INHIBITOR

RELATED APPLICATION

[0001] This present application claims benefit of priority from U.S. Provisional Patent Application No. 62/052,361, filed September 18, 2014, which is herein incorporated by reference in its entirety.

BACKGROUND

[0002] Bruton's tyrosine kinase (Btk), a member of the Tec family of non-receptor tyrosine kinases, is a key signaling enzyme expressed in all hematopoietic cells types except T lymphocytes and natural killer cells. Btk plays an essential role in the B-cell signaling pathway linking cell surface B-cell receptor (BCR) stimulation to downstream intracellular responses. BTK is also expressed by murine dendritic cells (DCs) and has been observed to augment TLR- 4 signaling in myeloid cells. Further, BTK enables DCs to regulate T cell proliferation and differentiation.

[0003] Ibrutinib is an irreversible BTK and ITK inhibitor and has been shown to be effective at treating several B cell malignancies. l-((R)-3-(4-amino-3-(4-phenoxyphenyl)-lH- pyrazolo[3,4-d]pyrimidin-l-yl)piperidin-l-yl)prop-2-en-l-one is also known by its IUPAC name as l-{(3R)-3-[4-amino-3-(4-phenoxyphenyl)-lH-pyrazolo[3,4-JJpyr imidin-l-yl]piperidin-l- yl}prop-2-en-l-one or 2-Propen-l-one, l-[(3i?)-3-[4-amino-3-(4-phenoxyphenyl)-lH- pyrazolo[3,4-if|pyrimidin-l-yl]-l-piperidinyl-, and has been given the USAN name, Ibrutinib. The various names given for Ibrutinib are used interchangeably herein.

SUMMARY OF THE INVENTION

[0004] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising an effective amount of a BTK inhibitor for promoting Thl7 cell differentiation in a subject having cancer. In some embodiments, the Thl7 cell differentiation leads to secretion of Thl7 associated cytokines. In some embodiments, the Thl7 associated cytokines include IL17A, IL17F, IL21, and IL22. In some embodiments, the Thl7 cell differentiation leads to an increase in secretion of Thl7 associated cytokines compared to reference levels. In some embodiments, the BTK inhibitor further induces dendritic cells to secrete co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and the cytokines promote Thl7 cell differentiation. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80. In some embodiments, the co -stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD86. In some embodiments, the cytokines are selected from IL-Ιβ, IL-6, IL-18, TGF-β, or a combination thereof. In some embodiments, the expression level of the cytokines are increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to control expression levels of the cytokines. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and

nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some

embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the BTK inhibitor is ibrutinib. In some embodiments, ibrutinib is administered once a day, two times per day, three times per day, four times per day, or five times per day. In some embodiments, ibrutinib is administered at a dosage of about 40 mg/day to about 1000 mg/day. In some embodiments, ibrutinib is administered orally.

[0005] Disclosed herein, in certain embodiments, is a method of treating cancer in a subject in need thereof by promoting Thl7 cell differentiation in the subject, comprising administering to the subject an amount of a BTK inhibitor effective to promote Thl7 cell differentiation in the subject. In some embodiments, the Thl7 cell differentiation leads to secretion of Thl7 associated cytokines. In some embodiments, the Thl7 associated cytokines include IL17A, IL17F, IL21 , and IL22. In some embodiments, the Thl7 cell differentiation leads to an increase in secretion of Thl7 associated cytokines compared to a reference level. In some embodiments, the BTK inhibitor further induces dendritic cells to secrete co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and the cytokines promote Thl7 cell differentiation. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80. In some embodiments, the co -stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or less relative to a control expression level of CD86. In some embodiments, the cytokines are selected from IL-Ιβ, IL-6, IL-18, TGF-β, or a combination thereof. In some embodiments, the expression level of the cytokines are increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%), 80%), 90%, 100%), or more relative to control expression levels of the cytokines. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and

nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some

embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the BTK inhibitor is ibrutinib. In some embodiments, ibrutinib is administered once a day, two times per day, three times per day, four times per day, or five times per day. In some embodiments, ibrutinib is administered at a dosage of about 40 mg/day to about 1000 mg/day. In some embodiments, ibrutinib is administered orally.

[0006] Disclosed herein, in certain embodiments, is a method of treating cancer in a subject in need thereof by increasing the expression of one or more cytokines selected from IL-17A, IL17F, IL21 , and IL-22 comprising administering to the subject an amount of a BTK inhibitor effective to increase the expression of the cytokines in the subject. In some embodiments, the expression of co-stimulatory molecules is increased with the administration of the BTK inhibitor. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80. In some embodiments, the expression of co-stimulatory molecules is decreased with the administration of the BTK inhibitor. In some embodiments, the co-stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or less relative to a control expression level of CD86. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a relapsed or refractory cancer. In some

embodiments, the BTK inhibitor is ibrutinib. In some embodiments, ibrutinib is administered once a day, two times per day, three times per day, four times per day, or five times per day. In some embodiments, ibrutinib is administered at a dosage of about 40 mg/day to about 1000 mg/day. In some embodiments, ibrutinib is administered orally. [0007] Disclosed herein, in certain embodiments, is a pharmaceutical composition comprising an effective amount of a BTK inhibitor for treating dendritic cells thereby inducing the dendritic cells to modulate the expression levels of co -stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80. In some embodiments, the co -stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or less relative to a control expression level of CD86. In some embodiments, the cytokines are selected from IL-Ιβ, IL-6, IL-18, TGF-β, or a combination thereof. In some embodiments, the expression level of the cytokines are increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%), 80%), 90%, 100%), or more relative to control expression levels of the cytokines. In some embodiments, the Thl7 cell differentiation leads to secretion of Thl 7 associated cytokines. In some embodiments, the Thl7 associated cytokines include IL17A, IL17F, IL21, and IL22. In some embodiments, the Thl7 cell differentiation leads to an increase in secretion of Thl7 associated cytokines compared to a reference level. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the BTK inhibitor is ibrutinib.

[0008] Disclosed herein, in certain embodiments, is a method of generating dendritic cells comprising treating the dendritic cells with an amount of a BTK inhibitor effective to induce the dendritic cells to modulate the expression levels of co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%), 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80. In some embodiments, the co -stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or less relative to a control expression level of CD86. In some embodiments, the cytokines are selected from IL-Ιβ, IL-6, IL-18, TGF-β, or a combination thereof. In some embodiments, the expression level of the cytokines are increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%), 90%, 100%), or more relative to control expression levels of the cytokines. In some embodiments, the Thl7 cell differentiation leads to secretion of Thl 7 associated cytokines. In some embodiments, the Thl7 associated cytokines include IL17A, IL17F, IL21, and IL22. In some embodiments, the Thl7 cell differentiation leads to an increase in secretion of Thl7 associated cytokines compared to a reference level. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a relapsed or refractory cancer. In some embodiments, the BT inhibitor is ibrutinib.

[0009] Disclosed herein, in certain embodiments, is a population of dendritic cells wherein the dendritic cells are treated with an amount of a BTK inhibitor effective to induce the dendritic cells to modulate the expression levels of co -stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80. In some embodiments, the co -stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or less relative to a control expression level of CD86. In some embodiments, the cytokines are selected from IL-Ιβ, IL-6, IL-18, TGF-β, or a combination thereof. In some embodiments, the expression level of the cytokines are increased by about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%), 80%), 90%, 100%), or more relative to control expression levels of the cytokines. In some embodiments, the BTK inhibitor is ibrutinib.

[0010] Disclosed herein, in certain embodiments, is a method of treating a cancer comprising administering to a patient in need thereof a therapeutic effective amount of ibrutinib treated- dendritic cells. In some embodiments, the ibrutinib treated-dendritic cells promote Thl7 cell differentiation. In some embodiments, the Thl7 cell differentiation leads to secretion of Thl7 associated cytokines. In some embodiments, the Thl7 associated cytokines include IL17A, IL17F, IL21 , and IL22. In some embodiments, the Thl7 cell differentiation leads to an increase in secretion of Thl7 associated cytokines compared to reference levels. In some embodiments, the ibrutinib treated-dendritic cells secrete co -stimulatory molecules and cytokines, wherein the co-stimulatory molecules and the cytokines promote Thl7 cell differentiation. In some embodiments, the co-stimulatory molecule is CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%), 90%, 100%), or more relative to a control expression level of CD80. In some embodiments, the co-stimulatory molecule is CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or less relative to a control expression level of CD86. In some embodiments, the cytokines are selected from IL-Ι β, IL-6, IL-18, TGF-β, or a combination thereof. In some embodiments, the expression level of the cytokines are increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to control expression levels of the cytokines. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a relapsed or refractory cancer.

[0011] In some embodiments, a pharmaceutical composition is provided. The pharmaceutical composition preferably comprises an effective amount of a BTK inhibitor for promoting maturation of dendritic cells in a subject having cancer. In some embodiments, the

pharmaceutical composition comprises an effective amount of a BTK inhibitor for promoting the activation of dendritic cells in a subject having cancer. In some embodiments, the BTK inhibitor modulates the expression level of one or more surface markers on dendritic cells that are associated with dendritic cell maturation and/or activation. In some embodiments, the BTK inhibitor increases the expression levels of surface markers on dendritic cells that are associated with dendritic cell maturation and/or activation. In some embodiments, the surface markers are CCR7, MHC-II, and/or CD1 lc. In some embodiments, the BTK inhibitor decreases the expression levels of surface markers on dendritic cells that are associated with dendritic cell maturation and/or activation. In some embodiments, the surface marker is Ly6C. In some embodiments, the BTK inhibitor induces dendritic cells to promote T-cell proliferation. In some embodiments, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. An exemplary hematological malignancy is a B-cell malignancy. Exemplary B-cell malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is relapsed or refractory cancer. In some embodiments, the cancer is treatment naive cancer. In some embodiments, the BTK inhibitor is ibrutinib. In some embodiments, ibrutinib is administered once per day, two times per day, three times per day, four times per day, or five times per day. In some embodiments, ibrutinib is administered at a dosage of about 40 mg/day to about 1000 mg/day. In some embodiments, ibrutinib is administered orally.

[0012] In some embodiments, a method of treating cancer in a subject in need thereof is provided. The method includes the step of promoting maturation of dendritic cells in the subject, comprising administering to the subject an amount of BTK inhibitor effective to promote dendritic cell maturation in the subject. In some embodiments, the method includes the step of promoting activation of dendritic cells in the subject, comprising administering to the subject an amount of BTK inhibitor effective to promote dendritic cell activation in the subject. In some embodiments, the BTK inhibitor modulates the expression level of one or more surface markers on dendritic cells that are associated with dendritic cell maturation and/or activation. In some embodiments, the BTK inhibitor increases the expression levels of surface markers on dendritic cells that are associated with dendritic cell maturation and/or activation. In some embodiments, the surface markers are CCR7, MHC-II, and/or CD1 lc. In some embodiments, the BTK inhibitor decreases the expression levels of surface markers on dendritic cells that are associated with dendritic cell maturation and/or activation. In some embodiments, the surface marker is Ly6C. In some embodiments, the BTK inhibitor induces dendritic cells to promote T- cell proliferation. In some embodiments, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is a hematological malignancy. An exemplary hematological malignancy is a B-cell malignancy. Exemplary B- cell malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some

embodiments, the cancer is relapsed or refractory cancer. In some embodiments, the cancer is treatment na ^' ive cancer. In some embodiments, the BTK inhibitor is ibrutinib. In some embodiments, ibrutinib is administered once per day, two times per day, three times per day, four times per day, or five times per day. In some embodiments, ibrutinib is administered at a dosage of about 40 mg/day to about 1000 mg/day. In some embodiments, ibrutinib is administered orally. In some embodiments, the BTK inhibitor further induces dendritic cells to secrete co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines promote dendritic cell maturation. In some embodiments, the co-stimulatory molecule is CD80.

[0013] In some embodiments, a method of treating a cancer in a subject in need thereof, by promoting dendritic-cell mediated T cell proliferation, is provided. The method preferably includes the step of administering to the subject an amount of BT inhibitor effective to promote dendritic-cell mediated T cell proliferation in the subject. In some embodiments, the BTK inhibitor modulates the secretion of cytokines. In some embodiments, the BTK inhibitor reduces the secretion of cytokines compared to reference levels. Exemplary cytokines include IL-12, IL-6, and TNF-a. In some embodiments, the BTK inhibitor increases the secretion of cytokines compared to reference levels. Exemplary cytokines include IFN-γ, IL-17, and IL-13.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Various aspects of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0015] Fig. lA-Fig. ID illustrate ibrutinib modulates murine dendritic cell activation upon LPS stimulation. Fig. 1A shows TNF-a, IL-10, IL-12 and NO production in control and Ibrutinib-treated DCs stimulated with LPS as determined by ELISA and Griess assay. The data are presented as mean + S.E.M of triplicate sample values from three independent experiments. Fig. IB shows real-time qPCR analysis of IL-6, TGF-β, IL-18 and IL-Ιβ mRNA induction in control and Ibrutinib-treated DCs upon LPS stimulation. The data are presented as mean + S.E.M of duplicates obtained by pooling three independent samples. Fig. 1C illustrates histogram plots showing expressions of MHC-II, CD86, CD80 and CD40 on control and Ibrutinib-treated DCs stimulated with LPS. Analyses were conducted by gating on CD1 lc ⁺ population. Numbers represent percentage of cells expressing the respective surface molecule on the gated population. The data presented are representative plots of three independent experiments. Fig. ID illustrates mean fluorescence intensities (MFIs) of MHC-II, CD86, CD80 and CD40 expression on control and Ibrutinib-treated DCs stimulated with LPS. The data are presented as mean + S.E.M of representative MFI values of three independent experiments.

