (CdA) and [A (2 ', 5') pA (3 ', 5') p] cyclic (ML-CDA) and Rp-monothio. Molecules derivatives of CDN (Rp, monothio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML R-CDa). The activity of CDN derivatives was evaluated after treatment with phosphodiesterase SVPD, which cleaves both 2'-5 'and 3'-5' phosphodiester bonds, or with NP1, which selectively digests the 3'-5 'phosphodiester bonds (Pino, et al, (2008) Journal of Biological Cheimistry, 283 , 36494 36503) Figure 12 shows that the compounds Rp, Rp-dithio, Rp, Rp-dithio- [A (3 ', 5') pA (3 ', 5') p] cyclic (RR-CDA) and Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) retain their potency after treatment with SVPD and NP1, while oxo ([A ( 3 ', 5') pA (3 ', 5') p] cyclic (c Da) and [A (2 ' , 5 ') pA (3', 5 ') p] cyclic (ML-CDA) lost activity after digestion with SVPD and NP1. The Rp-monothio derivative (Rp, monothio [A (2 ', 5') pA (3 ', 5') p] cyclic (ML R-CDA) containing a single thio substitution at the 3'-5 phosphodiester bond retained activity upon NP1 digestion, but was susceptible to treatment with SVPD, which cleaves the 2'-5 'phosphodiester bond. The differential susceptibility of the oxo, Rp-monothio and Rp, Rp-dithium derivatives to SVPD or NP1 digestion confirms the structure of the Rp-monothio and Rp, Rpdithium derivatives. These results also demonstrate the usefulness of Rp, Rp-dithio derivatives due to their resistance to digestion with phosphodiesterases, present in sera and / or host cells, thus resulting in a more powerful activation of innate immune signaling and greater efficacy in vivo antitumor therapy , as shown herein.
[0383] Example 7. Molecules derived from synthetic CDN potently activate the signaling of all human STING alleles
[0385] To determine the responsiveness of the five known natural human STING variants (named WT, REF, HAQ, AQ, and Q) to native and derived molecules, a panel of human embryonic kidney (HEK) 293T cell lines expressing human STING alleles. The parenta1HEK 293T cell line does not express endogenous STING, so the responsiveness of exogenously expressed STING alleles can be evaluated. The MSCV2.2 plasmids encoding hSTING (REF) -GFP, hSTING (WT) -GFP, hSTING (HAQ) -GFP, hSTING (Q) -GFP, and mSTING (WT) -GFP were obtained from the Vance Laboratory at UC Berkeley. hSTING (AQ) -GFP was derived from hSTING (Q) -GFP using a QuickChange site-directed mutagenesis kit (Stratagene). The hSTING (REF) allele sequence is also known as the Barber allele (Ishikawa, H., and Barber, GN (2008). Nature 455, 674-678), and has the NCBI reference sequence NP_938023.1. The amino acid difference between hSTING (REF) and the rest of the human STING alleles WT, HAQ, AQ and Q is shown in Fig. 13, which is adapted from Yi et al., Plos One 8: e77846 (2013). Stable HEK 293T-derived cell lines expressing each of the individual human STING alleles were generated by FACS sorting of GFP-positive cells using a Mo Flo cell sorter at the UC Berkeley Cancer Research Laboratory Flow Cytometry Center. . 1 x 104 HEK293T STING cells were seeded in 96-well plates and transiently transfected (using Lipofectamine 2000) with 50 ng of a human IFN-p reporter plasmid (pLuc-IFN-p) expressing the human IFN-p promoter chain. above a luciferase indicator and 10 ng of TK-renilla for normalization. After 24 hours, cells were stimulated with synthetic and native CDN-derived molecules using digitonin permeabilization to ensure uniform absorption. Each STING cell line was stimulated with 10 pM of [G (3 ', 5') pA (3 ', 5') p] cyclic (cGAMP), [G (2 ', 5') pA (3 ', 5' ) p] cyclic (ML-cGAMP), Rp, Rp-dithio- [G (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-cGAMP), [A (3 ', 5 ') pA (3', 5 ') p] cyclic (CDA), Rp, Rp-dithio- [A (3', 5 ') pA (3', 5 ') p] cyclic (RR-CDA), [ A (2 ', 5') pA (3 ', 5') p] cyclic (ML-CDA), Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA), [G (3 ', 5') pG (3 ', 5') p] cyclic (CDG), Rp, Rp-dithio- [G (3 ', 5') pG (3 ', 5') p] cyclic (RR-CDG), [G (2 ', 5') pG (3 ', 5') p] cyclic (ML-CDG) or Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) in 25 ul digitonin buffer (50 mM HEp ES, 100 mM KCl, 3 mM MgCh, 0.1 mM Dt T, 85 mM sucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP, 10 ug / ml digitonin). After 20 min, the stimulation mixtures were removed and 200 ul of standard RPMI medium was added. After stimulation for 6 hours, cell lysates were prepared and reporter gene activity was measured using the dual luciferase assay system (Promega) on a Spectramax M3 luminometer.
