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  • C12N5/0646—Natural killers cells [NK], NKT cells
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  • A61K31/47—Quinolines; Isoquinolines
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  • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
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  • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
  • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
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  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
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  • C12N2501/20—Cytokines; Chemokines
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/999—Small molecules not provided for elsewhere

Definitions

• NK Natural Killer
• NK cells are innate lymphoid cells (ILCs), specifically large granular cytotoxic lymphocytes that bridge the innate and the adaptive arms of the immune response.
• ILCs innate lymphoid cells
• NK cells 15 also exhibit the highest level of cytotoxic activity within the immune system. Therefore, altered NK cell functionality or numbers impact the functioning of the immune system against infection and cancer. For example, a large scale study in Japan has shown that reduced levels of NK cells in a cohort of people aged over 40 is associated with a significantly higher incidence of cancer.
• CLP Common Lymphoid Progenitor 20
• HSCs Haematopoietic Stem Cells
• NK cells are different from B and T cells as they lack specific cell surface antigen receptors.
• NK cells may kill cancerous and pathogen ⁇ infected cells without prior sensitisation, making them part of the innate immune response. They also have a critical role in tumour immunosurveillance by directly influencing the adaptive immune response. 25 Activation of NK cells triggers them to release perforin and cytoplasmic granules containing granzymes. Perforin polymerises to form pores on target cells in the presence of Ca2+. Granzymes may enter these pores into target cells, causing DNA fragmentation and apoptosis. NK cells may also secrete cytokines, which trigger the action of other immune cells in the adaptive arm of the immunity. Numerous groups have worked on methods to increase the number of endogenous NK cells.
• cytokines that are essential for NK cell development.
• Administration of IL ⁇ 2 and IL ⁇ 15 was predicted to enhance NK cell development.
• IL ⁇ 2 promotes the proliferation and cytotoxicity of NK cells
• IL ⁇ 15 promotes the development and expansion of NK cells.
• the cytokines were found only stimulate a minimal expansion of NK cells with reduced half ⁇ life, even at a very high dose.
• administered cytokines often leads to systemic toxicity due to inappropriate activation of immune responses and the induction of NK cell apoptosis.
• producing large numbers of NK cells is difficult, and producing fully functional NK cells with high cytotoxicity is even harder.
• NK cells have a critical role in the immune system where they destroy 10 cancerous, pathogen ⁇ infected or damaged cells.
• Boosting NK cell number or functionality is predicted to increase the killing of these cells.
• Existing therapies such as NK cell adoptive transfer and cytokine enhancement of endogenous NK cells are not very successful in terms of their efficacy.
• NK cells are differentiated from the HSCs in the bone marrow and distributed throughout lymphoid and non ⁇ lymphoid tissues including lymph nodes, spleen, peripheral blood, lungs and liver.
• cytokines and transcription factors are needed to encourage HSCs to develop into NK cells. Each cytokine and transcription factor must be present at a precise time and concentration in order to push differentiation from HSCs into NK cells. However, the precise hierarchy of cytokines and transcription factors governing NK cell maturation is still incompletely understood.
• the present inventors have previously shown that inhibiting the action of REV ⁇ ERB increases 20 NK cell production.
• the inventors demonstrated that inhibiting the action of REV ⁇ ERB, e.g. using the REV ⁇ ERB antagonist SR8278, increases E4bp4 expression, which in turn increases NK cell production.
• the structure of SR8278 is shown below: S Many synthetic ligands for REV ⁇ ERB have been generated in the art.
• the compounds in this library all possess an alkyl ester group or similar attached to the tetrahydroisoquinoline (or open ⁇ ring equivalent) core at position 3 in structure shown below: 10
• an ethyl ester group is present in this position. Therefore, the research thus far has strongly suggested that an ester moiety, or similar, is important to the REV ⁇ ERB inhibitory action of the compounds.
• the present inventors have now developed a new library of compounds in which the group previously attached to the tetrahydroisoquinoline (or open ⁇ ring equivalent) core at position 3 has 15 been replaced for instance with a carboxylic acid group.
• Acid ⁇ form compounds of the present invention have surprisingly been shown to possess activity as REV ⁇ ERB antagonists. Even more surprising is that the activities of these acid ⁇ form compounds have been shown to be enhanced relative to their ethyl ester counterparts. Further beneficial and surprising properties have been shown using acid ⁇ form compounds of 20 the present invention, including good plasma stability, high levels of target engagement and low levels of off ⁇ target effects. Acid ⁇ form compounds of the present invention have also been used to expand an NK cell population.
• the present invention provides ex vivo method for expanding an NK cell population, comprising the steps of: 25 a) culturing an haematopoietic progenitor cell (HPC) comprising sample obtained from an individual; b) adding a compound that inhibits the action of REV ⁇ ERB to said sample; and c) expanding said cells in vitro to produce an NK cell population; wherein the compound that inhibits the action of REV ⁇ ERB has the formula (I): I) where: represents bonds that are all either present or absent; R 1 is hydrogen; 5 R 2 is selected from 5 ⁇ 10 membered heterocyclyl rings and C 1 ⁇ 6 hydrocarbyl, and is optionally substituted with one or more groups independently selected from C 1 ⁇ 4 hydrocarbyl, ⁇ OR’, ⁇ OC(O)R’, ⁇ C(O)OR’, ⁇ SR’, ⁇ S(O)R’, ⁇ S(O) 2 R’, ⁇ NR’ 2 , ⁇ NR’C(O)R’
• the method of therapy in which the pharmaceutical composition or products may be used may be: (a) a method of treating a disease or disorder selected from cancer, an infectious disease (acute or chronic), an autoimmune disease or a disease or disorder related to female infertility or pregnancy; or (b) a method of treatment of a viral infection, a bacterial infection, a protest infection, a fungal infection and/or a helminth infection.
• the invention further provides a method of treatment by increasing the number of NK cells in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a compound which inhibits the action of REV ⁇ ERB according to the invention, and optionally a Notch ligand, wherein preferably the Notch ligand is delta ⁇ like ligand 4 (DLL4), or a fragment thereof which retains the function of DLL4.
• a compound which inhibits the action of REV ⁇ ERB according to the invention and optionally a Notch ligand, wherein preferably the Notch ligand is delta ⁇ like ligand 4 (DLL4), or a fragment thereof which retains the function of DLL4.
• DLL4 delta ⁇ like ligand 4
• the pharmaceutical composition or pharmaceutical products for use of the invention, or the method of treatment of the invention may be used in combination with antibody ⁇ mediated immunotherapy, wherein optionally said compound or products is for administration before, simultaneously with, or after administration of the antibody ⁇ mediated immunotherapy.
• the invention also provides an isolated compound of formula (I) as defined herein, provided 30 that the compound is not: , 5 , , a .
• the invention also provides the use of a compound of formula (I) as defined herein as a REV ⁇ ERB inhibitor, provided that the compound is not: 5 .
• the invention further provides a pharmaceutical composition comprising a compound of formula (I) as defined herein, and a pharmaceutically acceptable carrier, diluent and/or excipient, provided that the compound is not: 10 , F , , NH 2 ,
• a composition comprising a compound of formula (I) as defined herein, wherein the composition comprises the compound of formula (I) in an amount of at least 0.01%, preferably at least 0.1%, more preferably at least 1%, such as at least 10% by weight, 10 provided that the compound is not: , or .
• BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 NK cell developmental pathway. NK cells are differentiated from Hematopoietic Stem Cells (HSCs).
• NK cells develop from HSC into Common Lymphoid Progenitor (CLP) cells, NK progenitor 5 (NKP) cells, immature NK (iNK) cells, mature NK (mNK) cells and finally into conventional NK (cNK) cells, which circulate in the bloodstream.
• CLP Common Lymphoid Progenitor
• iNK immature NK
• mNK mature NK
• cNK conventional NK
• FIG. 1 Schematic representation of example two ⁇ stage culture of NK cell development using a REV ⁇ ERB 15 inhibitory compound and a Notch ligand.
• an embodiment, description, aspect or disclosure comprises the recited feature(s)
• the present invention also encompasses said embodiment, description, aspect or disclosure consisting of said feature(s).
• chemically feasible means a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim.
• the structures disclosed herein, in all of their embodiments are intended to include only “chemically feasible” structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed herein and do not form part of the present invention.
• an "analogue” of a chemical structure refers to a chemical 5 structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure.
• a related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative.”
• a substituent is specified to be an atom or atoms of specified identity, "or a bond”, a configuration is referred to when the substituent is "a bond” that the groups that are immediately 10 adjacent to the specified substituent are directly connected to each other in a chemically feasible bonding configuration. All chiral, diastereomeric, racemic forms of a structure are intended, unless a particular stereochemistry or isomeric form is specifically indicated.
• Hydrocarbyl groups are groups that consist only of carbon and hydrogen, though the groups may be substituted one or more times, as defined herein. Hydrocarbyl groups include straight chain and branched groups.
• hydrocarbyl groups typically have from 1 to 6 carbon atoms, particularly from 1 to 4 carbon atoms or from 1 to 3 carbon atoms.
• hydrocarbyl encompasses aromatic and non ⁇ aromatic groups.
• Preferred hydrocarbyl groups include alkyl, alkenyl and alkynyl groups which are described further below.
• Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons, from 1 to 8 carbon atoms, from 5 1 to 6 carbon atoms.
• straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n ⁇ propyl, n ⁇ butyl, n ⁇ pentyl, n ⁇ hexyl, n ⁇ heptyl, and n ⁇ octyl groups.
• branched alkyl groups include, but are not limited to, isopropyl, iso ⁇ butyl, sec ⁇ butyl, t ⁇ butyl, neopentyl, isopentyl, and 2,2 ⁇ dimethylpropyl groups.
• alkyl encompasses n ⁇ alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. 10 Representative substituted alkyl groups can be substituted one or more times, as defined herein. Cycloalkyl groups are cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8 ⁇ 12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7.
• Cycloalkyl groups further include polycyclic cycloalkyl 15 groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups can be mono ⁇ substituted or substituted more than once, as defined herein. 20 Alkenyl groups are alkyl groups, e.g. as described above, but which comprise at least one carbon ⁇ carbon double bond.
• alkenyl groups include straight chain and branched alkenyl groups and non ⁇ aromatic cycloalkenyl groups.
• Alkenyl groups are preferably, but not necessarily, bonded to the rest of a molecule through a carbon which forms part of a double bond.
• Representative substituted alkenyl groups can be mono ⁇ substituted or substituted more than once, as defined herein.
• 25 Alkynyl groups are alkyl groups, e.g. as described above, but which comprise at least one carbon ⁇ carbon triple bond.
• alkynyl groups include straight chain and branched alkynyl groups.
