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1. WO2020002911 - NATURAL KILLER CELLS

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

1. An ex vivo method for expanding an IMK cell population, comprising the steps of:

d) culturing an haematopoietic progenitor cell (HPC) comprising sample obtained from an individual;

e) contacting said sample with a compound that inhibits the action of REV-ERB; and f) expanding said ceils in vitro to produce an NK cell population;

wherein the compound has formula (I):


where: - represents bonds that are all either present or absent;

R1 is selected from C1-6 hydrocarbyl, and is optionally substituted with one or more groups independently selected from C1-4 hydrocarbyl, -OR', -OC(0)R', -C(0)OR', -SR', -S(0)R', -S(0)2R', -NR' 2, -NR'C(0)R', -C(0)NR'2, -CN, -N02, -Ph, -CF3 and halogen;

R/ is selected from 5-10 membered heterocyclyl rings and Ci-6 hydrocarbyl, and is optionally substituted with one or more groups independently selected from Ci- hydrocarbyl, -OR', -OC(0)R', -C(0)OR', -SR', -S(0)R', -S(0)2R', -NR'2, -NR'C(0)R', - C(0)NR'2, -CN, -N02, -Ph, -CF3 and halogen;

X is selected from -O- and -NR'- or is absent;

Y is selected from -C(O)- or -CR'2-;

Z is selected from -O- and -NR'- or is absent;

each Ra is independently selected from H, Ci_4 hydrocarbyl, -OR', -OC(0)R', -C(0)OR', -SR', -S(OjR', -S(0)2R', -N R'2J -N R'C(0)R', -C(0)N R'2, -CN, -N02, -Ph, -CF3 and halogen; each Rb is independently selected from H, Ci.4 hydrocarbyl and -OR';

Rc is selected from H and Ci4 hydrocarbyl; and

each R' is independently selected from H, Ci.4 hydrocarbyl and -Ph;

or a pharmaceutically acceptable salt thereof, provided that the compound is not:


2. The method of claim 1, wherein - represents bonds that are all present and the compound is a closed ring structure according to formula la:


3. The method of claim 1 or 2, wherein R1 is unsubstituted and, as such, is preferably selected from Ci-6 alkyl and Ci_s alkenyl, more preferably, R1 is selected from Ci-6 alkyl, even more preferably from C1-4 alkyl, such as from methyl, ethyl and propyl.

4. The method of any one of claims 1 to 3, wherein:

R2 is selected from:

(i) optionally substituted 5-10 membered heterocyclyl rings, preferably optionally substituted 5-10 membered heteroaryl rings, and the 5-10 membered heteroaryl ring Is preferably an optionally substituted 5-, 6- or 9-membered heteroaryl rings, wherein optionally the 5-, 6-, or 9-membered heteroaryl ring is selected from optionally substituted: furany!, thiophenyl, oxazolyl, isooxazolyl, isothiazolyl, thiazo!yl, pyrazolyl, imidazo!y!, pyrrolyl and triazolyl, pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl, indolyl, isoindolyl, indazolyl, benzimidazolyl, azaindo!y!, henzofuranyl, isobenzofuranyl, benzisoxazolyl and benzoxazolyl; and

(ii) optionally substituted phenyl, preferably substituted phenyl; and/or

R2 is unsubstituted or substituted with one or two groups independently selected from Ci-4 alkyl, -OR', -OC(0)R', -C(0)OR', -SR', -S(0)R', -S(0)2R', -NR'2, -NR'C(0)R', -C{0)MR'2, -CN, -N02, -Ph, -CF3 and halogen, preferably R2 is unsubstituted or substituted with one or two groups independently selected from -Me, -OMe, -CM, -N02, -F, -Cl, -I and -SMe.

5. The method of any one of the preceding claims, wherein:

a) X is -0-;

b) Y is -C(O)-;

c) Z is -O- or is absent, preferably Z is absent;

d) each Ra is independently selected from
alkyl and -OR', preferably from

alkyl, and more preferably, each R3 is H;

e) each Rb is independently selected from H and Ci_4 alkyl, preferably, each Rb is H;

f) Rc is H; and/or

g) each R' is independently selected from H and Ci-4 alkyl, preferably from H, methyl and ethyl, and more preferably from methyl and ethyl.

6. The method of any one of the preceding claims, wherein RD and Rc are all H, such that the compound has the formula (II):


where R1, R2, Ra, X, Y and Z are defined in claims 1 to 5.