[0387] Fig. 14 depicts the stimulation of HEK293 cell lines encoding alleles of human STING variants by measuring the IFNp-LUC reporter induction factor (ULR plotted on the y-axis). As shown in Fig. 14, the compounds Rp, Rp-mixed-bond dithio, Rp, Rp-dithio- [G (2 ', 5') pA (3 ', 5') p] cyclic (ML RR- cGAMP), Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) and Rp, Rp-dithio- [A (2 ', 5' ) cyclic pA (3 ', 5') p] (ML Rr -CDA) strongly induce IFNp reporter activity by all human STING alleles. The refractory human STING alleles, hSTING (REF) and hSTING (Q), responded at a low level to stimulation with native molecules with canonical internucleotide phosphate bridge bonds: [G (3 ', 5') pA (3 ', 5 ') p] cyclic (cGAMP), [A (3', 5 ') pA (3', 5 ') p] cyclic (CDA); and [G (3 ', 5') pG (3 ', 5') p] cyclic (CDG). Surprisingly, in contrast, cell lines expressing refractory human STING alleles responded to stimulation with Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR -CDA) synthetic: ML RR-CDA; ML RR-CDG; and ML RR-cGAMP. Cells expressing mouse STING responded to all molecules tested, demonstrating that molecules derived from modified synthetic CDNs are relevant for the activation of the human STING signaling pathway. These results demonstrate that the compounds Rp, Rp-mixed-bond dithio, Rp, Rp-dithio- [G (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-cGAMP), Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) and Rp, Rp-dithio- [A (2 ', 5') pA (3 ' , 5 ') p] cyclic (ML RR-CDA) potently activate all tested human STING alleles, indicating that these molecules will effectively induce innate immunity in a wide selection of the human population.
[0389] To demonstrate that synthetic CDN-derived molecules induced human dendritic cell (DC) maturation, CD14 + monocytes from human PBMCs were treated for 6 days with 50 ng / ml GM-CSF and 25 ng / ml IL-4 . Seven days later, the monocyte derived DCs were stimulated with LPS (1 pg / ml) or CDN (50 pM) added directly to the media. After 48 h, the surface expression of MHC class I (HLA-ABc), CD80, CD83 and CD86 was determined by activated FACS in the population of DC CD11c +. Fig. 15A represents bar graphs indicating the mean mean fluorescence intensity (MFI) after stimulation with the CDN molecules indicated in the figure. Representative histograms of CD80, CD86, CD83, and MHC Class I (HLA-ABC) expression in human DC are also shown in Fig. 15B. The filled histograms correspond to unstimulated cells, the dotted line represents stimulation with LPS, and the solid line represents stimulation with Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA). These results demonstrate that the molecules of synthetic CDNs with structures comprising the Rp, Rp-dithium substitution of the loose oxygen atoms of the internucleotide phosphate bridge in combination with the non-canonical or mixed 2'-5, 3'-5 binding phosphate bridge structure '(ML) activates signaling in all human STING alleles, and potently activates maturation of human DCs.