• Alkynyl groups are preferably, but not necessarily, bonded to the rest of a molecule through a carbon which forms part of a triple bond.
• Heterocyclyl groups/rings or the term "heterocyclyl” includes aromatic and non ⁇ aromatic ring compounds containing 3 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S.
• a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof.
• heterocyclyl groups include 3 to about 20 ring members, whereas in compounds of formula (I) heterocyclyl rings typically have 5 to 10 ring members.
• a heterocyclyl ring can be a 5 ⁇ membered ring with one heteroatom, a 6 ⁇ membered ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms.
• a heterocyclyl ring can also include one or more double bonds.
• a heteroaryl ring is an embodiment of a heterocyclyl group.
• the phrase "heterocyclyl 5 group" includes fused ring species including those comprising fused aromatic and non ⁇ aromatic groups.
• a dioxolanyl ring and a benzdioxolanyl ring system are both heterocyclyl groups within the meaning herein.
• the phrase also includes polycyclic ring systems containing a heteroatom as described herein.
• Heterocyclyl groups can be unsubstituted, or can be substituted as discussed above.
• 10 Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8 ⁇ 12 ring members.
• heteroaryl rings typically have 5 to about 10 ring members.
• a heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. Likewise a heteroaryl can be a 5 ⁇ ring with one heteroatom, a 6 ⁇ ring with 15 two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms.
• Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, 20 thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups.
• Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed above. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed above.
• Halogen refers to fluorine, chlorine, bromine or iodine.
• a "salt" as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion.
• exhausted NK cells may exhibit altered marker expression, such as an increase in the expression of one or more inhibitory receptor (as described herein) and/or a decrease in the expression of one or more activatory receptor 30 (as described herein).
• increased expression of NKG2A and/or Tim3 may be used as a marker for NK cell exhaustion. Again, the expression of these markers may be quantified relative to any appropriate control as defined herein.
• the terms “functional” and “fully functional” in the context of NK cells means that an NK cell or expanded NK cell population has all of the expected effector functions when responding to a given immune challenge.
• an expanded NK cell population 30 of the invention at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more, up to 100% of the NK cells of an expanded NK cell population of the invention are (fully) functional.
• at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% or more of the NK cells of an expanded NK cell population of the invention are fully functional, according to any definition (e.g. marker and/or effector function definition) herein.
• An expanded NK cell population of the invention may be produced by any of the methods disclosed herein. Typically an expanded NK cell population of the invention is produced by an ex vivo 5 method as disclosed herein.
• E4bp4 E4bp4 (also known as Nfil3) is a basic leucine zipper protein transcription factor which is involved in the regulation of IL ⁇ 3 expression, and is involved in the coordinating the circadian clock. 10
• the genomic DNA sequence of the human E4bp4 gene is given in SEQ ID NO: 1 (Genbank Accession No. X64318, version X64318.1). As shown in Figure 1, E4bp4 is expressed in CLPs and is critical in the production of NK cells from blood stem cell progenitors.
• E4bp4 is a lineage commitment factor, 15 controlling the development of NKPs from HSCs ( Figure 1).
• E4bp4 is a lineage commitment factor, 15 controlling the development of NKPs from HSCs ( Figure 1).
• E4bp4 ’s critical function in NK cells is specific to the early stages of the developmental pathway, as specific ablation of E4bp4 in peripheral mNK cells does not affect NK cell number or response to cytomegalovirus infection.
• E4bp4 regulates other transcription factors that are essential in NK cell development, such as Id2 and Eomes.
• E4bp4 Transcription factors such as E4bp4 can be hard to target because of their structure and function. For example, they usually lack enzymatic activity or cofactor binding sites.
• E4bp4 expression can be increased using a compound which inhibits the activity of REV ⁇ ERB (see PCT/GB2018/050542, particularly the examples, which is herein incorporated by 25 reference in its entirety). Further, the present inventors have demonstrated that the use of a REV ⁇ ERB inhibitor to increase E4bp4 expression results in an increase in NK cell number.
• REV ⁇ ERB binds to porphyrin heme, and it is this characteristic that is believed to make REV ⁇ ERB a druggable target (see below).
• the inventors have shown that by targeting REV ⁇ ERB and inhibiting its activity, it is possible to increase E4bp4 expression and hence increase NK 30 cell number.
• the present invention is concerned with compounds which inhibit the action of REV ⁇ ERB, and their use in increasing E4bp4 expression, and hence NK cell number.
• Increase in E4bp4 expression Accor ent invention provides ex vivo methods for producing expanded NK cell populations, and therapeutic methods and applications for increasing NK cell number in a patient in need thereof.
• said methods and applications involve the use of a compound 5 which inhibits the action of REV ⁇ ERB.
• said compounds act by increasing E4bp4 expression.
• An increase in E4bp4 expression may be measured relative to a control.
• the expression of E4bp4 in a sample of HPCs, an expanded NK cell population or in a sample obtained from a patient to be treated according to the invention may be compared with the expression of E4bp4 in a control.
• Expression may be quantified in terms of gene and/or protein expression, and may be compared with 10 expression of a control (e.g. housekeeping gene or protein).
• the actual amount of the E4bp4 gene, mRNA transcript and/or protein such as the mass, molar amount, concentration or molarity of the E4bp4 gene, mRNA transcript and/or protein, or the number of mRNA molecules per cell in a sample of HPCs, an expanded NK cell population or in a sample obtained from a patient to be treated according to the invention and the control may be assessed and compared with the corresponding 15 value from the control.
• the expression of the E4bp4 gene and/or protein in a sample of HPCs, an expanded NK cell population or in a sample obtained from a patient to be treated according to the invention may be compared with that of the control without quantifying the mass, molar amount, concentration or molarity of the one or more gene and/or protein.
• the control is an equivalent population or sample in which no increase in E4bp4 20 expression has been effected.
• a suitable control would be a different individual to which the compound has not been administered or the same individual prior to administration of the compound.
• E4bp4 expression may be understood to mean that, the expression of E4bp4 is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200% compared with the control.
• E4bp4 30 expression is increased by at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90% or more compared with the control.
• a reference to increasing E4bp4 expression may be understood to mean that, the expression of E4bp4 is increased by at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 2.9 ⁇ fold, at least 3 ⁇ fold, at least 4 ⁇ fold, at least 5 ⁇ fold, at least 6 ⁇ fold, at least 7 ⁇ fold, at least 8 ⁇ fold, at least 9 ⁇ fold, at least 10 ⁇ fold or more relative to a control.
• E4bp4 gene expression is increased by at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 2.9 ⁇ fold, at least 3 ⁇ fold, or more compared 5 with the control.
• E4bp4 protein expression is increased by at least 2 ⁇ fold, at least 3 ⁇ fold, preferably at least 5 ⁇ fold, more preferably at least 6 ⁇ fold or more compared with the control.
• the expression of the E4bp4 gene and/or protein according to the invention may be determined by quantitative and/or qualitative analysis. Typically, gene expression may be expressed in terms of mRNA levels.
• the expression level of the E4bp4 gene and/or protein according to the invention encompasses the mass of the E4bp4 mRNA transcript and/or protein, the molar amount of the E4bp4 gene, mRNA transcript and/or protein, the concentration of the E4bp4 gene and/or protein and the molarity of the E4bp4 gene and/or protein.
• This expression level may be given in any appropriate units.
• the concentration of the E4bp4 gene and/or protein may be given in pg/ml, ng/ml 15 or ⁇ g/ml.
• the expression level of the E4bp4 gene and/or protein according to the invention may be measured directly or indirectly.
• the relative expression of the E4bp4 gene and/or protein according to the invention relative to a control may be determined using any appropriate technique. Suitable standard techniques are 20 known in the art, for example Western blotting, enzyme ⁇ linked immunosorbent assays (ELISAs) and RT ⁇ qPCR.
• the expression level of the E4bp4 gene and/or protein may be increased compared with a control for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, 25 at least 5 days, at least 6 days, at least 1 week.
• the expression level of the E4bp4 gene and/or protein is increased for at least 12 to 72 hours. Typically this is assessed relative to the last administration of the compound which inhibits REV ⁇ ERB activity.
• the expression level of the E4bp4 gene and/or protein may be increased compared with a control for at least one, at least two, at least three, at least four, at least five, at least ten, at least 20, 30 at least 30, at least 40 or more passages of the NK cell precursors in culture.
• the expression level of the E4bp4 gene and/or protein may be altered indefinitely.
• REV ⁇ ERB REV ⁇ ERB proteins are members of the nuclear receptor family of intracellular transcription factors.
• the mRNA sequence of the human REV ⁇ ERB ⁇ gene (Nr1d1) is given in SEQ ID NO: 3 (Genbank Accession No. NM_021724, version NM_021724.4).
• the mRNA sequence of the human REV ⁇ ERB ⁇ 5 gene (Nr1d2) is given in SEQ ID NO: 5 (Genbank Accession No. AB307693, version AB307693.1).
• REV ⁇ ERB regulates the circadian clock, and has also been implicated in the regulation of cartilage breakdown.
• the present inventors have previously demonstrated that inhibition of REV ⁇ ERB activity is sufficient to elicit a significant increase in E4bp4 expression, and that this in turn brings about an 10 expansion of NK cells, resulting in an increase in NK cell number (see PCT/GB2018/050542, particularly the examples, which is herein incorporated by reference in its entirety). Inhibition of REV ⁇ ERB activity can bring about an increase in NK cell number, and that typically the resulting NK cells are (fully) functional as defined herein. The effect of REV ⁇ ERB inhibition is mediated in an E4pb4 ⁇ dependent manner.
• the present invention relates to the use of compounds to inhibit the action of REV ⁇ ERB, i.e. compounds which inhibit REV ⁇ ERB activity.
• REV ⁇ REB activity may be inhibited by any appropriate means. Suitable standard techniques are known in the art. Inhibition may take place via any suitable mechanism, depending for example on the nature (see below) of the compound used, e.g. steric interference in any direct or indirect interaction or inhibition of REV ⁇ ERB.
• a REV ⁇ ERB inhibitor (interchangeably referred to herein as a REV ⁇ ERB antagonist) is any compound which inhibits, decreases, suppresses or ablates the action of REV ⁇ ERB, whether in part or 5 completely.
• a decrease in REV ⁇ ERB activity may be measured relative to a control.
• the activity of REV ⁇ ERB in a sample of NK precursor or progenitor cells, an expanded NK cell population or in a sample obtained from a patient to be treated according to the invention may be compared with the activity of REV ⁇ ERB in a control.
