WO2015128492A1

WO2015128492A1 - Monomethyl- and dimethylfumarate for nk cell activation

Info

Publication number: WO2015128492A1
Application number: PCT/EP2015/054202
Authority: WO
Inventors: Azzam A. MAGHAZACHI
Original assignee: Maghazachi Azzam A
Priority date: 2014-02-28
Filing date: 2015-02-27
Publication date: 2015-09-03

Abstract

Compositions and methods for enhancing NK cell mediated killing of target cells, e.g., cancer cells, virally infected cells, among others, are provided. In one aspect, compositions and methods for treatment of cancer and/or infectious disease are provided.

Description

MONOMETHYL- AND DIMETHYLFUMARATE FOR NK CELL ACTIVATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/946,301 , filed February 28, 2014, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates, inter alia, to the use of prodrugs and their drugs, e.g., dimethyl fumarate (DMF) and monomethyl fumarate (MMF), e.g., in the treatment of cancer, infectious disease and other conditions.

BACKGROUND OF THE INVENTION

A number of cancers are, at present, incurable. For others, chemotherapy is only partially effective and a significant proportion of patients relapse following treatment. Some

haematological malignancies are treatable by hematopoietic stem cell transplantation (HSCT), but fewer than 30% of patients requiring HSCT have a suitable donor and are the requisite age.

Infectious diseases, such as viral infections, are difficult to prevent and/or treat. For many infectious diseases, vaccinations and/or treatments are unavailable. In many cases, available treatments address the symptoms of the infection and do not treat the infection itself.

Natural Killer (NK) cells are large granular lymphocytes that possess the ability to spontaneously lyse certain target cells, including tumor cells. The activities of NK cells are regulated by activating and inhibitory receptors, which by intracellular integration of challenges and inhibition, determine the cell course of action. They are activated by cells that are in distress through the detection of stress-induced ligands on target cells by Natural Cytotoxicity Receptors (NCRs). In addition, NK cells express several receptors that inhibit activation, including members of the killer-cell immunoglobulin-like receptors (KIRs) family and CD94-NKG2A.

A need exists for identifying agents which appropriately activate NK cells to enhance NK cell mediated killing of cells in a subject, e.g., tumor cells in a cancer patient and/or infected cells in a patient with an infectious disease. SUMMARY OF THE INVENTION

The present invention provides, at least in part, compositions and methods for treating a subject, e.g., a subject having cancer, a subject having an infectious disease. In certain embodiments, provided compositions and methods provide enhanced NK cell mediated killing of a target cell, e.g., a cancer cell or a virally infected cell. Applicants have identified that DMF and/or MMF treatment of NK cells, e.g., NK cells which are phenotypically non-cytolytic, e.g., CD56+/^bnght NK cells, leads to enhanced killing of cells, e.g., tumor cells. Applicants have further identified that DMF and/or MMF activate NK cells to lyse cells that are generally resistant to NK cell killing, e.g., a B cell lymphoma cell line, e.g., RAJI cells. Without wishing to be bound by any particular theory, the data presented in the examples herein indicate that DMF and/or MMF can mediate NK cell activity for enhanced cell lysis of cancer cells, infected cells, e.g., virally infected cells, among others. Thus, the invention can, therefore, be used, for example: to treat cancer in a subject, to treat an infectious disease in a subject, and/or to generally enhance cell lysis of a target cell by a natural killer (NK) cell. Many of the methods and other inventions provided herein are described for use with the prodrug DMF and its active metabolite MMF. However, it should be understood that the methods and other inventions can be used with, or apply generically to, dialkyl fumarate prodrugs, e.g., as shown in Formula A below, other prodrugs, e.g., as shown in Formulas I-VI, and their active metabolites (e.g., MMF), and monoalkyl fumarate drugs, e.g., as shown in Formula B below.

Formula A

wherein R^1§ and R^2§, which may be the same or different, independently represent a linear, branched or cyclic, saturated or unsaturated C_1-20 alkyl radical which may be optionally substituted with halogen (CI, F, I, Br), hydroxy, Ci-4 alkoxy, nitro or cyano.

In an embodiment, R^1§ and R^2§, which may be the same or different, independently are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, cyclopentyl, 2 -ethyl hexyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, vinyl, allyl, 2-hydroxy ethyl, 2 or 3-hydroxy propyl, 2-methoxy ethyl, methoxy methyl or 2- or 3-methoxy propyl. In an embodiment, R^1§ and R^2§ are identical and are methyl or ethyl.

In an embodiment, R^1§ and R^2§ are methyl.

Formula B

wherein R^lh represents a linear, branched or cyclic, saturated or unsaturated C_1-20 alkyl radical which may be optionally substituted with halogen (CI, F, I, Br), hydroxy, C_1-4 alkoxy, nitro or cyano;

or a pharmaceutically acceptable salt thereof.

In an embodiment, R^lh is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, cyclopentyl, 2-ethyl hexyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, vinyl, allyl, 2- hydroxy ethyl, 2 or 3-hydroxy propyl, 2-methoxy ethyl, methoxy methyl or 2- or 3-methoxy propyl.

In an embodiment, R^lh is methyl or ethyl.

In an embodiment, R^lh is methyl.

Accordingly, in one aspect, the invention features a method of treating cancer in a subject in need thereof comprising administering to the subject a solid dosage form comprising a therapeutically effective amount of dimethyl fumarate (DMF), or a prodrug thereof. In some embodiments the subject is administered DMF.

In some embodiments, the cancer is a hematological malignancy, e.g., leukemia, lymphoma, or myeloma. In certain embodiments, the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL). In certain embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma. In certain embodiments, the myeloma is multiple myeloma.

In some embodiments, the cancer is a solid tumor, such as breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and combinations thereof. In another aspect, the invention features a method of treating an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of dimethyl fumarate (DMF), or a prodrug thereof. In some embodiments, the subject is administered a therapeutically effective amount of DMF.

In some embodiments, the infectious disease is a viral infection selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

In some embodiments, the subject is a human.

In particular embodiments, DMF, or prodrug thereof is administered orally. In some embodiments, DMF, or prodrug thereof is administered daily. In a particular embodiment, DMF is administered orally. In a more particular embodiment, DMF is administered daily in an oral dosage form. DMF, or prodrug thereof may be administered in any appropriate dose. For example, in some embodiments, DMF, or prodrug thereof is administered in a dose of approximately 50-2000 mg/day to the subject. In some embodiments, DMF, or a prodrug thereof is administered in a dose of approximately 240-1000 mg/day. In some embodiments, DMF is administered in a dose of approximately 240-720 mg/day. In a particular embodiment, DMF is administered in a dose of approximately 480 mg/day or approximately 720 mg/day. In some embodiments, DMF is administered in a dose of approximately 720 mg/day in three equal doses. In some embodiments, DMF is administered in a dose of approximately 480 mg/day in two equal doses. In some embodiments, DMF, or a prodrug thereof is administered in a dose of less than 500 mg, less than 400 mg, less than 300 mg, less than 200 mg or less than 100 mg per dose to the subject and the dose is administered 1 , 2, 3, 4, 5, or 6 times daily. In some embodiments, DMF, or a prodrug thereof is administered in a dose of more than 100 mg, more than 200, more than 300 mg, more than 400 mg, or more than 500 mg per dose to the subject and the dose is administered 1 , 2, 3, 4, 5, or 6 times daily. Preferably, DMF is administered 3 times daily. More preferably DMF is administered 2 times daily. Most preferably DMF is administered once daily.

In some embodiments, the solid dosage form is selected from the group consisting of tablets, micro-tablets, pellets, granulates, capsules (e.g., soft or hard gelatin capsules), sachets, powders and lozenges. In a preferred embodiment, preparations are in the form of micro-tablets or pellets, optionally filled in capsules or sachets. The size or mean diameter of the pellets or microtablets can range from 300 to 4000 μπι, e.g., 500 to 3500 μπι, 1000 to 3000 μπι, or 1500 to 2500 μπι. In some embodiments, the size or mean diameter of the pellets or microtablets are about 2000 μπι, e.g., 2000 μπι.

In some embodiments, an oral dosage form is prepared with an enteric coating, e.g., to delay the release of the drug from the dosage forms. In some embodiments, the enteric coating is selected from the group consisting of waxes, shellacs, polymers, and plant fibers.

In certain embodiments, the method further includes administering one or more cancer therapeutic agents in combination with the DMF, or prodrug thereof (preferably with DMF). In some embodiments, cancer therapeutic agent is a chemotherapeutic agent, e.g., aclarubicin, alemtuzumab, amsacrine, asparaginase, azacitidine, busulphan, chlorambucil, cladribine, clofarabine, cytabarine, daunorubicin, doxorubicin, filgrastim, fiudarabine, interferon alpha 2A, mercaptopurine, methotrexate, mitoxantrone, nelarabine, nilotinib, pentostatin, rituximab, teniposide, thioguanine, vincristine, and combinations thereof.

In certain embodiments, the method further includes administering an antiviral agent in combination with DMF, or prodrug thereof (preferably with DMF). In some embodiments, the antiviral agent is selected from the group consisting of abacavir, acyclovir, afefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, boceprevirertet, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, efuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, tea tree oil, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifiuridine, trizivir, tromantadine, truvada, traporved, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. In related aspect, the invention features a method of enhancing cell lysis of a target cell by a natural killer (NK) cell comprising contacting the NK cell with a composition comprising dimethyl fumarate (DMF), or a prodrug thereof (preferably with DMF) in an amount effective to increase the capacity of the NK cell to lyse the target cell. In particular embodiments, the contacting step is performed in vivo. In particular embodiments, the contacting step is performed ex vivo.

In some embodiments, the target cell is a cancer cell. For example, in certain

embodiments, the cancer cell is a hematological malignancy cell, e.g., a leukemia, lymphoma, or myeloma. In certain embodiments, the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia

(AMoL). In certain embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma. In certain embodiments, the myeloma is multiple myeloma.

In some embodiments, the cancer is a solid tumor, such as breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and combinations thereof.

In particular embodiments, the target cell is a virally-infected cell. For example, in some embodiments, the target cell is infected by hepatitis B (HBV), hepatitis C (HCV), human T- lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human

cytomegalovirus (CMV), or a combination thereof.

In some embodiments, the target cell is a human cell. In certain embodiments, the NK cell is a human NK cell. In particular embodiments, the NK cell is a CD56+/^bnsht NK cell. In certain embodiments, the NK cell is a Οϋ56-/^Λιη NK cell. In particular embodiments, the target cell is a cell that was previously resistant to NK cell lysis. In particular embodiments, the NK cell has an increased capacity to lyse Raji cells when contacted with the composition comprising DMF, or prodrug thereof (preferably with DMF) as compared to the capacity of the NK cell to lyse Raji cells before being contacted with the composition comprising DMF, or prodrug thereof. In another aspect, the invention features a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of

monomethyl fumarate (MMF), or a metabolite thereof.

In another aspect, the invention features a method of treating an infectious disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of monomethyl fumarate (MMF), or a metabolite thereof. In some embodiments, the infectious disease is a viral infection selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

In some embodiments, the subject is a human.

In certain embodiments, MMF, or metabolite thereof is administered orally. In particular embodiments, MMF, or metabolite thereof is administered daily. The MMF or active metabolite thereof may be administered in any appropriate dose. For example, in some embodiments, the MMF, or metabolite thereof is administered in a dose of approximately 50-2000 mg/day to the subject. In some embodiments, MMF, or metabolite thereof is administered in a dose of approximately 240-1000 mg/day. In some embodiments, MMF is administered in a dose of approximately 240-720 mg/day. In a particular embodiment, MMF is administered in a dose of approximately 480 mg/day or approximately 720 mg/day. In some embodiments, MMF is administered in a dose of approximately 720 mg/day in three equal doses. In some embodiments, MMF is administered in a dose of approximately 480 mg/day in two equal doses. In some embodiments, MMF, or a prodrug thereof is administered in a dose of less than 500 mg, less than 400 mg, less than 300 mg, less than 200 mg or less than 100 mg per dose to the subject and the dose is administered 1 , 2, 3, 4, 5, or 6 times daily. In some embodiments, MMF, or a prodrug thereof is administered in a dose of more than 100 mg, more than 200, more than 300 mg, more than 400 mg, or more than 500 mg per dose to the subject and the dose is administered 1, 2, 3, 4, 5, or 6 times daily. Preferably, MMF is administered 3 times daily. More preferably MMF is administered 2 times daily. Most preferably MMF is administered once daily.

In some embodiments, the solid dosage form is selected from the group consisting of tablets, micro -tablets, pellets, granulates, capsules (e.g., soft or hard gelatin capsules), sachets, powders and lozenges. In a preferred embodiment, preparations are in the form of micro-tablets or pellets, optionally filled in capsules or sachets. The size or mean diameter of the pellets or microtablets can range from 300 to 4000 μπι, e.g., 500 to 3500 μπι, 1000 to 3000 μπι, or 1500 to 2500 μπι. In some embodiments, the size or mean diameter of the pellets or microtablets are about 2000 μπι, e.g., 2000 μπι.

In certain embodiments, the method further includes administering a cancer therapeutic agent in combination with the MMF, or metabolite thereof. In some embodiments, cancer therapeutic agent is a chemotherapeutic agent, e.g., aclarubicin, alemtuzumab, amsacrine, asparaginase, azacitidine, busulphan, chlorambucil, cladribine, clofarabine, cytabarine, daunorubicin, doxorubicin, filgrastim, fludarabine, interferon alpha 2A, mercaptopurine, methotrexate, mitoxantrone, nelarabine, nilotinib, pentostatin, rituximab, teniposide, thioguanine, vincristine, and combinations thereof.

In certain embodiments, the method further includes administering an antiviral agent in combination with the MMF, or metabolite thereof.

In a related aspect, the invention features a method of enhancing cell lysis of a target cell by a natural killer (NK) cell comprising contacting the NK cell with a composition comprising monomethyl fumarate (MMF), or a metabolite thereof in an amount effective to increase the capacity of the NK cell to lyse the target cell. In particular embodiments, the contacting step is performed in vivo. In particular embodiments, the contacting step is performed ex vivo.

In some embodiments, the target cell is a cancer cell. For example, in certain

cytomegalovirus (CMV), or a combination thereof.

In some embodiments, the target cell is a human cell. In certain embodiments, the NK cell is a human NK cell. In particular embodiments, the NK cell is a CD56+/^bnsht NK cell. In certain embodiments, the NK cell is a Οϋ56-/^Λπι NK cell. In particular embodiments, the target cell is a cell that was previously resistant to NK cell lysis. In particular embodiments, the NK cell has an increased capacity to lyse Raji cells when contacted with the composition comprising DMF, or prodrug thereof as compared to the capacity of the NK cell to lyse Raji cells before being contacted with the composition comprising DMF, or prodrug thereof.

In another aspect, the invention features an in vitro or ex vivo method for preparing an activated natural killer (NK) cell, the method comprising a step of contacting an isolated NK cell with dimethyl fumarate (DMF) in an amount effective to activate the NK cell. In some embodiments, the activated NK cell has an increased capacity to lyse Raji cells as compared to the isolated NK cell before activation. In some embodiments, the DMF is present in an amount of about 500 μηι, about 400 μηι, about 300 μηι, about 200 μηι, about 100 μηι, about 50 μηι, about 10 μηι, about 5 μηι, about 1 μηι, or about 0.1 μηι.

In certain embodiments, the method further includes administering the activated NK cell to a subject in need thereof.

In yet another aspect, the invention features an in vitro or ex vivo method for preparing an activated natural killer (NK) cell, the method comprising a step of contacting an isolated NK cell with monomethyl fumarate (MMF) in an amount effective to activate the NK cell. In some embodiments, the activated NK cell has an increased capacity to lyse Raji cells as compared to the isolated NK cell before activation. In some embodiments, MMF is present in an amount of about 500 μηι, about 400 μηι, about 300 μηι, about 200 μηι, about 100 μηι, about 50 μηι, about 10 μηι, about 5 μηι, about 1 μηι, or about 0.1 μηι.

In a related aspect, the invention features a population of activated NK cells prepared by the methods described herein.