[0390] Example 8. CDN-induced antigen-specific T-lymphocyte response
[0391] To determine the OVA-specific CD8 T lymphocyte response induced by the different cyclic dinucleotide molecules, C57BL / 6 mice (n = 5) were immunized subcutaneously with 0 pg (no CDN) or with 5 pg or 25 pg of [G (2 ', 5') pG (3 ', 5') p] (mixed bond or ML-CDG) formulated in 2% squalene and water with 10 pg ovalbumin protein. Seven days after vaccination, blood was drawn from each animal and PBMC were prepared. 5 x 10 ⁴ PBMC were stimulated overnight in an IFNy ELISpot assay with medium alone (no stimulation) or with peptide OVA257-2641 pM in the presence of 1 x 10 ⁵ naive splenocytes as feeder cells. IFNy ELISpots were developed and quantified using a CTL plate reader and ImmunoSpot software.
[0392] As shown in Fig. 16, both doses of cyclic [G (2 ', 5') pG (3 ', 5') p] (ML-CDG) induce OVA-specific CD8 immune responses in C57BL / 6 mice. . These responses are significantly higher than the responses induced by the unstimulated controls and by a control group without CDN. These results demonstrate that formulations of [G (2 ', 5') pG (3 ', 5') p] cyclic (ML-CDG) with an antigen can stimulate antigen-specific CD8 T cell responses in vivo.
[0393] To determine whether STING signaling is required for c [G (2 ', 5') pG (3 ', 5') p] (ML-CDG) to induce an OVA-specific c D8 T cell response, we immunized C57BL / 6 mice (n = 3 or 5) and goldenticket mice (n = 3) subcutaneously with 0 pg (no CDN) or 25 pg of c [G (2 ', 5') pG (3 ', 5 ') p] (ML-CDG) formulated in 2% squalene and water with 10 pg of ovalbumin protein. Seven days after vaccination, blood was drawn from each animal and PBMC were prepared. 5 x 10 ⁴ PBMC were stimulated overnight in an IFNy ELISpot assay with medium alone (no stimulation) or with peptide OVA257-2641 pM in the presence of 1 x 10 ⁵ naive splenocytes as feeder cells. IFNy ELISpots were developed and quantified using a CTL plate reader and ImmunoSpot software.
[0394] Fig. 17 shows that c [G (2 ', 5') pG (3 ', 5') p] (ML-CDG) induces an OVA-specific CD8 T-cell response that depends on the presence of a STING molecule. functional. In wild-type C57BL / 6 mice with a functional STING molecule, the formulation of c [G (2 ', 5') pG (3 ', 5') p] (ML-CDG) and ovalbumin protein induces immune responses Specific OVA257-264 significant compared to the control without stimulation and a control without CDN. In goldenticket mice, which do not express a functional STING molecule (Sauer, "Infection and Immunity" 2011), the OVA-specific responses induced by c [G (2 ', 5') pG (3 ', 5') p] ( ML-CDG) are not significantly different from the OVA-specific responses induced by a control formulation that does not include CDN (no CDN). These results indicate that the immune response induced by c [G (2 ', 5') pG (3 ', 5') p] (ML-CDG) requires a functional STING molecule.
[0395] Example 9. Comparative data with different derivatives of CDN
[0396] To assess the ability of the derived molecules to enhance antitumor immunity, B16 melanoma cells (5 x 10 ⁴ cells in 100 µl PBS) were implanted subcutaneously in the lumbar part of 6-8 week old female C57BL / 6 mice. (8 mice per group). Treatments began when tumors reached a volume of approximately 75 mm ³ , on day 14 after tumor implantation. The CDN compounds were administered (25 µg in a total volume of 40 µl of HBSS) by subcutaneous injection into the center of the tumor using a 27-gauge needle. Injections were repeated every three days, for a total of three intratumoral injections. The CDNs tested were [G (3 ', 5') pG (3 ', 5') p] cyclic (CDG); [G (2 ', 5') pG (3 ', 5') p] cyclic (mixed bond or ML CDG); Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG); [A (3 ', 5') pA (3 ', 5') p] cyclic (CDA); [A (2 ', 5') pA (3 ', 5') p] cyclic (mixed bond or Ml CdA); and Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CdA).