• Activity may be quantified in any appropriate terms, for example binding of 10 REV ⁇ ERB to the E4bp4 gene, or in terms of E4bp4 expression as defined herein. Any appropriate technique or method may be used for quantifying REV ⁇ ERB activity. Suitable techniques are known in the art, for example luciferase assays for quantifying expression of a reporter gene. Typically the control is an equivalent population or sample in which no REV ⁇ ERB inhibitory compound has been added, for example a sample obtained from a different individual to which the 15 compound has not been administered, or the same individual the prior to administration of the compound. Conventional methods for the ex vivo expansion of NK cells, including known methods may be considered control methods according to the present invention.
• a reference to inhibiting REV ⁇ ERB activity may be understood to mean that, the activity of REV ⁇ ERB is decreased by at least 10%, at least 20%, at least 20 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to total (100%) inhibition of REV ⁇ ERB activity, as compared with the control.
• REV ⁇ ERB activity is decreased by at least 50%, preferably at least 70%, more preferably at least 80%, more preferably at least 90%, even more preferably at least 95% or more compared with 25 the control.
• the activity of REV ⁇ ERB may be determined by quantitative and/or qualitative analysis, and may be measured directly or indirectly.
• the activity of REV ⁇ ERB relative to a control may be determined using any appropriate technique. Suitable standard techniques are known in the art, such as by quantifying E4bp4 30 expression, and/or luciferase assays.
• the activity of REV ⁇ ERB may be inhibited compared with a control for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 1 week.
• the activity of REV ⁇ ERB is decreased for at least 12 to 72 hours. Typically this is assessed relative to the last administration of the compound which inhibits REV ⁇ ERB activity.
• the invention relates to compounds 10 which inhibit REV ⁇ ERB activity, including compounds which inhibit REV ⁇ ERB ⁇ activity (i.e. REV ⁇ ERB ⁇ inhibitors, also referred to as REV ⁇ ERB ⁇ antagonists) and/or to compounds which inhibit REV ⁇ ERB ⁇ activity (i.e. REV ⁇ ERB ⁇ inhibitors, also referred to as REV ⁇ ERB ⁇ antagonists).
• the invention relates to compounds which inhibit the activity of both REV ⁇ ERB ⁇ and REV ⁇ ERB ⁇ (i.e. REV ⁇ ERB ⁇ and REV ⁇ ERB ⁇ inhibitors, also referred to as REV ⁇ ERB ⁇ and REV ⁇ ERB ⁇ 15 antagonists).
• R REV ⁇ ERB inhibitory compounds of the invention may be specific for REV ⁇ ERB.
• the compound binds to REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ , with no significant cross ⁇ 20 reactivity to any other molecule, particularly any other protein.
• modulator that is specific for REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ will show no significant cross ⁇ reactivity with human neutrophil elastase. Cross ⁇ reactivity may be assessed by any suitable method.
• Cross ⁇ reactivity of REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ inhibitor with a molecule other than REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ may be considered significant if the inhibitor binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 25 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ .
• An inhibitor that is specific for REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ may bind to another molecule such as human neutrophil elastase at less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that it binds to REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ .
• the inhibitor binds to the other molecule at less than 20%, less than 15%, less than 10% or 30 less than 5%, less than 2% or less than 1% the strength that it binds to REV ⁇ ERB ⁇ and/or REV ⁇ ERB ⁇ .
• REV ⁇ ERB inhibitory compounds of the invention may have off ⁇ target effects.
• an off ⁇ target effect is activity against a target other than REV ⁇ ERB.
• compounds with off ⁇ target effects are encompassed by the present invention if the activity against the non ⁇ REV ⁇ ERB target is not significant compared with the activity against REV ⁇ ERB.
• an off ⁇ target effect is significant may depend on the intended use of the compound.
• a compound which may exert an off ⁇ target effect on the central nervous system would not be significant for a compound used in an ex vivo method as disclosed herein, but may be significant (depending on the magnitude of the off ⁇ target effect) for an in vivo therapeutic indication as disclosed herein.
• the presence and magnitude of any 5 potential off target effects can be readily assessed using standard methods known in the art.
• a compound that inhibits the action of REV ⁇ ERB according to the present invention is a compound which binds to the porphyrin heme moiety of REV ⁇ ERB, and hence inhibits the activity of REV ⁇ ERB.
• the small molecule may act via a different mechanism, for example, by binding to a non ⁇ heme portion of REV ⁇ ERB. Standard techniques are known in the art for the production of small molecules, which can then readily be tested for REV ⁇ ERB inhibitory activity as described herein 20 25 30 Structure of porphyrin heme The inventors have generated new library of compounds that inhibit REV ⁇ ERB activity (referred to interchangeably herein as inhibiting the action of REV ⁇ ERB).
• the compounds used in the present invention have formula (I): I) 10 where: represents bonds that are all either present or absent; R 1 is hydrogen; R 2 is selected from 5 ⁇ 10 membered heterocyclyl rings and C 1 ⁇ 6 hydrocarbyl, and is optionally substituted with one or more groups independently selected from C 1 ⁇ 4 hydrocarbyl, ⁇ OR’, ⁇ OC(O)R’, ⁇ C(O)OR’, ⁇ SR’, ⁇ S(O)R’, ⁇ S(O) 2 R’, ⁇ NR’ 2 , ⁇ NR’C(O)R’, 15 ⁇ C(O)NR’ 2 , ⁇ CN, ⁇ NO 2 , ⁇ Ph, ⁇ CF 3 and halogen; X is selected from ⁇ O ⁇ and NR’; Y is selected from ⁇ C(O) ⁇ and ⁇ CR’ 2 ⁇ ; Z is selected from ⁇ O ⁇ and ⁇ NR’ ⁇ or is absent; each R a is independently selected from H, C 1 ⁇ 4 hydrocarbyl
• R 25 As described above, represents bonds that are all either present or absent. Accordingly, the present invention relates to both closed ring structures of formula (Ia) and open ring structures of formula (Ib): ) R R ). 5 Preferably, represents bonds that are all present and the compound is a closed ring structure of formula (Ia).
• R 1 is hydrogen.
• R 2 is selected from 5 ⁇ 10 membered heterocyclyl rings and C 1 ⁇ 6 hydrocarbyl, and is optionally substituted. Preferably, R 2 is selected from optionally substituted 5 ⁇ 10 10 membered heterocyclyl rings.
• Non ⁇ limiting examples of 5 ⁇ membered heteroaryl rings include furanyl, thiophenyl, oxazolyl, isooxazolyl, isothiazolyl, thiazolyl, pyrazolyl, imidazolyl, pyrrolyl and triazolyl.
• Non ⁇ limiting examples of 6 ⁇ membered heteroaryl rings include pyridinyl, pyridazinyl, 20 pyrimidinyl and pyrazinyl.
• Non ⁇ limiting examples of 9 ⁇ membered heteroaryl rings include indolyl, isoindolyl, indazolyl, benzimidazolyl, azaindolyl, benzofuranyl, isobenzofuranyl, benzisoxazolyl and benzoxazolyl.
• Optionally substituted 5 ⁇ membered heteroaryl rings are particularly preferred, in particular optionally substituted furanyl and oxazolyl.
• R 2 is optionally substituted C 1 ⁇ 6 hydrocarbyl, it is preferably selected from optionally substituted phenyl and optionally substituted C 1 ⁇ 6 alkyl. 5 Where R 2 is optionally substituted phenyl, the phenyl group is preferably substituted.
• R 2 is optionally substituted C 1 ⁇ 6 alkyl
• the alkyl group is preferably unsubstituted, and as such R 2 is preferably selected from C 2 ⁇ 4 alkyl, such as from ethyl, propyl (e.g. ⁇ iPr) and butyl (e.g. ⁇ tBu).
• R 2 is optionally substituted with one or more groups independently selected from C 1 ⁇ 4 10 hydrocarbyl (preferably C 1 ⁇ 4 alkyl), ⁇ OR’, ⁇ OC(O)R’, ⁇ C(O)OR’, ⁇ SR’, ⁇ S(O)R’, ⁇ S(O) 2 R’, ⁇ NR’ 2 , ⁇ NR’C(O)R’, ⁇ C(O)NR’ 2 , ⁇ CN, ⁇ NO 2 , ⁇ Ph, ⁇ CF 3 and halogen.
• R 2 is unsubstituted or substituted with one or two of these groups.
• substituents for R 2 include ⁇ Me, ⁇ OMe, ⁇ CN, ⁇ NO 2 , ⁇ F, ⁇ Cl, ⁇ I and ⁇ SMe.
• X is selected from ⁇ O ⁇ and ⁇ NR’ ⁇ .
• X is preferably ⁇ O ⁇ .
• Y is selected from ⁇ C(O) ⁇ and ⁇ CR’ 2 ⁇ .
• Y is preferably ⁇ C(O) ⁇ .
• Z is selected from ⁇ O ⁇ and ⁇ NR’ ⁇ or is absent.
• Z is selected from ⁇ O ⁇ or is absent.
• each R a is independently selected from H, C 1 ⁇ 4 alkyl, ⁇ OR’ and ⁇ NR’ 2 , and more preferably from H and C 1 ⁇ 4 alkyl. More preferably, each R a is preferably H.
• each R b is independently selected from H, C 1 ⁇ 4 hydrocarbyl (preferably C 1 ⁇ 4 alkyl) and ⁇ OR’.
• each R b is independently selected from H and C 1 ⁇ 4 alkyl, 25 and more preferably is H.
• R c is selected from H and C 1 ⁇ 4 hydrocarbyl (preferably C 1 ⁇ 4 alkyl).
• R c is H.
• each R’ is independently selected from H and C 1 ⁇ 4 hydrocarbyl (preferably C 1 ⁇ 4 alkyl) and ⁇ Ph.
• each R’ is independently selected from H and C 1 ⁇ 4 alkyl, more 30 preferably from H, methyl and ethyl, and more preferably from methyl and ethyl.
• X is ⁇ O ⁇ and the compounds used in the present invention have the formula (II): I) where R 1 , R 2 , R a , R b , R c , Y and Z are as defined previously.
• R b and R c are all H, and so the compounds used in the present invention have the formula (III): I) where R 1 , R 2 , R a , Y and Z are as defined previously.
• Y is ⁇ C(O) ⁇ and the compounds used in the present 10 invention have the formula (IV): (IV) where R 1 , R 2 , R a and Z are as defined previously.
• the compounds used in the present invention are closed ring structures having the formula (V): V) e R 1 , R 2 wher , R a and Z are as defined previously.
• R a are also all H, and so the compounds used in the present invention have the formula (VI): I) 10 where R 1 , R 2 and Z are as defined previously. It will be appreciated that compounds of formulas (II) ⁇ (VI) may also be used in the form of a pharmaceutically acceptable salt. Specific examples of compounds according to the present invention are set out in Table 1 below: 15 acid7 O OH N O acid66 O OH N O HN acid72 O OH N O O acid73 Table 1: Exemplary compounds of the invention It will be appreciated that each of the compounds depicted in Table 1 may be used in the 5 form of a pharmaceutically acceptable salt.