7. The method of claim 6, wherein Ra are all H, such that the compound has the formula (ill):


where R1, R2, X, Y and Z are defined in claims 1 to 5;

preferably X is -0-, such that the compound has the formula (IV):


where R1, R2, Y and Z are as defined in claims 1 to 5;

more preferably, Y is -C(0)-, such that the compound has the formula (V):


where R1, R2 and Z are as defined in claims 1 to 5;

still more preferably, R1 is ethyl, such that the compound has the formula (VI):

where R2 and Z are as defined in claims 1 to 5;

yet still more preferably, the compound is a closed ring structure with the formula (VII):


where R2 and Z are as defined in claims 1 to 5.

8. The method of any one of the preceding claims, wherein the compound has a formula selected from:

5
PCT/GB2019/051803

5 wherein preferably the compound has the formula:


wherein more preferably the compound has the formula:

9. The method of any one of claims 1 to 8, wherein said compound increases E4bp4 expression by decreasing REV-ERB activity.

10. The method of any one of claims 1 to 9, wherein said compound decreases the activity of REV-ERB-a and/or REV-ERB-b, preferably REV-ERB-b, and more preferably REV-ERB-a and REV-ERB-b.

11. The method of any one of claims 1 to 9, wherein said compound is a REV-ERB antagonist, preferably an antagonist of REV-ERB-a and REV-ERB-b.

12. The method of any one of claims 1 to 11, which further comprises a step of culturing the HPCs in the presence of a Notch ligand, wherein optionally the vessel in which the HPCs are cultured is coated with the Notch ligand.

13. The method of claim 12, wherein:

a) the Notch ligand is delta-like ligand 4 (DLL4), or a fragment thereof which retains the function of DLL4; and/or

b) the Notch ligand is present on or from 4 days after isolating said HPCs.

14. The method of claim 12 or 13, wherein:

a) the ceils are cultured in the presence of IL-15 after the step of culturing in the presence of the Notch ligand; and/or

b) the HPCs are cultured in the presence of the Notch ligand in combination with IL-7, Flt3L and/or stem cell factor (SCF), preferably the Notch ligand in combination with IL-7, FitBL and SCF;

wherein optionally either or both of the step of culturing in the cells in the presence of the Notch ligand and the step of culturing in the presence of IL-15 are carried out in the absence of a stroma! support cell, and preferably wherein both steps are carried out in the absence of a stromal support cell.

15. The method of any one of the preceding claims, which further comprises the step of contacting the HPCs with a compound which results in alteration of the post-translational modification of E4bp4, thereby causing an increase in E4bp4 activity;

wherein optionally the alteration of post -translational modification of E4bp4 is a reduction in SUMOylation and/or phosphorylation of E4bp4, and preferably the compound which results in the alteration of post-translational modification of E4bp4:

a) reduces SUMOylation at one or more of residues K1Q, K116, K219, K337 and/or K394 of E4bp4, or a residue corresponding thereto, or any combination thereof; and/or b) reduces phosphorylation at one or more of residues S286, S301 and/or S454 of E4bp4, or a residue corresponding thereto, or any combination thereof.

16. The method of any one of the preceding claims, wherein the sample of HPCs is obtained from bone marrow, cord blood and/or peripheral blood.

17. An expanded NK cell population obtained by the method of any one of claims 1 to 16, wherein at least 85% of the NK cells are CD56+ and CD45+.

18. A composition comprising an expanded NK cel! population as defined in claim 17 and a pharmaceutically acceptable carrier, diluent and/or excipient.

19. A compound which inhibits the action of REV-ERB activity for use in a method of therapy by increasing production of natural killer (NK) cells in a patient, wherein said compound is as defined in any one of claims 1 to 11.

20. Products containing a compound which inhibits the action of REV-ERB and a Notch ligand as a combined preparation for simultaneous, separate or sequential use in a method of therapy by increasing production of natural killer (NK) cells in a patient, wherein said compound is as defined in any one of claims 1 to 11, and optionally said Notch ligand is delta-like ligand 4 (DLL4), or a fragment thereof which retains the function of DLL4.

21 The compound for use of claim 19 or the products for use of claim 20, wherein said method of therapy is:

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.

22. 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 as defined in any one of claims 1 to 11, 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.

23 The compound for use of any one of claims 19 or 21, the products for use of claim 20 or 21 or the method of treatment of claim 22, which is 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.

24. A compound of formula (I) as defined in any one of claims 1 to 11, provided that the compound is not: