SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 28, 2017, is named 593474 ACT-045_SL.txt and is 70,787 bytes in size.

BACKGROUND

Histone deacetylase (HDAC) inhibition can cause cancer cell growth arrest. However, pan-HDAC inhibition leads to significant adverse effects, and an alternative HDAC inhibition profile is desirable.

HDAC6 is a Class IIb HDAC and is known to remove acetyl groups from many cellular proteins, including α-tubulin and HSP90. It has been reported that HSP90 hyperacetylation destabilizes its target proteins, including ER and EGFR. Inhibitors of HDAC6 have demonstrated anti-cancer proliferative activity in various cancer types. Blocking HDAC6 activity has been shown to cause cancer cell growth inhibition through various mechanisms.

B-lymphocyte antigen CD20 (CD20) is a glycosylated phosphoprotein expressed on the surface of all B-cells. CD20 enables a B-cell immune response against antigens. CD20 is targeted by CD20 inhibitory antibodies. CD20 inhibitory antibodies can treat a B-cell disorder, including cancer, an autoimmune disorder, or a transplant rejection.

Due to the dose-limiting toxicities of current pan-selective HDAC inhibitors, there is an ongoing need for improved methods for the treatment of cancer.

SUMMARY

In order to provide alternative efficacious and less toxic cancer treatments, provided herein are methods for the treatment of a B-cell disorder (e.g., cancer), and pharmaceutical combinations comprising an HDAC inhibitor and a CD20 inhibitory antibody. The pharmaceutical combinations and methods disclosed herein are well tolerated and do not exhibit the dose-limiting toxicities of prior therapies.

In one aspect, provided herein are methods for treating a B-cell disorder, such as cancer, an autoimmune disorder, or a transplant rejection, in a subject in need thereof. In particular, provided herein are methods for treating a B-cell disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an HDAC6 selective inhibitor and a therapeutically effective amount of a CD20 inhibitory antibody. In another embodiment, provided herein are methods for treating a B-cell disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound A


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody.

In another embodiment, provided herein are methods for treating a B-cell disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound B


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody, wherein the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In another embodiment, provided herein are methods for treating a B-cell disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof,whereinring B is aryl or heteroaryl;R1is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C1-6-alkyl;andR is H or C1-6-alkyl; and

b) a CD20 inhibitory antibody. In an embodiment, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In an embodiment of Formula I, R1is an aryl that is substituted by OH, halo, or C1-6-alkyl.

In some embodiments of the methods disclosed herein, the B-cell disorder is an autoimmune disorder or transplant rejection.

In other embodiments of the methods disclosed herein, the autoimmune disorder is selected from anti-N-methyl D-aspartate (NMDA) receptor encephalitis, autoimmune anemia, autoimmune pancreatitis, bullous skin disorders, chronic inflammatory demyelinating polyneuropathy, Evans syndrome, Graves' ophthalmopathy, idiopathic thrombocytopenic purpura (ITP), IgG4-related disease, pure red cell aplasia, multiple sclerosis, neuromyelitis optica (Devic's disease), opsoclonus myoclonus syndrome (OMS), rheumatoid arthritis, Sjogren's syndrome, systemic lupus erythematosus, thrombotic thrombocytopenic purpura (TTP), type 1 diabetes mellitus, and vasculitis. In another embodiment of the methods disclosed herein, the vasculitis is granulomatosis with polyangiitis (GPA) (Wegner's granulomatosis) or microscopic polyangiitis (MPA).

In some embodiments of the methods disclosed herein, the B-cell disorder is cancer. In other embodiments, provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound A


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody.

In other embodiments, provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound B


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody, wherein the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In another embodiment, provided herein are methods for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof,whereinring B is aryl or heteroaryl;R1is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C1-6-alkyl;andR is H or C1-6-alkyl; and

b) a CD20 inhibitory antibody. In an embodiment, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In an embodiment of Formula I, R1is an aryl that is substituted by OH, halo, or C1-6-alkyl.