• the compound of formula (I) is also not disclosed in either WO 2013/033310 or WO 2015/103527.
• the compound of formula (I) is preferably not selected from: 10 , , , , , , .
• a compound in an isolated form is equivalent to a composition which consists 15 substantially of the compound.
• an isolated compound of formula (I) will contain less than 10%, and typically less than 1% by weight of impurities, i.e. substances other than compounds of formula (I).
• impurities i.e. substances other than compounds of formula (I).
• compounds of the present invention contain one or more chiral centres, the compounds may exist in, and may be isolated as pure enantiomeric or 20 diastereomeric forms or as racemic mixtures.
• the present invention therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of the compounds of the invention.
• compounds of formula (I) may be used in a racemic mixture, or as single enantiomers, i.e.: .
• the compounds of formula (I) may have rotameric forms, or may not have rotational activity.
• Rotameric forms include slow rotating forms and fast rotating forms.
• fast rotating forms of the compounds of formula (I) are preferred.
• a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a 10 hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms.
• Compounds may be used in the form of salts, hydrates and solvate forms, as defined in the Definitions section herein. It will be appreciated that salt forms of the compound may themselves be in the form of hydrates or solvate forms.
• the compounds of the present invention typically have improved REV ⁇ ERB inhibitory activity compared with SR8278. This inhibitory activity may be measured by any appropriate means, such as conventional means known in the art and/or the methods described herein.
• a reference to a compound with improved REV ⁇ ERB inhibitory activity may be 5 understood to mean that, the compound decreases REV ⁇ ERB activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200% more than the decrease in REV ⁇ ERB activity obtained using a control REV ⁇ ERB inhibitory compound, such as SR8278.
• a reference to a compound with improved REV ⁇ ERB inhibitory activity may be understood to mean that, said compound is at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 15 2.9 ⁇ fold, at least 3 ⁇ fold, at least 4 ⁇ fold, at least 5 ⁇ fold, at least 6 ⁇ fold, at least 7 ⁇ fold, at least 8 ⁇ fold, at least 9 ⁇ fold, at least 10 ⁇ fold or more, more effective in inhibiting REV ⁇ ERB activity relative to a control REV ⁇ ERB inhibitory compound, such as SR8278.
• an improved REV ⁇ ERB inhibitory compound is at least 1.5 ⁇ fold, at least 2 ⁇ fold or more effective in inhibiting REV ⁇ ERB activity relative to a control REV ⁇ ERB inhibitory compound, such as SR8278.
• the small molecules of the invention may be used in the form of proteolysis targeting 25 chimeras (also referred to as PROTACs or PROTAC reagents).
• PROTACs are heterobifunctional small molecules that simultaneously bind a target protein and ubiquitin ligase, enabling ubiquitination and degradation of the target.
• a PROTAC reagent typically comprises a ligand for the target protein (in the case of the present invention, REV ⁇ ERB) and a ligand for an E3 ligase recognition domain.
• REV ⁇ ERB a ligand for the target protein
• E3 ligase recognition domain an E3 ligase is recruited to the PROTAC ⁇ bound REV ⁇ ERB, 30 inducing ubiquitin transfer from the E3 ligase complex to the target protein (in the case of the present invention, REV ⁇ ERB).
• a PROTAC reagent may be produced by conjugating a ligand for an E3 ⁇ ligase to a small molecule inhibitor as described herein via a linker.
• a PROTAC reagent comprises a ligand for the E3 RING Cullin ligase von ⁇ Hippel Lindau protein (VHL) or cereblon ⁇ a part of a CRL4 E3 RING Cullin ligase complex, connected to a small molecule inhibitor of the invention via a linker.
• VHL von ⁇ Hippel Lindau protein
• 5 Notch ligand The Notch signalling pathway is primarily associated with promoting T cell development and repressing concomitant B cell development.
• Mammals have four types of Notch receptor – Notch1, Notch2, Notch3 and Notch4, all of which are single ⁇ pass heterodimeric transmembrane proteins. Mammals have two types of canonical Notch ligands – Delta type and Jagged type, collectively known 10 as DSL ligands. There are three delta ⁇ like ligands (DLLs), DLL1, DLL3 and DLL4 and two jagged (JAG) ligands, JAG1 and JAG2. DLL and JAG ligands typically comprise the following domains: a module at the N ⁇ terminus of Notch ligand (MNNL) domain and a Delta/Serrate/Lag ⁇ 2 (DSL) domain, together with a number of EGF repeats.
• MNNL N ⁇ terminus of Notch ligand
• DSL Delta/Serrate/Lag ⁇ 2
• DLL3 comprises six EGF repeats.
• DLL1 and DLL4 comprise eight EGF repeats.
• JAG1 and JAG2 comprise 16 EGF repeats.
• a reference herein to a Notch ligand is a reference to any Notch ligand, such as a ligand of Notch1, Notch2, Notch3 and/or Notch 4, preferably a ligand of at least Notch1.
• the protein sequence of human Notch1 is given in SEQ ID NO: 10 (GenBank Accession No. CR457221, version CR457221.1).
• the Notch ligand of use in the present invention is a 20 canonical Notch ligand.
• the Notch ligand is a DLL, more preferably DLL4.
• the protein sequence of human DLL4 is given in SEQ ID NO: 8 (GenBank Accession No. AF253468, version AF253468.1).
• a reference herein to a Notch ligand also embraces fragments thereof, provided said fragment retains the Notch ⁇ binding and activatory activity of the Notch ligand from which it is derived.
• Notch ligands suitable for use in the present invention have previously been described by the present inventors (see PCT/GB2018/050818, which is herein incorporated by reference in its entirety, particularly pages 15 and 16 and the Examples).
• Preferred examples of Notch ligand fragments include Notch ligand (N ⁇ EGF1) and Notch ligand (N ⁇ EGF2), such as DLL4 (N ⁇ EGF1) and DLL4 (N ⁇ EGF2).
• N ⁇ EGF1 and Notch ligand such as DLL4 (N ⁇ EGF1) and DLL4 (N ⁇ EGF2).
• a Notch ligand, fragment thereof, or molecule that mimics the effect e.g.
• a Notch ligand such as DLL4 may comprise modifications, such as amino acid mutations which alter, typically increase, the affinity of the ligand/fragment/mimetic for its Notch receptor. Techniques for identifying such modifications are known in the art. For example, amino acids which increase the affinity of a Notch ligand/fragment/mimetic can be identified using yeast surface display. Again, such modifications have previously been described by the present inventors (see PCT/GB2018/050818, which is herein incorporated by reference in its entirety, particularly page 16).
• the DLL4 ligand of the invention comprises the amino acid substitutions, G28S, F107L and L206P, more preferably 5 G28S, F107L, N118I, I143F, H194Y, L206P and/or K215E.
• a functional fragment of DLL4 comprises at least residues 65 to 114 and 179 to 219 of full ⁇ length DLL4, preferably held in the correct conformation to allow interaction with the Notch ligand.
• the invention encompasses the use of molecules that would mimic the effect (e.g. 10 activity/function) of a Notch ligand (also referred to herein as mimetics).
• Peptidomimetics may have advantages over peptides in terms of stability and bioavailability associated with a natural peptide. Peptidomimetics can have main ⁇ or side ⁇ chain modifications of the parent peptide designed for 15 biological function. Examples of classes of peptidomimetics include, but are not limited to, peptoids and ⁇ peptides, as well as peptides incorporating D ⁇ amino acids.
• peptidomimetics such as peptoids
• sequences of canonical and non ⁇ canonical Notch ligands are known in the art, as are the sequences of canonical and non ⁇ canonical Notch ligands.
• suitable molecules which mimic the effect of a desired 20 Notch ligand using known techniques and based on the known Notch ligand sequences.
• peptidomimetics may be designed to interact with key residues of Notch (e.g.
• Notch1 that are known to be involved in binding to DLL4, such as one or more of residues 415 (E415), 418 (L418), 420 (A420), 421 (N421), 422 (P422), 424 (E424), 425 (H425), 436 (F436), 447 (P447), 448 (R448), 450 (E450), 452 (D452), 469 (D469), 477 (I477), 480 (P480) of Notch (Notch1), or any 25 combination thereof.
• methods of the present invention may further comprise a step of contacting an haematopoietic progenitor cell (HPC) comprising sample obtained from an individual/patient with a compound which results in the alteration of post ⁇ translational modification of E4bp4, thereby causing 20 an increase in E4bp4 activity.
• HPC haematopoietic progenitor cell
• compounds which alter the post ⁇ translational modification of E4pb4 as described herein may be used in combination with the methods and compounds of the invention which inhibit REV ⁇ ERB activity. This combination may further be used in combination with the use of a Notch ligand (e.g. DLL4) as described herein.
• a compound which alters or affects the post ⁇ translational modification of E4bp4 may 25 therefore be used according to the invention for increasing production of NK cells in a patient, wherein said compound increases E4bp4 activity, or for use in a method of treatment by increasing the number of NK cells in a patient in need thereof, together with the indications disclosed herein relating to increased E4bp4 expression by decreasing REV ⁇ ERB activity, and optionally the indications disclosed herein relating to increasing NK cell number by culturing HPCs in the presence of a Notch ligand.
• any of the disclosure herein in relation to methods of increasing NK cell number, methods of expanding NK cells in the context of compounds which inhibit the action of REV ⁇ ERB, and/or Notch ligands, expanded NK cell populations produced by said methods and therapeutic indications relating to said compounds and populations applies inter alia to the disclosed methods of increasing E4bp4 activity to increase NK cell number.
• the feeder cell layers, growth factors and/or other culture conditions and diseases to be treated may be the same in relation to the post ⁇ translational modification aspects as for the REV ⁇ ERB inhibition and/or Notch ligand aspects disclosed herein.
• the REV ⁇ ERB inhibitor compound, Notch ligand and/or E4bp4 post ⁇ translational modifier may be used simultaneously, separately or sequentially.
• a compound which alters the post ⁇ 5 translational modification of E4bp4 is used in combination with a compound which inhibits the action of REV ⁇ ERB
• the sample is contacted with REV ⁇ ERB inhibitory compound before being contacted with the post ⁇ translational modifier.
• REV ⁇ ERB inhibitory compound typically the E4bp4 post ⁇ translational modifier is used after the REV ⁇ ERB inhibitory compound and the Notch ligand; preferably the REV ⁇ ERB inhibitory compound are used together, or more preferably the REV ⁇ ERB inhibitory 10 compound is used before the Notch ligand (as described herein).