The present invention contemplates treatment with the prodrug DMF and its active metabolite MMF. However, it should be understood that the methods and other inventions can be used with, or apply generically to, dialkyl fumarate prodrugs, e.g., as shown in Formula A below, and other prodrugs, e.g., as shown in Formulas I- VI, and their active metabolites (e.g., MMF), and monoalkyl fumarate drugs, e.g., as shown in Formula B below. In an embodiment the drug is MMF and the prodrug is DMF. In an embodiment the drug is MMF and the prodrug is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein R^la and R ^a ' are independently chosen from hydrogen, Ci_6 alkyl, and substituted Ci_6 alkyl;

R^Ja and R^4a ' are independently chosen from hydrogen, Ci-₆ alkyl, substituted Ci-₆ alkyl, Ci-6 heteroalkyl, substituted Ci_6 heteroalkyl, C₄-₁₂ cycloalkylalkyl, substituted C₄-₁₂ cycloalkylalkyl, C₇-₁₂ arylalkyl, and substituted C₇-₁₂ arylalkyl; or R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C5-₁₀ heteroaryl, substituted C5-₁₀ heteroaryl, C5-₁₀ heterocycloalkyl, and substituted C5-₁₀ heterocycloalkyl; and

R^5a is chosen from methyl, ethyl, and C3-6 alkyl; wherein each substituent group is independently chosen from halogen, -OH,

-CN, -CF₃, =0, -NO2, benzyl, -C(0)NR^{l la} ₂, -R^{l la}, -OR^{l la}, -C(0)R^{l la}, -COOR^{l la}, and -NR^{l la} ₂ wherein each R^{l la} is independently chosen from hydrogen and C₁-4 alkyl; with the proviso that when R^5a is ethyl; then R^3a and R^4a are independently chosen from hydrogen, Ci_6 alkyl, and substituted Ci_6 alkyl.

In certain embodiments of a compound of Formula (I), each substituent group is independently chosen from halogen, -OH, -CN, -CF₃, -R^{l la}, -0R^{l la}, and

-NR^{l la} ₂ wherein each R^{l la} is independently chosen from hydrogen and C₁-4 alkyl. In certain embodiments, each substituent group is independently chosen from -OH, and

-COOH.

In certain embodiments of a compound of Formula (I), each substituent group is independently chosen from =0, C₁-4 alkyl, and -COOR^{l la} wherein R^{l la} is chosen from hydrogen and C ₁-4 alkyl.

In certain embodiments of a compound of Formula (I), each of R^la and R^2a is hydrogen.

In certain embodiments of a compound of Formula (I), one of R^la and R^2a is hydrogen and the other of R^la and R^2a is Ci_₄ alkyl.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. In certain embodiments of a compound of Formula (I), one of R^la and R^2a is hydrogen and the other of R^la and R^2a is methyl.

In certain embodiments of a compound of Formula (I), R^3a and R^4a are independently chosen from hydrogen and Ci_6 alkyl.

In certain embodiments of a compound of Formula (I), R^3a and R^4a are independently chosen from hydrogen and C_1-4 alkyl.

In certain embodiments of a compound of Formula (I), R^3a and R^4a are independently chosen from hydrogen, methyl, and ethyl.

In certain embodiments of a compound of Formula (I), each of R^3a and R^4a is hydrogen; in certain embodiments, each of R^3a and R^4a is methyl; and in certain embodiments, each of R^3a and R^4a is ethyl.

In certain embodiments of a compound of Formula (I), R^3a is hydrogen; and R^4a is chosen from Ci-4 alkyl, substituted C_1-4 alkyl wherein the substituent group is chosen from =0, -0R^{l la}, - COOR^{l la}, and -NR^lla ₂, wherein each R^{l la} is independently chosen form hydrogen and C_1-4 alkyl. In certain embodiments of a compound of Formula (I), R^3a is hydrogen; and R^4a is chosen from Ci-4 alkyl, benzyl, 2-methoxyethyl, carboxymethyl, carboxypropyl, 1 ,2,4-thiadoxolyl, methoxy, 2-methoxycarbonyl, 2-oxo(l ,3-oxazolidinyl), 2-(methylethoxy)ethyl, 2-ethoxyethyl, (tert- butyloxycarbonyl)methyl, (ethoxycarbonyl)methyl, carboxymethyl,

(methylethyl)oxycarbonylmethyl, and ethoxycarbonylmethyl.

In certain embodiments of a compound of Formula (I), R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring. In certain

embodiments of a compound of Formula (I), R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C5 heterocycloalkyl, substituted C5 heterocycloalkyl, C5 heteroaryl, and substituted C5 heteroaryl ring. In certain embodiments of a compound of Formula (I), R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C₆ heterocycloalkyl, substituted C₆ heterocycloalkyl, C₆ heteroaryl, and substituted C₆ heteroaryl ring. In certain embodiments of a compound of Formula (I), R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from piperazine, 1 ,3-oxazolidinyl, pyrrolidine, and morpholine ring. In certain embodiments of a compound of Formula (I), R^3a and R^4a together with the nitrogen to which they are bonded form a Cs_-10 heterocycloalkyl ring.

In certain embodiments of a compound of Formula (I), R^5a is methyl.

In certain embodiments of a compound of Formula (I), R^5a is ethyl.

In certain embodiments of a compound of Formula (I), R^5a is C3-6 alkyl.

In certain embodiments of a compound of Formula (I), R^5a is chosen from methyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

In certain embodiments of a compound of Formula (I), R^5a is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is Ci_6 alkyl; R^3a is hydrogen; R^4a is chosen from hydrogen, Ci_6 alkyl, and benzyl.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is Ci_6 alkyl; R^3a is hydrogen; R^4a is chosen from hydrogen, Ci_6 alkyl, and benzyl; and R^5a is methyl.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is chosen from hydrogen and Ci_6 alkyl; and each of R^3a and R^4a is Ci-6 alkyl.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is chosen from hydrogen and Ci_6 alkyl; each of R^3a and R^4a is Ci_6 alkyl; and R^5a is methyl. In certain embodiments of a compound of Formula (I), each of R^la and R^2a is hydrogen; each of R^3a and R^4a is Ci-₆ alkyl; and R^5a is methyl.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is chosen from hydrogen and C₁-4 alkyl; R^3a is hydrogen; R^4a is chosen from C1-4 alkyl, substituted C1-4 alkyl wherein the substituent group is chosen from =0, - OR^{l la}, -COOR^{l la}, and -NR^{l la} ₂, wherein each R^{l la} is independently chosen form hydrogen and Ci-4 alkyl; and R^5a is methyl. In certain embodiments of a compound of Formula (I), one of R^la and R^2a is hydrogen and the other of R^la and R^2a is methyl; R^3a is hydrogen; R^4a is chosen from Ci-4 alkyl, substituted C₁-4 alkyl wherein the substituent group is chosen from =0, -0R^{l la}, - COOR^{l la}, and

-NR^{l la} ₂, wherein each R^{l la} is independently chosen form hydrogen and C₁-4 alkyl; and R^5a is methyl. In certain embodiments of a compound of Formula (I), each of R^la and R^2a is hydrogen; R^3a is hydrogen; R^4a is chosen from C_1-4 alkyl, substituted C1-4 alkyl wherein the substituent group is chosen from =0, -0R^{l la}, -COOR^lla, and -NR^{l la} ₂, wherein each R^{l la} is independently chosen form hydrogen and C_1-4 alkyl; and R^5a is methyl.

In certain embodiments of a compound of Formula (I), R^3a and R^4a together with the nitrogen to which they are bonded form a Cs_-10 heterocycloalkyl ring.

In certain embodiments of a compound of Formula (I), one 0 f R^la and R^2a is hydro gen and the other of R^la and R^2a is chosen from hydrogen and Ci_6 alkyl; R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring; and R^5a is methyl. In certain embodiments of a compound of Formula (I), one of R^la and R^2a is hydrogen and the other of R^la and R^2a is methyl; R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring; and R^5a is methyl. In certain embodiments of a compound of Formula (I), each of R^la and R^2a is hydrogen; R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a C5-6 heterocycloalkyl, substituted C5-6 heterocycloalkyl, C5-6 heteroaryl, and substituted C5-6 heteroaryl ring; and R^5a is methyl.

In certain embodiments of a compound of Formula (I), one of R^la and R^2a is hydrogen and the other of R^la and R^2a is chosen from hydrogen and Ci_6 alkyl; and R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from morpholine, piperazine, and N-substituted piperazine.

In certain embodiments of a compound of Formula (I), one of R^la and R^2a is hydrogen and the other of R^la and R^2a is chosen from hydrogen and Ci_6 alkyl; R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from morpholine, piperazine, and N- substituted piperazine; and R^5a is methyl.

In certain embodiments of a compound of Formula (I), R^5a is not methyl.

In certain embodiments of a compound of Formula (I), R^la is hydrogen, and in certain embodiments, R^2ais hydrogen.

In certain embodiments of a compound of Formula (I), the compound is chosen from: ( ,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene-l ,4-dioate; methyl[N- benzylcarbamoyl]methyl(2E)but-2-ene- 1 ,4-dioate; methyl 2-morpholin-4-yl-2-oxoethyl(2E)but- 2-ene-l,4-dioate; ( -butylcarbamoyl)methyl methyl(2E)but-2-ene-l ,4-dioate; [N-(2- methoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-l ,4-dioate; 2-{2-[(2E)-3- (methoxycarbonyl)prop-2-enoyloxy] acetylamino } acetic acid; 4- { 2- [(2E)-3 - (methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid; methyl(N-(l ,3,4-thiadiazol-2- yl)carbamoyl)methyl(2E)but-2ene-l ,4-dioate; ( ,N-dimethylcarbamoyl)methyl methyl(2E)but- 2-ene-l,4-dioate; ( -methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-l ,4-dioate; bis-(2-methoxyethylamino)carbamoyl]methyl methyl(2E)but-2-ene- 1 ,4-dioate; [N- (methoxycarbonyl)carbamoyl]methyl methyl(2E)but-2ene-l,4-dioate; 4-{2-[(2E)-3- (methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid, sodium salt; methyl 2-oxo-2- piperazinylethyl(2E)but-2-ene-l ,4-dioate; methyl 2-oxo-2-(2-oxo(l ,3-oxazolidin-3- yl)ethyl(2E)but-2ene-l ,4-dioate; {N-[2-(dimethylamino)ethyl]carbamoyl}methyl

methyl(2E)but-2ene-l,4 dioate; methyl 2-(4-methylpiperazinyl)-2-oxoethyl(2E)but-2-ene-l,4- dioate; methyl {N-[(propylamino)carbonyl]carbamoyl}methyl(2E)but-2ene-l ,4-dioate; 2-(4- acetylpiperazinyl)-2-oxoethyl methyl(2E)but-2ene- 1 ,4-dioate; {N,N-bis[2- (methylethoxy)ethyl]carbamoyl}methyl methyl(2E)but-2-ene-l,4-dioate; methyl 2-(4- benzylpiperazinyl)-2-oxoethyl(2E)but-2-ene- 1 ,4-dioate; [N,N-bis(2- ethoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-l,4-dioate; 2-{(2S)-2-[(tert- butyl)oxycarbonyl]pyrrolidinyl} -2-oxoethyl methyl(2E)but-2ene-l ,4-dioate; 1-{2-{(2Ε)-3- (methoxycarbonyl)prop-2-enoyloxy]acetyl} (2S)pyrrolidine-2-carboxylic acid; (N- { [tert- butyl)oxycarbonyl]methyl} -N-methylcarbamoyl)methyl methyl(2E)but-2ene 1 ,4-dioate; {N- (ethoxycarbonyl)methyl]-N-methylcarbamoyl} methyl methyl(2E)but-2-ene-l ,4-dioate; methyl 1 -methyl-2-morpholin-4-yl-2-oxoethyl(2E)but-2-ene-l ,4-dioate; [N,N-bis(2- methoxyethyl)carbamoyl] ethyl methyl(2E)but-2-ene-l,4-dioate;

( ,N-dimethylcarbamoyl)ethyl methyl(2E)but-2-ene-l ,4-dioate; 2-{2-[(2E)-3-(methoxy carbonyl)prop-2-enoyloxyl]-N-methylacetylamino} acetic acid; (N- { [(tert- butyl)oxycarbonyl]methyl} carbamoyl)methyl methyl(2E)but-2-ene- 1 ,4-dioate; (2E)but-methyl- N- { [(methylethyl)oxycarbonyl]methyl} carbamoyl)methyl(2E)but-2-ene- 1 ,4-dioate; {N- [(ethoxycarbonyl)methyl]-N-benzylcarbamoyl}methyl methyl(2E)but-2-ene-l ,4-dioate; {N- [(ethoxycarbonyl)methyl]-N-benzylcarbamoyl} ethyl methyl(2E)but-2-ene-l ,4-dioate;

{N-[(ethoxycarbonyl)methyl]-N-methylcarbamoyl} ethyl methyl(2E)but-2-ene-l ,4-dioate; (1S)- l-methyl-2-morpholin-4-yl-2-oxo ethyl methyl(2E)but-2-ene-l,4-dioate; (lS)-l -[N,N-bis(2- methoxyethyl)carbamoyl] ethyl methyl(2E)but-2-ene- 1 ,4-dioate; ( 1 R)- 1 -(N,N- diethylcarbamoyl)ethyl methyl(2E)but-2-ene-l,4-dioate; and a phannaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (I), the compound is chosen from: ( ,N-diethylcarbamoyl)methyl methyl(2E)but-2-ene-l ,4-dioate; methyl[N- benzylcarbamoyl]methyl(2E)but-2-ene- 1 ,4-dioate; methyl 2-morpholin-4-yl-2-oxoethyl(2E)but- 2-ene-l,4-dioate; ( -butylcarbamoyl)methyl methyl(2E)but-2-ene-l ,4-dioate; [N-(2- methoxyethyl)carbamoyl]methyl methyl(2E)but-2-ene-l ,4-dioate; 2-{2-[(2E)-3- (methoxycarbonyl)prop-2-enoyloxy]acetylamino} acetic acid; {2-[(2E)-3- (methoxycarbonyl)prop-2-enoyloxy]acetylamino}butanoic acid; methyl( -(l ,3,4-thiadiazol-2- yl)carbamoyl)methyl(2E)but-2ene-l ,4-dioate; ( ,N-dimethylcarbamoyl)methyl methyl(2E)but- 2-ene-l,4-dioate; ( -methoxy-N-methylcarbamoyl)methyl methyl(2E)but-2-ene-l,4-dioate; bis- (2-methoxyethylamino)carbamoyl]methyl methyl(2E)but-2-ene-l ,4-dioate; [N- (methoxycarbonyl)carbamoyl]methyl methyl(2E)but-2ene-l,4-dioate; methyl 2-oxo-2- piperazinylethyl(2E)but-2-ene-l ,4-dioate; methyl 2-oxo-2-(2-oxo(l ,3-oxazolidin-3- yl)ethyl(2E)but-2ene-l ,4-dioate; {N-[2-(dimethylamino)ethyl]carbamoyl}methyl

methyl(2E)but-2ene-l ,4-dioate; ( -[(methoxycarbonyl)ethyl]carbamoyl)methyl methyl(2E)but- 2-ene-l,4-dioate; 2-{2-[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]acetylamino}propanoic acid; and a pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (I), R^3a and R^4a are independently chosen from hydrogen, Ci_6 alkyl, substituted Ci_6 alkyl, C₆-io aryl, substituted C₆-io aryl, C_4-12 cycloalkylalkyl, substituted C_4-12 cycloalkylalkyl, C_7-12 arylalkyl, substituted C_7-12 arylalkyl, Ci-₆ heteroalkyl, substituted Ci_6 heteroalkyl, C₆-io heteroaryl, substituted C₆-io heteroaryl, C_4-12 heterocycloalkylalkyl, substituted C4-12 heterocycloalkylalkyl, C_7-12 heteroarylalkyl, substituted C7-12 heteroarylalkyl; or R^3a and R^4a together with the nitrogen to which they are bonded form a ring chosen from a Cs_-10 heteroaryl, substituted Cs_-10 heteroaryl, Cs_-10 heterocycloalkyl, and substituted Cs_-10 heterocycloalkyl.