[0016] Fig. 2A-Fig. 2D illustrate ibrutinib treated DCs promote Thl7 differentiation. Fig. 2A shows the analysis of T cell proliferation upon co-culture with control, ibrutinib, LPS or LPS/ibrutinib-treated DCs. Production of T cell derived cytokines IL-17 (Fig. 2B), IFN-γ (Fig. 2C), and IL-13 (Fig. 2D) were determined by ELISA in co-culture experiments with DCs stimulated as mentioned in Fig. 2A. DCs were pulsed with OVA prior to stimulation and incubated with CFSE stained T cells. Analyses were conducted by gating on CD4 population. The data are presented as mean + S.E.M of duplicates and are representative of two independent experiments.

[0017] Figs. 3A-3C illustrate that ibrutinib treatment enhances the development and maturation of dendritic cells. Fig. 3 A illustrates dot plots showing the percentage of CD1 lc ⁺ DCs in untreated and ibrutinib -treated DC cultures. The numbers are presented as mean + S.E.M of representative values from three independent experiments. Fig. 3B illustrates histogram plots showing expressions of Ly6C, MHC-II, CD80 and CD40 on control (untreated) and ibrutinib- treated DCs stimulated with LPS. As shown, ibrutinib treatment increased the expression of surface markers MHC-II and CD80 on DCs and decreased the expression of Ly6C on DCs. Fig. 3C illustrates mean fluorescence intensities (MFIs) of Ly6C, MHC-II, and CD80 expression on control and ibrutinib-treated DCs stimulated with LPS. The data are presented as mean + S.E.M of representative MFI values from three independent experiments. Bone marrow cells from C57BL/6 mice were cultured in the presence of GMCSF + ΙμΜ ibrutinib for 7 days to generate untreated- and ibrutinib-treated DCs. At Day 7, DCs were stained with fluorescently labelled antibodies for CD1 lc, Ly6C, MHC-II and CD80 and the expressions of the respective surface markers were determined by flow cytometry.

[0018] Figs. 4A-4C illustrate that ibrutinib differentially regulates the expression of MHC-II, co-stimulatory molecules, and CCR7 on LPS-treated DCs. Fig. 4A illustrates histogram plots showing expressions of MHC-II, CD80, CD86, and CD40 on control (untreated) and ibrutinib- treated DCs stimulated with LPS. As shown, ibrutinib treatment increased the expression of surface markers MHC-II and CD80 on DCs and decreases percentage of CD86 DCs upon LPS stimulation. Fig. 4B illustrates the mean fluorescence intensities (MFIs) of MHC-II, CD80, CD86, and CD40 on control (untreated) and ibrutinib-treated DCs stimulated with LPS. The data are presented as mean + S.E.M of representative MFI values from three independent experiments. Fig. 4C illustrates dot plots of the percentage of CCR7+ DCs in LPS/ibrutinib- treated DC cultures compared to LPS/untreated DC cultures. The numbers represent mean + S.E.M of representative duplicates values from two independent experiments. Untreated and ibrutinib-treated DCs were treated with control (media) or LPS (1 μg/ml) for 24 hours. After 24 hours, cells were blocked, stained with fluorescently labelled antibody for the respective surface molecules and expressions of the surface molecules was determined by flow cytometry.

Analyses were conducted by gating on CD1 lc ⁺ DCs.

[0019] Fig. 5A-5F illustrate that ibrutinib differentially regulates cytokine production and nitric oxide (NO) responses by LPS-treated DCs. Figs. 5A-5F shows 11-10, IFN-β, IL-6, IL-12, NO, and TNF- production, respectively, in control (untreated) and ibrutinib-treated DCs stimulated with LPS. As shown, LPS/ibrutinib-treated DCs produced higher levels of IL-10 and IFN-β, and lower levels of IL-6, IL-12, and NO. Untreated- and ibrutinib-treated DCs were treated with control (media) or LPS (1 μg/ml). After 24 hours of LPS treatment, cytokine production was determined in the culture supernatants by ELISA. After 48 hours of LPS treatment, NO levels were determined in the culture supernatants by measuring nitrite concentrations using Griess assay. The data are presented as mean + S.E.M of triplicate sample values from two independent experiments.

[0020] Figs. 6A-6D illustrate that ibrutinib-treated DCs enhance T cell proliferation and production of T cell derived cytokines. Fig. 6A illustrates a graphical representation of T cell proliferation upon co-culture with untreated-, ibrutinib-, LPS/untreated- or LPS/ibrutinib-treated DCs. Untreated- and ibrutinib-treated DCs were pulsed with OVA (10 μg/ml) for 2 hours and treated with LPS (1 μ /ηιι) for 22 hours. After OVA/LPS stimulation, DCs were cultured in 1 :4 ratio with CFSE-stained T cells enriched from spleens of OT-II mice for 6 days. At Day 6, cells from co-culture were blocked, stained with anti-CD4 antibody and T cell proliferation was measured by flow cytometry. Analyses were conducted by gating on CD4 ⁺ population. The data are presented as mean + S.E.M of duplicates and are representative of two independent experiments. Figs. 6B-6D illustrate graphical representations of production of T cell cytokines IFN-γ, IL-17, and IL-13, respectively, in co-culture experiments as described above. At Day 7 of co-culture, cell culture supernatants were collected and the respective cytokines were measured by ELISA. The data are presented as mean + S.E.M of duplicates and are representative of two independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

Certain Terminology

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise.

Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0022] As used herein, ranges and amounts can be expressed as "about" a particular value or range. About also includes the exact amount. Hence "about 5 μί" means "about 5 μί" and also "5 μ " Generally, the term "about" includes an amount that would be expected to be within experimental error.

[0023] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0024] "Antibodies" and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. The terms are used synonymously. In some instances the antigen specificity of the immunoglobulin is known.

[0025] The term "antibody" is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind antigen (e.g., Fab, F(ab') ₂ , Fv, single chain antibodies, diabodies, antibody chimeras, hybrid antibodies, bispecific antibodies, humanized antibodies, and the like), and recombinant peptides comprising the forgoing.

[0026] The terms "monoclonal antibody" and "mAb" as used herein refer to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.

[0027] Native antibodies" and "native immunoglobulins" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy-chain variable domains.

[0028] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies. Variable regions confer antigen-binding specificity. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions, both in the light chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are celled in the framework (FR) regions. The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-pleated-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-pleated-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al. (1991) NIH PubL. No. 91-3242, Vol. I, pages 647-669). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as Fc receptor (FcR) binding, participation of the antibody in antibody-dependent cellular toxicity, initiation of complement dependent cytotoxicity, and mast cell degranulation.

[0029] The term "hypervariable region," when used herein, refers to the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a "complementarily determining region" or "CDR" (i.e., residues 24- 34 (LI), 50-56 (L2), and 89-97 (L3) in the light-chain variable domain and 31-35 (HI), 50-65 (H2), and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, Md.) and/or those residues from a "hypervariable loop" (i.e., residues 26-32 (LI), 50- 52 (L2), and 91 -96 (L3) in the light-chain variable domain and (HI), 53-55 (H2), and 96-101 (13) in the heavy chain variable domain; Clothia and Lesk, (1987) J. Mol. Biol., 196:901 -917). "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues, as herein deemed.

[0030] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen- binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab, F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 10: 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0031] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0032] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. Fab' fragments are produced by reducing the F(ab')2 fragment's heavy chain disulfide bridge. Other chemical couplings of antibody fragments are also known.

[0033] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0034] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, human IgGl and IgG3 isotypes have ADCC (antibody dependent cell-mediated cytotoxicity) activity.

[0035] As used herein, the terms "individual(s)", "subject(s)" and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician's assistant, an orderly or a hospice worker).

Methods of Use

[0036] Dendritic cells (DCs) are professional antigen presenting cells that initiate, promote, and regulate adaptive immune responses. DCs are present in the skin, and the inner linings of the nose, lungs, stomach and intestines. DCs are also present in an immature state in the blood. Once activated, they migrate to the lymph nodes where interaction with T and B cells lead to shaping of the adaptive immune response. In general, two subsets of DCs are present, conventional dendritic cells (or myeloid dendritic cells) (cDC or mDC) and plasmacytoid DC. In the blood, three types of DCs are present, including CD1 lc+ myeloid DCs, the CD 141+ myeloid DCs, and the CD303+ plasmacytoid DCs.

[0037] DCs are derived from hematopoietic bone marrow progenitor cells, where they exist has immature dendritic cells. Armed with pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), DCs constantly sample its surrounding area for pathogens such as viruses and bacteria. Upon establishing contact with a presentable antigen, they are then transformed into mature DCs and begin migration into the lymph nodes. The presentable antigen is presented on the surface of the DCs using major histone class (MHC) molecules. Further, DCs also upregulate cell-surface receptors that act as co-receptors in T-cell activation (CD8+, CD4+ T cells) such as co-stimulatory molecules such as CD80, CD86, and CD40, which enhances activation of T-cells, and cytokines. As used herein, cytokines include, for example, interlukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-22, IL-23, IL-35, IF a, IFNp, IFNy, TNF-a, chemokines, TGF-β, G-CSF, GM-GSF, and TSLP.

[0038] Bruton's tyrosine kinase is a key component in the B cell receptor (BCR) signaling pathway, and participates in regulation of cell proliferation and cell survival. One study had revealed that BTK is expressed in murine bone marrow derived dendritic cells (BMDCs).

Further, in some instances BTK enhances innate immune responses and participates in TLR-4 mediated myeloid cell activation. In some cases, BTK also enables DCs to regulate T cell proliferation and differentiation. Ibrutinib is an inhibitor of BTK. In some cases, ibrutinib has been shown to modulate B cell, T cell and NK cell functions. In some cases, ibrutinib also modulates DC cell functions.

[0039] DC-derived cytokines such as IL-6 and TGF-β are important for initiation of Thl7 immune response. Thl7 cells are a subset of activated CD4+ T cells which are responsive to IL- 1R1 and IL-23R signaling. They promote neutrophil activation, provide immunity against pathogens, and in some cases, induces inflammation. Thl7 cell differentiation is induced by cytokines such as IL-6, IL-21, IL-23, IL-Ιβ, IL-18, and TGF-β. Upon differentiation, Thl7 cells secret IL17A, IL17B, IL-21, and IL22.

[0040] In some embodiments, the levels of Thl7 secreted cytokines are elevated in certain types of cancers. In some cases, elevated levels of Thl7 secreted cytokines correspond to a better prognosis for a patient suffering from the cancer. In some cases, elevated levels of Thl7 secreted cytokines correspond to a poor prognosis for a patient suffering from the cancer.

[0041] In certain aspects, disclosed herein, are methods, compositions, and kits comprising an effective amount of a TEC inhibitor for treating dendritic cells thereby inducing the dendritic cells to modulate the expression levels of co -stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In some embodiments, the TEC inhibitor is an ITK inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor. In some embodiments, the BTK inhibitor is selected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-lOl/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI- 1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-1 1066 (also, CTK4I7891 , HMS3265G21 , HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13. In some embodiments, the BT inhibitor is ibrutinib.

[0042] In some embodiments, disclosed herein, are methods, compositions, and kits comprising an effective amount of a BTK inhibitor for treating dendritic cells thereby inducing the dendritic cells to modulate the expression levels of co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation.

[0043] In some embodiments, disclosed herein, are methods, compositions, and kits comprising an effective amount of ibrutinib for treating dendritic cells thereby inducing the dendritic cells to modulate the expression levels of co -stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation.

[0044] In some embodiments, methods, compositions, and kits comprising an effective amount of ibrutinib for promoting maturation of dendritic cells in a subject having cancer is provided.

[0045] In some embodiments, methods, compositions, and kits comprising an effective amount of ibrutinib for promoting dendritic-cell mediated T cell proliferation is provided.

[0046] Also disclosed herein, in certain embodiments, is a population of dendritic cells wherein the dendritic cells are treated with an amount of a TEC inhibitor effective to induce the dendritic cells to modulate the expression levels of co -stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In some embodiments, the TEC inhibitor is an ITK inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor. In some embodiments, the BTK inhibitor is selected from among ibrutinib (PCI-32765), PCI- 45292, PCI-45466, AVL-lOl/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC- 263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI- 1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-1 1066 (also, CTK4I7891 , HMS3265G21 , HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13. In some embodiments, the BTK inhibitor is ibrutinib.