• Types of post ⁇ translational modification encompasses any alteration of post ⁇ translational modification which results in an increase in E4bp4 activity.
• Non ⁇ limiting examples of post ⁇ translation modification include 15 phosphorylation, SUMOylation, the addition of a hydrophobic group (e.g. myristoylation, palmitoylation), addition of a cofactor, the addition of small chemical groups (e.g. acylation, alkylation, amidation, glycosylation), glycation, carbamylation, cabonylation, chemical modifications (e.g. deamidation) and/or structural changes.
• wild ⁇ type (unmodified) E4bp4 is typically SUMOylated at one or more of residues K10, K116, K219, K337 and/or K394 or residues corresponding 25 thereto, or any combination thereof.
• wild ⁇ type (unmodified) E4bp4 is SUMOylated at least at residue K219 (or a corresponding residue).
• wild ⁇ type (unmodified) E4bp4 is typically phosphorylated at residues S286, S301 and S454, or residues corresponding thereto, or any combination thereof.
• a compound which alters the post ⁇ translational modification of E4bp4 reduces, inhibits or ablates SUMOylation at residue K219 (or a 30 residue corresponding thereto), and/or reduces, inhibits or ablates phosphorylation at residues S286, S301 and S454 (or corresponding residues), or any combination thereof.
• Non ⁇ limiting examples of suitable kinase inhibitors include 4 ⁇ (4 ⁇ (2,3 ⁇ dihydrobenzo[1,4]dioxin ⁇ 6 ⁇ yl) ⁇ 5 ⁇ pyridin ⁇ 2 ⁇ yl ⁇ 1H ⁇ imidazol ⁇ 2 ⁇ yl)benzamide (D4476) and 4,5,6,7 ⁇ tetrabromo ⁇ 2 ⁇ azabenzimidazole, 4,5,6,7 ⁇ Tetrabromobenzotriazole (TBB).
• D4476 4 ⁇ (4 ⁇ (2,3 ⁇ dihydrobenzo[1,4]dioxin ⁇ 6 ⁇ yl) ⁇ 5 ⁇ pyridin ⁇ 2 ⁇ yl ⁇ 1H ⁇ imidazol ⁇ 2 ⁇ yl)benzamide
• TB 4,5,6,7 ⁇ tetrabromo ⁇ 2 ⁇ azabenzimidazole
• TAB 4,5,6,7 ⁇ Tetrabromobenzotriazole
• the activity of E4bp4 in a sample of NK precursor or progenitor cells, an expanded NK cell population or in a sample obtained from an individual/patient to be treated according to the invention may be compared with the activity of E4bp4 in a control.
• Activity may be quantified in any appropriate terms, for example an increase in the expression of any downstream target of E4bp4.
• Any appropriate technique or method may be used for quantifying E4bp4 activity. Suitable techniques are known in the art, for example luciferase assays for quantifying expression of a reporter gene.
• the control is an equivalent population or sample which has not been treated 25 according to the present invention.
• the corresponding control may be a population or sample in which no compound has been added to inhibit the action of REV ⁇ ERB or to alter or effect the post ⁇ translational modification of E4bp4.
• a control may be a sample obtained from a different individual treated according to the invention, or the same individual the prior to treatment. Conventional methods for the ex vivo expansion of NK cells, including known methods may be considered control methods according to the present invention.
• a reference to increasing E4bp4 activity may be understood to mean that, the activity of E4bp4 is increased by at least 1.25 ⁇ fold, at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 2.9 ⁇ fold, at least 3 ⁇ fold, at least 4 ⁇ fold, at least 5 ⁇ fold, at least 6 ⁇ fold, at least 7 ⁇ fold, at least 8 ⁇ fold, at least 9 ⁇ fold, at least 10 ⁇ fold or more relative to 10 a control.
• E4bp4 activity is increased by at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 2.9 ⁇ fold, at least 3 ⁇ fold, or more compared with the control.
• E4bp4 activity may be measured indirectly be determining the increase in NK cell number.
• the number of NK cells may be increased by at least 1.25 ⁇ fold, at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 2.5 ⁇ fold, at least 3 ⁇ fold, at least 15 4 ⁇ fold, at least 5 ⁇ fold, at least 6 ⁇ fold, at least 7 ⁇ fold, at least 8 ⁇ fold, at least 9 ⁇ fold, at least 10 ⁇ fold or more relative to a control.
• the number of NK cells may be increased by at least 10 ⁇ fold, at least 20 ⁇ fold, at least 30 ⁇ fold, at least 40 ⁇ fold, at least 50 ⁇ fold, at least 100 ⁇ fold, or more relative to a control.
• the number of NK cells is increased by at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 5 ⁇ fold, or more compared with the control.
• the activity of E4bp4 may be determined by quantitative and/or qualitative analysis, and may be measured directly or indirectly.
• the activity of E4bp4 relative to a control may be determined using any appropriate technique. Suitable standard techniques are known in the art.
• the activity of E4bp4 may be increased compared with a control for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at 25 least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 1 week.
• the activity of E4bp4 is increased for at least 12 to 72 hours.
• the methods of the invention allow for the rapid expansion of NK cells, reducing the time needed for their culture, and hence the risk of exhaustion, enhancing the cytotoxicity of the NK cells when transfused into a patient.
• said method is a therapeutic method as described herein.
• all the disclosure herein in relation to therapeutic indications and 10 applications of the invention is applicable to said methods.
• the method of the invention is ex vivo.
• the invention provides an ex vivo method for expanding an NK cell population comprising the steps of: (a) culturing an NK precursor cell comprising sample obtained from an individual; (b) adding a compound that inhibits the action of REV ⁇ ERB to said sample; and (c) expanding said cells in vitro to produce an NK cell population.
• step (b) the compound is actively added to the sample as a compound of formula (I) and, as such, the method of the invention does not cover embodiments in which the compound is added to the sample in a different form (such as a prodrug) and converts in situ into a compound of formula (I).
• the compound that inhibits the action of REV ⁇ ERB may be any compound of formula (I) as 20 described herein. Typically said compound increases E4bp4 expression by decreasing REV ⁇ ERB activity as described herein.
• the compound has the formula (II) as defined herein, more preferably the compound has the formula (III) as defined herein, even more preferably the compound has the formula (IV) as defined herein, and even more preferably, the compound has the formula (V) as defined herein. Examples of specific compounds which may be used in the methods of 25 the invention are described herein, with compounds acid7 and acid11 being particularly preferred. Additional external stimuli, such as growth factors and/or cytokines, may be used to further enhance the production of NK cells.
• IL ⁇ 7 may be used at a concentration of about 1 ng/ml to about 100 ng/ml, about 1 ng/ml to about 50 ng/ml, about 1 ng/ml to about 25 ng/ml, about 1 ng/ml to about 10 ng/ml or less. In some embodiments IL ⁇ 7 is used at a concentration of about 50 ng/ml, about 25 ng/ml, about 20 ng/ml, about 15 ng/ml, about 10 ng/ml or about 5 ng/ml, preferably about 10 ng/ml.
• Flt3L may be used at a concentration of about 1 ng/ml to about 100 ng/ml, about 1 ng/ml to about 50 ng/ml, about 1 ng/ml to about 25 ng/ml, about 1 ng/ml to about 10 ng/ml or less. In some embodiments Flt3L is used at a concentration of about 50 ng/ml, about 25 ng/ml, about 20 ng/ml, about 15 ng/ml, about 10 ng/ml or about 5 ng/ml, preferably about 10 ng/ml.
• IL ⁇ 15 may be used at a concentration of about 1 ng/ml to about 100 ng/ml, about 1 ng/ml to about 50 ng/ml, about 1 ng/ml to about 40 ng/ml, about 1 ng/ml to about 30 ng/ml, about 1 ng/ml to about 20 ng/ml, about 1 ng/ml to about 10 ng/ml or less.
• IL ⁇ 15 is used at a concentration of about 50 ng/ml, about 40 ng/ml, about 35 ng/ml, about 30 ng/ml, about 25 ng/ml, about 20 ng/ml or about 10 ng/ml, preferably about 30 ng/ml.
• the HPCs may be cultured on or with suitable support/stromal cells or cell layer. Any appropriate stromal cell may be used, including, but not limited to OP9 stromal cells and/or EL08 ⁇ 1D2 stromal cells.
• the ex vivo method comprises a single culturing stage.
• the HPCs in a sample obtained from a patient are cultured, a compound of the 20 invention is added, and the HPCs are expanded to form an NK cell population, typically under substantially constant culture conditions. Typically this involves incubating the HPCs with factors such as IL ⁇ 3, IL ⁇ 7, SCF, Flt3L and/or IL ⁇ 15, preferably all of these factors.
• the HPCs are preferably also cultured on or with stromal cells/ cell layer, such as EL08 ⁇ 1D2 stromal cells.
• the medium which induces differentiation of the HPCs to NK cells does not comprise 25 IL ⁇ 3.
• the ex vivo method comprises two culturing stages.
• the first is a lymphoid production stage, in which the HPCs in a sample obtained from a patient are cultured. Typically this involves incubating the HPCs with cytokines and growth factors associated with lymphoid production, such as Flt3L, IL ⁇ 7 and/or SCF. This stage may last for at least one, at least two, at least 30 three, at least four, or more days. In some embodiments, this stage lasts for two days.
• the second stage of the ex vivo method is a stage of NK cell expansion.
• the HPCs are cultured in medium which does not induce differentiation of the HPCs for between about 2 to about 8 days, such as about 2 days to about 6 days, optionally for between about 4 days to about 6 days.
• the REV ⁇ ERB inhibitor may be added for (i) all or part of the culture period in medium which does not induce 15 differentiation of the HPCs; and/or (ii) all or part of the culture period in medium which induces differentiation of the HPCs to NK cells.
• the medium which does not induce differentiation of the HPCs comprises at least one of Flt3L, GM ⁇ CSF, IL ⁇ 3, IL ⁇ 6, TPO and/or stem cell factor (SCF), preferably Flt3L, GM ⁇ CSF, IL ⁇ 3, IL ⁇ 6, TPO and SCF; and/or the medium which induces differentiation of 25 the HPCs to NK cells comprises IL ⁇ 7, Flt3L, IL ⁇ 15, and/or SCF, preferably IL ⁇ 7, Flt3L, IL ⁇ 15 and SCF.
• SCF stem cell factor
• the HPC comprising sample may be cultured ex vivo for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 30 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days or more.
• said sample is cultured for at least 9 days in order to produce an expanded NK cell population.
• These culture periods are for the total culture period of the ex vivo method, i.e. if there are two stages, these periods are for the total (stage 1 plus stage 2).