[0397] As shown in Fig. 18, ML RR-CDG and ML RR-CDA derivatives induced potent antitumor efficacy, compared to cyclic ML CDG and cyclic m L CDA cyclic dinucleotide molecules. The ML RR-CDA molecule induced significantly more tumor rejection than the ML CDA derivative (p = 0.0004, Student's t-test), and mice in the ML RR-CDG tumor group remained nearly tumor-free on day 44 after of tumor implantation. These data demonstrate the enhanced potency of ML RR-CDN derivatives compared to ML CDN derivative molecules and the significant antitumor efficacy of ML RR-CDN molecules in the B16 melanoma mouse model.
[0398] To further assess the ability of the derived molecules to enhance antitumor immunity, CT26 colon carcinoma cells (2 x 10 ⁵ cells in 100 μl of p Bs) were implanted by intravenous injection into 6-6 female BALB / c mice. 8 weeks old and overall survival was assessed. The CDN compounds (25 µg in a total volume of 100 µl HBSS) were administered one day after tumor implantation by subcutaneous injection at the base of the tail. The mice were boosted with an additional injection one week later for a total of two vaccines.
[0399] As shown in Fig. 19A, cyclic Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] (ML RR-CDG) induced significantly higher survival rates. high compared to the [G (2 ', 5') pG (3 ', 5') p] cyclic (ML CDG) molecule (p = 0.0018, log-rank test), and Rp, Rp-dithio - [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) induced significantly higher survival rates compared to the [A (2 ', 5') pA molecule (3 ', 5') p] cyclic (ML CDA) (p = 0.0005, logarithmic rank test). This demonstrates the significant antitumor efficacy of ML RR-CDN derivatives compared to ML CDN derivative molecules in a CT26 lung metastasis survival model. These results demonstrate that the CDN derivative molecules can be successfully administered subcutaneously.
[0401] To demonstrate that the activation of tumor-initiating T-lymphocyte sensitization and antitumor efficacy induced by CDN-derived molecules was not limited to a single tumor type and mouse genetic background, the ability of synthetic CDNs to enhance antitumor immunity in other tumor models. CT26 colon carcinoma cells (1 x 105 cells in 100 µl PBS) or 4T1 breast carcinoma cells (1 x 105 cells in 100 µl PBS) were implanted subcutaneously into the flank of female BALB / c mice from 6-8 weeks (8 mice per group). The treatments began when the tumors reached a volume of approximately 75 mm3, which was approximately day 14 after tumor implantation. The compounds Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) or Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) (25 pg in a total volume of 40 µl HBSS), or HBSS vehicle control, and Rp, Rp-dithio- [A (2 ', 5 ') cyclic pA (3', 5 ') p] cyclic (μL RR-CDA) (50 μg in a total volume of 40 μl HBSS) or HBSS vehicle control, were administered by subcutaneous injection in the center of the tumor using a 27 gauge needle. Injections were repeated every three days, for a total of three intratumoral injections.
[0403] As shown in Fig. 19B, ML RR-CDG completely inhibited tumor growth in 7 of the 8 mice, while ML RR-CDA completely inhibited tumor growth of all established CT26 tumors. As shown in Fig. 19C, the ML RR-CDA derivative completely inhibited the tumor growth of all established 4T1 breast tumors. These data demonstrate the surprising potency and long-lasting antitumor efficacy of synthetic derivatives of Rp, Rp-mixed-binding cyclic dinucleotide (ML RR-CDN) in multiple tumor models.
[0405] Example 10. The antitumor efficacy induced by CDN depends on STING
[0407] To determine whether the effects of the derived molecules are dependent on STING, B16 melanoma cells (5 x 104 cells in 100 µl PBS) were implanted into the right flank of 6-8 week old female STING '/ _ goldenticket mice or wild-type C57BL / 6 control mice (5 mice per group). Treatments began when the tumors reached a volume of approximately 75 mm3, on day 14 after tumor implantation. The compounds administered were Rp, Rp-dithio- [G (2 ', 5') pG (3 ', 5') p] cyclic (ML RR-CDG) (25 pg in a total volume of 40 µl of HBSS), Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CdA) (50 pg in a total volume of 40 µl HBSS), the TLR9 agonist CpG 1668 (50 pg in a total volume of 40 µl HBSS) or HBSS vehicle control. Mice were treated by subcutaneous injection into the center of the tumor using only a 27 gauge needle. Injections were repeated every three days, for a total of three intratumoral injections.