[0047] In some embodiments, is a population of dendritic cells wherein the dendritic cells are treated with an amount of a BTK inhibitor effective to induce the dendritic cells to modulate the expression levels of co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation.

[0048] In some embodiments, is a population of dendritic cells wherein the dendritic cells are treated with an amount of ibrutinib effective to induce the dendritic cells to modulate the expression levels of co-stimulatory molecules and cytokines, wherein the co-stimulatory molecules and cytokines are capable of inducing Thl7 cells to undergo differentiation.

[0049] In certain aspects, disclosed herein include a method of treating a cancer comprising administering to a patient in need thereof a therapeutic effective amount of a TEC inhibitor treated-dendritic cells. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In some embodiments, the TEC inhibitor is an ITK inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor. In some embodiments, the BTK inhibitor is selected from among ibrutinib (PCI-32765), PCI- 45292, PCI-45466, AVL-lOl/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC- 263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI- 1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-1 1066 (also, CTK4I7891 , HMS3265G21 , HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13. In some embodiments, the BTK inhibitor is ibrutinib.

[0050] In some embodiments, disclosed herein include a method of treating a cancer comprising administering to a patient in need thereof a therapeutic effective amount of a BTK inhibitor treated-dendritic cells. [0051] In some embodiments, disclosed herein include a method of treating a cancer comprising administering to a patient in need thereof a therapeutic effective amount of ibrutinib treated-dendritic cells.

[0052] In certain aspects, further disclosed herein include methods of generating a TEC inhibitor treated dendritic cell-based vaccines for treating a cancer in a subject in need thereof. Also described include methods of generating the TEC inhibitor treated dendritic cell-based vaccines. In some embodiments, described herein include methods of generating the TEC inhibitor treated dendritic cell-based vaccines for treating a pathogen associated with cancer. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In some embodiments, the TEC inhibitor is an ITK inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor. In some embodiments, the BTK inhibitor is selected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC- 101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY- 1 1066 (also, CTK4I7891 , HMS3265G21 , HMS3265G22, HMS3265H21 , HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13. In some embodiments, the BTK inhibitor is ibrutinib.

[0053] In some embodiments, further disclosed herein include methods of generating a BTK inhibitor treated dendritic cell-based vaccines for treating a cancer in a subject in need thereof. Also described include methods of generating the BTK inhibitor treated dendritic cell-based vaccines. In some embodiments, described herein include methods of generating the BTK inhibitor treated dendritic cell-based vaccines for treating a pathogen associated with cancer.

[0054] In some embodiments, further disclosed herein include methods of generating ibrutinib treated dendritic cell-based vaccines for treating a cancer in a subject in need thereof. Also described include methods of generating ibrutinib treated dendritic cell-based vaccines. In some embodiments, described herein include methods of generating ibrutinib treated dendritic cell- based vaccines for treating a pathogen associated with cancer.

[0055] In certain aspects, disclosed herein, are methods, compositions, and kits comprising an effective amount of a TEC inhibitor for promoting Thl7 cell differentiation in a subject having cancer. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In some embodiments, the TEC inhibitor is an ITK inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor. In some

embodiments, the BTK inhibitor is selected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-lOl/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI- 1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-1 1066 (also, CTK4I7891 , HMS3265G21 , HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13. In some embodiments, the BTK inhibitor is ibrutinib.

[0056] In some embodiments, disclosed herein, are methods, compositions, and kits comprising an effective amount of a BTK inhibitor for promoting Thl7 cell differentiation in a subject having cancer.

[0057] In some embodiments, disclosed herein, are methods, compositions, and kits comprising an effective amount of ibrutinib for promoting Thl7 cell differentiation in a subject having cancer.

[0058] As used herein, cancer refers to a solid tumor or a hematological cancer. In some embodiments, cancer refers to a solid tumor. In some embodiments, the solid tumor is a sarcoma or carcinoma. In some embodiments, the solid tumor is a sarcoma. In some embodiments, the solid tumor is a carcinoma. In some embodiments, the sarcoma is selected from alveolar rhabdomyosarcoma; alveolar soft part sarcoma; ameloblastoma; angiosarcoma;

chondrosarcoma; chordoma; clear cell sarcoma of soft tissue; dedifferentiated liposarcoma; desmoid; desmoplastic small round cell tumor; embryonal rhabdomyosarcoma; epithelioid fibrosarcoma; epithelioid hemangioendothelioma; epithelioid sarcoma; esthesioneuroblastoma; Ewing sarcoma; extrarenal rhabdoid tumor; extraskeletal myxoid chondrosarcoma; extraskeletal osteosarcoma; fibrosarcoma; giant cell tumor; hemangiopericytoma; infantile fibrosarcoma; inflammatory myofibroblastic tumor; Kaposi sarcoma; leiomyosarcoma of bone; liposarcoma; liposarcoma of bone; malignant fibrous histiocytoma (MFH); malignant fibrous histiocytoma (MFH) of bone; malignant mesenchymoma; malignant peripheral nerve sheath tumor;

mesenchymal chondrosarcoma; myxofibrosarcoma; myxoid liposarcoma; myxoinflammatory fibroblastic sarcoma; neoplasms with perivascular epitheioid cell differentiation; osteosarcoma; parosteal osteosarcoma; neoplasm with perivascular epitheioid cell differentiation; periosteal osteosarcoma; pleomorphic liposarcoma; pleomorphic rhabdomyosarcoma; PNET/extraskeletal Ewing tumor; rhabdomyosarcoma; round cell liposarcoma; small cell osteosarcoma; solitary fibrous tumor; synovial sarcoma; telangiectatic osteosarcoma. In some embodiments, the carcinoma is selected from an adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, or small cell carcinoma. In some embodiments, the carcinoma is selected from anal cancer; appendix cancer; bile duct cancer (i.e., cholangiocarcinoma); bladder cancer; brain tumor; breast cancer; cervical cancer; colon cancer; cancer of Unknown Primary (CUP); esophageal cancer; eye cancer; fallopian tube cancer; gastroenterological cancer; kidney cancer; liver cancer; lung cancer; medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreatic cancer; parathyroid disease; penile cancer; pituitary tumor; prostate cancer; rectal cancer; skin cancer; stomach cancer; testicular cancer; throat cancer; thyroid cancer; uterine cancer; vaginal cancer; or vulvar cancer. In some embodiments, the carcinoma is breast cancer. In some embodiments, the breast cancer is invasive ductal carcinoma, ductal carcinoma in situ, invasive lobular carcinoma, or lobular carcinoma in situ. In some embodiments, the carcinoma is pancreatic cancer. In some embodiments, the pancreatic cancer is adenocarcinoma, or islet cell carcinoma. In some embodiments, the carcinoma is colorectal (colon) cancer. In some embodiments, the colorectal cancer is adenocarcinoma. In some embodiments, the solid tumor is a colon polyp. In some embodiments, the colon polyp is associated with familial adenomatous polyposis. In some embodiments, the carcinoma is bladder cancer. In some embodiments, the bladder cancer is transitional cell bladder cancer, squamous cell bladder cancer, or adenocarcinoma. In some embodiments, the carcinoma is lung cancer. In some embodiments, the lung cancer is a non- small cell lung cancer. In some embodiments, the non-small cell lung cancer is adenocarcinoma, squamous-cell lung carcinoma, or large-cell lung carcinoma. In some embodiments, the lung cancer is a small cell lung cancer. In some embodiments, the carcinoma is prostate cancer. In some embodiments, the prostate cancer is adenocarcinoma or small cell carcinoma. In some embodiments, the carcinoma is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the carcinoma is bile duct cancer. In some embodiments, the bile duct cancer is proximal bile duct carcinoma or distal bile duct carcinoma.

[0059] In some embodiments, the cancer is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is nasopharyngeal carcinoma. [0060] In some embodiments, the cancer is a relapsed or refractory cancer. In some cases, the relapsed or refractory cancer is a solid tumor. In some embodiments, the relapsed or refractory cancer is selected from pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, and nasopharyngeal carcinoma. In some embodiments, the relapsed or refractory cancer is relapsed or refractory pancreatic cancer. In some embodiments, the relapsed or refractory cancer is relapsed or refractory breast cancer. In some embodiments, the relapsed or refractory cancer is relapsed or refractory ovarian cancer. In some embodiments, the relapsed or refractory cancer is relapsed or refractory thyroid cancer. In some embodiments, the relapsed or refractory cancer is relapsed or refractory nasopharyngeal carcinoma.

[0061] In some cases, the cancer is a hematological cancer. In some embodiments, the hematological cancer is a leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, a Hodgkin's lymphoma, or a B-cell malignancy.

[0062] In some embodiments, the cancer is a B-cell proliferative disorder. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, or a non-CLL/SLL lymphoma. In some embodiments, the cancer is follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some

embodiments, DLBCL is further divided into subtypes: activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) and germinal center diffuse large B-cell lymphoma (GCB DLBCL). In some embodiments, ABC-DLBCL is characterized by a CD79B mutation. In some embodiments, ABC-DLBCL is characterized by a CD79A mutation. In some embodiments, the ABC-DLBCL is characterized by a mutation in MyD88, A20, or a combination thereof. In some embodiments, the cancer is acute or chronic myelogenous (or myeloid) leukemia,

myelodysplastic syndrome, or acute lymphoblastic leukemia.

[0063] In some case, the B cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the B cell malignancy is acute lymphoblastic leukemia (ALL). In some embodiments, the B cell malignancy is acute myelogenous leukemia (AML). In some embodiments, the B cell malignancy is chronic lymphocytic leukemia (CLL). [0064] In some embodiments, the hematological cancer is a relapsed or refractory

hematological cancer. In some embodiments, the relapsed or refractory hematological cancer is a relapsed or refractory B cell malignancy. In some embodiments, the relapsed or refractory B cell malignancy is selected from acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), and chronic lymphocytic leukemia (CLL). In some embodiments, the relapsed or refractory B cell malignancy is relapsed or refractory acute lymphoblastic leukemia (ALL). In some embodiments, the relapsed or refractory B cell malignancy is relapsed or refractory acute myelogenous leukemia (AML). In some embodiments, the relapsed or refractory B cell malignancy is relapsed or refractory chronic lymphocytic leukemia (CLL).

[0065] In some embodiments, the cancer is a treatment na ^' ive cancer. In some embodiments, the treatment na ^' ive cancer is a treatment naive solid tumor. In some embodiments, the treatment na ^' ive cancer is a treatment na ^' ive hematological malignancy. In some embodiments, the treatment na ^' ive hematological malignancy is a treatment na ^' ive B cell malignancy.

[0066] As used herein, pathogen associated with cancer refers to any bacteria or viruses that are associated with cancer. In some embodiments, virus associated with cancer includes adenovirus, papilloma virus, parvovirus, herpes viruses, pox virus, hepatitis virus, influenza, and HIV. In some embodiments, bacteria associated with cancer include Listeria monocytogenes. TEC Family Kinase Inhibitors

[0067] BT is a member of the Tyrosine -protein kinase (TEC) family of kinases. In some embodiments, the TEC family comprises BTK, ITK, TEC, RLK and BMX. In some

embodiments, a covalent TEC family kinase inhibitor inhibits the kinase activity of BTK, ITK, TEC, RLK and BMX. In some embodiments, a covalent TEC family kinase inhibitor is a BTK inhibitor. In some embodiments, a covalent TEC family kinase inhibitor is an ITK inhibitor. In some embodiments, a covalent TEC family kinase inhibitor is a TEC inhibitor. In some embodiments, a covalent TEC family kinase inhibitor is a RLK inhibitor. In some

embodiments, a covalent TEC family kinase inhibitor is a BMK inhibitor.

BTK Inhibitor Compounds Including Ibrutinib, and Pharmaceutically Acceptable Salts Thereof

[0068] The BTK inhibitor compound described herein (i.e. Ibrutinib) is selective for BTK and kinases having a cysteine residue in an amino acid sequence position of the tyrosine kinase that is homologous to the amino acid sequence position of cysteine 481 in BTK. The BTK inhibitor compound can form a covalent bond with Cys 481 of BTK (e.g., via a Michael reaction).