• An example culture scheme for HPCs for the production of NK cells is set out in Figure 2A. Preferred examples of culture schemes are those set out in UK Patent Application No.
• any reference to a REV ⁇ ERB inhibitor compound in said application may be a REV ⁇ ERB inhibitor compound of the present invention.
• a compound which inhibits REV ⁇ ERB activity may be added to the medium which does not induce differentiation of the HPCs after a period of time of between about 0 to about 2 days, 5 particularly the compound which inhibits REV ⁇ ERB activity may be added to the medium which does not induce differentiation of the HPCs after about 1 day.
• the HPCs may be contacted with a REV ⁇ ERB inhibitory compound for at least part of the first culture period in medium which does not induce differentiation of the HPCs, up to the entirety of said first culture period.
• the HPCs may be contacted with a REV ⁇ ERB inhibitory compound for 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 days 10 of this culture period may or a period of any duration between 2 and 8 days.
• a REV ⁇ ERB inhibitory compound for between about 2 days to about 6 days (e.g. 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 or 6 days or a period of any duration between 2 and 6 days, such as between about 4 to 6 days) of the first culture period in medium which does not induce differentiation of the HPCs.
• the HPCs may be contacted with a REV ⁇ ERB inhibitory compound for between 15 about 2 days to about 4 days (e.g. 2, 2.5, 3, 3.5 or 4 days or a period of any duration between 2 and 4 days) of the first culture period in medium which does not induce differentiation of the HPCs.
• the REV ⁇ ERB inhibitor compound of the invention may be added to the sample comprising HPCs within one week, within six days, within five days, within four days, within three days, within two days, within one day of isolating the HPCs in the sample, or on the same day as isolating the NK cell 20 precursors. Typically this is the same day that the sample is obtained from the patient.
• the compound of the invention is added to the sample within two days of isolating the HPCs in the sample, even more preferably on day one or two following isolation of the HPCs.
• the compound is added at multiple time points, for example when the culture medium is changed.
• the compound of the invention may be added two days after isolating the HPCs, 25 and then added again at day five post ⁇ isolation of the HPCs.
• This disclosure applies to all methods of the invention, e.g. for one stage and two stage methods as described herein, and/or methods which also use a Notch ligand and/or a compound which alters the posttranslational modification of E4bp4 as described herein.
• a compound of the invention may be used at a final concentration of about 2 to about 20 ⁇ M, about 2 to about 15 ⁇ M, about 5 to about 15 ⁇ M, about 5 to about 14 ⁇ M, about 4 to about 13 ⁇ M, about 5 to about 12 ⁇ M, about 5 to about 11 ⁇ M, or preferably about 5 to about 10 ⁇ M.
• the present inventors have previously demonstrated that combining the use of a Notch ligand (such as DLL4) and REV ⁇ ERB inhibition results in a potent means for enhancing NK cell production, allowing for the production of large numbers of functional NK cells that are suitable for in vivo therapeutic use more rapidly than the current methods.
• a Notch ligand such as DLL4
• REV ⁇ ERB inhibition results in a potent means for enhancing NK cell production, allowing for the production of large numbers of functional NK cells that are suitable for in vivo therapeutic use more rapidly than the current methods.
• the invention provides an ex vivo method for expanding an NK cell population comprising the steps of: (a) adding a compound that inhibits the action of REV ⁇ ERB (as described herein) to an HPC comprising sample obtained from an individual/patient and culturing the sample with the compound; (b) culturing said cells in the presence of a Notch ligand (such as DLL4); and (c) expanding said cells in vitro to produce an NK cell population.
• Step (a) and (b) may be carried out 10 concurrently or in any order. For example, step (a) may be carried out first, followed by step (b), such that the cells are first exposed to a REV ⁇ ERB inhibitory compound and then cultured in the presence of a Notch ligand.
• step (b) may be carried out first, followed by step (a), such that the cells are first cultured in the presence of a Notch ligand and then in the presence of a REV ⁇ ERB inhibitory compound.
• steps (a) and (b) may be carried out concurrently, such that the 15 cells are simultaneously cultured in the presence of a REV ⁇ ERB inhibitory compound and a Notch ligand.
• step (a) may be carried out first, followed by step (b), such that the cells are first cultured in the presence of a REV ⁇ ERB inhibitory compound and then in the presence of a Notch ligand.
• the invention provides an ex vivo method 20 for expanding an NK cell population comprising the steps of: (a) adding a compound that inhibits the action of REV ⁇ ERB (as described herein) and culturing the sample with the compound; (b) culturing said cells in the presence of a Notch ligand (such as DLL4); and (c) expanding said cells in vitro to produce an NK cell population.
• step (b) may be carried out first, followed by step (a), such that the cells are first cultured in the presence of a Notch ligand and then in 25 the presence of a REV ⁇ ERB inhibitory compound.
• the invention provides an ex vivo method for expanding an NK cell population comprising the steps of: (a) adding a Notch ligand (such as DLL4) and culturing the sample with said ligand; (b) culturing said cells in the presence of a compound that inhibits the action of REV ⁇ ERB (as described herein); and (c) expanding said cells in vitro to produce an NK cell population.
• a Notch ligand such as DLL4
• culturing said cells in the presence of a compound that inhibits the action of REV ⁇ ERB (as described herein); and (c) expanding said cells in vitro to produce an NK cell population.
• both the REV ⁇ ERB inhibitory 30 compound and the Notch ligand are present for at least part of the first culture period in medium which does not induce differentiation of the HPCs, and are not present for at least part of, preferably all of the culture period in medium which induces differentiation of the HPCs to NK cells.
• the Notch ligand is a Notch ligand as described herein.
• the Notch ligand is DDL4, or a fragment thereof which retains the function of DLL4, as described herein.
• the Notch ligand e.g. DLL4
• the Notch ligand may be added for all or part of the culture period in medium which does not induce differentiation of the HPCs; but is not added for 5 all or part of, preferably all of, the culture period in medium which induces differentiation of the HPCs to NK cells.
• the Notch ligand (such as DLL4) may be present in solution (e.g.
• the Notch ligand e.g. DLL4 is used to coat the vessel in which the HPCs are cultured.
• the Notch ligand e.g. DLL4 may be used at a concentration of about 1 ⁇ g/ml to about 100 ⁇ g/ml, about 1 ⁇ g/ml to about 50 ⁇ g/ml, about 1 ⁇ g/ml to about 25 ⁇ g/ml, about 1 ⁇ g/ml to about 10 ⁇ g/ml or less.
• the Notch ligand e.g.
• DLL4 is used at a concentration of about 50 ⁇ g/ml, about 25 ⁇ g/ml, about 20 ⁇ g/ml, about 15 ⁇ g/ml, about 10 ⁇ g/ml, or about 5 ⁇ g/ml, preferably about 10 ⁇ g/ml.
• Additional substrates and/or linkers may be 15 used to facilitate the attachment of the Notch ligand (such as DLL4) to the surface of the culture vessels. Examples of such substrates are known in the art, such as poly ⁇ L ⁇ lysine.
• HPCs may be cultured in the presence or absence of a stromal support cell or feeder cell, or population thereof.
• the cells are cultured in the absence of a stromal support cell or population thereof.
• the ex vivo method comprises a single culturing stage in which the HPCs in a sample obtained from an individual/patient are cultured, a compound of the invention is added, the HPCs are contacted with said compound and a Notch ligand and expanded to form an NK cell population, typically under substantially constant culture conditions (i.e. steps (a) and (b) of the method are carried out concurrently). Typically this involves incubating the HPCs with factors such as 25 IL ⁇ 3, IL ⁇ 7, SCF, Flt3L and/or IL ⁇ 15, preferably all of these factors.
• the HPCs may be cultured in the presence or absence of stromal cells/ cell layer, such as EL08 ⁇ 1D2 stromal cells.
• the ex vivo method comprises two culture stages (analogous to the scheme shown in Figure 2).
• the first is a lymphoid production stage, in which the HPCs in a sample obtained from an individual/patient are cultured. Typically this involves incubating the HPCs with 30 cytokines and growth factors associated with lymphoid production, such as Flt3L, IL ⁇ 7 and/or SCF.
• This stage may last for at least one, at least two, at least three, at least four, or more days. In some preferred embodiments, this stage lasts for two days.
• that stage may be further divided so that: (i) the REV ⁇ ERB inhibitory compound is added before the Notch ligand; or (ii) the Notch ligand is added before the REV ⁇ ERB inhibitory compound.
• the Notch ligand and REV ⁇ ERB inhibitory compound may be 20 added simultaneously in the same stage.
• Figure 2B illustrates some embodiments of the different method schemes for the REV ⁇ ERB inhibitory compound and Notch ligand combination aspects of the invention.
• the timings of administration included in Figure 2B are non ⁇ limiting; any appropriate timings for administration, such as those described herein, may be used.
• the REV ⁇ ERB inhibitor is added in stage 1 (e.g. at day 0 or on day 2), with the Notch 25 ligand being added later (e.g. at day 2 or 4 respectively).
• the REV ⁇ ERB inhibitory compound may be added during the first stage, and the Notch ligand is added during the second stage, and preferably at the start of this second stage.
• both the REV ⁇ ERB inhibitory compound and the Notch ligand are present for at least part of the first culture period in medium 30 which does not induce differentiation of the HPCs, and are not present for at least part of, preferably all, of the culture period in medium which induces differentiation of the HPCs to NK cells. This is described in more detail in UK Patent Application No. 2212838.3, which is herein incorporated by reference in its entirety.
• the HPC comprising sample may be cultured ex vivo for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days or more.
• said sample is cultured for at least 9 days in order to produce an expanded NK cell population. 5
• These culture periods are for the total culture period of the ex vivo method, i.e. if there are two stages, these periods are for the total (stage 1 plus stage 2).
• the REV ⁇ ERB inhibitory compound of the invention may be added to the sample comprising HPCs within one week, within six days, within five days, within four days, within three days, within two days, within one day of isolating the HPCs in the sample, or on the same day as isolating the NK cell 10 precursors. Typically this is the same day that the sample is obtained from the patient.
• the REV ⁇ ERB inhibitory compound of the invention is added to the sample within two days of isolating the HPCs in the sample, such as on the day of isolation of the HPCs, or one day after isolation of the HPCs.
• the REV ⁇ ERB inhibitory compound of the invention is added to the sample one or two days post isolation of the HPCs.
• the compound is added at multiple time points, 15 for example when the culture medium is changed.
• the compound of the invention may be added one or two days after isolating the HPCs, and then added again at day five post ⁇ isolation of the HPCs.