[0409] As shown in Fig. 20A, ML RR-CDN derivatives induced drastic tumor inhibition in wild-type C57BL / 6 mice compared to the HBSS control, and significantly more tumor inhibition than the TLR9 agonist CpG 1668 (p = 0.03, Student's t test). In Fig. 20B, tumor growth was not inhibited by ML RR-CDG or ML RR-CDa, demonstrating that the antitumor efficacy observed in wild-type C57BL / 6 mice (Fig. 20a) was completely dependent on one pathway. signaling STING functional. On the contrary, the tumor growth of CpG 1668 was similar both in wild-type mice and in STING '/ _ mice, compared to the HBSS control (p = 0.03, Student's t-test), showing that the action of this compound is independent of STING.
[0411] Example 11. Derivatives of CDN induce long-lasting and effective immunity against tumor-specific T lymphocytes
[0412] To determine whether synthetic derived CDN molecules produce long-lasting and effective immunity against tumor-specific T lymphocytes, CT26 colon carcinoma cells (1 x 105 cells in 100 µl of PBS) were implanted into 6-7 female BALB / c mice. 8 weeks (8 mice per group). Mice were treated with Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic compound (ML RR-CdA) (50 pg in a total volume of 40 μl of HBSS) or HBSS vehicle control, and tumor growth was controlled according to the above example. Mice were bled on day 18 after tumor implantation and PBMC were isolated by Ficoll gradient (Miltenyi Biotech). 5 x 10 4 PBMC were stimulated overnight in an IFNy ELISpot assay with medium alone (no stimulation) or with 1 pM of the H-2 Ld restricted endogenous tumor rejection peptide antigen AH1 in the presence of 1 x 10 5 splenocytes without prior treatment as feeder cells. IFN-y ELISpot plates were developed and quantified using a CTL plate reader and ImmunoSpot software. On day 55 after tumor implantation, CT26 or 4T1 tumor cells (both 1 x 105 cells in 100 µl PBS) were implanted into surviving mice and age-naïve control mice on the contralateral flank (4 mice per group), and tumor growth was monitored.
[0413] As shown in Fig. 21A, all ML RR-CDA treated mice rejected the growth of established CT26 colon carcinomas. To demonstrate that the effect was mediated by the CDN-mediated induction of an adaptive immune response of T lymphocytes, PBMCs were evaluated on day 18 after tumor induction for IFN-y production by the ELISpot assay, in response to stimulation with endogenous tumor antigen AH1. As shown in Fig. 21B, PBMC isolated from ML RR-CDA treated mice generated a significantly higher IFN- y level in response to stimulation of the AH1 peptide, compared to the control group treated with HBSS ( p = 0.003, Student's t test). Furthermore, in Fig. 21C, surviving mice that were re-exposed to a contralateral tumor exhibited complete protection against the same CT26 tumor type, without inhibiting the growth of the 4T1 tumor type. These data demonstrate the ability of ML RR-CDA to generate long-lasting and effective tumor-specific T-cell mediated antitumor immunity that produces both rejection of the treated tumor and a stable population of tumor antigen-specific memory T cells that can reject tumor. tumor exposure.
[0415] To determine whether the CDN-derived molecules induce effective and long-lasting antitumor immunity in an alternative tumor model, 6-8 week old female BALB / c mice (8 mice per group) were implanted with 4T1 breast carcinoma cells (1 x 105 cells in 100 µl PBS). Mice were treated with Rp compound, Rpdithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) (50 pg in a total volume of 40 µl of HBSS) or HBSS vehicle control, according to the previous experiment. On day 35 after tumor implantation, CT26 or 4T1 tumor cells (both 1x10 5 cells in 100 µl PBS) were implanted into surviving mice and age-naïve control mice on the contralateral flank (4 mice per group), and tumor growth was monitored.