[0069] In some embodiments, the BTK inhibitor is a compound of Formula (A) having the structure:

Formula (A);

wherein:

A is N;

Ri is phenyl-O-phenyl or phenyl-S-phenyl;

R ₂ and 3 are independently H;

R4 is L3-X-L4-G, wherein,

L3 is optional, and when present is a bond, optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted cycloalkyl, optionally substituted or unsubstituted alkenyl, optionally substituted or unsubstituted alkynyl;

X is optional, and when present is a bond, -0-, -C(=0)-, -S-, -S(=0)-, -S(=0) ₂ -, -NH-, - NR ₉ -, -NHC(O)-, -C(0)NH-, -NR ₉ C(0)-, -C(0)NR ₉ -, -S(=0) ₂ NH-, -NHS(=0) ₂ -, -S(0) ₂ NR ₉ -, - NR ₉ S(=0) ₂ -, -OC(0)NH-, -NHC(0)0-, -OC(0)NR ₉ -, -NR ₉ C(0)0-, -CH=NO-, -ON=CH-, - NRioC(0)NRio-, heteroaryl-, aryl-, -NRi ₀ C(=NR _n )NRio-, -NRi ₀ C(=NR _n )-, -C(=NRn)NRi ₀ -, - OC(=NRii)-, or -C(=NRn)0-;

L ₄ is optional, and when present is a bond, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle;

or L ₃ , X and L ₄ taken together form a nitrogen containing heterocyclic ring;

G is , wherein,

R6, R ₇ and Rg are independently selected from among H, halogen, CN, OH, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl or substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl;

each R ₉ is independently selected from among H, substituted or unsubstituted lower alkyl, and substituted or unsubstituted lower cycloalkyl;

each Rio is independently H, substituted or unsubstituted lower alkyl, or substituted or unsubstituted lower cycloalkyl; or two Rio groups can together form a 5-, 6-, 7-, or 8-membered heterocyclic ring; or Rio and Rn can together form a 5-, 6-, 7-, or 8-membered heterocyclic ring; or each Rn is independently selected from H or substituted or unsubstituted alkyl; or a pharmaceutically acceptable salt thereof. In some embodiments, L ₃ , X and L ₄ taken together form a nitrogen containing heterocyclic ring. In some embodiments, the nitrogen containing heterocyclic ring is

a piperidine group. In some embodiments, G is

embodiments, the compound of Formula (A) is l-[(3R)-3-[4-amino-3-(4- phenoxyphenyl)pyrazolo [3 ,4-d]pyrimidin- 1 -yljpiperidin- 1 -yl]prop-2-en- 1 -one.

[0070] "Ibrutinib" or "l -((R)-3-(4-amino-3-(4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrim idin- l-yl)piperidin-l-yl)prop-2-en-l-one" or "l-{(3i?)-3-[4-amino-3-(4-phenoxyphenyl)-lH- pyrazolo[3,4-ii]pyrimidin-l-yl]piperidin-l-yl}prop-2-en-l -one" or "2-Propen-l-one, l-[(3R)-3- [4-amino-3-(4-phenoxyphenyl)- lH-pyrazolo[3 ,4-JJpyrimidin- 1 -y 1] - 1 -piperidinyl-" or Ibrutinib or any other suitable name refers to the compound with the following structure:

[0071] A wide variety of pharmaceutically acceptable salts is formed from Ibrutinib and includes:

[0072] - acid addition salts formed by reacting ibrutinib with an organic acid, which includes aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyl alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, amino acids, etc. and include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like;

[0073] - acid addition salts formed by reacting Ibrutinib with an inorganic acid, which includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. [0074] The term "pharmaceutically acceptable salts" in reference to Ibrutinib refers to a salt of Ibrutinib, which does not cause significant irritation to a mammal to which it is administered and does not substantially abrogate the biological activity and properties of the compound.

[0075] It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms (solvates). Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and are formed during the process of product formation or isolation with pharmaceutically acceptable solvents such as water, ethanol, methanol, methyl tert-butyl ether (MTBE), diisopropyl ether (DIPE), ethyl acetate, isopropyl acetate, isopropyl alcohol, methyl isobutyl ketone (MIBK), methyl ethyl ketone (MEK), acetone, nitromethane, tetrahydrofuran (THF), dichloromethane (DCM), dioxane, heptanes, toluene, anisole, acetonitrile, and the like. In one aspect, solvates are formed using, but limited to, Class 3 solvent(s). Categories of solvents are defined in, for example, the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), "Impurities: Guidelines for Residual Solvents, Q3C(R3), (November 2005). Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. In some embodiments, solvates of ibrutinib, or pharmaceutically acceptable salts thereof, are conveniently prepared or formed during the processes described herein. In some embodiments, solvates of ibrutinib are anhydrous. In some embodiments, ibrutinib, or pharmaceutically acceptable salts thereof, exist in unsolvated form. In some embodiments, ibrutinib, or pharmaceutically acceptable salts thereof, exist in unsolvated form and are anhydrous.

[0076] In yet other embodiments, ibrutinib, or a pharmaceutically acceptable salt thereof, is prepared in various forms, including but not limited to, amorphous phase, crystalline forms, milled forms and nano-particulate forms. In some embodiments, ibrutinib, or a

pharmaceutically acceptable salt thereof, is amorphous. In some embodiments, ibrutinib, or a pharmaceutically acceptable salt thereof, is amorphous and anhydrous. In some embodiments, ibrutinib, or a pharmaceutically acceptable salt thereof, is crystalline. In some embodiments, ibrutinib, or a pharmaceutically acceptable salt thereof, is crystalline and anhydrous.

[0077] In some embodiments, ibrutinib is prepared as outlined in US Patent no. 7,514,444.

[0078] In some embodiments, the Btk inhibitor is PCI-45292, PCI-45466, AVL-l Ol/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY- 1 1066 (also, CTK4I7891 , HMS3265G21 , HMS3265G22, HMS3265H21 , HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13.

[0079] In some embodiments, the BTK inhibitor is 4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6- ((4-(morpholine-4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropy razin-2-yl)phenyl)benzamide (CGI- 1746); 7-benzyl-l-(3-( iperidin-l-yl)propyl)-2-(4-( yridin-4-yl)phenyl)-m-imidazo[4,5- g]quinoxalin-6(5H)-one (CTA-056); (R)-N-(3-(6-(4-(l ,4-dimethyl-3-oxopiperazin-2- yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-me thylphenyl)-4,5,6,7- tetrahydrobenzo[b]thiophene-2-carboxamide (GDC-0834); 6-cyclopropyl-8-fluoro-2-(2- hydroxymethyl-3- { l-methyl-5-[5-(4-methyl-piperazin- 1 -yl)-pyridin-2-ylamino]-6-oxo- 1 ,6- dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-l-one (RN-486); N-[5-[5-(4-acetylpiperazine-l- carbonyl)-4-methoxy-2-methylphenyl] sulfanyl- 1 ,3 -thiazol-2-yl] -4- [(3 ,3 -dimethylbutan-2- ylamino)methyl]benzamide (BMS-509744, HY-11092); or N-(5-((5-(4-Acetylpiperazine-l- carbonyl)-4-methoxy-2-methylphenyl)thio)thiazol-2-yl)-4-(((3 -methylbutan-2- yl)amino)methyl)benzamide (HY11066); or a pharmaceutically acceptable salt thereof.

[0080] In some embodiments, the BTK inhibitor is:

; or a pharmaceutically acceptable salt thereof. ITK Inhibitors

[0081] In some embodiments, the ITK inhibitor covalently binds to Cysteine 442 of ITK. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in

WO2002/0500071, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2005/070420, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2005/079791, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2007/076228, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2007/058832, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2004/016610, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2004/01661 1 , which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2004/016600, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2004/016615, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2005/026175, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2006/065946, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2007/027594, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2007/017455, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2008/025820, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2008/025821, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2008/025822, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2011/017219, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO201 1/090760, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2009/158571 , which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in WO2009/051822, which is incorporated by reference in its entirety. In some embodiments, the ITK inhibitor is an ITK inhibitor compound described in US 13/177657, which is incorporated by reference in its entirety.

[0082] In some embodiments, the ITK inhibitor has a structure selected from:

Combination Therapy

[0083] In some embodiments, a TEC inhibitor is administered in combination with an additional therapeutic agent for the treatment of cancer. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RLK inhibitor, or a BMX inhibitor. In certain embodiments, an ITK inhibitor is administered in combination with an additional therapeutic agent for the treatment of cancer. In certain embodiments, a BTK inhibitor (e.g. ibrutinib) is administered in combination with an additional therapeutic agent for the treatment of cancer. In some embodiments, the additional therapeutic agent is an inhibitor of LYN, SY , JAK, PI3K, PLCy, MAP , HDAC, NF _K B, or MEK. In some embodiments, the additional therapeutic agent is selected from a chemotherapeutic agent, a biologic agent, radiation therapy, bone marrow transplant or surgery.

[0084] In some embodiments, the additional therapeutic agent is selected from among a chemotherapeutic agent, a biologic agent, radiation therapy, bone marrow transplant or surgery. In some embodiments, the chemotherapeutic agent is selected from among chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, lenalidomide, temsirolimus, everolimus, fludarabine, fostamatinib, paclitaxel, docetaxel, ofatumumab, rituximab, dexamethasone, prednisone, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, or a combination thereof.

Pharmaceutical Compositions and Formulations

[0085] In some embodiments, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients can be used as suitable and as understood in the art. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and

Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999), herein incorporated by reference in their entirety.

[0086] A pharmaceutical composition, as used herein, refers to a mixture of a compound described herein, such as, for example, ibrutinib, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. In practicing the methods of treatment or use provided herein, therapeutically effective amounts of compounds described herein are administered in a pharmaceutical composition to a mammal having a disease, disorder, or condition to be treated. Preferably, the mammal is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The compounds can be used singly or in combination with one or more therapeutic agents as components of mixtures. [0087] In certain embodiments, compositions also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0088] In other embodiments, compositions also include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0089] The term "pharmaceutical combination" as used herein, means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g. a compound described herein and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed

combination" means that the active ingredients, e.g. a compound described herein and a co- agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more active ingredients.

[0090] The pharmaceutical formulations described herein can be administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0091] In some embodiments, pharmaceutical compositions including a compound described herein are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes. [0092] "Antifoaming agents" reduce foaming during processing which can result in coagulation of aqueous dispersions, bubbles in the finished film, or generally impair processing. Exemplary anti-foaming agents include silicon emulsions or sorbitan sesquoleate.

[0093] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. In certain embodiments, antioxidants enhance chemical stability where required.

[0094] In certain embodiments, compositions provided herein also include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0095] In some embodiments, formulations described herein benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0096] "Binders" impart cohesive qualities and include, e.g., alginic acid and salts thereof; cellulose derivatives such as carboxymethylcellulose, methylcellulose (e.g., Methocel ^® ), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel ^® ), ethylcellulose (e.g., Ethocel ^® ), and microcrystalline cellulose (e.g., Avicel ^® ); microcrystalline dextrose; amylose; magnesium aluminum silicate; polysaccharide acids; bentonites; gelatin; polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac ^® ), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab ^® ), and lactose; a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, polyvinylpyrrolidone (e.g., Polyvidone ^® CL, Kollidon ^® CL, Polyplasdone ^® XL- 10), larch arabogalactan, Veegum ^® , polyethylene glycol, waxes, sodium alginate, and the like.

[0097] A "carrier" or "carrier materials" include any commonly used excipients in

pharmaceutics and should be selected on the basis of compatibility with compounds disclosed herein, such as, compounds of ibrutinib, and the release profile properties of the desired dosage form. Exemplary carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "Pharmaceutically compatible carrier materials" include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa. : Mack Publishing Company, 1995); Hoover, John E., Remington 's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and

Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999).

[0098] "Dispersing agents," and/or "viscosity modulating agents" include materials that control the diffusion and homogeneity of a drug through liquid media or a granulation method or blend method. In some embodiments, these agents also facilitate the effectiveness of a coating or eroding matrix. Exemplary diffusion facilitators/dispersing agents include, e.g., hydrophilic polymers, electrolytes, Tween ^® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone ^® ), and the carbohydrate-based dispersing agents such as, for example, hydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,

hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630), 4-(l , l ,3,3-tetramethylbutyl)- phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68 ^® , F88 ^® , and F108 ^® , which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908 ^® , also known as Poloxamine 908 ^® , which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, N.J.)), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (S-630), polyethylene glycol, e.g., the

polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans and combinations thereof. Plasticizers such as cellulose or triethyl cellulose can also be used as dispersing agents.

Dispersing agents particularly useful in liposomal dispersions and self-emulsifying dispersions are dimyristoyl phosphatidyl choline, natural phosphatidyl choline from eggs, natural phosphatidyl glycerol from eggs, cholesterol and isopropyl myristate.

[0099] Combinations of one or more erosion facilitator with one or more diffusion facilitator can also be used in the present compositions.

[00100] The term "diluent" refers to chemical compounds that are used to dilute the compound of interest prior to delivery. Diluents can also be used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution. In certain embodiments, diluents increase bulk of the composition to facilitate compression or create sufficient bulk for homogenous blend for capsule filling. Such compounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,

microcrystalline cellulose such as Avicel ^® ; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar, such as Di-Pac ^® (Amstar); mannitol,

hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents, confectioner's sugar; monobasic calcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; powdered cellulose, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

[00101] The term "disintegrate" includes both the dissolution and dispersion of the dosage form when contacted with gastrointestinal fluid. "Disintegration agents or disintegrants" facilitate the breakup or disintegration of a substance. Examples of disintegration agents include a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel ^® , or sodium starch glycolate such as Promogel ^® or Explotab ^® , a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel ^® , Avicel ^® PH101, Avicel ^® PH102, Avicel ^® PH105, Elcema ^® P100, Emcocel ^® , Vivacel ^® , Ming Tia ^® , and Solka-Floc ^® ,

methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol ^® ), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

[00102] "Drug absorption" or "absorption" typically refers to the process of movement of drug from site of administration of a drug across a barrier into a blood vessel or the site of action, e.g., a drug moving from the gastrointestinal tract into the portal vein or lymphatic system.