In another embodiment of the methods provided herein, the cancer is a hematologic malignancy. In yet another embodiment, the cancer is a leukemia, a lymphoma, or a myeloma. In still another embodiment, the cancer is selected from the group consisting of acute lymphoblastic leukemia, B-cell leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, B-cell lymphoma, Burkitt lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, lymphocyte predominant subtype of Hodgkin's lymphoma, mantle cell lymphoma, and multiple myeloma. In a further embodiment, the cancer is selected from the group consisting of chronic lymphocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma. In another embodiment, the cancer is multiple myeloma.

In other aspects, provided herein are methods for upregulating antibody-dependent cell-mediated cytotoxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound A


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody.

In another aspect, provided herein are methods for upregulating antibody-dependent cell-mediated cytotoxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound B


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody, wherein the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In another embodiment, provided herein are methods for upregulating antibody-dependent cell-mediated cytotoxicity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof,whereinring B is aryl or heteroaryl;R1is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C1-6-alkyl;andR is H or C1-6-alkyl; and

b) a CD20 inhibitory antibody. In an embodiment, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In an embodiment of Formula I, R1is an aryl that is substituted by OH, halo, or C1-6-alkyl.

In yet another aspect, provided herein are methods for upregulating lymphocyte functional activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound A


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody.

In still another aspect, provided herein are methods for upregulating lymphocyte functional activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound B


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody, wherein the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In another embodiment, provided herein are methods for upregulating lymphocyte functional activity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof,whereinring B is aryl or heteroaryl;R1is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C1-6-alkyl;andR is H or C1-6-alkyl; and

b) a CD20 inhibitory antibody. In an embodiment, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In an embodiment of Formula I, R1is an aryl that is substituted by OH, halo, or C1-6-alkyl.

In some embodiments of the methods disclosed herein, the lymphocyte is a natural killer cell. In other embodiments, the lymphocyte is a T lymphocyte. In another embodiment, the lymphocyte is a B lymphocyte. In yet another embodiment, the B lymphocyte is a follicular B lymphocyte. In still another embodiment, the B lymphocyte is a diffuse large B-cell.

In other embodiments of the methods disclosed herein, the expression of CD20 on a cancer cell is upregulated. In some embodiments of the methods disclosed herein, the expression of CD20 on a chronic lymphocytic leukemia cell is upregulated. In another embodiment of the methods disclosed herein, the expression of CD20 on a non-Hodgkin's lymphoma cell is upregulated. In still another embodiment of the methods disclosed herein, the expression of CD20 on a multiple myeloma cell is upregulated.

In yet another aspect, provided herein are methods of upregulating the expression of CD20 in a cell comprising contacting the cell with an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound A


or a pharmaceutically acceptable salt thereof.

In still another aspect, provided herein are methods of upregulating the expression of CD20 in a cell comprising contacting the cell with an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound B



or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein are methods for upregulating the expression of CD20 in a cell comprising contacting the cell with an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof,whereinring B is aryl or heteroaryl;R1is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C1-6-alkyl;andR is H or C1-6-alkyl. In an embodiment, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In an embodiment of Formula I, R1is an aryl that is substituted by OH, halo, or C1-6-alkyl.

In some embodiments of the methods disclosed herein, the cell is a cancer cell. In another embodiment, the cell is a chronic lymphocytic leukemia cell. In still another embodiment, the cell is a non-Hodgkin's lymphoma cell. In yet another embodiment, the cell is a multiple myeloma cell.

In other embodiments of the methods disclosed herein, the viability of a peripheral blood mononuclear cell is maintained. In another embodiment, the peripheral blood mononuclear cell is a CD20+ B lymphocyte, a CD20+ follicular B lymphocyte, or a CD20+ diffuse large B-cell.

In some embodiments of the methods and the pharmaceutical combinations disclosed herein, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, rituximab, SBI-087, TRU-015, ublituximab, and veltuzumab.