[0417] As shown in Fig. 22A, and previously demonstrated, ML RR-CDA treatment completely inhibited tumor growth of established 4T1 breast carcinomas. Furthermore, in Fig. 22B, repeated exposure to 4T1 tumor cells on the contralateral side induced complete protection. Repeated exposure to the more immunogenic CT26 tumor also generated complete protection, indicating that these tumors share similar tumor antigens, providing further evidence for the potency of synthetic CDN-derived molecules.
[0419] Example 12. Activation of tumor initiation T cell sensitization by intratumoral injection with synthetic derivatives of CDN induces abscopal tumor inhibition.
[0421] The examples shown herein demonstrate that intratumoral (IT) injection of synthetic derivatives of CDN produces surprising and long-lasting tumor kill, due to STING-dependent activation of pro-inflammatory cytokines, to facilitate the development of effective T-cell immunity. tumor specific. STING-dependent induction of tumor-specific T-lymphocyte immunity protects animals against subsequent exposure to the autologous tumor. It will be apparent to those of skill in the art that advanced cancer is metastatic and that, to be effective, therapies must inhibit the growth of distal masses. Treatment of a selected lesion or lesions that inhibits tumor outgrowth of untreated distal tumor masses is known as the abscopal effect. To test whether IT injection of a selected tumor with synthetic CDN-derived molecules inhibited tumor outgrowth of an untreated distal tumor, (A) CT26 colon carcinoma cells were implanted subcutaneously (1 x 105 cells in 100 µl of PBS) or (B) 4T1 breast carcinoma cells (1 x 105 cells in 100 µl PBS) in the contralateral flank of 6-8 week old female BALB / c mice (8 mice per group). Treatments began when the tumors reached a volume of approximately 75 mm3, on day 13 after tumor implantation. The compound of Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) (50 pg in a total volume of 40 μl of HBSS) or control of HBSS vehicle, was administered by subcutaneous injection into the center of the primary tumor (right side) with a 27 gauge needle alone. Injections were repeated every three days, for a total of three intratumoral injections.
[0423] As shown in Fig. 23, the compound Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic (ML RR-CDA) induced complete inhibition of the primary tumor treated in both CT26 (Fig. 23A) and 4T1 (Fig. 23B) tumor bearing animals, compared to the HBSS vehicle control. In addition, contralateral (untreated) tumor growth in both tumor models was also significantly inhibited, compared to HBSS controls (Fig. 23A, p = 0.0011, Fig. 23B, p = 0.0019, t-test Student). These data demonstrate the significant antitumor efficacy of the ML RR-CDA derivative when injected into the primary tumor, as well as its significant abscope anti-tumor immune effects.
[0425] To determine whether synthetic CDN-derived molecules enhance abscopal antitumor immunity in an alternative tumor model and other mouse genetic backgrounds, B16 melanoma cells were implanted into 6-8 week-old female C57BL / 6 mice (8 mice per group) (5 x 104 cells in 100 µl PBS) on the right side. One week later, mice were implanted intravenously with 1 x 10 5 B16 melanoma cells to colonize the lung, along with a group of naïve control mice of the same age. When the subcutaneous flank tumor had reached approximately 75 mm3 on day 13, mice were treated intratumorally with Rp, Rp-dithio- [A (2 ', 5') pA (3 ', 5') p] cyclic ( ML RR-CDA) (50 pg in a total volume of 40 μl Hb Ss) or HBSS vehicle control, for three injections according to the above protocol. On day 28 after subcutaneous tumor implantation (day 21 after iv implantation), the mice were sacrificed, and the mice were harvested and fixed. lungs (10% neutral buffered formalin), and the number of lung tumor nodes was counted using a dissecting microscope.