• the Notch ligand of the invention may be added to the sample comprising HPCs within one week, within six days, within five days, within four days, within three days, within two days, within 20 one day of isolating the HPCs in the sample, or on the same day as isolating the NK cell precursors. Typically this is the same day that the sample is obtained from the patient.
• the Notch ligand of the invention is added to the sample within four days of isolating the HPCs in the sample, such as on day one or two following isolation of the HPCs. Most preferably the Notch ligand of the invention is added to the sample two or four days post isolation of the HPCs. Thus, typically the Notch ligand is 25 present on or from 4 days after isolating the HPCs.
• Preferred embodiments of the invention comprise (i) adding the REV ⁇ ERB inhibitory compound and the Notch ligand to the sample on the day of isolation of the HPCs; (ii) adding the REV ⁇ ERB inhibitory compound to the sample on the day of isolation of the HPCs and adding the Notch ligand to the sample on day two post isolation of the HPCs; or (iii) adding the REV ⁇ ERB inhibitory 30 compound to the sample on day two post isolation of the HPCs and adding the Notch ligand to the sample on day four post isolation of the HPCs; with option (iii) being particularly preferred.
• the cells may be cultured in the presence of additional external stimuli (as described herein) in combination with a Notch ligand.
• a Notch ligand may be used with IL ⁇ 15.
• the cells may be first exposed to a Notch ligand and then IL ⁇ 15.
• the cells may first be cultured in the presence of IL ⁇ 15 and then in the presence of a 5 Notch ligand.
• the cells may be simultaneously cultured in the presence of a Notch ligand and IL ⁇ 15.
• the cells are first cultured in the presence of a Notch ligand and then IL ⁇ 15.
• the HPCs may be cultured in the presence of a Notch ligand (such as DLL4) for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 10 days, at least 1 week. Typically for 72 hours to 1 week.
• a Notch ligand such as DLL4
• the cells may be cultured in the presence of IL ⁇ 15 for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks or longer, until the desired number of NK cells is produced.
• the step of culturing in Il ⁇ 15 may be is 1 week 15 or more in length, 7 to 9 days in length, or about two weeks in length. Alternatively, these durations may be measured in terms of the number of cell passages.
• At least one, at least two, at least three, at least four, at least five, at least ten, at least 20, at least 30, at least 40 or more passages of the cells (either in vivo, or cultured ex vivo or in vitro).
• the durations of exposure to Notch ligand and IL ⁇ 15 are independent, and any duration for 20 Notch culture may be used in combination with any duration of IL ⁇ 15 culture.
• Notch exposure/culture is 72 hours to 1 week in length and IL ⁇ 15 exposure/culture is 1 week (or more) in length.
• IL ⁇ 7, Flt3L and/or SCF are used together with the Notch ligand.
• the HPCs are cultured in the presence of IL ⁇ 7, Flt3L and SCF together with the Notch 25 ligand.
• the methods of the invention may further comprise a step of contacting the HPCs with a compound which results in the alteration of post ⁇ translational modification of E4bp4, thereby causing an increase in E4bp4 activity, as described herein.
• the alteration of post ⁇ translational modification of 30 E4bp4 is a reduction in SUMOylation and/or phosphorylation of E4bp4 as described herein.
• the compound which results in the alteration of post ⁇ translational modification of E4bp4 reduces SUMOylation at one or more of residues K10, K116, K219, K337 and/or K394 of E4bp4, or a residue corresponding thereto, or any combination thereof; and/or reduces phosphorylation at one or more of residues S286, S301 and/or S454 of E4bp4, or a residue corresponding thereto, or any combination thereof.
• Any appropriate concentration of a compound which results in the alteration of post ⁇ translational modification of E4bp4 may be used, provided that it increases the activity of E4bp4 as 5 described herein and has utility in expanding an NK cell population.
• a compound which results in the alteration of post ⁇ translational modification of E4bp4 may be used at a final concentration of about 0.5 to about 5 ⁇ M, more preferably of about 0.5 to about 2 ⁇ M, even more preferable of about 0.5 to about 1 ⁇ M.
• the REV ⁇ ERB inhibitor compound, Notch ligand and/or compound which alters E4bp4 post ⁇ translational modification may be used simultaneously, separately or sequentially as described herein.
• Each of the REV ⁇ ERB inhibitor compound, Notch ligand and/or compound which alters E4bp4 post ⁇ translational modification may independently be used as a single treatment or application or in multiple treatments or applications (in both in vitro, ex vivo or in vivo methods as described herein).
• at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or more applications may be used.
• the multiple applications may 20 be applied at any appropriate time points according to a method or treatment of the invention.
• a REV ⁇ ERB inhibitory compound of the invention may each independently be applied twice a day, once daily, every other day, once every three days or weekly.
• the REV ⁇ ERB inhibitory compounds of the invention may independently be applied as necessary when the culture medium is changed.
• the method of the invention may further comprise modulating (increasing or decreasing the expression and/or activity of one or more additional gene and/or protein in the HPCs in order to enhance NK cell expansion.
• the sample comprising HPCs obtained from an individual/patient may be a sample obtained from bone marrow, cord blood and/or peripheral blood.
• the sample may be a cord or peripheral blood sample, or a bone marrow sample or biopsy.
• a method of the invention may result in an increase in, the number of NK cells of at least 1.5 ⁇ fold, at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 2.9 ⁇ fold, at least 3 ⁇ fold, at least 4 ⁇ fold, 15 at least 5 ⁇ fold, at least 6 ⁇ fold, at least 7 ⁇ fold, at least 8 ⁇ fold, at least 9 ⁇ fold, at least 10 ⁇ fold or more relative to a control.
• the number of NK cells is increased by at least 2 ⁇ fold, at least 2.1 ⁇ fold, at least 2.2 ⁇ fold, at least 2.3 ⁇ fold, at least 2.4 ⁇ fold, at least 2.5 ⁇ fold, at least 2.6 ⁇ fold, at least 2.7 ⁇ fold, at least 2.8 ⁇ fold, at least 2.9 ⁇ fold, at least 3 ⁇ fold, or more compared with the control.
• a method of the invention may accelerate the production of phenotypically mature NK cells. 20 In other words, the method of the invention may reduce the time taken to arrive at a population of mature NK cells. A reduction in the run time of the method offers a further advantage over the conventional methods for NK cell expansion known in the art.
• a method of the invention may 25 achieve an equivalent population in 10 days or less, preferably in one week or less.
• a method of the invention may achieve a population of at least 40% mature NK cells, preferably at least 45%, at least 46%, at least 47%, at least 48%, or at least 49% mature NK cells, even more preferably at least 50% mature NK cells in three weeks or less, 20 days or less, 19 days or less, 18 days or less, 17 days or less, 16 days or less, 15 days or less, two weeks or less, 13 days or less, or 12 days or less.
• the % of NK cells in the final cell 5 population may be very high (typically greater than 85%, preferably greater than 90%, more preferably greater than 95%, and may approach 100%). In such instances, a final purification step may optionally be omitted.
• Therapeutic indications 10 The invention provides a REV ⁇ ERB inhibitor, or a pharmaceutic composition which comprises said inhibitor, for use in a method of therapy by increasing the production of NK cells in a patient. Pharmaceutical compositions are those which, with reasonable medical judgment, are suitable for use in contact with the tissues of a patient.
• the invention also provides pharmaceutical products containing a compound which inhibits the action of REV ⁇ ERB and a Notch ligand as a combined preparation for simultaneous, separate or 25 sequential use in a method of therapy by increasing the production of NK cells in a patient.
• pharmaceutical products are also, with reasonable medical judgment, products suitable for use in contact with the tissues of a patient.
• the Notch ligand for use in said method of therapy may be any Notch ligand as described herein.
• the Notch ligand is DLL4 or a fragment thereof which retains 30 the function of DLL4. Any REV ⁇ ERB inhibitor and any Notch ligand of the invention may be used in combination.
• a method of therapy relating to said REV ⁇ ERB inhibitor and Notch ligand products comprises administering the products (as described herein) to a patient or subject.
• the Notch ligand and REV ⁇ ERB inhibitor may be administered simultaneously, separately or sequentially.
• the Notch ligand may be administered first, followed by the REV ⁇ ERB inhibitor, or vice versa.
• Sequential administration may mean that the two products are administered immediately one after the other, or that the second product is administered within 1 minute, within two minutes, within 5 three minutes, within four minutes, within five minutes, within 10 minutes, within 15 minutes, within 20 minutes, within 25 minutes, within 30 minutes, within 45 minutes, within one hour, or more of the first product being administered.
• an increase in the number of NK cells and/or increase in NK cell 30 production may be defined in terms of the absolute number of NK cells in a sample or patient, such as the percentage of NK cells, for example the percentage of NK cells in the circulating lymphocyte population.
• a compound of the invention may cause an increase in NK number, resulting in a percentage of NK cells of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or more.
• the number of NK cells may be determined by quantitative and/or qualitative analysis, and may be measured directly or indirectly.
• the number of NK cells relative to a control may be determined 5 using any appropriate technique. Suitable standard techniques, such as flow cytometry, FACS and MACS, are known in the art.
• the number of NK cells may be increased compared with a control for at least 6 hours, at least 12 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at 10 least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or more. Typically this is assessed relative to the last administration of the compound which inhibits REV ⁇ ERB activity.
• the number of NK cells may be quantified in terms of the total number of NK cells in a sample from a patient or culture sample (from an ex vivo method of the invention).
• a “subject” or “patient” 15 (these terms are used interchangeably herein) is any animal patient that would benefit from an increase in the number of NK cells.
• Typical animal patients are mammals, such as primates.
• the patient is a human.
• the present invention provides a method of treatment by increasing the number of NK cells in a patient in need thereof, comprising administering to said patient a therapeutically effective 20 amount of a compound which inhibits the action of REV ⁇ ERB (as described herein), or a pharmaceutical composition comprising said compound.
• the present invention provides the use of a compound which inhibits the action of REV ⁇ ERB in the manufacture of a medicament.
• Said medicament increases the number of NK cells in a patient.
• the present invention provides the use of a compound which inhibits the action of REV ⁇ ERB and a Notch ligand in the manufacture of a medicament.
• Said medicament 30 increases the number of NK cells in a patient.
• the term “preventing” includes inducing or providing protective immunity against such 10 diseases or disorders, particularly infectious diseases as described herein. Immunity may be quantified using any appropriate technique, examples of which are known in the art.
• a compound, pharmaceutic compositions or pharmaceutical products of the invention may be administered to a patient already having a disease or disorder which may be treated by increasing NK cell number.
• the patient may be suspected of having an infectious disease or cancer 15 as described herein, and may or may not be showing symptoms of said disease or disorder.
• the compound, compositions or products of the invention can cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/or prolong the survival of a subject beyond that expected in the absence of such treatment.