In some embodiments, the compound that metabolizes to MMF is a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein

R^6b is chosen from Ci_6 alkyl, substituted Ci_6 alkyl, Ci_6 heteroalkyl, substituted Ci_6 heteroalkyl, C₃_8 cycloalkyl, substituted C₃_8 cycloalkyl, C₆-8 aryl, substituted C₆-8 aryl, and -OR^10b wherein R^10b is chosen from Ci_6 alkyl, substituted Ci_6 alkyl, C₃_io cycloalkyl, substituted C₃-io cycloalkyl, C₆-io aryl, and substituted C₆-io aryl;

R^7b and R^8b are independently chosen from hydrogen, Ci_6 alkyl, and substituted Ci_6 alkyl; and

R^9b is chosen from Ci-₆ alkyl and substituted Ci-₆ alkyl;

wherein each substituent group is independently chosen from halogen, -OH, -CN, -CF₃, =0, -NO2, benzyl, -C(0)NR^llb ₂, -R^{l lb}, -0R^{l lb}, -C(0)R^llb, -COOR^llb, and -NR^{l lb} ₂ wherein each R^llb is independently chosen from hydrogen and C_1-4 alkyl.

In certain embodiments of a compound of Formula (II), each substituent group is independently chosen from halogen, -OH, -CN, -CF₃, -R^{l lb}, -0R^{l lb}, and -NR^llb ₂ wherein each

R^{l lb} is independently chosen from hydrogen and C_1-4 alkyl.

In certain embodiments of a compound of Formula (I), each substituent group is independently chosen from =0, C_1-4 alkyl, and -COOR^llb wherein R^{l lb} is chosen from hydrogen and Ci-4 alkyl.

In certain embodiments of a compound of Formula (II), one of R^7b and R^8b is hydrogen and the other of R^7b and R^8b is Ci_6 alkyl. In certain embodiments of a compound of Formula (II), one of R^7b and R^8b is hydrogen and the other of R⁷¹¹ and R^8b is C_1-4 alkyl.

In certain embodiments of a compound of Formula (II), one of R^7b and R^8b is hydrogen and the other of R^7b and R^8b is chosen from methyl, ethyl, n-propyl, and isopropyl. In certain

embodiments of a compound of Formula (II), each of R^7b and R^8b is hydrogen.

In certain embodiments of a compound of Formula (II), R^9b is chosen from substituted Ci-6 alkyl and -0R^{l lb} wherein R^llb is independently C_1-4 alkyl.

In certain embodiments of a compound of Formula (II), R^9b is Ci_6 alkyl, in certain embodiments, R^9b is C_1-3 alkyl; and in certain embodiments, R^9b is chosen from methyl and ethyl.

In certain embodiments of a compound of Formula (II), R^9b is methyl. In certain embodiments of a compound of Formula (II), R is chosen from ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and tert-butyl.

In certain embodiments of a compound of Formula (II), R^9b is chosen from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

In certain embodiments of a compound of Formula (II), R^6b is C_1-6 alkyl; one of R^7b and R^8b is hydrogen and the other of R^7b and R^8b is Ci_6 alkyl; and R^9b is chosen from Ci_6 alkyl and substituted Ci_6 alkyl.

In certain embodiments of a compound of Formula (II), R^6b is -OR^10b.

In certain embodiments of a compound of Formula (II), R^10b is chosen from

Ci-4 alkyl, cyclohexyl, and phenyl.

In certain embodiments of a compound of Formula (II), R^6b is chosen from methyl, ethyl, n-propyl, and isopropyl; one of R^7b and R^8b is hydrogen and the other of R^7b and R^8b is chosen from methyl, ethyl, n-propyl, and isopropyl.

In certain embodiments of a compound of Formula (II), R^6b is substituted Ci_₂ alkyl, wherein each of the one or more substituent groups are chosen from -COOH,

-NHC(0)CH₂NH₂, and -NH₂.

In certain embodiments of a compound of Formula (II), R^6b is chosen from ethoxy, methylethoxy, isopropyl, phenyl, cyclohexyl, cyclohexyloxy,

-CH( H₂CH₂COOH, -CH₂CH( H₂)COOH, -CH( HC(0)CH₂NH₂)-CH₂COOH, and - CH₂CH( HC(0)CH₂NH₂)-COOH.

In certain embodiments of a compound of Formula (II), R^9b is chosen from methyl and ethyl; one of R^7b and R^8b is hydrogen and the other of R^7b and R^8b is chosen from hydrogen, methyl, ethyl, n-propyl, and isopropyl; and is chosen from C1-3 alkyl, substituted Ci_₂ alkyl wherein each of the one or more substituent groups are chosen -COOH, -NHC(0)CH₂NH₂, and - NH₂, -OR^10b wherein R^10b is chosen from C1-3 alkyl and cyclohexyl, phenyl, and cyclohexyl. In certain embodiments of a compound of Formula (II), the compound is chosen from:

ethoxycarbonyloxyethyl methyl(2E)but-2-ene-l ,4-dioate;

methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene- 1 ,4-dioate;

(cyclohexyloxycarbonyloxy)ethyl methyl(2E)but-2-ene-l,4-dioate; and a pharmaceutically acceptable salt of any of the foregoing. In certain embodiments of a compound of Formula (II), the compound is chosen from: methyl(2-methylpropanoyloxy)ethyl(2E)but-2-ene- 1 ,4-dioate; methyl

phenylcarbonyloxyethyl(2E)but-2-ene-l ,4-dioate; cyclohexylcarbonyloxybutyl methyl(2E)but-2- ene-1 ,4-dioate; [(2E)-3-(methoxycarbonyl)prop-2-enoyloxy] ethyl methyl(2E)but-2-ene-l ,4- dioate; methyl 2-methyl-l-phenylcarbonyloxypropyl(2E)but-2-ene-l,4-dioate; and a

pharmaceutically acceptable salt of any of the foregoing.

In certain embodiments of a compound of Formula (II), the compound is chosen from: ethoxycarbonyloxyethyl methyl(2E)but-2-ene-l ,4-dioate;

methyl(methylethoxycarbonyloxy)ethyl(2E)but-2-ene- 1 ,4-dioate; methyl(2- methylpropanoyloxy)ethyl(2E)but-2-ene-l ,4-dioate; methyl phenylcarbonyloxyethyl(2E)but-2- ene-l ,4-dioate; cyclohexylcarbonyloxybutyl methyl(2E)but-2-ene-l,4-dioate; [(2E)-3- (methoxycarbonyl)prop-2-enoyloxy]ethyl methyl(2E)but-2-ene-l ,4-dioate;

(cyclohexyloxycarbonyloxy)ethyl methyl(2E)but-2-ene-l,4-dioate; methyl 2-methyl-l- phenylcarbonyloxypropyl(2E)but-2-ene-l ,4-dioate; 3-({[(2E)-3-(methoxycarbonyl)prop-2- enoyloxy]methyl}oxycarbonyl)(3S)-3-aminopropanoic acid, 2,2,2-trifluoroacetic acid; 3-({[(2E)- 3-(methoxycarbonyl)prop-2-enoyloxy]methyl} oxycarbonyl)(2S)-2-aminopropanoic acid, 2,2,2- trifluoroacetic acid; 3-({[(2E)-3-(methoxycarbonyl)prop-2-enoyloxy]methyl}oxycarbonyl)(3S)- 3-(2- -aminoacetylamino)propanoic acid, 2,2,2-trifluoroacetic acid; 3-({[(2E)-3- (methoxycarbonyl)prop-2-enoyloxy]methyl} oxycarbonyl)(2S)-2-aminopropanoic acid, 2,2,2- trifluoroacetic acid; 3-{[(2E)-3-(methoxycarbonyl)prop-2enoyloxy]ethoxycarbonyloxy}(2S)-2- aminopropanoic acid, chloride; and a pharmaceutically acceptable salt of any of the foregoing. The compounds of Formulae (I)-(II) may be prepared using methods known to those skilled in the art, or the methods disclosed in U.S. Pat. No. 8,148,414 B2.

In another embodiment is provided silicon-containing compounds, which like DMF and the compounds of Formulae (I)-(II), can metabolize into MMF upon administration.

In some embodiments, the compound that metabolizes to MMF is a compound of Formula (III):

(III) or a pharmaceutically acceptable salt thereof, wherein:

R^2c is Ci-Cio alkyl, C5-C15 aryl, hydroxyl, -O-Ci-Cio alkyl, or -O-Cs-Cis aryl; each of R^3c, R^4c, and R^5c, independently, is C1-C10 alkyl, C5-C15 aryl, hydroxyl, -

O-Ci-Cio alkyl, -O-C5-C15 aryl, or

wherein R^lc is C1-C24 alkyl or C5-C50 aryl; each of which can be optionally substituted; and

each of m, n, and r, independently, is 0-4;

provided that at least one of R^3c, R^4c, and R^5c is

Another group of compounds of Formula III include compounds wherein R^lc is optionally substituted C1-C24 alkyl. Another group of compounds of Formula III include compounds wherein R^lc is optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula III include compounds wherein R^lc is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula III include compounds wherein R^lc is optionally substituted C5- C50 aryl. Another group of compounds of Formula III include compounds wherein R^lc is optionally substituted C5-C10 aryl. Another group of compounds of Formula III include compounds wherein R^2c is C1-C10 alkyl. Another group of compounds of Formula III include compounds wherein R^2c is optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula III include compounds wherein R^2c is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula III include compounds wherein R^2c is optionally substituted C5-C15 aryl. Another group of compounds of Formula III include compounds wherein R^2c is optionally substituted C5-C10 aryl.

In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein

R^2c is Ci-Cio alkyl, C₆-Cio aryl, hydroxyl, -O-Ci-Cio alkyl, or -O-C6-C10 aryl;

each of R^3c, R^4c, and R^5c, independently, is C1-C10 alkyl, C₆-Cio aryl, hydroxyl,

-O-Ci-Cio alkyl, -O-C₆-Ci₀ aryl, or

wherein R^lc is C1-C24 alkyl or C₆-Cio aryl; each of which can be optionally substituted; and

each of m, n, and r, independently, is 0-4;

provided that at least one of R^3c, R^4c, and R^5c is

In some embodiments, the compound that metabolizes to MMF is chosen from

(dimethylsilanediyl)dimethyl difumarate; methyl ((trimethoxysilyl)methyl) fomarate; methyl ((trihydroxysilyl)methyl) fumarate; trimethyl (methylsilanetriyl) trifumarate; and a

pharmaceutically acceptable salt of any of the foregoing.

In some embodiments, the compound that metabolizes to MMF is a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

each R^ld is independently optionally substituted C1-C24 alkyl or C5-C50 aryl; each of, independently, R and R , is Ci-Cio alkyl or C5-C15 aryl.

R and R^JC can be the same or different, can be optionally substituted, and independently can be selected from the group consisting of C1-C10 alkyl or C5-C15 aryl.

In another embodiment, compounds of Formula IV include compounds wherein each R^ld is independently optionally substituted C1-C24 alkyl or C₆-Cio aryl. In another embodiment, compounds of Formula IV include compounds wherein R^ldis optionally substituted C1-C24 alkyl. Another group of compounds of Formula IV include compounds wherein R^ld is optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula IV include compounds wherein R^ld is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula IV include compounds wherein R^ld is optionally substituted C5-C50 aryl. Another group of compounds of Formula IV include compounds wherein R^ld is optionally substituted C5- C10 aryl. Another group of compounds of Formula IV include compounds wherein each of R^2d and R^3d is, independently, optionally substituted C1-C10 alkyl. Another group of compounds of Formula IV include compounds wherein each of R^2d and R^3d is, independently, optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula IV include compounds wherein each of R^2d and R^3d is, independently, optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula IV include compounds wherein each of R^2d and R^3d is, independently, optionally substituted C5-C15 aryl. Another group of compounds of Formula IV include compounds wherein each of R^2d and R^3d is, independently, optionally substituted C5-C10 aryl.

In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R^ld is C1-C24 alkyl or C₆-Cio aryl; and

each of, independently, R^2d and R^3d, is C1-C10 alkyl or C In some embodiments, the compound that metabolizes to MMF is a compound of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein:

R^le is Ci-C₂₄ alkyl or C5-C50 aryl;

each of R^2e, R^3e, and R^5e, independently, is hydroxyl, C1-C10 alkyl, C5-C15 aryl, -O-Ci-Cio alkyl, or -O-Cs-Cis aryl; and

n is 1 or 2.

In another embodiment, compounds of Formula V include compounds wherein R^le is optionally substituted C1-C24 alkyl. Another group of compounds of Formula V include compounds wherein R^le is optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula V include compounds wherein R^le is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula V include compounds wherein R^le is optionally substituted C5-C50 aryl. Another group of compounds of Formula V include compounds wherein R^le is optionally substituted C5-C10 aryl. Another group of compounds of Formula V include compounds wherein each of R^2e, R^3e, and R^5e is, independently, hydroxyl. Another group of compounds of Formula V include compounds wherein each of R^2e, R^3e, and R^5e is,

independently, optionally substituted C1-C10 alkyl. Another group of compounds of Formula V include compounds wherein each of R^2e, R^3e, and R^5e is, independently, optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula V include compounds wherein each of R^2e, R^3e, and R^5e is, independently, optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula V include compounds wherein each of R^2e, R^3e, and R^5e is, independently, optionally substituted C5-C15 aryl. Another group of compounds of Formula V include compounds wherein each of R^2e, R^3e, and R^5e is, independently, optionally substituted C5-C10 aryl.

In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein:

R^le is Ci-C₂₄ alkyl or C₆-Ci₀ aryl;

each of R^2e, R^3e, and R^5e, independently, is hydroxyl, C1-C10 alkyl, C6-Cio aryl, -O-Ci-Cio alkyl, or -0-C6-Cio aryl; and

n is 1 or 2.

In some embodiments, the compound that metabolizes to MMF is a compound of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

R^lf is C1-C24 alkyl or C5-C50 aryl; and

R^2f is C1-C10 alkyl.

In another embodiment, compounds of Formula VI include compounds wherein R^lf is optionally substituted C1-C24 alkyl. Another group of compounds of Formula VI include

If

compounds wherein R is optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula VI include compounds wherein R^lf is optionally substituted methyl, ethyl, or isopropyl. Another group of compounds of Formula VI include compounds wherein R^lf is optionally substituted C5-C50 aryl. Another group of compounds of Formula VI include compounds

If

wherein R is optionally substituted C5-C10 aryl. Another group of compounds of Formula VI include compounds wherein R is optionally substituted Ci-C₆ alkyl. Another group of compounds of Formula VI include compounds wherein R is optionally substituted methyl, ethyl, or isopropyl.

In a further embodiment, the compound that metabolizes to MMF is a compound of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

If

R is C1-C24 alkyl or C₆-Cio aryl; and

R^2f is C1-C10 alkyl.

In an embodiment, the dialkyl fumarate is:

Formula A

wherein R^1§ and R^2§, which may be the same or different, independently represent a linear, branched or cyclic, saturated or unsaturated C_1-20 alkyl radical which may be optionally substituted with halogen (CI, F, I, Br), hydroxy, Ci-₄ alkoxy, nitro or cyano.

In an embodiment, R^1§ and R^2§, which may be the same or different, independently are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, pentyl, cyclopentyl, 2 -ethyl hexyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, vinyl, allyl, 2-hydroxy ethyl, 2 or 3-hydroxy propyl, 2-methoxy ethyl, methoxy methyl or 2- or 3-methoxy propyl.

In an embodiment, R^1§ and R^2§ are identical and are methyl or ethyl.

In an embodiment, R^1§ and R^2§ are methyl. In an embodiment, the compound is a monoalkyl fumarate. In an embodiment, the monoalkyl fumarate is:

Formula B

or a pharmaceutically acceptable salt thereof.

In an embodiment, R^lh is methyl or ethyl.

In an embodiment, R^lh is methyl.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the

accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

FIGURE 1 depicts exemplary DMF or MMF augmentation of NK cell lysis of K562 tumor target cells. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and then incubated with K562 cells in the 4 h NK cell assay. Mean ± SEM of 4 or 5 experiments done on different donors. E:T target cell ratio shown is 10: 1. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). Cytotoxicity assay was done as described in A and B. P values comparing the percent of cytotoxicity in the presence of the drugs vs. their absence (Control=C), are placed on top of columns.

FIGURE 2 depicts exemplary DMF or MMF augmentation of NK cell lysis of RAJI tumor target cells. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and then incubated with K562 cells in the 4 h NK cell assay. Mean ± SEM of 4 or 5 experiments done on different donors. E:T target cell ratio shown is 10:1. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). Cytotoxicity assay was done as described in A and B. P values comparing the percent of cytotoxicity in the presence of the drugs vs. their absence (Control=C), are placed on top of columns.