[00103] An "enteric coating" is a substance that remains substantially intact in the stomach but dissolves and releases the drug in the small intestine or colon. Generally, the enteric coating comprises a polymeric material that prevents release in the low pH environment of the stomach but that ionizes at a higher pH, typically a pH of 6 to 7, and thus dissolves sufficiently in the small intestine or colon to release the active agent therein.

[00104] "Erosion facilitators" include materials that control the erosion of a particular material in gastrointestinal fluid. Erosion facilitators are generally known to those of ordinary skill in the art. Exemplary erosion facilitators include, e.g., hydrophilic polymers, electrolytes, proteins, peptides, and amino acids.

[00105] "Filling agents" include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[00106] "Flavoring agents" and/or "sweeteners" useful in the formulations described herein, include, e.g., acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate (MagnaSweet ^® ), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet ^® Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin, sylitol, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate - mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and mixtures thereof.

[00107] "Lubricants" and "glidants" are compounds that prevent, reduce or inhibit adhesion or friction of materials. Exemplary lubricants include, e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex ^® ), higher fatty acids and their alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet ^® , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or a methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil ^® , a starch such as corn starch, silicone oil, a surfactant, and the like.

[00108] A "measurable serum concentration" or "measurable plasma concentration" describes the blood serum or blood plasma concentration, typically measured in mg, μg, or ng of therapeutic agent per mL, dL, or L of blood serum, absorbed into the bloodstream after administration. As used herein, measurable plasma concentrations are typically measured in ng/ml or μg/ml.

[00109] "Pharmacodynamics" refers to the factors which determine the biologic response observed relative to the concentration of drug at a site of action.

[00110] "Pharmacokinetics" refers to the factors which determine the attainment and maintenance of the appropriate concentration of drug at a site of action.

[00111] "Plasticizers" are compounds used to soften the microencapsulation material or film coatings to make them less brittle. Suitable plasticizers include, e.g., polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethyl cellulose and triacetin. In some embodiments, plasticizers can also function as dispersing agents or wetting agents.

[00112] "Solubilizers" include compounds such as triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N- methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide and the like.

[00113] "Stabilizers" include compounds such as any antioxidation agents, buffers, acids, preservatives and the like. [00114] "Steady state," as used herein, is when the amount of drug administered is equal to the amount of drug eliminated within one dosing interval resulting in a plateau or constant plasma drug exposure.

[00115] "Suspending agents" include compounds such as polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,

hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[00116] "Surfactants" include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic ^® (BASF), and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g. , polyoxyethylene (60)

hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. In some embodiments, surfactants are included to enhance physical stability or for other purposes.

[00117] "Viscosity enhancing agents" include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,

hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.

[00118] "Wetting agents" include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts and the like.

Dosage Forms

[00119] The compositions described herein can be formulated for administration to a subject via any conventional means including, but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), buccal, intranasal, rectal or transdermal administration routes. As used herein, the term "subject" is used to mean an animal, preferably a mammal, including a human or non-human. As used herein, the terms patient and subject are used interchangeably.

[00120] Moreover, the pharmaceutical compositions described herein, which include ibrutinib can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[00121] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In some embodiments, disintegrating agents are added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[00122] Dragee cores are provided with suitable coatings. For this purpose, in some embodiments, concentrated sugar solutions are used, which, in particular embodiments, optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. In some embodiments, dyestuffs or pigments are added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[00123] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In some embodiments, in soft capsules, the active compounds are dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, in some embodiments, stabilizers are added. All formulations for oral administration should be in dosages suitable for such administration. [00124] In some embodiments, the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid- disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder) a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or "sprinkle capsules"), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet, including but not limited to, a fast-melt tablet. Additionally, in some embodiments,

pharmaceutical formulations described herein are administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation is administered in two, or three, or four, capsules or tablets.

[00125] In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of ibrutinib, with one or more pharmaceutical excipients to form a bulk blend composition. When referring to these bulk blend compositions as homogeneous, it is meant that the particles of ibrutinib are dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. In some embodiments, the individual unit dosages also include film coatings, which disintegrate upon oral ingestion or upon contact with diluent. These formulations can be manufactured by conventional pharmacological techniques.

[00126] Conventional pharmacological techniques include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. See, e.g., Lachman et al., The Theory and Practice of

Industrial Pharmacy (1986). Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.

[00127] The pharmaceutical solid dosage forms described herein can include a compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof. In still other aspects, using standard coating procedures, such as those described in Remington 's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of ibrutinib. In another embodiment, some or all of the particles of ibrutinib, are not microencapsulated and are uncoated.

[00128] Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose,

hydroxypropylmethylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

[00129] Suitable filling agents for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, hydroxypropylmethycellulose (HPMC),

hydroxypropylmethycellulose phthalate, hydroxypropylmethylcellulose acetate stearate

(HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[00130] In order to release the compound of ibrutinib, from a solid dosage form matrix as efficiently as possible, disintegrants are often used in the formulation, especially when the dosage forms are compressed with binder. Disintegrants help rupturing the dosage form matrix by swelling or capillary action when moisture is absorbed into the dosage form. Suitable disintegrants for use in the solid dosage forms described herein include, but are not limited to, natural starch such as corn starch or potato starch, a pregelatinized starch such as National 15 1 or Amijel ^® , or sodium starch glycolate such as Promogel ^® or Explotab ^® , a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel ^® , Avicel ^® PH101 , Avicel ^® PH102, Avicel ^® PH105, Elcema ^® P100, Emcocel ^® , Vivacel ^® , Ming Tia ^® , and Solka-Floc ^® ,

methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol ^® ), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross-linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum ^® HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like.

[00131] Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to,

carboxymethylcellulose, methylcellulose (e.g., Methocel ^® ), hydroxypropylmethylcellulose (e.g. Hypromellose USP Pharmacoat-603, hydroxypropylmethylcellulose acetate stearate (Aqoate HS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel ^® ), ethylcellulose (e.g., Ethocel ^® ), and microcrystalline cellulose (e.g., Avicel ^® ), microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonites, gelatin,

polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac ^® ), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab ^® ), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone (e.g., Povidone ^® CL, Kollidon ^® CL, Polyplasdone ^® XL-10, and Povidone ^® -12), larch arabogalactan, Veegum ^® , polyethylene glycol, waxes, sodium alginate, and the like.

[00132] In general, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fillers which itself can act as moderate binder. Formulators skilled in art can determine the binder level for the formulations, but binder usage level of up to 70% in tablet formulations is common.

[00133] Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet ^® , boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

[00134] Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrin), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins and the like.

[00135] The term "non water-soluble diluent" represents compounds typically used in the formulation of pharmaceuticals, such as calcium phosphate, calcium sulfate, starches, modified starches and microcrystalline cellulose, and microcellulose (e.g., having a density of about 0.45 g/cm , e.g. Avicel, powdered cellulose), and talc. [00136] Suitable wetting agents for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (e.g., Polyquat 10 ^® ), sodium oleate, sodium lauryl sulfate, magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and the like.

[00137] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic ^® (BASF), and the like.

[00138] Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,

polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 7000 to about 5400, vinyl pyrrolidone/vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy- propylmethylcellulose, polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium

carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[00139] Suitable antioxidants for use in the solid dosage forms described herein include, for example, e.g., butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol.

[00140] It should be appreciated that there is considerable overlap between additives used in the solid dosage forms described herein. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in solid dosage forms described herein. The amounts of such additives can be readily determined by one skilled in the art, according to the particular properties desired.

[00141] In other embodiments, one or more layers of the pharmaceutical formulation are plasticized. Illustratively, a plasticizer is generally a high boiling point solid or liquid. Suitable plasticizers can be added from about 0.01% to about 50% by weight (w/w) of the coating composition. Plasticizers include, but are not limited to, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, triacetin, polypropylene glycol, polyethylene glycol, triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, and castor oil. [00142] Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, compressed tablets which are designed to dissolve in the mouth will include one or more flavoring agents. In other embodiments, the compressed tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating can provide a delayed release of ibrutinib or the second agent, from the formulation. In other embodiments, the film coating aids in patient compliance (e.g., Opadry ^® coatings or sugar coating). Film coatings including Opadry ^® typically range from about 1% to about 3% of the tablet weight. In other embodiments, the compressed tablets include one or more excipients.

[00143] In some embodiments, a capsule is prepared, for example, by placing the bulk blend of the formulation of ibrutinib or the second agent, described above, inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule can be swallowed whole or the capsule can be opened and the contents sprinkled on food prior to eating. In some embodiments, the therapeutic dose is split into multiple (e.g., two, three, or four) capsules. In some

embodiments, the entire dose of the formulation is delivered in a capsule form.

[00144] In various embodiments, the particles of ibrutinib, and one or more excipients are dry blended and compressed into a mass, such as a tablet, having a hardness sufficient to provide a pharmaceutical composition that substantially disintegrates within less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, or less than about 60 minutes, after oral administration, thereby releasing the formulation into the gastrointestinal fluid.

[00145] In another aspect, in some embodiments, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[00146] Materials useful for the microencapsulation described herein include materials compatible with ibrutinib, which sufficiently isolate the compound of any of ibrutinib, from other non-compatible excipients. Materials compatible with compounds of any of ibrutinib, are those that delay the release of the compounds of any of ibrutinib, in vivo. [00147] Exemplary microencapsulation materials useful for delaying the release of the formulations including compounds described herein, include, but are not limited to,

hydroxypropyl cellulose ethers (HPC) such as Klucel ^® or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Pharmacoat ^® , Metolose SR, Methocel ^® -E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methylcellulose polymers such as Methocel ^® -A,

hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose ^® , Ethylcelluloses (EC) and mixtures thereof such as E461 , Ethocel ^® , Aqualon ^® -EC, Surelease ^® , Polyvinyl alcohol (PVA) such as Opadry AMB, hydroxyethylcelluloses such as Natrosol ^® , carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) such as Aqualon ^® -CMC, polyvinyl alcohol and polyethylene glycol co-polymers such as Kollicoat IR ^® , monoglycerides (Myverol), triglycerides ( LX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit ^® EPO, Eudragit ^® L30D- 55, Eudragit ^® FS 30D Eudragit ^® L100-55, Eudragit ^® L100, Eudragit ^® S 100, Eudragit ^® RD100, Eudragit ^® E100, Eudragit ^® L12.5, Eudragit ^® S12.5, Eudragit ^® NE30D, and Eudragit ^® NE 40D, cellulose acetate phthalate, sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins, and mixtures of these materials.

[00148] In still other embodiments, plasticizers such as polyethylene glycols, e.g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, and triacetin are incorporated into the microencapsulation material. In other embodiments, the microencapsulating material useful for delaying the release of the pharmaceutical compositions is from the USP or the National Formulary (NF). In yet other embodiments, the microencapsulation material is Klucel. In still other embodiments, the microencapsulation material is methocel.

[00149] In some embodiments, microencapsulated compounds of any of ibrutinib, are formulated by methods known by one of ordinary skill in the art. Such known methods include, e.g., spray drying processes, spinning disk-solvent processes, hot melt processes, spray chilling methods, fluidized bed, electrostatic deposition, centrifugal extrusion, rotational suspension separation, polymerization at liquid-gas or solid-gas interface, pressure extrusion, or spraying solvent extraction bath. In addition to these, several chemical techniques, e.g., complex coacervation, solvent evaporation, polymer-polymer incompatibility, interfacial polymerization in liquid media, in situ polymerization, in-liquid drying, and desolvation in liquid media could also be used. Furthermore, in some embodiments, other methods such as roller compaction, extrusion/spheronization, coacervation, or nanoparticle coating are used. [00150] In one embodiment, the particles of compounds of any of ibrutinib, are microencapsulated prior to being formulated into one of the above forms. In still another embodiment, some or most of the particles are coated prior to being further formulated by using standard coating procedures, such as those described in Remington 's Pharmaceutical Sciences, 20th Edition (2000).

[00151] In other embodiments, the solid dosage formulations of the compounds of any of ibrutinib, are plasticized (coated) with one or more layers. Illustratively, a plasticizer is generally a high boiling point solid or liquid. Suitable plasticizers can be added from about 0.01% to about 50% by weight (w/w) of the coating composition. Plasticizers include, but are not limited to, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, triacetin, polypropylene glycol, polyethylene glycol, triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, and castor oil.

[00152] In other embodiments, a powder including the formulations with a compound of any of ibrutinib, described herein, is formulated to include one or more pharmaceutical excipients and flavors. In some embodiments, such a powder is prepared, for example, by mixing the formulation and optional pharmaceutical excipients to form a bulk blend composition.