• a compound, pharmaceutical compositions or pharmaceutical products of the 20 invention may be administered to a patient who may ultimately be infected with a particular infectious disease, or develop a disease or disorder as described herein, in order to cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/or prolong the survival of a subject beyond that expected in the absence of such treatment, or, in the case of infectious diseases help prevent that patient from transmitting said disease.
• the treatments and preventative therapies of the present invention are applicable to a variety of different subjects of different ages. In the context of humans, the therapies are applicable to children (e.g. infants, children under 5 years old, older children or teenagers) and adults. In the context of other animal subjects (e.g.
• Cancers that may be treated according to the present invention include bladder cancer, blood cancers, leukaemia, 5 bone cancers, bowel cancer, brain tumours, breast cancer, kidney cancer, liver cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer and uterine cancer.
• Autoimmune diseases that may be treated according to the present invention include systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and obesity ⁇ induced insulin resistance.
• the term diseases or disorders related to female infertility 10 or pregnancy includes, but is not limited to, fetal growth restriction, preterm labour, defects in uterine vascular remodelling and preeclampsia.
• the compounds, pharmaceutical compositions or pharmaceutical products of the invention may be used in combination with one or more additional therapeutic agents or treatments, which typically may be selected from a conventional treatment for the disease or disorder to be treated.
• additional therapeutic agents or treatments typically may be selected from a conventional treatment for the disease or disorder to be treated.
• a compound, compositions or products of the invention are for use in the treatment of a cancer, such as lung cancer, then said compound, compositions or products may be used in combination with conventional treatments for lung cancer, such as radiotherapy, chemotherapy or surgery.
• a compound, compositions or products of the invention may be administered before, 20 simultaneously with, or after the administration of the one or more additional therapeutic agent or treatment.
• a compound, pharmaceutical compositions or pharmaceutical products of the invention is for use in combination with antibody ⁇ mediated immunotherapy.
• Antibody ⁇ mediated immunotherapy involves the administration of antibodies to a 25 patient to target disease ⁇ specific antigens. Such antibodies could be used to increase the specificity and killing activity of NK cells, which express receptors for the F C regions of IgG antibodies. Activation of these F C receptors, leads to NK cell activation, resulting in cytokine secretion and release of cytotoxic granules by the activated NK cell, causing lysis of the cell expressing the disease antigen.
• Such combination therapy is particularly preferred for the treatment of cancer (using antibodies to tumour ⁇ 30 specific antigens). Any antibody used in immunotherapy may be used in combination with a compound of the invention.
• the present invention provides an expanded NK cell population (as described herein) for use in a method of therapy, for example in the treatment of cancer, an infectious diseases, an autoimmune disease or 10 a disease or disorder related to female infertility or pregnancy.
• the invention provides a method of treatment by increasing the number of NK cells in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of an expanded NK cell population.
• compositions and formulations The “compound” and products described herein may be comprised in a “therapeutic/prophylactic composition”, “formulation” or “medicament” of the invention.
• the compound or expanded NK cell population of the invention (as defined above) can be combined or administered in addition to a pharmaceutically acceptable carrier, diluent and/or 20 excipient.
• the compound or expanded NK cell population of the invention can further be combined with one or more of a salt, excipient, diluent, adjuvant, immunoregulatory agent and/or antimicrobial compound.
• the compound of formula (I) may be in the form of a salt, particularly a pharmaceutically acceptable salt.
• Pharmaceutically acceptable salts include acid addition salts formed with inorganic 25 acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2 ⁇ ethylamino ethanol, histidine, procaine, and the like.
• inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2 ⁇ ethylamino ethanol, histidine, procaine, and the like.
• compositions, therapeutic formulations, medicaments and prophylactic formulations are generally by conventional routes e.g. intravenous, subcutaneous, intraperitoneal, or mucosal routes.
• the administration may be by parenteral injection, for example, a subcutaneous, intradermal or intramuscular injection.
• formulations comprising antibodies or expanded NK cell populations of the invention may be particularly suited to administration intravenously, intramuscularly, intradermally, or subcutaneously.
• composition comprises a compound of the invention
• this may be in lyophilized form, in which case it may include a stabilizer, such as BSA.
• a preservative such as thiomersal or sodium azide
• additional adjuvants which may be effective include but are not limited to: 25 complete Freunds adjuvant (CFA), Incomplete Freunds adjuvant (IFA), Saponin, a purified extract fraction of Saponin such as Quil A, a derivative of Saponin such as QS ⁇ 21, lipid particles based on Saponin such as ISCOM/ISCOMATRIX, E.
• coli heat labile toxin (LT) mutants such as LTK63 and/ or LTK72, aluminium hydroxide, N ⁇ acetyl ⁇ muramyl ⁇ L ⁇ threonyl ⁇ D ⁇ isoglutamine (thr ⁇ MDP), N ⁇ acetyl ⁇ nor ⁇ muramyl ⁇ L ⁇ alanyl ⁇ D ⁇ isoglutamine (CGP 11637, referred to as nor ⁇ MDP), N ⁇ acetylmuramyl ⁇ L ⁇ alanyl ⁇ D ⁇ 30 isoglutaminyl ⁇ L ⁇ alanine ⁇ 2 ⁇ (1' ⁇ 2' ⁇ dipalmitoyl ⁇ sn ⁇ glycero ⁇ 3 ⁇ hydroxyphosphoryl oxy) ⁇ ethylamine (CGP 19835A, referred to as MTP ⁇ PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2 % squalene/
• Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium 10 carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
• the dosage ranges for administration of the compounds or products of the present invention are those which produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the compound, the route of administration, the nature of 15 the formulation, the age of the patient, the nature, extent or severity of the patient’s condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
• compositions of the present invention are preferably free from compounds disclosed in either WO 2013/033310 or WO 2015/103527.
• SEQ ID NOs SEQ ID NO: 1 – E4bp4 gene sequence (X64318.1) 1 gcccctttct ttctcctcgt cggcccgaga gcaggaacac gataacgaag gaggcccaac 10 61 ttcattcaat aaggagcctg acggatttat cccagacggt agaacaaaag gaagaatatt 121 gatggatttt aaaccagagt ttttaaagag cttgagaata cggggaaatt aattgttct 181 cctacacacaca tagatagggt aaggttgtt ctgatgcagc
• FuGENE was used as transfection reagent in a 5:2 ratio FuGENE:DNA, and after addition, mixture was gently mixed and left to incubate for 20 min at RT. Afterwards, 20 ⁇ L of the DNA mix was 15 transferred into the 24 ⁇ well plate containing the pre ⁇ seeded cells, with each condition in triplicate. 5 h after transfection, cells were treated with compounds 7, 11, 7acid, 11acid, SR8278, GSK1362 or DMSO as a vehicle. For that, 2X stock solutions of the compounds (or DMSO vehicle control) in DMEM were prepared. 0.5 mL of these 2X stock solutions were added to each well.
• Results The resulting data are shown in Figure 4, expressed as the average and standard error 15 obtained from three independent experiments carried out in triplicate.
• compounds acid7 and acid11 produced the greatest increase in luciferase expression of all the compounds tested.
• Compounds acid7 and acid11 also produced a greater increase in luciferase expression than their ethyl ester analogues (compounds 7 and 11) in the REV ⁇ ERB ⁇ experiments.
• 20 The same experiments were repeated, but with the tested compounds used at concentrations of 5 and 50 ⁇ M.
• Plasma protein binding experiments were carried out on the compounds of the present invention. Once again, for comparison, tests were also carried out on compounds 7 and 11 and SR8278, as well as warfarin as a literature control.
• Plasma protein binding 1 mM stock solutions of test compounds were prepared in DMSO and diluted 200 ⁇ fold in mice plasma to prepare a concentration of 5 ⁇ M, with a final DMSO concentration in plasma of 0.5%. Rapid equilibrium dialysis was performed with a rapid equilibrium dialysis (RED) device containing a dialysis membrane with a molecular weight cut ⁇ off of 8,000 Daltons.
• RED rapid equilibrium dialysis
• Example 5 screening of acid ⁇ form compounds for off ⁇ target effect
• Prior art REV ⁇ ERB ligands have been known to exhibit clear off ⁇ target effects, and in particular to activate LXR ⁇ , a nuclear receptor closely related to REV ⁇ ERB and involved in numerous biological pathways. Activity of REV ⁇ ERB ligands on LXR ⁇ would ideally be avoided.
• a reporter gene assay similar 25 to that described in Example 2 was carried out using LXR ⁇ and compounds of the present invention. For comparison, tests were also carried out using compound 7 and compound 11, SR8278 and GSK1362, and with DMSO as a control.
• Reporter gene assay for LXR ⁇ activity HEK293T cells were seeded and transfected following the protocol previously described in Example 2.
• the DNA master mix used was composed of 50 ng 5 LXR ⁇ or pcLXR ⁇ as the empty vector, 100 ng of pGL3 ⁇ LXR REX2 ⁇ luc, 10 ng of Renilla luciferase and 240 ng of BSM. 5 h after transfection compounds (or DMSO vehicle control) were added at 10 ⁇ M concentration. GW3965 was used as LXR ⁇ activator control at 5 ⁇ M. After 24 h, cells were lysed and activity was measured as previously described using Dual ⁇ Luciferase Reporter Assay System. Results: The resulting data are shown in Figure 5, expressed as the average and standard error 10 obtained from three independent experiments carried out in triplicate.
• Example 6 effect of acid ⁇ form compounds on expression of E4BP4
• the effect of compounds of the present invention on endogenous REV ⁇ ERB was assessed by 20 looking at the expression of E4BP4 in different cell lines.
• a first set of experiments was conducted using HepG2 and a second set of experiments was conducted using NK ⁇ 92 cells.
• Sample preparation HepG2 cells were seeded in 6 ⁇ well plates in DMEM media supplemented with 10% FBS and 1% PSG (2.5 ⁇ 10 6 cells in 3 mL media per well) at 37°C and 5% CO 2 .
• RNA extraction was isolated using RNeasy Plus Mini Kit (QIAGEN, cat. 74134) according to the manufacture’s protocol. HepG2 cells were harvested directly from the well, by removing media, washing with EDTA, lysed by adding 350 ⁇ L of RLT plus 1% ⁇ ME and homogenized by vortex. NK ⁇ 92 cells were collected into tubes, centrifuged, and the pellets were lysed by adding 350 ⁇ L RLT plus 1% 5 ⁇ ME and homogenized by vortex. Cell lysate was transferred to gDNA Eliminator spin column and centrifuged to remove genomic DNA. 350 ⁇ L 70% ethanol was added and samples were transferred to RNeasy MinElute spin column and centrifuged.
• cDNA Complementary DNA

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