FIGURE 3 depicts exemplary up-regulation of the expression of NKp30 on the surface of NK cells by DMF or MMF. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and the expression of NKp30 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). The cells were washed and the expression of NKp30 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. P values comparing the percent of positive cells in the presence of the drugs vs. their absence

(Control=C), are placed on top of columns. Shown are percentages of positive cells expressing the particular marker. A similar pattern was observed when mean fluorescence intensity (MFI) was examined (not shown). FIGURE 4 depicts exemplary up-regulation of the expression of Kp46 on the surface of NK cells by DMF or MMF. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and the expression of NKp46 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). The cells were washed and the expression of NKp46 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. P values comparing the percent of positive cells in the presence of the drugs vs. their absence

(Control=C), are placed on top of columns. Shown are percentages of positive cells expressing the particular marker. A similar pattern was observed when mean fluorescence intensity (MFI) was examined (not shown).

FIGURE 5 depicts exemplary up-regulation of the expression of CD 107a on the surface of NK cells by DMF or MMF. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and the expression of CD 107a on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). The cells were washed and the expression of CD 107a on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. P values comparing the percent of positive cells in the presence of the drugs vs. their absence

FIGURE 6 depicts exemplary increase of the release of Granzyme B from NK cells by MMF. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. Supernatants were collected and the levels of Granzyme B were measured. Mean ± SEM of 4 experiments done from different donors. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). Supernatants were collected and the levels of Granzyme B were measured. Mean ± SEM of 4 experiments done from different donors. P values comparing the percent of positive cells in the presence of the drugs vs. their absence (Control=C), are placed on top of columns.

FIGURE 7 depicts exemplary anti-NKP46 inhibition of MMF-induced cytotoxicity, Granzyme B release and CD 107a expression in CD56 NK cells. CD56 NK cells were incubated with 10 μg/mL anti-NKP46, anti-NKp30 or isotype control antibody for 30 min at 37°C (similar results were observed after incubation at 4°C; not shown). The cells were either left intact (Control=C), or incubated with 100 μΜ MMF, washed and the expression of NKp46 molecule was determined by binding the cells to PE-conjugated mouse anti-human NKp46 (A). CD56⁺ NK cells were either not incubated with MMF (Control=C) but were incubated with 10 μg/mL of isotype IgG control forNKp46, isotype IgG control forNKp30, anti-NKp46 or anti-NKp46, or were incubated with the same antibodies in the presence of 100 μΜ MMF for 24 h. All preparations were washed and then incubated with K562 cells at 10: 1 E:T cell ratio in the 4 h NK cytotoxicity assay (B). CD56 NK cells were either not incubated with MMF (Control=C) but were incubated with 10 μg/mL of isotype IgG control for NKp46, isotype IgG control for NKp30, anti-NKp46 or anti-NKp46, or were incubated with the same antibodies in the presence of 100 μΜ MMF for 24 h. All preparations were washed and then incubated with RAJI cells at 10:1 E:T cell ratio in the 4 h NK cytotoxicity assay (C). The cells were treated as in panels B and C, but the supernatants were collected after 24 h, and examined for the levels of Granzyme B (D). Similar treatments were performed in panel (E), and the expression of CD 107a was determined by flow cytometric analysis 24 h post-incubation. P values are shown on top of panels.

FIGURE 8 depicts exemplary expression of NKp44 on the surface of NK cells treated with DMF or MMF. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and the expression of NKp44 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). The cells were washed and the expression of NKp44 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. Shown are percentages of positive cells expressing the particular marker. A similar pattern was observed when mean fluorescence intensity (MFI) was examined (not shown). FIGURE 9 depicts exemplary expression of NKG2D on the surface of NK cells. CD56 NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and the expression of NKG2D on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. CD56 NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). The cells were washed and the expression of NKG2D on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. Shown are percentages of positive cells expressing the particular marker. A similar pattern was observed when mean fluorescence intensity (MFI) was examined (not shown).

FIGURE 10 depicts exemplary expression of KIR CD 158 on the surface of NK cells. CD56⁺ NK cells were treated for 4 h (A) or 24 h (B) with media (Control=C) or 100, 10, 1 and 0.1 μΜ of DMF or MMF. The cells were washed and the expression of CD 158 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. CD56^" NK cells were also incubated with various concentrations of DMF or MMF for 4 h (C) or 24 h (D). The cells were washed and the expression of CD 158 on the surface of these cells was detected by flow cytometric analysis. Mean ± SEM of 4 or 5 experiments done on different donors. Shown are percentages of positive cells expressing the particular marker. A similar pattern was observed when mean fluorescence intensity (MFI) was examined (not shown).

DETAILED DESCRIPTION

Definitions

As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

"About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. "Acquire" or "acquiring" as the terms are used herein, refer to obtaining possession of a physical entity, or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a physical process (e.g., performing a synthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a

substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non covalent bond, between a first and a second atom of the reagent.

"Acquiring a sample" as the term is used herein, refers to obtaining possession of a sample, e.g., a tissue sample or nucleic acid sample, by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample). Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starting material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient. Exemplary changes include making a physical entity from a starting material, dissecting or scraping a tissue; separating or purifying a substance (e.g., a sample tissue or a nucleic acid sample); combining two or more separate entities into a mixture; performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a sample includes performing a process that includes a physical change in a sample or another substance, e.g., as described above.

The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

"Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result.

As used herein, the term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. In another example, a cell naturally present in a living animal is not "isolated," but the same cell partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated cell can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell preparation.

As used herein, the term "prodrug" or "pro-drug," refers to a compound that is processed, in the body of a subject, into a drug. In an embodiment the processing comprises the breaking or formation of a bond, e.g. , a covalent bond. Typically, breakage of a covalent bond releases the drug.

As used herein, the term "metabolite" or "active metabolite" refers to a biologically active compound that results from the processing of a prodrug, e.g., in the body of a subject, into a drug. In an embodiment the processing comprises the breaking or formation of a bond, e.g., a covalent bond. Typically, breakage of a covalent bond releases the drug.

"Sample," "tissue sample," "subject or patient sample," "subject or patient cell or tissue sample" or "specimen" each refers to a biological sample obtained from a tissue, e.g., a bodily fluid, of a subject or patient. The source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents (e.g., serum, plasma); bodily fluids such as cerebral spinal fluid, whole blood, plasma and serum. The sample can include a non-cellular fraction (e.g., plasma, serum, or other non-cellular body fluid). In one embodiment, the sample is a serum sample. In other embodiments, the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood). In certain embodiments, the blood can be further processed to obtain plasma or serum. In another

embodiment, the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)). In an embodiment the sample includes NK cells. For example, the sample can be a fine needle biopsy sample, an archival sample (e.g., an archived sample with a known diagnosis and/or treatment history), a histological section (e.g., a frozen or formalin-fixed section, e.g., after long term storage), among others. The term sample includes any material obtained and/or derived from a biological sample, including a polypeptide, and nucleic acid (e.g., genomic DNA, cDNA, R A) purified or processed from the sample. Purification and/or processing of the sample can involve one or more of extraction, concentration, antibody isolation, sorting, concentration, fixation, addition of reagents and the like. The sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.

The term "alkyl" as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 24 carbons. Alkyl groups include straight-chained and branched C1-C24 alkyl groups, e.g., C1-C10 alkyl groups. C1-C10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, 1 -methylhexyl, 2- ethylhexyl, 1 ,4-dimethylpentyl, octyl, nonyl, and decyl. Unless otherwise indicated, all alkyl groups described herein include both unsubstituted and substituted alkyl groups. Further, each alkyl group can include its deuterated counterparts. The term "heteroalkyl" is an alkyl group in which one to five carbons in the alkyl chain are replace by an independently selected oxygen, nitrogen or sulfur atom.

The term "aryl" as employed herein by itself or as part of another group refers to monocyclic, bicyclic, or tricyclic aromatic hydrocarbon containing from 5 to 50 carbons in the ring portion. Aryl groups include Cs-is aryl, e.g., phenyl, p-tolyl,

4-methoxyphenyl, 4-(tert-butoxy)phenyl, 3-methyl-4-methoxyphenyl, 4-fluorophenyl, 4- chlorophenyl, 3-nitrophenyl, 3-aminophenyl, 3-acetamidophenyl,

4-acetamidophenyl, 2-methyl-3-acetamidophenyl, 2-methyl-3-aminophenyl,

3- methyl-4-aminophenyl, 2-amino-3-methylphenyl, 2,4-dimethyl-3-aminophenyl,

4- hydroxyphenyl, 3-methyl-4-hydroxyphenyl, 1-naphthyl, 3-amino-naphthyl,

2-methyl-3-amino-naphthyl, 6-amino-2-naphthyl, 4,6-dimethoxy-2-naphthyl, indanyl, biphenyl, phenanthryl, anthryl, and acenaphthyl. Unless otherwise indicated, all aryl groups described herein include both unsubstituted and substituted aryl groups.

The term "arylalkyl" refers to an alkyl group which is attached to another moiety through an alkyl group.

"Halogen" or '¾alo" may be fiuoro, chloro, bromo or iodo.

The term "cycloalkyl" refers to completely saturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms, preferably 3-9, or more preferably 3-8 carbon atoms. Exemplary monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Exemplary bicyclic cycloalkyl groups include bornyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, 6,6-dimethylbicyclo[3.1.l ]heptyl, 2,6,6-trimethylbicyclo[3.1.1]heptyl, or bicyclo[2.2.2]octyl. Exemplary tricyclic carbocyclyl groups include adamantyl.

The term "cycloalkylalkyl" refers to a cycloalkyl group which is attached to another moiety through an alkyl group.

The term "heterocycloalkyl" refers to completely saturated monocyclic, bicyclic or tricyclic heterocyclyl comprising 3-15 ring members, at least one of which is a heteroatom, and up to 10 of which may be heteroatoms, wherein the heteroatoms are independently selected from O, S and N, and wherein N and S can be optionally oxidized to various oxidation states.

Examples of heterocycloalkyl groups include [l,3]dioxolane, 1,4-dioxane, 1,4-dithiane, piperazinyl, 1,3-dioxolane, imidazolidinyl, imidazolinyl, pyrrolidine, dihydropyran, oxathiolane, dithiolane, 1,3-dioxane, 1 ,3-dithianyl, oxathianyl, thiomorpholinyl, oxiranyl, aziridinyl, oxetanyl, azetidinyl, tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, and piperazinyl.

As used herein, the term 'Tieteroaryl" refers to a 5-14 membered monocyclic-, bicyclic-, or tricyclic-ring system, having 1 to 10 heteroatoms independently selected from N, O or S, wherein N and S can be optionally oxidized to various oxidation states, and wherein at least one ring in the ring system is aromatic. In one embodiment, the heteroaryl is monocyclic and has 5 or 6 ring members. Examples of monocyclic heteroaryl groups include pyridyl, thienyl, furanyl, pyrrolyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl and tetrazolyl. In another embodiment, the heteroaryl is bicyclic and has from 8 to 10 ring members. Examples of bicyclic heteroaryl groups include indolyl, benzofuranyl, quinolyl, isoquinolyl indazolyl, indolinyl, isoindolyl, indolizinyl, benzamidazolyl, quinolinyl, 5,6,7, 8-tetrahydroquinoline and 6,7-dihydro-5H-pyrrolo[3,2-d]pyrimidine.

The term 'Tieteroarylalkyl" refers to an alkyl group which is attached to another moiety through an alkyl group.

Description

Provided herein are compositions and methods for, inter alia, treating cancer in a subject, treating an infectious disease in a subject, and/or generally enhancing cell lysis of a target cell by a natural killer (NK) cell.

Cells

Natural Killer (NK) cells can be obtained from any appropriate source, including from a subject. Examples of subjects include animals, such as a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey); rodent (e.g., rat, mouse, guinea pig); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys, that will be or has been the object of treatment, observation, and/or experiment.

Cells can be isolated from any appropriate sample, including but not limited to, a biological sample obtained from a tissue, e.g., a bodily fluid, of a subject or patient. The source of the tissue sample can be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, or aspirate; blood or any blood constituents (e.g., serum, plasma); bodily fluids such as cerebral spinal fluid, whole blood, plasma and serum. The sample can include a non- cellular fraction (e.g., plasma, serum, or other non-cellular body fluid). In one embodiment, the sample is a serum sample. In other embodiments, the body fluid from which the sample is obtained from an individual comprises blood (e.g., whole blood). In certain embodiments, the blood can be further processed to obtain plasma or serum. In another embodiment, the sample contains a tissue, cells (e.g., peripheral blood mononuclear cells (PBMC)). In an embodiment the sample includes NK cells. For example, the sample can be a fine needle biopsy sample, an archival sample (e.g., an archived sample with a known diagnosis and/or treatment history), a histological section (e.g., a frozen or formalin-fixed section, e.g., after long term storage), among others. The term sample includes any material obtained and/or derived from a biological sample, including a polypeptide, and nucleic acid (e.g., genomic DNA, cDNA, R A) purified or processed from the sample. Purification and/or processing of the sample can involve one or more of extraction, concentration, antibody isolation, sorting, concentration, fixation, addition of reagents and the like. The sample can contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like.

In some embodiments, cells are isolated, e.g., altered or removed from the natural state. For example, a cell naturally present in a living animal is not "isolated," but the same cell partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated cell can exist in substantially purified form, or can exist in a non-native environment such as, for example, in a cell preparation. As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a

homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

NK cell phenotype and activity can be assessed using any appropriate assay, such as those described in the Examples herein. For example, NK killing assays can be used to evaluate the cytolytic activity of an NK cell on a cell line, such as a tumor cell line. Exemplary NK killing assays disclosed in the Examples herein include killing by NK cells of a leukemia cell line and/or a B cell lymphoma cell line.

In vitro and/or ex vivo methods are also contemplated. In vitro and ex vivo procedures are well known in the art. Briefly, NK cells may be isolated from a mammal (e.g., a human) and treated (e.g., activated with a composition described herein). The activated NK cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian

recipient may be a human and the activated NK cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

Treatment

Provided compositions and methods may be used for the treatment of cancer in a subject in need thereof. The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. In some embodiments, a cancer is a hematological malignancy, such as a leukemia, lymphoma, or myeloma. Examples of leukemias include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL). Examples of lymphomas include, but are not limited to, Hodgkin's lymphoma and Non-Hodgkin's lymphoma. A non-limiting example of a myeloma is multiple myeloma. In some embodiments, a cancer is a solid tumor. Examples of solid tumors include, but are not limited to, breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, among others.

Compositions and methods provided herein may be used for treatment of infectious diseases, such as viral infections. Examples of viral infections include, but are not limited to, hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human

papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell

polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), among others.

"Treat," "treatment," and other forms of this word refer to the administration of an agent, e.g., DMF, MMF, a prodrug or active metabolite thereof, alone or in combination with one or more symptom management agents, to a subject, e.g., a cancer patient or a patient with an infectious disease, to impede progression of the cancer, to induce remission, to extend the expected survival time of the subject and or reduce the need for medical interventions (e.g., hospitalizations). In those subjects, treatment can include, but is not limited to, inhibiting or reducing one or more symptoms such as increasing remission rate, reducing relapse rate, reducing size or number of malignant lesions; inhibiting or retarding the development of new malignant lesions; prolonging survival, or prolonging progression-free survival, and/or enhanced quality of life.

As used herein, unless otherwise specified, the terms "prevent," "preventing" and

"prevention" contemplate an action that occurs before a subject begins to suffer from the cancer and/or infectious disease and/or which inhibits or reduces the severity of the disease. In some embodiments, a subject is infected with a virus, e.g., an oncovirus. In an embodiment, a subject infected with a virus is treated with a composition described herein, e.g., DMF or MMF. In an embodiment, a subject infected with a virus is treated to prevent cancer formation in the subject.

As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or

management of cancer and/or infectious disease, or to delay or minimize one or more symptoms associated with cancer and/or infectious disease. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of cancer and/or infectious disease. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease, or enhances the therapeutic efficacy of another therapeutic agent.

As used herein, the term "patient" or "subject" refers to an animal, typically a human (i.e., a male or female of any age group, e.g., a pediatric patient (e.g., infant, child, adolescent) or adult patient (e.g., young adult, middle-aged adult or senior adult) or other mammal, such as a primate (e.g., cynomolgus monkey, rhesus monkey); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys, that will be or has been the object of treatment, observation, and/or experiment. When the term is used in conjunction with administration of a compound or drug, then the patient has been the object of treatment, observation, and/or administration of the compound or drug.