[0427] As shown in Fig. 24A, and in previous experiments, ML RR-CDA treatment significantly inhibited tumor growth of the primary flank tumor, compared to the HBSS control group (p <0.001, Student's t-test ). In addition, in Fig. 24B and as depicted in Fig. 24C, treatment with the CDN derivative significantly inhibited the growth of distal tumor nodes in the lung, compared to the HBSS and treatment-naïve tumor groups ( only iv). The results shown in the present document demonstrate that the intratumoral (IT) injection of synthetic derivatives of CDN produces an abscopal antitumor effect, as demonstrated by the destruction of the treated tumor, due to the STING-dependent activation of pro-inflammatory cytokines and the development of effective tumor-specific T-lymphocyte immunity, which then inhibits the outgrowth of untreated distal tumors.

权利要求:
Claims (15)
[1]
1. A compound of formula

[2]
2. A composition comprising a compound according to claim 1 and a pharmaceutically acceptable excipient.
[3]
3. A composition according to claim 2, comprising a delivery vehicle that enhances cell uptake and / or stability of the compound.
[4]
4. A composition according to claim 3, wherein the delivery vehicle comprises one or more agents selected from the group consisting of lipids, interlayer cross-linked multilamellar vesicles, nanoparticles or biodegradable microparticles based on poly (D, L- lactic-co-glycolic) [PLGA] or based on polyanhydride, and lipid bilayers supported by nanoporous particles.
[5]
A composition according to any one of claims 2 to 4, further comprising a CTLA-4 antagonist, a TLR agonist, CpG, monophosphoryl lipid A and / or an inactivated tumor cell expressing and secreting one or more cytokines that stimulate the induction, recruitment and / or maturation of dendritic cells.
[6]
6. A composition according to claim 5, wherein the inactivated tumor cell expresses and secretes GM-CSF, CCL20, CCL3, IL-12p70 or FLT-3 ligand.
[7]
7. A composition according to any one of claims 2 to 4, further comprising one or more antigens selected for the purpose of inducing an immune response against the antigen / s when the composition is administered to an individual.
[8]
8. A compound according to claim 1 or a composition according to any one of claims 2 to 7 for use in a method of treatment.
[9]
9. A compound according to claim 1 or a composition according to any one of claims 2 to 7 for use in a method of treating an individual suffering from cancer.
[10]
10. A compound or composition for use according to claim 9 in a method of treating an individual suffering from a cancer selected from the group consisting of:
a colorectal cancer, a lung cancer, a brain cancer, a liver cancer, a stomach cancer, a sarcoma, a leukemia, a lymphoma, a multiple myeloma, an ovarian cancer, a uterine cancer, a breast cancer , a melanoma, a prostate cancer, a pancreatic carcinoma, a kidney carcinoma, head and neck cancer, and cervical cancer.
[11]
11. A compound or composition for use according to any one of claims 9 to 10 in a method of treatment, wherein said treatment comprises:
induce an immune response in the individual;
inducing STING-dependent type I interferon production in the individual;
administering said compound or said composition to the individual, wherein the individual expresses a cancer antigen, before or after a primary therapy administered to kill or destroy cancer cells expressing said cancer antigen in the individual;
parenterally administer said compound or said composition to the individual, wherein the individual suffers from cancer; or
administering said compound or said composition together with one or more therapeutic antibodies that induce antibody-dependent cellular cytotoxicity in order to stimulate antibody-dependent cellular cytotoxicity in the individual.
[12]
12. A compound or composition for use according to claim 11, wherein the compound or composition is administered after primary therapy.
[13]
A compound or composition for use according to claim 11 or claim 12, wherein the primary therapy comprises surgery to kill cancer cells in a mammal, radiation therapy to kill cancer cells in the mammal, or surgery and radiation therapy. .
[14]
14. A compound or composition for use according to claim 11, wherein parenteral administration is subcutaneous, intramuscular, or intradermal, and optionally, directly into a tumor mass.
[15]
15. A compound or composition for use according to any one of claims 11 to 14, wherein the method further comprises administering to the mammal one or more of a CTLA-4 antagonist, an antagonist of the PD- pathway 1, a TLR agonist, or one or more therapeutic antibodies.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201361825005P| true| 2013-05-18|2013-05-18|

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US201361902125P| true| 2013-11-08|2013-11-08|

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