Additional embodiments also include a suspending agent and/or a wetting agent. This bulk blend is uniformly subdivided into unit dosage packaging or multi-dosage packaging units.

[00153] In still other embodiments, effervescent powders are also prepared in accordance with the present disclosure. Effervescent salts have been used to disperse medicines in water for oral administration. Effervescent salts are granules or coarse powders containing a medicinal agent in a dry mixture, usually composed of sodium bicarbonate, citric acid and/or tartaric acid. When salts of the compositions described herein are added to water, the acids and the base react to liberate carbon dioxide gas, thereby causing "effervescence." Examples of effervescent salts include, e.g., the following ingredients: sodium bicarbonate or a mixture of sodium bicarbonate and sodium carbonate, citric acid and/or tartaric acid. Any acid-base combination that results in the liberation of carbon dioxide can be used in place of the combination of sodium bicarbonate and citric and tartaric acids, as long as the ingredients were suitable for pharmaceutical use and result in a pH of about 6.0 or higher.

[00154] In some embodiments, the solid dosage forms described herein can be formulated as enteric coated delayed release oral dosage forms, i.e., as an oral dosage form of a pharmaceutical composition as described herein which utilizes an enteric coating to affect release in the small intestine of the gastrointestinal tract. In some embodiments, the enteric coated dosage form is a compressed or molded or extruded tablet/mold (coated or uncoated) containing granules, powder, pellets, beads or particles of the active ingredient and/or other composition components, which are themselves coated or uncoated. In some embodiments, the enteric coated oral dosage form is a capsule (coated or uncoated) containing pellets, beads or granules of the solid carrier or the composition, which are themselves coated or uncoated.

[00155] The term "delayed release" as used herein refers to the delivery so that the release can be accomplished at some generally predictable location in the intestinal tract more distal to that which would have been accomplished if there had been no delayed release alterations. In some embodiments the method for delay of release is coating. Any coatings should be applied to a sufficient thickness such that the entire coating does not dissolve in the gastrointestinal fluids at pH below about 5, but does dissolve at pH about 5 and above. It is expected that any anionic polymer exhibiting a pH-dependent solubility profile can be used as an enteric coating in the methods and compositions described herein to achieve delivery to the lower gastrointestinal tract. In some embodiments the polymers described herein are anionic carboxylic polymers. In other embodiments, the polymers and compatible mixtures thereof, and some of their properties, include, but are not limited to:

[00156] Shellac, also called purified lac, a refined product obtained from the resinous secretion of an insect. This coating dissolves in media of pH >7;

[00157] Acrylic polymers. The performance of acrylic polymers (primarily their solubility in biological fluids) can vary based on the degree and type of substitution. Examples of suitable acrylic polymers include methacrylic acid copolymers and ammonium methacrylate copolymers. The Eudragit series E, L, S, RL, RS and NE (Rohm Pharma) are available as solubilized in organic solvent, aqueous dispersion, or dry powders. The Eudragit series RL, NE, and RS are insoluble in the gastrointestinal tract but are permeable and are used primarily for colonic targeting. The Eudragit series E dissolve in the stomach. The Eudragit series L, L-30D and S are insoluble in stomach and dissolve in the intestine;

[00158] Cellulose Derivatives. Examples of suitable cellulose derivatives are: ethyl cellulose; reaction mixtures of partial acetate esters of cellulose with phthalic anhydride. The performance can vary based on the degree and type of substitution. Cellulose acetate phthalate (CAP) dissolves in pH >6. Aquateric (FMC) is an aqueous based system and is a spray dried CAP psuedolatex with particles <1 μηι. Other components in Aquateric can include pluronics, Tweens, and acetylated monoglycerides. Other suitable cellulose derivatives include: cellulose acetate trimellitate (Eastman); methylcellulose (Pharmacoat, Methocel); hydroxypropylmethyl cellulose phthalate (HPMCP); hydroxypropylmethyl cellulose succinate (HPMCS); and hydroxypropylmethylcellulose acetate succinate (e.g., AQOAT (Shin Etsu)). The performance can vary based on the degree and type of substitution. For example, HPMCP such as, HP-50, HP-55, HP-55S, HP-55F grades are suitable. The performance can vary based on the degree and type of substitution. For example, suitable grades of hydroxypropylmethylcellulose acetate succinate include, but are not limited to, AS-LG (LF), which dissolves at pH 5, AS-MG (MF), which dissolves at pH 5.5, and AS-HG (HF), which dissolves at higher pH. These polymers are offered as granules, or as fine powders for aqueous dispersions; Poly Vinyl Acetate Phthalate (PVAP). PVAP dissolves in pH >5, and it is much less permeable to water vapor and gastric fluids.

[00159] In some embodiments, the coating can, and usually does, contain a plasticizer and possibly other coating excipients such as colorants, talc, and/or magnesium stearate, which are well known in the art. Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin

(glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. In particular, anionic carboxylic acrylic polymers usually will contain 10-25% by weight of a plasticizer, especially dibutyl phthalate,

polyethylene glycol, triethyl citrate and triacetin. Conventional coating techniques such as spray or pan coating are employed to apply coatings. The coating thickness must be sufficient to ensure that the oral dosage form remains intact until the desired site of topical delivery in the intestinal tract is reached.

[00160] In some embodiments, colorants, detackifiers, surfactants, antifoaming agents, lubricants (e.g., carnuba wax or PEG) are added to the coatings besides plasticizers to solubilize or disperse the coating material, and to improve coating performance and the coated product.

[00161] In other embodiments, the formulations described herein, which include ibrutinib, are delivered using a pulsatile dosage form. A pulsatile dosage form is capable of providing one or more immediate release pulses at predetermined time points after a controlled lag time or at specific sites. Many other types of controlled release systems known to those of ordinary skill in the art and are suitable for use with the formulations described herein. Examples of such delivery systems include, e.g., polymer-based systems, such as polylactic and polyglycolic acid, plyanhydrides and polycaprolactone; porous matrices, nonpolymer-based systems that are lipids, including sterols, such as cholesterol, cholesterol esters and fatty acids, or neutral fats, such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide -based systems; wax coatings, bioerodible dosage forms, compressed tablets using conventional binders and the like. See, e.g., Liberman et al., Pharmaceutical Dosage Forms, 2 Ed., Vol. 1 , pp. 209-214 (1990); Singh et al, Encyclopedia of Pharmaceutical Technology, 2 ^nd Ed., pp. 751-753 (2002); U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509, 5,461 ,140, 5,456,923, 5,516,527, 5,622,721, 5,686,105, 5,700,410, 5,977,175, 6,465,014 and 6,932,983. [00162] In some embodiments, pharmaceutical formulations are provided that include particles of ibrutinib, described herein and at least one dispersing agent or suspending agent for oral administration to a subject. In some embodiments, the formulations are a powder and/or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

[00163] Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2 ^nd Ed., pp. 754-757 (2002). In addition, in some embodiments, the liquid dosage forms include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions can further include a crystalline inhibitor.

[00164] The aqueous suspensions and dispersions described herein can remain in a

homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The homogeneity should be determined by a sampling method consistent with regard to determining homogeneity of the entire composition. In one

embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

[00165] Examples of disintegrating agents for use in the aqueous suspensions and dispersions include, but are not limited to, a starch, e.g., a natural starch such as corn starch or potato starch, a pregelatinized starch such as National 1551 or Amijel ^® , or sodium starch glycolate such as Promogel ^® or Explotab ^® ; a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel ^® , Avicel ^® PH101, Avicel ^® PH102, Avicel ^® PH105, Elcema ^® P100, Emcocel ^® , Vivacel ^® , Ming Tia ^® , and Solka-Floc ^® , methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol ^® ), cross-linked

carboxymethylcellulose, or cross-linked croscarmellose; a cross-linked starch such as sodium starch glycolate; a cross-linked polymer such as crospovidone; a cross-linked

polyvinylpyrrolidone; alginate such as alginic acid or a salt of alginic acid such as sodium alginate; a clay such as Veegum ^® HV (magnesium aluminum silicate); a gum such as agar, guar, locust bean, araya, pectin, or tragacanth; sodium starch glycolate; bentonite; a natural sponge; a surfactant; a resin such as a cation-exchange resin; citrus pulp; sodium lauryl sulfate; sodium lauryl sulfate in combination starch; and the like.

[00166] In some embodiments, the dispersing agents suitable for the aqueous suspensions and dispersions described herein are known in the art and include, for example, hydrophilic polymers, electrolytes, Tween ^® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone ^® ), and the carbohydrate-based dispersing agents such as, for example, hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers (e.g. HPMC K100, HPMC 4M, HPMC K15M, and HPMC K100M), carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate,

hydroxypropylmethyl-cellulose acetate stearate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer (Plasdone ^® , e.g., S-630), 4-(l,l,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68 ^® , F88 ^® , and F 108 ^® , which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908 ^® , also known as Poloxamine 908 ^® , which is a tetrafunctional block copolymer derived from sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, N.J.)). In other embodiments, the dispersing agent is selected from a group not comprising one of the following agents: hydrophilic polymers; electrolytes; Tween ^® 60 or 80; PEG; polyvinylpyrrolidone (PVP);

hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC, HPC-SL, and HPC-L); hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers (e.g. HPMC K100, HPMC 4M, HPMC K15M, HPMC 100M, and Pharmacoat ^® USP 2910 (Shin-Etsu));

carboxymethylcellulose sodium; methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl- cellulose phthalate; hydroxypropylmethyl-cellulose acetate stearate; non-crystalline cellulose; magnesium aluminum silicate; triethanolamine; polyvinyl alcohol (PVA); 4-(l ,l,3,3- tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics F68 ^® , F88 ^® , and F108 ^® , which are block copolymers of ethylene oxide and propylene oxide); or poloxamines (e.g., Tetronic 908 ^® , also known as Poloxamine 908 ^® ).

[00167] Wetting agents suitable for the aqueous suspensions and dispersions described herein are known in the art and include, but are not limited to, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fatty acid esters (e.g., the commercially available Tweens ^® such as e.g., Tween 20 ^® and Tween 80 ^® (ICI Specialty Chemicals)), and polyethylene glycols (e.g., Carbowaxs 3350 ^® and 1450 ^® , and Carbopol 934 ^® (Union Carbide)), oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, vitamin E TPGS, sodium taurocholate, simethicone, phosphotidyl choline and the like.

[00168] Suitable preservatives for the aqueous suspensions or dispersions described herein include, for example, potassium sorbate, parabens (e.g., methylparaben and propylparaben), benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl alcohol or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. Preservatives, as used herein, are incorporated into the dosage form at a concentration sufficient to inhibit microbial growth.

[00169] Suitable viscosity enhancing agents for the aqueous suspensions or dispersions described herein include, but are not limited to, methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, Plasdon ^® S-630, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof. The concentration of the viscosity enhancing agent will depend upon the agent selected and the viscosity desired.

[00170] Examples of sweetening agents suitable for the aqueous suspensions or dispersions described herein include, for example, acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate (MagnaSweet ^® ), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet ^® Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, e.g., anise-menthol, cherry-anise, cinnamon- orange, cherry-cinnamon, chocolate -mint, honey-lemon, lemon-lime, lemon-mint, menthol- eucalyptus, orange-cream, vanilla-mint, and mixtures thereof. In one embodiment, the aqueous liquid dispersion can comprise a sweetening agent or flavoring agent in a concentration ranging from about 0.001% to about 1.0% the volume of the aqueous dispersion. In another

embodiment, the aqueous liquid dispersion can comprise a sweetening agent or flavoring agent in a concentration ranging from about 0.005%> to about 0.5% the volume of the aqueous dispersion. In yet another embodiment, the aqueous liquid dispersion can comprise a sweetening agent or flavoring agent in a concentration ranging from about 0.01% to about 1.0% the volume of the aqueous dispersion.

[00171] In addition to the additives listed above, the liquid formulations can also include inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifiers. Exemplary emulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1 ,3 -butyl eneglycol,

dimethylformamide, sodium lauryl sulfate, sodium doccusate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholme, oils, such as cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, or mixtures of these substances, and the like.

[00172] In some embodiments, the pharmaceutical formulations described herein can be self- emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase can be added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provide improvements in the bioavailability of hydrophobic active ingredients. Methods of producing self-emulsifying dosage forms are known in the art and include, but are not limited to, for example, U.S. Pat. Nos.

5,858,401, 6,667,048, and 6,960,563, each of which is specifically incorporated by reference.

[00173] It is to be appreciated that there is overlap between the above-listed additives used in the aqueous dispersions or suspensions described herein, since a given additive is often classified differently by different practitioners in the field, or is commonly used for any of several different functions. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in formulations described herein. The amounts of such additives can be readily determined by one skilled in the art, according to the particular properties desired.