In some embodiments, dialkyl fumarates, e.g., those of Formula A, can be used to treat NK function related disorders and conditions, e.g., with the proviso that such disorders do not include: cancer, e.g., hematopoietic malignancies, e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, and lymphoma; solid tumors, e.g., gastrointestinal sarcoma,

neuroblastoma, and kidney cancer; viral infection; autoimmune disorders; inflammation; and/or transplantation, e.g., solid organ transplantation, and GVHD.

Pharmaceutical Compositions

In certain embodiments, DMF, MMF, or a prodrug or active metabolite thereof (e.g., DMF, MMF, prodrugs, e.g., as shown in Formulas I- VI, and their active metabolites), or pharmaceutically acceptable salt thereof, is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable excipient. In particular embodiments, the

pharmaceutical composition is configured in a unit dosage form. In some embodiments, the pharmaceutical composition is configured in a solid dosage form. In certain embodiments, the solid dosage form is selected from the group consisting of tablets, micro-tablets, pellets, granulates, capsules (e.g., soft or hard gelatin capsules), sachets, powders and lozenges. In a preferred embodiment, preparations are in the form of micro-tablets or pellets, optionally filled in capsules or sachets. The size or mean diameter of the pellets or microtablets can range from 300 to 4000 μιη, e.g., 500 to 3500 μιη, 1000 to 3000 μιη, or 1500 to 2500 μιη. In some embodiments, the size or mean diameter of the pellets or microtablets are about 2000 μηι, e.g., 2000 μιη.

In some embodiments, the pharmaceutical composition is configured in a liquid dosage form.

Pharmaceutically acceptable carriers can be sterile liquids, e.g., water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the oral dosage form is a liquid. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Oral dosage forms may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, surface deposition, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Further techniques for formulation and administration of active ingredients may be found in "Remington's

Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is

incorporated herein by reference as if fully set forth herein. Oral dosage forms for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used

pharmaceutically.

In some embodiments, the pharmaceutical composition is administered via oral, subcutaneous, intravenous, intramuscular, intranasal, transdermal, transmucosal, bucal, sublingual, or lung administration. In particular embodiments, the pharmaceutical composition is administered via oral administration.

In some embodiments, oral preparations are provided with an enteric coating, e.g., to delay the release of the drug from the dosage forms (e.g., as described in US 6,509,376, the contents of which are incorporated herein by reference). Depending on the composition and/or thickness, the enteric coatings are resistant to acidic gastric fluids but are soluble at higher pH in the intestine. Therefore, enteric coated oral dosage forms do not generally release the drug in the acidic gastric fluids where the drug is susceptible to degradation. The enteric coating polymer may be selected from polymers soluble at pH existing in the upper part of the small intestine or in the latter part of the small intestine and accordingly the release of the drug is delayed by a time period required for the dosage form to transit to these parts of the intestine. A variety of enteric coatings are known, including, but not limited to waxes, shellacs, polymers, and plant fibers. Specific enteric coatings include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate, polyvinyl acetate phthalate, methyl methacrylate-methacrylic acid copolymers, sodium alginate, and stearic acid. An enteric coating may be applied to a solid oral dosage form, e.g., a capsule or tablet, using a variety of known techniques, e.g., spray coating or pan coating. In some embodiments, the enteric coating can be: methylcellulose; ethylcellulose

hydroxyethylcellulose; hydroxypropylmethylcellulose (HPMC); sodium carboxymethylcellulose; agar-agar; carob gum; alginates; molasses; polysaccharides of mannose and galactose; chitosan; modified starches; aliphatic poly (esters); poly anhydrides; polyhydroxyethyle methylacrylate (PHEMA); cross-linked polyvinyl alcohol (PVA); cross-linked polyvinyl pyrrolidone (PVP); polyethylene oxide (PEO); polyacrylamide (PA); polyethylene glycol (PEG); polyvinyl alcohol (PVA); polyvinyl pyrrolidone (PVP); hydroxypropyl methyl cellulose (HPMC); polylactic acid (PLA); polyglycolic acid (PGA); polycaprolactone (PCL); polyanhydrides; polyortho esters; polyethylene vinyl acetate (PVA); polydimethyl siloxane (PDS); polyether urethane (PEU); polyvinyl chloride (PVC); cellulose acetate (CA); ethyl cellulose (EC); polycarbophil; sodium carboxymethyl cellulose; polyacrylic acid; tragacanth; methyl cellulose; pectin; xanthan gum; guar gum; and Karaya gum.

The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1). Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject's disposition to the disease, condition or symptoms, and the judgment of the treating physician. For example, in some embodiments, a dosage may range from 10 to 5000 mg of DMF, or molar equivalent thereof, e.g., 50 to 2000 mg of DMF, or molar equivalent thereof, or 100 to 1000 mg of DMF, or molar equivalent thereof. In some embodiments, a dosage may range from 50-2000 mg of DMF, or molar equivalent thereof, per day. In some embodiments, a dosage may range from 240-1000 mg of DMF, or molar equivalent thereof, per day. In some embodiments, a compound, e.g., DMF, MMF, prodrugs or metabolite described herein, is administered in a dose of approximately 240-720 mg/day. In a particular embodiment, a compound, e.g., DMF, MMF, prodrugs or metabolite described herein, is administered in a dose of approximately 480 mg/day or approximately 720 mg/day. In some embodiments, a compound, e.g., DMF, MMF, prodrugs or metabolite described herein, is administered in a dose of approximately 720 mg/day in three equal doses. In some embodiments, a compound, e.g., DMF, MMF, prodrugs or metabolite described herein, is administered in a dose of approximately 480 mg/day in two equal doses. In some embodiments, a dosage may be less than 500 mg, less than 400 mg, less than 300 mg, less than 200 mg or less than 100 mg of a compound, e.g., DMF, MMF, prodrugs or metabolite described herein,, or molar equivalent thereof, per dose. In some embodiments, a dosage may be more than 100 mg, more than 200, more than 300 mg, more than 400 mg, or more than 500 mg a compound, e.g., DMF, MMF, prodrugs or metabolite described herein,, or molar equivalent thereof, per dose. In some embodiments, a pharmaceutical composition is administered daily or multiple times per day, e.g., 1 , 2, 3, 4, 5, 6 or more times per day. In some embodiments, a pharmaceutical composition is administered three times daily. In some embodiment, a pharmaceutical composition is administered twice daily. In some embodiments, a

pharmaceutical composition is administered once daily.

In some embodiments, a dosage is 120 mg. In some embodiments, a dosage is 120 mg twice per day. In some embodiments, a dosage is 240 mg. In some embodiments, a dosage is 240 mg twice per day. In some embodiments, a dosage is 240 mg three times per day.

Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Subjects may, however, require intermittent treatment on a long- term basis upon any recurrence of disease symptoms. In some embodiments, a lower dose is administered to the subject initially, with an increase in the dosage to reach a maintenance dose after a given period of time, e.g., after 1, 2, 3, 4, 5, 6, 7 or more days of administration of the initial dose, or e.g., after 1 , 2, 3, 4, 5, 6 or more weeks of administration of the initial dose. For example, in some embodiments, an initial treatment is given at a dosage is 120 mg twice per day for seven days. In some such embodiments, the dosage may be increased to 240 mg twice per day after the initial seven day treatment.

Combination Therapies

In one embodiment, combination treatment of an individual with cancer and/or viral infection is contemplated. It will be appreciated that the therapies, as described above and herein, can be administered in combination with one or more additional therapies to treat and/or reduce the symptoms of cancer and/or infectious disease. The pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other additional therapies or therapeutic agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutic agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the pharmaceutical composition with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved. In general, it is expected that additional therapeutic agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some

embodiments, the levels utilized in combination will be lower than those utilized individually.

For example, compositions disclosed herein may be administered in combination with a chemotherapeutic agent, radiation treatment, surgical treatment, or other acceptable cancer treatments. Exemplary chemotherapeutic agents include, but are not limited to, aclarubicin, alemtuzumab, amsacrine, asparaginase, azacitidine, busulphan, chlorambucil, cladribine, clofarabine, cytabarine, daunorubicin, doxorubicin, filgrastim, fiudarabine, interferon alpha 2A, mercaptopurine, methotrexate, mitoxantrone, nelarabine, nilotinib, pentostatin, rituximab, teniposide, thioguanine, vincristine, and combinations thereof.

Compositions disclosed herein may be administered with an antiviral agent. Exemplary antiviral agents include, but are not limited to, agents which mimic the virus-associated protein (VAP) to bind to cellular receptors, agents which mimic the cellular receptor and bind to the VAP, agents which inhibit viral entry, agents which inhibit viral uncoating (e.g., amantadine, rimantidine, or pleconaril, among others), agents which inhibit reverse transcription (e.g., acyclovir, zidovudine, lamivudine, among others), agents which target viral integrase, agents which inhibit viral transcription, translation, and/or protein processing (e.g., antisense oligonucleotides, e.g., fomivirsen), agents which inhibit viral assembly (e.g., rifampicin), agents which inhibit viral release (e.g., zanamivir, oseltamivir), agents which stimulate the immune system of the host (e.g., interferons). Exemplary available antiviral drugs include, but are not limited to, abacavir, acyclovir, afefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, balavir, boceprevirertet, cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, efuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I,

lamivudine, lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sofosbuvir, stavudine, tea tree oil, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, traporved, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine, among others.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, figures, sequence listing, patents and published patent applications cited throughout this application are hereby incorporated by reference.

EXAMPLES Example 1: Monomethyl Fumarate Activates NK Cells and Converts resting Non-Cytolytic CD56⁺ NK Cells into Robust Anti-Tumor Effectors Through Degranulation and Up- Regulation of NKp46 and CD107a.

Dimethyl fumarate (DMF) is a drug for treating multiple sclerosis (MS) patients. In the present example, we examined the effects of DMF and its metabolite monomethyl fumarate (MMF) on various activities of natural killer (NK) cells. The present example demonstrates that DMF and MMF induce primary CD56+ NK cell lysis of K562 cells after 4 h, and MMF induces it after 24 h incubation, whereas both molecules enhance CD56- NK cell lysis after 24 h incubation. Surprisingly, DMF and MMF induce CD56+ NK cell lysis of the NK-resistant RAJI cells after 4 and 24 h. DMF induces NKp30 expression on CD56+, and MMF increases it on CD56- NK cells after 4 h. Intriguingly, MMF up-regulates the expression of NKp46 on the surface of CD56+ cells 24 h post-incubation. This effect is closely correlated with the ability of this metabolite to up-regulate the expression of CD 107a on the surface of CD56+ NK cells and to induce the release of Granzyme B from these cells. Anti-NKp46 antibody inhibits MMF- induced up-regulation of CD 107a, Granzyme B release and the ability ofCD56+ NK cells to lyse tumor cells 24 h post-incubation. Taken together, these results show that DMF and MMF convert the non-cytolytic CD56+ NK cells into robust anti-tumor effector cells, an effect mediated by NKp46 for MMF.

Natural Killer (NK) cells are large granular lymphocytes that possess the ability to spontaneously lyse target cells (Wood SM, et al. (201 1) Cell Mol Life Sci 68: 3479-3493).

These cells can be divided into several subsets; the most studied are those that express CD56 known as CD56⁺/^bright cells which are regulatory secreting IFN-γ and other cytokines, but are less cytolytic than CD56^~/dim cells which are highly cytolytic but secrete cytokines with less intensity than the former cells. Phenotypically, CD56^~/dim cells are killer inhibitory receptors (KIR)⁺, natural cytotoxicity receptors (NCRs)⁺ and perforin⁺, whereas CD56^+/bri8ht cells are KIR^dim, NC^low and perforin^"/low (Chiesa MD et al. (2003) Eur J Immunol 33: 1657-1666 ; Moretta L, et al. (2002) Eur J Immunol 32 : 1205-1211). NK cells also have immunoregulatory features including secretion of cytokines, chemokines and cell to cell cross-talk (Fauriat C et al. (2010) Blood 115: 2167-2176; Moretta L, Ferlazzo G, et al. (2006) Immunol. Rev 214: 219-228), and are important in defending against viral infections as well as controlling tumor growths Maghazachi AA et al. (1998) FASEB J 12: 913-924; Maghazachi AA (2005) Pharmacol Rev 57: 339-357). The activities of NK cells are regulated by activating and inhibitory receptors, which by intracellular integration of challenges and inhibition, determine the cell course of action Moretta A, et al. (2008) Immunol Rev 224: 58-69). They are activated by cells that are in distress through the detection of stress-induced ligands on target cells by NCRs which include NKp46, NKp44, and NKp30, as well as C-type lectin receptors such as NKG2D (Raulet DH, et al. (2013) Annu Rev Immunol 31 : 413-441). In addition, NK cells express several receptors that inhibit activation, including members of the killer-cell immunoglobulin-like receptors (KIRs) family that interact with HLA-I molecules, and CD94-NKG2A that interacts with HLA-E. In the absence of these "self ligands, NK cells are activated and kill target cells (Ljunggren HG et al. (1990) Immunol Today 1 1 : 237-244).

Dimethyl fumarate (DMF) is currently used to treat patients with multiple sclerosis (MS), under the name Tecfidera (Biogen-Idec, CA). This drug was found to be safe when used in 257 MS patients receiving high dose three times daily (Kappos L et al. (2008) Lancet 372: 1463- 1472). DMF mechanism of action is attributed to activating the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an anti-oxidative factor (Linker RA et al. (2008) Expert Rev Neurother 8: 1683-1690; Gold R et al. (2012) Clin Immunol 142: 44-48). Consequently, DMF protects neurons and astrocytes against oxidative stress which induces cellular injury and loss (Scannevin RH et al. (2012) J Pharmacol Exp Ther 341 : 274-284). It is also observed that DMF reduces the nuclear factor NF-κΒ in astrocytes and C6 cells, and protects ΙκΒα by inhibiting its degradation, and reducing nitric oxide synthase 2 (Lin SX et al. (2006) ASN Neuro 3: 75-84).

In the experimental autoimmune encephalomyelitis (EAE) model, DMF exerts clinical effects by reducing macrophage inflammation in the spinal cord (Schilling S et al. (2006) Clin Exp Immunol 145: 101 -107). In addition, DMF inhibits dendritic cells (DCs) maturation by reducing the release of the inflammatory cytokines IL-6 and IL-12 (Peng H et al. (2012) J Biol Chem 287: 28017-28026). Ghoreschi et al. observed that fumarates switch the immune system towards Th2 type of response (Ghoreschi K et al. (201 1) J Exp Med 208: 2291-2303). They observed that DMF suppresses STAT1 phosphorylation in DCs, shuts down DCs type I and generates type II DCs which favor the activation of Th2 cells, while inhibiting cells that express T-bet (Thl cells) or RORyt (Thl7 cells). Interestingly, it was reported that DMF may have anti- tumor effects. In an experimental model of melanoma, Loewe et al. observed that DMF reduces metastases in SCID mice and inhibits the in vitro proliferation of human melanoma cells A375 and M24met (Loewe R et al. (2006) Cancer Res 66: 1 1888-1 1896). DMF metabolite

monomethyl fumurate (MMF) has not been studied in detail.

It has been reported that another drug used for treating MS patients, glatiramer acetate (GA) enhances NK cell killing of K562 cells (Ftoglund RA et al. (2013) PLoS One 8: e62237). Here, we investigated the effects of DMF and MMF on human NK cell lysis of tumor target cells and their ability to release cytolytic molecules. We report that DMF and its derivative MMF activate and induce resting NK cells to lyse K562 and RAJI tumor target cells. We also observed that MMF converts resting CD56⁺ NK cells into cells that lyse RAJI targets through the up- regulation of CD 107a and the release of Granzyme B. These activities are inhibited by anti- NKp46 suggesting that NKp46 mediates the activities of this metabolite.

Materials and Methods

Culture medium

Culture medium contained RPMI 1640 supplemented with 100 U/ mL penicillin, 100 μg/ml streptomycin, 2 mM L-Glutamine, 1% nonessential amino acids, 50 μΜ 2- mercaptoethanol and 10% fetal calf serum (Sigma- Aldrich, St. Lois, MO, USA).