In other embodiments of the methods and the pharmaceutical combinations disclosed herein, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In one embodiment of the methods and the pharmaceutical combinations disclosed herein, the CD20 inhibitory antibody comprises three heavy chain complementarity determining regions (CDRs) set forth in SEQ ID NOs: 1-3 and three light chain CDRs set forth in SEQ ID NOs: 4-6. In another embodiment, the CD20 inhibitory antibody comprises a heavy chain variable region set forth in SEQ ID NO: 7, and a light chain variable region set forth in SEQ ID NO: 8. In yet another embodiment, the CD20 inhibitory antibody comprises a heavy chain variable region set forth in SEQ ID NO: 62, and a light chain variable region set forth in SEQ ID NO: 8. In still another embodiment, the CD20 inhibitory antibody comprises a heavy chain set forth in SEQ ID NO: 9, and a light chain set forth in SEQ ID NO: 10.

In yet another embodiment of the methods and the pharmaceutical combinations disclosed herein, the CD20 inhibitory antibody comprises three heavy chain CDRs set forth in SEQ ID NOs: 11-13 and three light chain CDRs set forth in SEQ ID NOs: 14-16. In some embodiments, the CD20 inhibitory antibody comprises a heavy chain variable region set forth in SEQ ID NO: 17, and a light chain variable region set forth in SEQ ID NO: 18. In another embodiment, the CD20 inhibitory antibody comprises a heavy chain set forth in SEQ ID NO: 19, and a light chain set forth in SEQ ID NO: 20.

In still another embodiment of the methods and the pharmaceutical combinations disclosed herein, the CD20 inhibitory antibody comprises three heavy chain CDRs set forth in SEQ ID NOs: 21-23 and three light chain CDRs set forth in SEQ ID NOs: 24-26. In another embodiment, the CD20 inhibitory antibody comprises a heavy chain variable region set forth in SEQ ID NO: 27, and a light chain variable region set forth in SEQ ID NO: 28. In some embodiments, the CD20 inhibitory antibody comprises a heavy chain set forth in SEQ ID NO: 29, and a light chain set forth in SEQ ID NO: 30.

In some embodiments of the methods disclosed herein, the HDAC6 selective inhibitor and the CD20 inhibitory antibody are administered at different times. In another embodiment, the HDAC6 selective inhibitor is administered orally, and the CD20 inhibitory antibody is administered intravenously.

In other embodiments of the methods disclosed herein, the cancer is resistant or refractory to treatment with the CD20 inhibitory antibody. In another embodiment of the methods disclosed herein, the chronic lymphocytic leukemia is resistant or refractory to treatment with the CD20 inhibitory antibody. In still another embodiment of the methods disclosed herein, the non-Hodgkin's lymphoma is resistant or refractory to treatment with the CD20 inhibitory antibody. In yet another embodiment of the methods disclosed herein, the multiple myeloma is resistant or refractory to treatment with the CD20 inhibitory antibody. In further embodiments of the methods disclosed herein, the cancer is resistant or refractory to treatment with a CD20 inhibitory antibody selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, rituximab, SBI-087, TRU-015, ublituximab, and veltuzumab.

In another aspect, provided herein are pharmaceutical combinations, wherein the combination comprises a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound A


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody.

In still another aspect, provided herein are pharmaceutical combinations, wherein the combination comprises a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is Compound B


or a pharmaceutically acceptable salt thereof; and

b) a CD20 inhibitory antibody, wherein the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In another embodiment, provided herein are pharmaceutical combinations, wherein the combination comprises a therapeutically effective amount of:

a) an HDAC6 selective inhibitor, wherein the HDAC6 selective inhibitor is a compound of Formula I

or a pharmaceutically acceptable salt thereof,whereinring B is aryl or heteroaryl;R1is an aryl or heteroaryl, each of which may be optionally substituted by OH, halo, or C1-6-alkyl;andR is H or C1-6-alkyl; and

b) a CD20 inhibitory antibody. In an embodiment, the CD20 inhibitory antibody is selected from the group consisting of FBTA05, MT-3724, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, PRO131921, SBI-087, TRU-015, ublituximab, and veltuzumab.

In an embodiment of Formula I, R1is an aryl that is substituted by OH, halo, or C1-6-alkyl.

In some embodiments of the pharmaceutical combinations disclosed herein, the combination further comprises one or more pharmaceutically acceptable carriers.

Also provided herein are uses of the pharmaceutical combinations disclosed herein for the manufacture of a pharmaceutical preparation or medicament for the treatment of cancer.

Other objects, features, and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the disclosure.