Intranasal Formulations

[00174] Intranasal formulations are known in the art and are described in, for example, U.S. Pat. Nos. 4,476, 1 16, 5,1 16,817 and 6,391,452, each of which is specifically incorporated by reference. Formulations that include ibrutinib, which are prepared according to these and other techniques well-known in the art are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. These ingredients are known to those skilled in the preparation of nasal dosage forms and some of these can be found in Remington: The Science and Practice of Pharmacy, 21st edition, 2005, a standard reference in the field. The choice of suitable carriers is highly dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. In some embodiments, minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are also present. The nasal dosage form should be isotonic with nasal secretions.

[00175] In some embodiments, for administration by inhalation described herein, the pharmaceutical compositions are in a form as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In some embodiments, in the case of a pressurized aerosol, the dosage unit is determined by providing a valve to deliver a metered amount. In some embodiments, capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator are formulated containing a powder mix of the compound described herein and a suitable powder base such as lactose or starch.

Buccal Formulations

[00176] In some embodiments, buccal formulations are administered using a variety of formulations known in the art. For example, such formulations include, but are not limited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739, 136, each of which is specifically incorporated by reference. In addition, the buccal dosage forms described herein can further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. The buccal dosage form is fabricated so as to erode gradually over a predetermined time period, wherein the delivery is provided essentially throughout. Buccal drug delivery, as will be appreciated by those skilled in the art, avoids the disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and/or first-pass inactivation in the liver. With regard to the bioerodible (hydrolysable) polymeric carrier, it will be appreciated that virtually any such carrier can be used, so long as the desired drug release profile is not compromised, and the carrier is compatible with ibrutinib, and any other components that are present in the buccal dosage unit. Generally, the polymeric carrier comprises hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the buccal mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers and co, e.g., those known as "carbomers"

(Carbopol ^® , which can be obtained from B.F. Goodrich, is one such polymer). In some embodiments, other components are also incorporated into the buccal dosage forms described herein include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. In some embodiments, for buccal or sublingual administration, the compositions are in the form of tablets, lozenges, or gels formulated in a conventional manner.

Transdermal Formulations

[00177] In some embodiments, transdermal formulations described herein are administered using a variety of devices which have been described in the art. For example, such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731 ,683, 3,742,951, 3,814,097, 3,921 ,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307, 4,077,407, 4,201 ,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and 6,946,144, each of which is specifically incorporated by reference in its entirety.

[00178] In some embodiments, the transdermal dosage forms described herein incorporate certain pharmaceutically acceptable excipients which are conventional in the art. In one embodiments, the transdermal formulations described herein include at least three components: (1) a formulation of a compound of ibrutinib; (2) a penetration enhancer; and (3) an aqueous adjuvant. In addition, transdermal formulations can include additional components such as, but not limited to, gelling agents, creams and ointment bases, and the like. In some embodiments, the transdermal formulation can further include a woven or non-woven backing material to enhance absorption and prevent the removal of the transdermal formulation from the skin. In other embodiments, the transdermal formulations described herein can maintain a saturated or supersaturated state to promote diffusion into the skin.

[00179] In some embodiments, formulations suitable for transdermal administration of compounds described herein employ transdermal delivery devices and transdermal delivery patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. In some embodiments, such patches are constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery of the compounds described herein can be accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches can provide controlled delivery of ibrutinib. The rate of absorption can be slowed by using rate-controlling membranes or by trapping the compound within a polymer matrix or gel. Conversely, absorption enhancers can be used to increase absorption. An absorption enhancer or carrier can include absorbable pharmaceutically acceptable solvents to assist passage through the skin. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

Injectable Formulations

[00180] In some embodiments, formulations that include a compound of ibrutinib, suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some embodiments, it is also desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[00181] In some embodiments, for intravenous injections, compounds described herein are formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal

administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. In some embodiments, for other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally known in the art.

[00182] In some embodiments, parenteral injections involve bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. In some embodiments, the pharmaceutical composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contains formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, in some embodiments, suspensions of the active compounds are prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. In some embodiments, aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, in some embodiments, the suspension also contains suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, in some embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

Other Formulations

[00183] In certain embodiments, delivery systems for pharmaceutical compounds are employed, such as, for example, liposomes and emulsions. In certain embodiments,

compositions provided herein can also include an mucoadhesive polymer, selected from among, for example, carboxymethylcellulose, carbomer (acrylic acid polymer),

polymethylmethacrylate), polyacrylamide, polycarbophil, acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.

[00184] In some embodiments, the compounds described herein are administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[00185] In some embodiments, the compounds described herein are formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted. Dosing and Treatment Regiments

[00186] In some embodiments, the amount of a TEC inhibitor that is administered from 10 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of a TEC inhibitor that is administered is from about 40 mg/day to 70 mg/day. In some embodiments, the amount of a TEC inhibitor that is administered per day is about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, or about 140 mg.

[00187] In some embodiments, the amount of an ITK inhibitor that is administered from 10 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of an ITK inhibitor that is administered is from about 40 mg/day to 70 mg/day. In some embodiments, the amount of an ITK inhibitor that is administered per day is about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, or about 140 mg.

[00188] In some embodiments, the amount of a BTK inhibitor that is administered from 10 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of a BTK inhibitor that is administered is from about 40 mg/day to 70 mg/day. In some embodiments, the amount of a BTK inhibitor that is administered per day is about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, or about 140 mg.

[00189] In some embodiments, the amount of ibrutinib that is administered from 10 mg/day up to, and including, 1000 mg/day. In some embodiments, the amount of ibrutinib that is administered is from about 40 mg/day to 70 mg/day. In some embodiments, the amount of ibrutinib that is administered per day is about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 1 10 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 400 mg, about 420 mg, about 440 mg, about 460 mg, about 480 mg, about 500 mg, about 520 mg, about 540 mg, about 560 mg, about 580 mg, about 600 mg, about 700 mg, or about 840 mg. In some embodiments, the amount of ibrutinib that is administered is about 40 mg/day. In some embodiments, the amount of ibrutinib that is administered is about 50 mg/day. In some embodiments, the amount of ibrutinib that is administered is about 60 mg/day. In some embodiments, the amount of ibrutinib that is administered is about 70 mg/day.

[00190] In some embodiments, ibrutinib is administered once per day, twice per day, or three times per day. In some embodiments, ibrutinib is administered once per day. In some embodiments, ibrutinib is administered as a maintenance therapy.

[00191] In some embodiments, the compositions disclosed herein are administered for prophylactic, therapeutic, or maintenance treatment. In some embodiments, the compositions disclosed herein are administered for therapeutic applications. In some embodiments, the compositions disclosed herein are administered for therapeutic applications. In some embodiments, the compositions disclosed herein are administered as a maintenance therapy, for example for a patient in remission.

[00192] In some embodiments, in the case wherein the patient's status does improve, upon the doctor's discretion the administration of the compounds is given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday can vary between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some embodiments, the dose reduction during a drug holiday is from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[00193] Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. Patients can, however, require intermittent treatment on a long- term basis upon any recurrence of symptoms.

[00194] The amount of a given agent that will correspond to such an amount will vary depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but can nevertheless be routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated. In general, however, doses employed for adult human treatment will typically be in the range of 0.02-5000 mg per day, or from about 1 -1500 mg per day. In some embodiments, the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[00195] In some embodiments, the pharmaceutical composition described herein is in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compound. In some embodiments, the unit dosage is in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single- dose non-reclosable containers. Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, in some embodiments, formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative.

[00196] The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. In some embodiments, such dosages are altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[00197] Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. In some embodiments, the dosage is varied within this range depending upon the dosage form employed and the route of administration utilized. Dendritic cell-based vaccine preparation/formulation

[00198] Dendritic cell-based vaccine can be prepared by any methods well known in the art. In some cases, dendritic cell-based vaccines is prepared through an ex vivo or in vivo method. In some instances, the ex vivo method comprises the use of autologous DCs pulsed ex vivo with tumor antigens or derived peptides, to activate or load the DCs prior to administration into the patient. In some instances, the in vivo method comprises targeting specific DC receptors using antibodies coupled with antigens. In some embodiments, the DC-based vaccine further comprises DC activators such as TLR3, TLR-7-8, and CD40 agonists. In some embodiments, the DC-based vaccine further comprises adjuvants, and a pharmaceutically acceptable carrier.

[00199] In some embodiments, an adjuvant is used to enhance the immune response (humoral and/or cellular) elicited in a patient receiving the vaccine. In some embodiments, the adjuvant is selected from bacteria toxoids, polyoxypropylene-polyoxyethylene block polymers, aluminum salts, liposomes, CpG polymers, oil-in-water emulsions, or a combination thereof. In some embodiments, the adjuvant comprises cytokines.In some embodiments, the cytokines are selected from IL-2, IL-4, IL-5, IL-6, IL-12, TNF, INFy, INFoc, GM-CSF, or a combination thereof. In some embodiments, the adjuvant is an oil-in-water emulsion. In some embodiments, the oil-in-water emulsion include at least one oil and at least one surfactant, with the oil(s) and surfactant(s) being biodegradable (metabolisable) and biocompatible. The oil droplets in the emulsion are generally less than 5 μιη in diameter, and may even have a sub-micron diameter, with these small sizes being achieved with a micro fluidiser to provide stable emulsions. Droplets with a size less than 220 nm are preferred as they can be subjected to filter sterilization.

[00200] In some embodiments, the oils used include such as those from an animal (such as fish) or vegetable source. Sources for vegetable oils include nuts, seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil, the most commonly available, exemplify the nut oils. Jojoba oil can be used e.g. obtained from the jojoba bean. Seed oils include safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil, etc. In the grain group, corn oil is the most readily available, but the oil of other cereal grains such as wheat, oats, rye, rice, teff, triticale, etc. may also be used. 6-10 carbon fatty acid esters of glycerol and 1 ,2-propanediol, while not occurring naturally in seed oils, may be prepared by hydrolysis, separation and esterification of the appropriate materials starting from the nut and seed oils. Fats and oils from mammalian milk are metabolizable and may therefore be used in the practice of this invention. The procedures for separation, purification, saponification and other means necessary for obtaining pure oils from animal sources are well known in the art. Most fish contain metabolizable oils which may be readily recovered. For example, cod liver oil, shark liver oils, and whale oil such as spermaceti exemplify several of the fish oils which may be used herein. A number of branched chain oils are synthesized biochemically in 5-carbon isoprene units and are generally referred to as terpenoids. Shark liver oil contains a branched, unsaturated terpenoid known as squalene, 2,6,10,15,19,23-hexamethyl-2,6,10,14, 18,22-tetracosahexaene. Squalane, the saturated analog to squalene, can also be used. Fish oils, including squalene and squalane, are readily available from commercial sources or may be obtained by methods known in the art.

[00201] Other useful oils are the tocopherols, which are advantageously included in vaccines for use in elderly patients (e.g. aged 60 years or older) because vitamin E has been reported to have a positive effect on the immune response in this patient group. They also have antioxidant properties that may help to stabilize the emulsions. Various tocopherols exist (α, β, γ, δ, ε or ξ) but a is usually used. A preferred -tocopherol is DL-a-tocopherol. a-tocopherol succinate is known to be compatible with influenza vaccines and to be a useful preservative as an alternative to mercurial compounds.

[00202] Mixtures of oils can be used e.g. squalene and -tocopherol. An oil content in the range of 2-20% (by volume) is typical.

[00203] Surfactants can be classified by their 'HLB' (hydrophile/lipophile balance). In some embodiments, surfactants of the invention have a HLB of at least 10, preferably at least 15, and more preferably at least 16. The invention can be used with surfactants including, but not limited to: the polyoxyethylene sorbitan esters surfactants (commonly referred to as the Tweens), especially polysorbate 20 and polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers; octoxynols, which can vary in the number of repeating ethoxy (oxy- 1 ,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40);

phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such as the Tergitol™ NP series; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethyleneglycol monolauryl ether (Brij 30); and sorbitan esters (commonly known as the SPANs), such as sorbitan trioleate (Span 85) and sorbitan monolaurate. Non-ionic surfactants are preferred. The most preferred surfactant for including in the emulsion is polysorbate 80 (polyoxyethylene sorbitan monooleate; Tween 80).

[00204] Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. A combination of a polyoxyethylene sorbitan ester and an octoxynol is also suitable. Another useful combination comprises laureth 9 plus a polyoxyethylene sorbitan ester and/or an octoxynol.

[00205] In some embodiments, amounts of surfactants (% by weight) are: polyoxyethylene sorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or other detergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

[00206] Specific oil-in- water emulsion adjuvants include, but are not limited to:

[00207] A submicron emulsion of squalene, polysorbate 80, and sorbitan trioleate. The composition of the emulsion by volume can be about 5% squalene, about 0.5% polysorbate 80 and about 0.5% Span 85. In weight terms, these ratios become 4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant is known as 'MF59'. The MF59 emulsion advantageously includes citrate ions e.g. 10 mM sodium citrate buffer.