Cell preparations

Peripheral blood cells were collected from blood bank healthy volunteers (Ulleval Hospital, Oslo, Norway). NK cells were isolated using RosetteSep Human NK cell Enrichment Cocktail (StemCell Technologies SARL, Grenoble, France) which removes CD3, CD4, CD19, CD36, CD66b and glycophorin A positive cells. These NK cells were further sorted into CD56⁺ and CD56 cells by magnetic separation with EasySep CD56 positive selection kit (StemCell Technologies SARL). The cells were counted and resuspended to a cell concentration of lxl0⁶/mL. CD56⁺ and CD56^" NK cells were incubated at 37°C with 5% C0₂ at a cell concentration of lxl0⁶/mL with 0.1 μΜ, 1 μΜ, 10 μΜ or 100 μΜ of DMF or MMF (Sigma- Aldrich), or with culture medium as a control for 4 or 24 h. After incubation, the cells were harvested and the cell suspensions were centrifuged at 1000 x g for 8 min before the supernatants were collected. The supernatants from 4 or 24 h incubation were kept in -80°C freezer until further investigation.

NK cell cytotoxicity assay

The human myeloid leukemia cell line K562 cells (CCL-243 obtained from American Type Culture Collection "ATCC", Manassas, Virginia, USA) or RATI human lymphoma cells (CCL-86, ATCC), were used as target cells. Target cells were incubated with 5 μg/mL calcein- AM (Sigma-Aldrich) for 1 h at 37° C, washed and then plated at 12,500 cells/well in 96 well plates. Total viability was measured in target cells incubated with culture medium only, and total cytotoxicity was measured in target cells incubated for 30 min with 2.5% Triton X-100. Pre- treated effector cells were plated onto 96-well plates at the indicated effector: target (E:T) cell ratios in triplicate. The plates were spun down at 500 rpm for 5 min and incubated for 4 h at 37°C and 5% C0₂. After incubation, the cells were centrifuged, supernatants removed and 200 μL PBS added to each well. The fluorescence intensity of the calcein-AM loaded cells was measured in a BioTek FLX TBI plate reader, using 485/528 nm fluorescence filters. Percentage cytotoxicity was calculated as previously described (Damaj BB et al. (2007) J Immunol 179: 7907-7915).

Flow cytometric analysis

CD56⁺ and CD56^" NK cells treated with 0.1 μΜ, 1 μΜ, 10 μΜ or 100 μΜ DMF or MMF, or with culture medium as a control, were washed extensively before staining with antibodies for surface molecules. For each treatment, 3x10⁵ cells were stained for 45 min at 4°C with 1 μg/mL FrTC-conjugated mouse anti-human CD158, 1 μg/mL PE-conjugated mouse anti- human NKp30 (CD337), 1 μg/mL PE-conjugated mouse anti-human NKp44 (CD336), 1 μg/mL PE-conjugated mouse anti-human NKp46 (CD335), 1 μg/mL PE-conjugated mouse anti-human NKG2D (CD314), FITC-conjugated or PE-conjugated mouse isotype controls (all antibodies were obtained R&D systems Europe, Abingdon, UK). The cells were also stained with FrTC- conjugated CD 107a or FrTC-conjugated IgGl isotype control (Beckton-Dickinson Pharmingen, San Diego, California, USA). They were washed twice, and examined in the flow cytometry (FACSCanto II, Becton Dickinson Biosciences, San Jose, CA). Gating was performed according to the isotype control. Analysis was done by FlowJo (Flow cytometry analysis software, Ashland, OR, USA).

ELISA assay to determine the levels of Granzyme B

Levels of Granzyme B were measured using human Granzyme B ELISA kit (Bender Med systems, Burlingame, CA, USA) according to the manufacturer's instructions. After adding TBM substrate solution, the microwell stripes were incubated at room temperature in the dark for 10 min before adding stop solution. Absorbance was read on a BioTek Powerwave XS plate reader with 450 nm wavelength. Standard curves and concentrations were calculated using Gen5™ Data Analysis Software (BioTek Instruments, VT, USA).

Treatment with anti-NKp46 or anti-NKp30 antibodies

CD56⁺NK cells (lX10⁶/mL) were either left untreated but were incubated with 10 μg/mL anti-NKp30 or 10 μg/mL anti-NKp46 and as a control with 10 μg/mL isotype IgG antibodies. CD56 NK cells (lXlOVmL) were also incubated with 100 μΜ MMF in the absence or presence of the same antibodies. After 24 h, the cells were washed and examined for lysis of K562 or RAJI cells and for the expression of CD 107a molecule. In addition, supernatants were collected from these cells and the levels of Granzyme B were measured in the ELISA assay. Viability was more than 90% after the incubation period as determined by trypan blue (Sigma- Aldrich) exclusion test.

Results

DMF and MMF increase NK cell lysis of K562 tumor target cells

Freshly isolated resting human blood NK cells were separated into CD56⁺ and CD56^", incubated with various concentrations of DMF or MMF for 4 or 24 h, washed extensively to remove the drugs and then examined for killing of the leukemic cell line K562. Results in Figure 1A show that all concentrations of DMF or MMF used enhanced CD56⁺ NK cell lysis of K562 cells 4 h post incubation (P<0.04 and <0.01 , respectively). Although there was an increased lysis with CD56 NK cells treated with DMF after 24 h, this killing did not reach significance.

However, treating CD56⁺NK cells for 24 h with MMF significantly augmented their killing of K562 cells (P<0.04, Figure IB). On the other hand, CD56^" NK cell killing of K562 was twice more potent than CD56 at the same 10:1 E:T cell ratio. The 100 μΜ concentration of DMF or 10 and 100 μΜ of MMF enhanced CD56^" lysis of K562 cells after 4 h incubation (Figure 1C). None of DMF or MMF concentrations significantly increased CD56^~ cell lysis of K562 cells after 24 h incubation, albeit a trend for increased killing (Figure ID).

DMF and MMF induce NK cell lysis of RAJI tumor cells

The B cell lymphoma RAJI cells are resistant to lysis by primary NK cells. However, we were encouraged by the effects of DMF and MMF enhancement of NK cell lysis against K562 cells, and sought to determine whether these drugs might also activate NK cells to lyse the NK resistant RAJI cells. Similar to what was done above, CD56 and CD56^" NK cells were treated with 0.1 , 1 , 10 and 100 μΜ of DMF or MMF for 4 or 24 h, washed and then incubated with RAJI cells in the 4 h NK cytotoxicity assay. Background lysis of CD56⁺ NK cells against RAJI cells was minimal, if any. However, the 10 or 100 μΜ concentrations of DMF or MMF significantly induced killing of RAJI cells after 4 h incubation (Figure 2A). The background cytotoxicity of CD56⁺ NK cells against RAJI was close to zero after 24 h incubation. However, all concentrations of the two drugs significantly induced CD56⁺ NK cells to lyse RAJI cells (Figure 2B). CD56^" NK cells killed about 15% of RAJI cells when used at 10:1 E:T ratio, and there was no effect for DMF on this activity 24 h post incubation (Figure 2C), but the 100 μΜ concentration of MMF significantly enhanced CD56 NK cell lysis of RAJI cells (P<0.04, Figure 2C). On the other hand, the 10 and 100 μΜ concentrations of MMF but not DMF significantly augmented CD56^" NK cell killing of RAJI cells after 24 h incubation (Figure 2D).

Effects of DMF and MMF on the expression of NK cytotoxicity receptors

To correlate the killing activities with the effects of the drugs on the expression of NK cytotoxicity receptors NKp30, NKp44 and NKp46, we investigated the expression of these molecules on the surface of NK cells after 4 or 24 h incubation with the drugs. The 100 μΜ concentration of DMF significantly up-regulated the expression of NKp30 on CD56⁺ NK cells 4 h post-incubation (P<0.05, Figure 3A), and there was a tendency of increased expression with the 10 μΜ concentration. In contrast, none of the MMF concentrations used increased this expression (Figure 3A). The effect of augmented expression disappeared after incubating CD56⁺ NK cells with DMF for 24 h (Figure 3B). There was a significant up-regulation of NKp30 on the surface of CD56^" NK after 4 h incubation with 100 and 10 μΜ concentrations of MMF but not DMF (P<0.02 and <0.05, respectively, Figure 3C). However, this effect disappeared after 24 h incubation with the drug (Figure 3D). A low expression of NKp44 on the surface of un-activated CD56 or CD56 NK cells was observed, a finding well within what has been described regarding the expression of this molecule on NK cells (Vitale M et al. (1998) J Exp Med 187: 2065-2072). In addition, neither DMF nor MMF affected such expression (Figure 8).

On the other hand, the 100 μΜ concentration of DMF significantly up-regulated the expression of NKp46 on the surface of CD56⁺ NK cells after 4 h incubation (P<0.05, Figure 4A), an effect that disappeared after 24 h incubation (Figure 4B). MMF did not significantly up- regulate the expression of NKp46 on CD56⁺ NK cells after 4 h incubation, albeit a statistically non-significant increased expression after incubation with the 100 μΜ concentration (Figure 4A). However, incubating CD56⁺ NK cells for 24 h with 10 or 100 μΜ concentrations of MMF significantly up-regulated the expression ofNKp46 (Figure 4B). There was no effect of the various concentrations of DMF or MMF on the expression of NKp46 on the surface of CD56^" NK cells after 4 h (Figure 4C), or 24 h (Figure 4D). Also, there was no effect of the drugs on the expression of the C-type lectin receptor NKG2D after 4 or 24 h incubation (Figure 9). Further, none of the concentrations of MMF or DMF affected the expression of KIR CD 158 on the surface of CD56⁺ or CD56 NK cells, 4 or 24 h post incubation (Figure 10).

MMF increases the expression of CD 107a and induces the release of Granzyme B from CD56⁺ NK cells

To get further insights into the effects of DMF and MMF on NK cell mediated cytotoxicity, we pursue to investigate the effects of these drugs on the expression of CD 107, a molecule that is expressed on the surface of cells due to the release of cytolytic molecules (Alter G (2004) J Immunol Methods 294: 15-22). The results demonstrate that various concentrations of DMF or MMF did not affect the expression of CD 107a on CD56⁺ NK cells after 4 h incubation (Figure 5A). Intriguingly, the 100 and 10 μΜ concentrations of MMF significantly induced the expression of this molecule on the surface of these cells 24 h post-incubation (P<0.05, Figure 5B). Of note, the 1 and 0.1 μΜ concentrations of MMF also tended to up- regulate the expression of this molecule (Figure 5B). In contrast, there was no effect of DMF or MMF on the expression of CD 107 on the surface of CD56^" NK cells after 4 h (Figure 5C) or 24 h (Figure 5D) incubation.

To correlate these results with the release of cytolytic granules, we examined the effects of the drugs on the release of Granzyme B from NK cells. None of DMF or MMF concentrations significantly induce the release of this molecule form CD56⁺ NK cells upon 4 h incubation (Figure 6A). Similarly, DMF did not significantly induce this release from these cells after 24 h incubation (Figure 6B). Surprisingly, all concentrations of MMF used significantly induced the release of Granzyme B from CD56⁺ NK cells after incubating with these cells for 24 h (Figure 6B). On the other hand, CD56^" NK cells secreted three times the amount of Granzyme B as compared to CD56⁺ NK cells after 4 h, and there were no significant effects of DMF or MMF on such release after 4 h (Figure 6C), or 24 h incubation with CD56^" NK cells (Figure 6D).

MMF induces the cytolytic activity, expression of CD 107 a and Granzyme B release through NKp46 up-regulation

The fact that MMF induced CD56⁺ NK cell lysis of K562 and RAJI cells after 24 h incubation corroborated with the expression of NKp46, CD 107a and Granzyme B release at the same time, suggests that MMF might utilize NKp46 to activate NK cells. To investigate this issue, we incubated CD56⁺ NK cells with anti-NKp46 and as controls with anti-NKp30 or with isotype antibodies. First we determined that treatment with anti-NKp46 down-regulated the expression of NKp46 molecule. Results in Figure 7 A demonstrate that such treatment resulted in the inability to recognize NKp46 by fluorescently labeled anti-NKp46. MMF increased the percentages of cells expressing NKp46, and whereas incubating the cells with isotype control antibody did not affect the expression of this molecule, incubating the cells for 30 min with anti- NKp46 prior to activation with MMF inhibited the effect of MMF (Figure 7A). These results demonstrate that anti-NKp46 is successful in blocking the expression of NKp46. Next the effect of anti-NKp46 on CD56⁺ NK cell lysis of K562 cells was investigated. Upon 24 h incubation, MMF increased CD56⁺ NK cell killing of K562 (P<0.03), and treatment with anti-NKp46 but not isotype control antibodies or anti-NKp30 inhibited this activity (P<0.001, Figure 7B).

Similarly, MMF induced CD56⁺ NK cell killing of RAJI cells after 24 h incubation (P<0.05 as compared to the control, Figure 7C) and that only anti-NKp46 but not anti-NKp30 or isotype control antibodies abolished this lysis (P<0.02, Figure 7C). Similarly, MMF increased Granzyme B release by these cells 24 h post incubation (P<0.04 as compared to the control, Figure 7D) and only anti-NKp46 reversed this increase (P<0.04, Figure 7D). In addition, MMF treatment up- regulated the expression of CD 107a on the surface of these cells (P<0.04 as compared to the control, Figure 7D), and this activity was abrogated by anti-NKp46 (P<0.03, Figure 7E).

Discussion

DMF has been previously used to treat psoriasis, but was recently approved in the USA to treat MS patients (Lee DH et al. (2012) Int J Mol Sci 13: 1 1783-1 1803). In the DEFINE and CONFIRM randomized, double blind, placebo -controlled studies involving patients with relapsing-remitting MS, it was observed that dimethyl fumarate or dimethyl fumarate significantly reduced relapse rate and improved neuroradiologic outcome when compared to placebo (Gold R et al. (2012) N Engl J Med 367: 1098-1107; Fox RJ et al. (2012) N Engl J Med 367: 1087-1097). Several mechanisms of action (MOA) were attributed to the effects of fumurates, such as the activation of anti-oxidnats, factor Nrf2 (Linker RA et al. (2008) Expert Rev Neurother 8: 1683-1690; Gold R et al. (2012) Clin Immunol 142: 44-48; Scannevin RH et al. (2012) J Pharmacol Exp Ther 341 : 274-284; Lee DH et al. (2012) Int J Mol Sci 13: 11783- 1 1803), glutathione and heam oxygenase-1 (Lin SX et al. (2006) ASN Neuro 3: 75-84). In addition, DMF was found to switch the immune system towards a Th2 anti-inflammatory type of response through the activation of DCs type II, and the suppression of DCs type I which consequently inhibit the generation of inflammatory Thl or Thl7 cells (Ghoreschi K et al. (2011) J Exp Med 208: 2291 -2303).

Further, it was reported that DMF inhibits the proliferation of melanoma cells (Loewe R et al. (2006) Cancer Res 66: 11888-11896), and synergizes with another drug, Dacarbazine for influencing the migration of tumor cells (Valero T et al. (2010) J Invest Deramtol 130: 1087- 194). Whether DMF or its derivative MMF exerts any effect on NK cells has not been previously investigated. Because drugs used to treat MS patients, such as GA or FTY720 "fingolimod" were found to activate these cells to kill K562 cells (Ftoglund RA et al. (2013) PLoS One 8: e62237; Al-Jaderi Z et al. (2013) Toxins 5: 1932-1947, respectively), we hypothesized that DMF or MMF might activate the anti-tumor effector NK cells. We observed that both drugs augmented NK cell cytolytic activity against the human chronic myelogenous leukemic cells K562. These cells are NK-sensitive and have been used as prototypes for measuring NK cell lytic effects against tumors. Intriguingly DMF and MMF also induced NK cell cytolysis of the B lymphoma RAJI cells, which are resistant to resting NK cell-mediated cytotoxicity due to their expression of HLA ligands engaged by NK cell inhibitory receptors. Even more interesting is the ability of these drugs to convert resting CD56⁺ NK cells that are not cytolytic cells and do not kill RAJI cells, into robust killers of K562 and RAJI cells.

Although neither DMF nor MMF down-regulated the KIR CD 158 expression on NK cells, these drugs up-regulated the expression of NK cytotoxicity receptors, in particular NKp30 and NKp46, hence shifting the threshold towards activation rather than inhibition. This way, the fumarates resemble activation molecules such as IL-2 which induces NK cell lysis of RAJI cells.

NKp30 and NKp46 are important forNK cells recognition and destruction of tumor cells. For example, in acute myelogenous leukemia, NK cells express low levels of these receptors resulting in the evasion of leukemic cells and the development of the disease, whereas up- regulation of these receptors leads to NK cells lysis of leukemic cells (Sanchez-Correa B et al. (2011) Cancer Immunol Immunother 60: 1 195-1205). The tumor ligand that binds NKp30 has been described and it belongs to the B7 family of molecules, and is consequently named B7-H6 (Brandt CS et al. (2009) J Exp Med 206: 1495-1503). This molecule is up-regulated during tumor transformation and its mRNA is absent from normal tissues or peripheral blood cells. Of note, NKp30 has several isoforms: "NKP30a and NKP30b", which are immunoregulatory and the immunosuppressive "NKP30c" (Delahaye NF et al. (201 1) Nat Med 17: 700-707). On the other hand, the ligand for NKp46 (and NKp44) is up-regulated on human glioblastoma cells infected with oncolytic Herpes simplex virus (Alvarez-Breckenridge CA et al. (2012) Nat Med 18: 1827-1834). This up-regulation results in killing the infected glioblastoma cells by NK cells expressing NKp46 [32]. NKp46 has been implicated in controlling tumor metastases in animals carrying the B16 melanoma or the Lewis lung carcinoma D122 (Glasner A et al. (2012) J Immunol 188: 2509-2515). Also the absence of NKp46 impaired eradication of lymphoma cells (Halfteck GG et al. (2009) J Immunol 182: 2221-2230). Our results suggest that NKp46 is involved in the activities exerted by MMF on NK cells, particularly after incubating CD56⁺ NK cells with this metabolite for 24 h. Consequently, we sought to understand the relationship among NKp46 and MMF, and investigated the role of NKp46 in MMF-induced CD56⁺ induced lysis of the NK-sensitive K562 cells and the NK- resistant RAJI cells. We observed that anti-NKp46 but not anti-NKp30 inhibited MMF-induced lysis of both tumor cell lines. Similarly, anti-NKp46 reversed MMF-induced expression of CD107a in these cells and their ability to release Granzyme B. The functions of NKC receptors are not redundant since they respond differentially to multiple stimuli to eliminate their targets (Hudspeth K et al. (2013) Frontiers Immunol 4 : 1-15). However, it is intriguing that NKp46 and not NKp30 is involved in mediating MMF various activities. Whether NKp46 recognizes MMF is an interesting possibility; MMF is the metabolite of DMF, where one methyl group is lost due to hydrolysis by esterases in the intestines following oral administration. It was observed that free DMF could not be detected in plasma of the portal vein blood after oral application of DMF in rats, due largely to its conversion into MMF, and due to the formation of adducts with glutathione "GST" (Dibbert S et al. (2013) Arch Dermatol Res 305: 447-451). For this reason, it is suggested that MMF could be the more active molecule.

NK cells contain several proteins such as members of Granzyme and perforin which are released upon contacting target cells leading to the death of the latter cells. CD 107a (also known as lysosomal-associated membrane protein- 1 "LAMP-1") is present in the membranes of cytolytic granules. This molecule starts to be expressed on the surface of CD8⁺ T cells upon degranulation (Betts MR et al. (2003) J Immunol Methods 281 :65-78) or on NK cells following stimulation (Alter G et al. (2004) J Immunol Methods 294: 15-22). Consequently, it was considered a distinct marker for cell-mediated lysis of target cells (Alter G et al. (2004) J Immunol Methods 294: 15-22). This molecule plays an important role for the survival of NK cells against suicide as it protects these cells from direct killing by cytolytic granules upon degranulation (Cohnen A et al. (2013) Blood 122: 141 1-1418). In our hands, CD107a was expressed on cytolytic CD56^" NK cells more than on CD56⁺ cells. However, we observed increased expression of this molecule on the surface of CD56⁺ NK cells 24 h post-incubation with MMF, and this activity correlated closely with the effect of MMF on the degranulation and the release of Granzyme B from CD56⁺ cells. The expression of CD 107a and the release of Granzyme B from CD56 NK cells 24 h after incubation with MMF could explain the ability of this drug to induce killing of K562 cells and RAJI cells by these cells that are considered immunoregulatory more than cytolytic. Hence, MMF converts the cells that normally release cytokines into cells that are highly cytolytic against tumor cells. Previous results also indicate that another drug for potential treatment of MS, daclizumab enhances the release of Granzyme K from NK cells which mediates apoptosis in activated T cells (Jiang W et al. (201 1) J Immunol 187: 781-790).

In summary we describe a novel mechanism of action for fumarates, and in particular MMF which activates NK cells to kill tumor target cells that include both the classical NK cell sensitive target K562 cells and the NK resistant target RAJI cells. What is intriguing is the ability of MMF to convert the low cytolytic CD56⁺ NK cells into highly anti-tumor effector cells most probably via NKp46. This effect has not been previously described and only few cytokines such as IL-2 and to a lesser effect IL- 15 and/or IL- 18 perform a similar effect, although chemokines have also been shown to activate NK cells to become cytolytic (Jiang W et al. (201 1) J Immunol 187: 781-790). This novel effect should encourage further studies on DMF and MMF-induced activation of NK cells in cancer treatment.

Example 2: Preparation of compounds (III)-(VI)

The compounds of Formulae (III)-(VI) may be prepared using methods known to those skilled in the art, or the methods disclosed in the present invention.

Specifically, the compounds of this invention of Formula IV may be prepared by the exemplary reaction in Scheme 1.

Scheme 1

wherein R , R , and R are each defined above for Formula IV.

Reaction of fumaric acid ester 1 with silane diacetate intermediate 2 in a refluxing organic solvent such as diethyl ether, toluene, or hexane to give the desired siloxane 3.

Some of the fumaric acid esters 1 are commercially available. Fumaric acid ester 1' can be prepared, for example, using synthetic methods known by one of ordinary skill in the art. For example, fumaric acid can be converted by reacting alcohol (R^lc-OH) with a catalytic amount of p-toluene sulfonic acid at room temperature for a few hours to overnight as shown in Scheme 2.

Scheme 2

wherein R^lc is defined above for Formula III.

Alternatively, fumaric acid ester 1 ' can be prepared by reacting alcohol

(R^lc-OH) under the coupling conditions of hydro xybenzotriazole (HOBT), l -ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI), and diisopropyl amine (DIPEA) as shown in Scheme 3.

Scheme 3

wherein R^lc is defined above for Formula III.

Some of the silanes that can be used in the present invention are commercially available. Commercially available silyl halides include trimethylsilyl chloride, dichloro- methylphenylsilane, dimethyldichlorosilane, methyltrichlorosilane,

(4-aminobutyl)diethoxymethylsilane, trichloro(chloromethyl)silane,

trichloro(dichlorophenyl)silane, trichloroethylsilane, trichlorophenylsilane, and

tnmethylchlorosilane. Commercial sources for silyl halides include Sigma Aldrich and Acros Organics.

Silanes used in the present invention can be prepared, for example, using synthetic methods known by one of ordinary skill in the art. For example, trichlorosilane may be prepared by the exemplary reaction in Scheme 4.

Scheme 4

R ,CI

+ HSiCI ^"Si

CI ^sci

The silylation of styrene derivatives catalyzed by palladium is described in Zhang, F. and Fan, Q.-H., Organic & Biomolecular Chemistry 7 ΑΊ0-ΑΑΊΑ (2009) and in Bell, J.R., et al., Tetrahedron 65:9368-9372 (2009).

Diacetate intermediate 2 may be prepared by treatment of dichloro substituted silicon compound 4 with sodium acetate in diethyl ether under reflux as shown in Scheme 5.

Scheme 5

wherein R^2d and R^3d are each defined above for Formula IV.

Specifically, the compounds of this invention of Formula V may be prepared by the exemplary reaction in Scheme 6.

Scheme 6

wherein R^le, R^2e, R^3e, and R^5e are as defined above for Formula V.

Fumaric acid ester 1" can be converted to the sodium salt 5 using, for example, sodium methoxide in methanol at room temperature. Removal of the solvent would afford sodium salt 5. Treatment of the sodium salt 5 with silane 6 in an organic solvent such as dimethylformamide under reflux would generate ester 7. The synthesis of structurally related (trimethoxysilyl)- methyl esters is described in Voronkov, M.G., et al., Zhurnal Obshchei Khimii 52:2052-2055 (1982).

Alternatively, the compounds of this invention of Formula V may be prepared by the exemplary reaction in Scheme 7.

Scheme 7

wherein R^le, R^4e, R^5e, R^6e, and n are as defined above for Formula V.

Treatment of the sodium salt 5 with silane 6 in an organic solvent such as

dimethylformamide under heating with or without an acid scavenger would generate ester 7.

Scheme 8

CH₂CI₂, 0°C to rt

wherein R^le, R^4e, R^5e, R^6e, and n are as defined above for Formula V.

Reaction of fumaric acid ester 1" with tri-substituted silane alcohol 8 in methylene chloride with mild base such as triethyl amine and 4-N,N-dimethyl amino pyridine (DMAP) at room temperature generates fumarate 7. See Coelho, P.J., et al., Eur. J. Org. Chem. 3039-3046 (2000).

Specifically, the compounds of this invention of Formula VI can be prepared by the exemplary reaction in Scheme 9.

Scheme 9

10

wherein R^lf and R^2f are as defined above for Formula VI.

Reaction of fumaric acid 1 "' with trichlorosilane 9 in a refiuxing organic solvent such as hexane or toluene using a catalytic amount of a base such as triethylamine generates the trifumarate silane 10. The reaction of acetic and methacrylic acids with 1 -silyladamantanes is described in Fedotov, N.S., et al., Zhurnal Obshchei Khimii 52:1837-1842 (1982). Example 3: Synthesis of (E)-Q.,0'-(dimethylsilanediyl)dimethyl difumarate (Compound 11)

Step 1 : Preparation of dimethylsilanediyl diacetate 11B

To a slurry of sodium acetate (8.2 g, 100 mmol, 2.0 equiv.) in anhydrous diethyl ether (40 mL) was slowly added a solution of dimethyldichloro silane 11A (6.45 g, 50 mmol, 1.0 equiv.) in anhydrous diethyl ether (10 mL). After addition was completed, the mixture was heated at reflux for 2 hours, and then filtered under N₂. The filtrate was concentrated under vacuum at 40 °C to give diacetate 11B as a colorless oil (6.1 g, 70%). 1H NMR (400 MHz, CDC1₃) δ ppm: 2.08 (s, 6H), 0.48 (s, 6H).

Step 2: Preparation of (E)-0,0 '-(dimethylsilanediyl)dimethyl difumarate 11

11 B 11

A mixture of 11B (2.0 mL, 12 mmol, 1.5 equiv.) and 11C (1.04 g, 8.0 mmol, 1.0 equiv.) in a sealed tube was heated at 170 °C with stirring under microwave condition for 1 hour. After cooling to 50 °C, the mixture was transferred to a round bottom flask and the excess silica reactant 11B was removed under vacuum at 100 °C to afford compound 11 as brown oil (1.47 g, 60%). 1H NMR (400 MHz, CDC1₃) δ ppm: 6.82-6.80 (m, 4H), 3.79 (s, 6H), 0.57 (s, 6H).

Example 4: Synthesis of methyl ((trimethoxysilvDmethyl) fumarate (Compound 12)

12A 12B 12

To a stirred solution of monomethyl fumarate (3.5 g, 27 mmol, 1.0 equiv.) in anhydrous THF (35 mL) at room temperature was added sodium hydride (1.08 g, 27 mmol, 1.0 equiv.) in small portions. After addition, the mixture was heated to reflux for 3 hours, and then cooled to room temperature. The solid was collected by filtration and washed twice with diethyl ether, and further dried in vacuo to give 3.8 g of 12B (93%).

To a suspension of 12B (760 mg, 5.0 mmol, 1.0 equiv.) in dry DMA (5 mL) at 100 °C under nitrogen was added a solution of 12A (1.03 g, 6.0 mmol, 1.2 equiv.) in dry DMA (1 mL) dropwise. The resulting mixture was heated to 160 °C and stirred for 1 hour, and then cooled to room temperature. The solid was filtered, and the filtrate was evaporated under reduced pressure to give the titled compound 12, 513 mg (37%), as a red viscous liquid.

1H NMR (400 MHz, CDC1₃) δ ppm: 6.90-6.86 (m, 2H), 3.97 (s, 2H), 3.82 (s, 3H), 3.62 (s, 9H).

Example 5: Synthesis of methyl ((trihydroxysilvDmethyl fumarate (Compound 13)

12 13 To a solution of 12 (1.0 g, 3.8 mmol, 1.0 equiv., prepared in Example 2) in MeOH (10 mL) at room temperature was added water (341 mg, 19.0 mmol, 5.0 equiv.) dropwise. After addition, the mixture was stirred at room temperature for 30 minutes, with white solids precipitated out. The solids were collected through filtration, washed with methanol three times, and dried at 60 °C in vacuo, to provide the titled compound 13, 500 mg (59%), as a white solid.

Ή NMR (400 MHz, DMSO-i/6) δ ppm: 6.79-6.74 (m, 2H), 3.91 -3.58 (m, 6H), 3.18-3.15 (m, 2H).

Example 6: Synthesis of trimethyl (methylsilanetrivD trifumarate (Compound 14)

Following the procedure described in Scheme 9, monomethyl fumarate 14 A would react with trichloromethane-silane 14B in refluxing toluene or hexanes with a catalytic amount of triethylamine to provide (2 Έ, 2 "E)-trimethyl Ο,Ο ', O "-(methylsilanetriyl) trifumarate 14C.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed.

Claims

What is claimed is:

1. Dimethyl fumarate (DMF), or a prodrug thereof, for use in treating cancer in a subject in need thereof, wherein the DMF or prodrug thereof is administered to the subject as a solid dosage form in a therapeutically effective amount.

2. DMF or prodrug thereof for use according to claim 1, wherein the subject is administered DMF.

3. DMF or prodrug thereof for use according to claim 1 or claim 2, wherein the cancer is a hematological malignancy.

4. DMF or prodrug thereof for use according to claim 3, wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.

5. DMF or prodrug thereof for use according to claim 4, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL).

6. DMF or prodrug thereof for use according to claim 4, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma.

7. DMF or prodrug thereof for use according to claim 4, wherein the myeloma is multiple myeloma.

8. DMF or prodrug thereof for use according to claim 1 , wherein the cancer is a solid tumor.

9. DMF or prodrug thereof for use according to claim 8, wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and combinations thereof.

10. Dimethyl fumarate (DMF), or a prodrug thereof, for use in treating an infectious disease in a subject in need thereof, wherein the DMF or prodrug thereof is administered to the subject in a therapeutically effective amount.

11. DMF or prodrug thereof for use according to claim 10, wherein the subject is administered DMF.

12. DMF or prodrug thereof for use according to claim 10 or 11 , wherein the infectious disease is a viral infection selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma- associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

13. DMF or prodrug thereof for use according to any one of the preceding claims, wherein the subject is a human.

14. DMF or prodrug thereof for use according to any one of the preceding claims, wherein DMF, or prodrug thereof is administered orally.

15. DMF or prodrug thereof for use according to any one of the preceding claims, wherein DMF, or prodrug thereof is administered daily.

16. DMF or prodrug thereof for use according to any one of the preceding claims, wherein DMF is administered daily in an oral dosage form.

17. DMF or prodrug thereof for use according to any one of the preceding claims, wherein DMF is administered in a dose of approximately 50-2000 mg/day to the subject.

18. DMF or prodrug thereof for use according to any one of the preceding claims, wherein the DMF is administered in a dose of approximately 240-1000 mg/day to the subject.

19. DMF or prodrug thereof for use according to claim 8, wherein 720 mg of DMF is administered daily to a subject.

20. DMF or prodrug thereof for use according to claim 19, wherein the 720 mg of DMF is administered in 3 equal doses to a subject.

21. DMF or prodrug thereof for use according to claim 18, wherein 480 mg of DMF is administered daily.

22. DMF or prodrug thereof for use according to claim 21 , wherein the 480 mg of DMF is administered in 2 equal doses.

23. DMF or prodrug thereof for use according to any of the preceding claims, wherein DMF is administered 1 , 2, 3, 4, 5 or 6 times daily.

24. DMF or prodrug thereof for use according to claim 23, wherein DMF is administered 3 times daily.

25. DMF or prodrug thereof for use according to claim 23, wherein DMF is administered 2 times daily.

26. DMF or prodrug thereof for use according to claim 23, wherein DMF is administered once daily.

27. DMF or prodrug thereof for use according to any of one of the preceding claims, wherein the solid dosage form is selected from the group consisting of tablets, micro-tablets, pellets, granulates, capsules, sachets, powders and lozenges.

28. DMF or prodrug thereof for use according to claim 27, wherein the solid dosage form is in the form of micro-tablets or pellets optionally filled in capsules or sachets.

29. DMF or prodrug thereof for use according to claim 28, wherein the mean diameter of the pellets or micro tablets is from 300 to 4000 μπι.

30. DMF or prodrug thereof for use according to claim 29, wherein the mean diameter of the pellets or micro tablets is about 2000 μπι.

31. DMF or prodrug thereof for use according to claim 14, wherein the oral dosage form is prepared with an enteric coating.

32. DMF or prodrug thereof for use according to any one of claims 1-8 or 13-31 , wherein one or more cancer therapeutic agents are administered in combination with the DMF, or prodrug thereof.

33. DMF or prodrug thereof for use according to claim 32, wherein the cancer therapeutic agent is a chemotherapeutic agent.

34. DMF or prodrug thereof for use according to claim 33, wherein the chemotherapeutic agent is selected from the group consisting of aclarubicin, alemtuzumab, amsacrine,

asparaginase, azacitidine, busulphan, chlorambucil, cladribine, clofarabine, cytabarine, daunorubicin, doxorubicin, filgrastim, fludarabine, interferon alpha 2A, mercaptopurine, methotrexate, mitoxantrone, nelarabine, nilotinib, pentostatin, rituximab, teniposide, thioguanine, vincristine, and combinations thereof.

35. DMF or prodrug thereof for use according to any one of claims 9-31 , wherein one or more antiviral agent are administered in combination with the DMF, or prodrug thereof.

36. A composition comprising dimethyl fumarate (DMF), or a prodrug thereof, for use in treating a condition that is susceptible of being improved by enhancing cell lysis of a target cell by a natural killer (NK) cell, wherein the NK cell is contacted with the composition in an amount effective to increase the capacity of the NK cell to lyse the target cell.

37. DMF or prodrug thereof for use according to claim 36, wherein the composition comprises DMF.

38. DMF or prodrug thereof for use according to claim 36, wherein the contacting step is performed in vivo.

39. DMF or prodrug thereof for use according to claim 36, wherein the contacting step is performed ex vivo.

40. DMF or prodrug thereof for use according to claim 36, wherein the target cell is a cancer cell.

41. DMF or prodrug thereof for use according to claim 40, wherein the cancer cell is a hematological malignancy cell.

42. DMF or prodrug thereof for use according to claim 41 , wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.

43. DMF or prodrug thereof for use according to claim 42, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL).

44. DMF or prodrug thereof for use according to claim 42, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma.

45. DMF or prodrug thereof for use according to claim 42, wherein the myeloma is multiple myeloma.

46. DMF or prodrug thereof for use according to claim 40, wherein the cancer is a solid tumor.

47. DMF or prodrug thereof for use according to claim 46, wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and

combinations thereof.

48. DMF or prodrug thereof for use according to claim 36, wherein the target cell is a virally-infected cell.

49. DMF or prodrug thereof for use according to claim 48, wherein the target cell is infected by a virus selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma- associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

50. DMF or prodrug thereof for use according to any one of claims 36-49, wherein the target cell is a human cell.

51. DMF or prodrug thereof for use according to any one of claims 36-50, wherein the NK cell is a human NK cell.

52. DMF or prodrug thereof for use according to any one of claims 36-51 , wherein the NK cell is a CD56+/^bright NK cell.

53. DMF or prodrug thereof for use according to any one of claims 36-51 , wherein the NK cell is a CD56-/^dim NK cell.

54. DMF or prodrug thereof for use according to any one of claims 36-53, wherein the target cell is a cell that was previously resistant to NK cell lysis.

55. DMF or prodrug thereof for use according to any one of claims 36-54, wherein the NK cell has an increased capacity to lyse Raji cells when contacted with the composition comprising DMF, or prodrug thereof as compared to the capacity of the NK cell to lyse Raji cells before being contacted with the composition comprising DMF, or prodrug thereof.

56. Monomethyl fumarate (MMF), or a metabolite thereof, for use in treating cancer in a subject in need thereof, wherein the MMF or metabolite thereof is administered to the subject as a solid dosage form in a therapeutically effective amount.

57. MMF or prodrug thereof for use according to claim 56, wherein the cancer is a hematological malignancy.

58. MMF or prodrug thereof for use according to claim 57, wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.

59. MMF or prodrug thereof for use according to claim 58, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL).

60. MMF or prodrug thereof for use according to claim 58, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma.

61. MMF or prodrug thereof for use according to claim 58, wherein the myeloma is multiple myeloma.

62. MMF or prodrug thereof for use according to claim 56, wherein the cancer is a solid tumor.

63. MMF or prodrug thereof for use according to claim 62, wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and

combinations thereof.

64. Monomethyl fumarate (MMF), or a metabolite thereof, for use in treating an infectious disease in a subject in need thereof, wherein the MMF or metabolite thereof is administered to the subject in a therapeutically effective amount.

65. MMF or prodrug thereof for use according to claim 64, wherein the infectious disease is a viral infection selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma- associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

66. MMF or prodrug thereof for use according to any one of claims 56-65, wherein the subject is a human.

67. MMF or prodrug thereof for use according to any one of claims 56-66, wherein MMF, or metabolite thereof is administered orally.

68. MMF or prodrug thereof for use according to any one of claims 56-67, wherein MMF, or metabolite thereof is administered daily.

69. MMF or prodrug thereof for use according to any one of claims 56-68, wherein MMF is administered daily in an oral dosage form.

70. MMF or prodrug thereof for use according to any one of claims 56-69, wherein MMF is administered in a dose of approximately 50-2000 mg/day to the subject.

71. MMF or prodrug thereof for use according to any one of claims 56-69, wherein MMF is administered in a dose of approximately 240-1000 mg/day to the subject.

72. MMF or prodrug thereof for use according to any one of claims 56-71 , wherein MMF is administered in 3 equal doses to the subject.

73. MMF or prodrug thereof for use according to any one of claims 56-71 , wherein MMF is administered in 2 equal doses to the subject.

74. MMF or prodrug thereof for use according to any one of claims 56-71 , wherein MMF is administered 1, 2, 3, 4, 5 or 6 times daily.

75. MMF or prodrug thereof for use according to claim 74, wherein MMF is administered 3 times daily.

76. MMF or prodrug thereof for use according to claim 74, wherein MMF is administered 2 times daily.

77. MMF or prodrug thereof for use according to claim 74, wherein MMF is administered once daily.

78. MMF or prodrug thereof for use according to any of one of claims 56-77, wherein the solid dosage form is selected from the group consisting of tablets, micro-tablets, pellets, granulates, capsules, sachets, powders and lozenges.

79. MMF or prodrug thereof for use according to claim 78, wherein the solid dosage form is in the form of micro-tablets or pellets optionally filled in capsules or sachets.

80. MMF or prodrug thereof for use according to claim 79, wherein the mean diameter of the pellets or microtablets is from 300 to 4000 μπι.

81. MMF or prodrug thereof for use according to claim 80, wherein the mean diameter of the pellets or microtablets is about 2000 μηι.

82. MMF or prodrug thereof for use according to claim 67, wherein the oral dosage form is prepared with an enteric coating.

83. MMF or prodrug thereof for use according to any one of claims 56-63 or 66-82, wherein one or more cancer therapeutic agents are administered in combination with MMF, or metabolite thereof.

84. MMF or prodrug thereof for use according to claim 83, wherein the cancer therapeutic agent is a chemotherapeutic agent.

85. MMF or prodrug thereof for use according to claim 84, wherein the

chemotherapeutic agent is selected from the group consisting of aclarubicin, alemtuzumab, amsacrine, asparaginase, azacitidine, busulphan, chlorambucil, cladribine, clofarabine, cytabarine, daunorubicin, doxorubicin, filgrastim, fludarabine, interferon alpha 2A,

mercaptopurine, methotrexate, mitoxantrone, nelarabine, nilotinib, pentostatin, rituximab, teniposide, thioguanine, vincristine, and combinations thereof.

86. MMF or prodrug thereof for use according to any one of claims 64-82, wherein one or more antiviral agents are administered in combination with MMF, or metabolite thereof.

87. A composition comprising (MMF), or a metabolite thereof, for use in treating a condition that is susceptible of being improved by enhancing cell lysis of a target cell by a natural killer (NK) cell, wherein the NK cell is contacted with the composition in an amount effective to increase the capacity of the NK cell to lyse the target cell.

88. MMF or prodrug thereof for use according to claim 87, wherein the contacting step is performed in vivo.

89. MMF or prodrug thereof for use according to claim 87, wherein the contacting step is performed ex vivo.

90. MMF or prodrug thereof for use according to claim 87, wherein the target cell is a cancer cell.

91. MMF or prodrug thereof for use according to claim 90, wherein the cancer cell is a hematological malignancy cell.

92. MMF or prodrug thereof for use according to claim 91 , wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.

93. MMF or prodrug thereof for use according to claim 92, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL).

94. MMF or prodrug thereof for use according to claim 92, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma.

95. MMF or prodrug thereof for use according to claim 92, wherein the myeloma is multiple myeloma.

96. MMF or prodrug thereof for use according to claim 90, wherein the cancer is a solid tumor.

97. MMF or prodrug thereof for use according to claim 96, wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and

combinations thereof.

98. MMF or prodrug thereof for use according to claim 87, wherein the target cell is a virally-infected cell.

99. MMF or prodrug thereof for use according to claim 98, wherein the target cell is infected by a virus selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma- associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

100. MMF or prodrug thereof for use according to any one of claims 87-99, wherein the target cell is a human cell.

101. MMF or prodrug thereof for use according to any one of claims 87-100, wherein the NK cell is a human NK cell.

102. MMF or prodrug thereof for use according to any one of claims 87-101 , wherein the NK cell is a CD56+/^bright NK cell.

103. MMF or prodrug thereof for use according to any one of claims 87-101 , wherein the NK cell is a CD56-/^dim NK cell.

104. MMF or prodrug thereof for use according to any one of claims 87-103, wherein the target cell is a cell that was previously resistant to NK cell lysis.

105. MMF or prodrug thereof for use according to any one of claims 87-104, wherein the NK cell has an increased capacity to lyse Raji cells when contacted with the composition comprising MMF or a metabolite thereof as compared to the capacity of the NK cell to lyse Raji cells before being contacted with the composition comprising MMF, or a metabolite thereof.

106. An in vitro or ex vivo method for preparing an activated natural killer (NK) cell, the method comprising a step of contacting an isolated NK cell with dimethyl fumarate (DMF) in an amount effective to activate the NK cell.

107. The method of claim 106, wherein the activated NK cell has an increased capacity to lyse Raji cells as compared to the isolated NK cell before activation.

108. The method of claim 106 or 107, wherein the DMF is present in an amount of about 500 μηι, about 400 μηι, about 300 μηι, about 200 μηι, about 100 μηι, about 50 μηι, about 10 μηι, about 5 μηι, about 1 μηι, or about 0.1 μηι.

109. The method of any one of claims 106-108, further comprising administering the activated NK cell to a subject in need thereof.

1 10. A population of activated NK cells prepared by the method of any one of claims 106-108.

1 11. An in vitro or ex vivo method for preparing an activated natural killer (NK) cell, the method comprising a step of contacting an isolated NK cell with monomethyl fumarate (MMF) in an amount effective to activate the NK cell.

1 12. The method of claim 1 11 , wherein the activated NK cell has an increased capacity to lyse Raji cells as compared to the isolated NK cell before activation.

1 13. The method of claim 1 1 1 or 112, wherein the DMF is present in an amount of about 500 μηι, about 400 μηι, about 300 μηι, about 200 μηι, about 100 μηι, about 50 μηι, about 10 μηι, about 5 μηι, about 1 μηι, or about 0.1 μηι.

1 14. The method of any one of claims 11 1-113, further comprising administering the activated NK cell to a subject in need thereof.

1 15. A population of activated NK cells prepared by the method of any one of claims 1 11-113.

1 16 An in vitro or ex vivo method of enhancing cell lysis of a target cell by a natural killer (NK) cell comprising contacting the NK cell with a composition comprising dimethyl fumarate (DMF), or a prodrug thereof, in an amount effective to increase the capacity of the NK cell to lyse the target cell.

1 17. The method of claim 1 16, wherein the composition comprises DMF.

1 18. The method of claim 1 16, wherein the target cell is a cancer cell.

1 19. The method of claim 1 18, wherein the cancer cell is a hematological malignancy cell.

120. The method of claim 1 19, wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.

121. The method of claim 120, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL).

122. The method of claim 120, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma.

123. The method of claim 120, wherein the myeloma is multiple myeloma.

124. The method of claim 1 18, wherein the cancer is a solid tumor.

125. The method of claim 124, wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and combinations thereof.

126. The method of claim 1 16, wherein the target cell is a virally-infected cell.

127. The method of claim 126, wherein the target cell is infected by a virus selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lymphotropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

128. The method of any one of claims 1 16-127, wherein the target cell is a human cell.

129. The method of any one of claims 1 16-128, wherein the NK cell is a human NK cell.

130. The method of any one of claims 1 16-129, wherein the NK cell is a CD56+/^bright NK cell.

131. The method of any one of claims 1 16-129, wherein the NK cell is a Οϋ56-/^Λι11 ΝΚ cell.

132. The method of any one of claims 1 16-131 , wherein the target cell is a cell that was previously resistant to NK cell lysis.

133. The method of any one of claims 1 16-132, wherein the NK cell has an increased capacity to lyse Raji cells when contacted with the composition comprising DMF, or prodrug thereof, as compared to the capacity of the NK cell to lyse Raji cells before being contacted with the composition comprising DMF, or prodrug thereof.

134. An in vitro or ex vivo method of enhancing cell lysis of a target cell by a natural killer (NK) cell comprising contacting the NK cell with a composition comprising monomethyl fumarate (MMF), or a metabolite thereof, in an amount effective to increase the capacity of the NK cell to lyse the target cell.

135. The method of claim 134, wherein the target cell is a cancer cell.

136. The method of claim 135, wherein the cancer cell is a hematological malignancy cell.

137. The method of claim 136, wherein the hematological malignancy is selected from the group consisting of leukemia, lymphoma, and myeloma.

138. The method of claim 137, wherein the leukemia is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphoblastic leukemia (CLL), chronic myelogenous leukemia (CML), and acute monocytic leukemia (AMoL).

139. The method of claim 137, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma and Non-Hodgkin's lymphoma.

140. The method of claim 137, wherein the myeloma is multiple myeloma.

141. The method of claim 135, wherein the cancer is a solid tumor.

142. The method of claim 141 , wherein the solid tumor is selected from the group consisting of breast cancer, prostate cancer, lung cancer, gastrointestinal cancer, brain cancer, liver cancer, kidney cancer, pancreatic cancer, melanoma, and combinations thereof.

143. The method of claim 134, wherein the target cell is a virally-infected cell.

144. The method of claim 98, wherein the target cell is infected by a virus selected from the group consisting of hepatitis B (HBV), hepatitis C (HCV), human T-lympho tropic virus (HTLV), human papillomavirus (HPV), Kaposi's sarcoma-associated herpesvirus (HHV-8), merkel cell polyomavirus, Epstein-Barr virus (EBV), human cytomegalovirus (CMV), and combinations thereof.

145. The method of any one of claims 134-144, wherein the target cell is a human cell.

146. The method of any one of claims 134-145, wherein the NK cell is a human NK cell.

147. The method of any one of claims 134-146, wherein the NK cell is a CD56+/^Dngm NK cell.

148. The method of any one of claims 134-146, wherein the NK cell is a CD56-/^dim NK cell.

149. The method of any one of claims 134-148, wherein the target cell is a cell that was previously resistant to NK cell lysis.

150. The method of any one of claims 134-149, wherein the NK cell has an increased capacity to lyse Raji cells when contacted with the composition comprising MMF or a metabolite thereof as compared to the capacity of the NK cell to lyse Raji cells before being contacted with the composition comprising MMF, or a metabolite thereof.