[00208] A submicron emulsion of squalene, a tocopherol, and polysorbate 80. These emulsions may have from 2 to 10% squalene, from 2 to 10% tocopherol and from 0.3 to 3% polysorbate 80, and the weight ratio of squalene:tocopherol is preferably≤1 (e.g. 0.90) as this can provide a more stable emulsion. Squalene and polysorbate 80 may be present at a volume ratio of about 5 :2 or at a weight ratio of about 11 :5. One such emulsion can be made by dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml of this solution with a mixture of (5g of DL-a- tocopherol and 5 ml squalene), then microfluidising the mixture. The resulting emulsion has submicron oil droplets e.g. with an average diameter of between 100 and 250 nm, preferably about 180 nm. The emulsion may also include a 3-de-O-acylated monophosphoryl lipid A (3d- MPL). Another useful emulsion of this type may comprise, per human dose, 0.5-10 mg squalene, 0.5-1 1 mg tocopherol, and 0.1 -4 mg polysorbate 80.

[00209] An emulsion of squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see below). The emulsion may contain a phosphate buffer.

[00210] An emulsion comprising a polysorbate (e.g. polysorbate 80), a Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an a-tocopherol succinate). The emulsion may include these three components at a mass ratio of about 75 : 11 : 10 (e.g. 750 μιηΐ polysorbate 80, 1 10 μιηΐ Triton X-100 and 100 μ/ml α-tocopherol succinate), and these concentrations should include any contribution of these components from antigens. The emulsion may also include squalene. The emulsion may also include a 3d-MPL. The aqueous phase may contain a phosphate buffer.

[00211] An emulsion of squalane, polysorbate 80 and poloxamer 401 ("Pluronic™ L121"). The emulsion can be formulated in phosphate buffered saline, pH 7.4. This emulsion is a useful delivery vehicle for muramyl dipeptides, and has been used with threonyl-MDP in the "SAF-1" adjuvant [84] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can also be used without the Thr-MDP, as in the "AF" adjuvant (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80). [00212] An emulsion comprising squalene, an aqueous solvent, a polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic surfactant (e.g. a sorbitan ester or mannide ester, such as sorbitan monoleate or 'Span 80'). The emulsion is preferably thermoreversible and/or has at least 90% of the oil droplets (by volume) with a size less than 200 nm. The emulsion may also include one or more of: alditol; a cryoprotective agent (e.g. a sugar, such as dodecylmaltoside and/or sucrose); and/or an alkylpolyglycoside. The emulsion may include a TLR4 agonist. Such emulsions may be lyophilized.

[00213] An emulsion of squalene, poloxamer 105 and Abil-Care [88]. The final concentration (weight) of these components in adjuvanted vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and 2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG- 16/ 16 dimethicone; caprylic/capric triglyceride).

[00214] An emulsion having from 0.5-50% of an oil, 0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant. As described in reference 89, preferred phospholipid components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, sphingomyelin and cardiolipin. Submicron droplet sizes are advantageous.

[00215] A submicron oil-in-water emulsion of a non-metabolisable oil (such as light mineral oil) and at least one surfactant (such as lecithin, Tween 80 or Span 80). Additives may be included, such as QuilA saponin, cholesterol, a saponin-lipophile conjugate (such as GPI-0100, described in reference 90, produced by addition of aliphatic amine to desacylsaponin via the carboxyl group of glucuronic acid), dimethyldioctadecylammonium bromide and/or N,N- dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.

[00216] An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are associated as helical micelles.

[00217] An emulsion comprising a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol, and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene - polyoxypropylene block copolymer).

[00218] An emulsion comprising a mineral oil, a non-ionic hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant (e.g. an ethoxylated fatty alcohol and/or polyoxyethylene-polyoxypropylene block copolymer).

[00219] In some embodiments, the vaccine includes preservatives such as thiomersal or 2- phenoxyethanol. In some embodiments, the vaccine is substantially free from (e.g. <10 μ /πι1) mercurial material e.g. thiomersal-free. In some embodiments, a-tocopherol succinate is used as an alternative to mercurial compounds. [00220] In some embodiments, for controlling the tonicity, a physiological salt such as sodium salt is included in the vaccine. In some embodiments, other salts include potassium chloride, potassium dihydrogen phosphate, disodium phosphate, and/or magnesium chloride, or the like.

[00221] In some embodiments, the vaccine has an osmolality of between 200 mOsm/kg and 400 mOsm/kg, between 240-360 mOsm/kg, or within the range of 290-310 mOsm/kg.

[00222] In some embodiments, the vaccine comprises one or more buffers, such as a Tris buffer; a borate buffer; a succinate buffer; a histidine buffer (particularly with an aluminum hydroxide adjuvant); or a citrate buffer. Buffers, in some cases, are included in the 5-20 mM range.

[00223] In some embodiments, the pH of the vaccine is between about 5.0 and 8.5, between about 6.0 and 8.0, between about 6.5 and 7.5, or between about 7.0 and 7.8.

[00224] In some embodiments, the vaccine is preferably sterile. The vaccine is preferably non- pyrogenic e.g. containing <1 EU (endotoxin unit, a standard measure) per dose, and preferably <0.1 EU per dose. The composition is preferably gluten free.

[00225] In some embodiments, the vaccine also includes detergent e.g. a polyoxyethylene sorbitan ester surfactant (known as 'Tweens'), an octoxynol (such as octoxynol-9 (Triton X-100) or t-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide ('CTAB'), or sodium deoxycholate, particularly for a split or surface antigen vaccine. The detergent may be present only at trace amounts. Thus the vaccine may includ less than 1 mg/ml of each of octoxynol- 10 and polysorbate 80. Other residual components in trace amounts could be antibiotics (e.g. neomycin, kanamycin, polymyxin B).

[00226] In some embodiments, the vaccine includes material for a single immunisation, or may include material for multiple immunisations (i.e. a 'multidose' kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptic adaptor for removal of material.

[00227] In some embodiments, the vaccine is administered in a dosage volume of about 0.5 mL, although a half dose (i.e. about 0.25 mL) may be administered to children. In some embodiments of the invention a composition may be administered in a higher dose e.g. about 1 ml e.g. after mixing.

[00228] In some embodiments, the vaccine and kit are stored at between 2°C and 8°C. In some embodiments, the vaccine is not stored frozen. In some embodiments, the vaccine is stored away from sunlight. Diagnostic and Therapeutic Methods

[00229] Disclosed herein are methods of determining Thl 7 cell differentiation based on the presence, absences, increase or decrease of the expression of biomarkers described herein. In some embodiments, the biomarkers include CD80, CD86, CD40, IL-Ιβ, IL-6, IL-18, TGF-β, IL17A, IL17F, IL21, and IL22.

[00230] In some embodiments, methods of determining maturation and/or activation of dendritic cells, and/or promoting dendritic-cell mediated T cell proliferation is based on the presence, absence, increase, or decrease of the expression of biomarkers as described herein. In some embodiments, the biomarkers include CCR7, MHC-II, CD1 lc, Ly6C, CD80, IFN-γ, IL- 17, IL-13, IL-12, IL-16, CD86, CD40, and/or TNF-a.

[00231] In some embodiments, the expression level of CD80 is compared to a control expression level of CD80. In some embodiments, the expression level of CD80 is increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD80.

[00232] In some embodiments, the expression level of CD86 is compared to a control expression level of CD86. In some embodiments, the expression level of CD86 is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to a control expression level of CD86.

[00233] In some embodiments, the expression level of the cytokines such as IL-Ιβ, IL-6, IL-18, and TGF-β are compared to control expression levels of the cytokines. In some embodiments, the expression level of the cytokines are increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to control expression levels of the cytokines.

[00234] In some embodiments, the expression level of the Thl 7 associated cytokines such as IL17A, IL17F, IL21, and IL22 are compared to a reference level. In some embodiments, expression level of the Thl 7 associated cytokines are increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to the reference level.

[00235] In some embodiments, the expression level of surface markers such as CCR7, MHC-II, and CD1 lc are compared to a control expression level of the surface markers. In some embodiments, the expression level of the surface markers are increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to the reference level.

[00236] In some embodiments, the expression of a surface marker such as Ly6C is compared to a control expression level of the surface marker. In some embodiments, the expression level of the surface marker is decreased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to the reference level.

[00237] In some embodiments, the expression level of cytokines such as IFN-γ, IL-17, and IL- 13 are compared to a control expression level of cytokines. In some embodiments, the expression level of the cytokines are increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to the reference level.

[00238] In some embodiments, the expression level of cytokines such as IL-12, IL-6, and TNF- α are compared to a control expression level of the cytokines. In some embodiments, the expression level of the cytokines are increased by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or more relative to the reference level.

[00239] Methods for determining the expression or presence of biomarkers such as CD80, CD86, CD40, IL-Ιβ, IL-6, IL-18, TGF-β, IL17A, IL17F, IL21, and IL22 are well known in the art. Mutations or modifications and expression levels of biomarkers are measured by RT - PCR, Qt-PCR, microarray, Northern blot, or other similar technologies. Circulating levels of biomarkers in a blood sample obtained from a candidate subject are measured, for example, by ELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Western blot, multiplexing technologies, or other similar methods. Cell surface expression of biomarkers are measured, for example, by flow cytometry, immunohistochemistry, Western Blot, immunoprecipitation, magnetic bead selection, and quantification of cells expressing either of these cell surface markers.

[00240] As disclosed herein, determining the presence, modifications, or expression of the biomarker of interest at the protein or nucleotide level are accomplished using any detection method known to those of skill in the art. By "determining the modification(s)" is intended to determine a mutation within the biomarker gene or a biomarker protein. As used herein, "modification" and "mutation" are used interchangeably. The term "biomarker" refers to in some cases the protein of interest. In some cases, "biomarker" refers to the gene of interest. In some cases, the terms "biomarker" and "biomarker gene" are used interchangeably. By "detecting expression" or "detecting the level of is intended determining the expression level or presence of a biomarker protein or gene in the biological sample. Thus, "detecting expression" encompasses instances where a biomarker is determined not to be expressed, not to be detectably expressed, expressed at a low level, expressed at a normal level, or overexpressed.

[00241] In certain aspects of the method provided herein, the one or more subpopulation of lymphocytes are isolated, detected or measured. In certain embodiments, the one or more subpopulation of lymphocytes are isolated, detected or measured using immunophenotyping techniques. In other embodiments, the one or more subpopulation of lymphocytes are isolated, detected or measured using fluorescence activated cell sorting (FACS) techniques.

[00242] In certain aspects, the modifications, expression, or presence of these various biomarkers and any clinically useful prognostic markers in a biological sample are detected at the protein or nucleic acid level, using, for example, immunohistochemistry techniques or nucleic acid-based techniques such as in situ hybridization and RT-PCR. In one

embodiments, the modifications, expression, or presence of one or more biomarkers is carried out by a means for nucleic acid amplification, a means for nucleic acid sequencing, a means utilizing a nucleic acid microarray (DNA and RNA), or a means for in situ hybridization using specifically labeled probes.

[00243] In some embodiments, the determining the modification, expression, or presence of one or more biomarkers is carried out through gel electrophoresis. In one embodiment, the determination is carried out through transfer to a membrane and hybridization with a specific probe.

[00244] In other embodiments, the determining the modification, expression, or presence of one or more biomarkers carried out by a diagnostic imaging technique.

[00245] In still other embodiments, the determining the modification, expression, or presence of one or more biomarkers carried out by a detectable solid substrate. In one embodiment, the detectable solid substrate is paramagnetic nanoparticles functionalized with antibodies.

[00246] In another aspect, provided herein are methods for detecting or measuring residual lymphoma following a course of treatment in order to guide continuing or discontinuing treatment or changing from one therapeutic regimen to another comprising determining the expression or presence of one or more biomarkers from one or more subpopulation of lymphocytes in a subject wherein the course of treatment is treatment with a Btk inhibitor (e.g., ibrutinib).

[00247] Methods for detecting the modification and expression of the biomarkers described herein, within the test and control biological samples comprise any methods that determine the quantity or the presence of these markers either at the nucleic acid or protein level. Such methods are well known in the art and include but are not limited to western blots, northern blots, ELISA, immunoprecipitation, immunofluorescence, flow cytometry,

immunohistochemistry, nucleic acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In some embodiments, expression of a biomarker is detected on a protein level using, for example, antibodies that are directed against specific biomarker proteins. These antibodies are used in various methods such as Western blot, ELISA, multiplexing technologies, immunoprecipitation, or immunohistochemistry techniques. In some embodiments, detection of biomarkers is accomplished by ELISA. In some

embodiments, detection of biomarkers is accomplished by electrochemiluminescence (ECL).

[00248] In some embodiments, the modification, expression, or presence of one or more of the biomarkers described herein are determined at the nucleic acid level. Nucleic acid-based techniques for assessing expression are well known in the art and include, for example, determining the level of biomarker mRNA in a biological sample. Many expression detection methods use isolated RNA. Any RNA isolation technique that does not select against the isolation of mRNA is utilized for the purification of RNA (see, e.g., Ausubel et al., ed. (1987- 1999) Current Protocols in Molecular Biology (John Wiley & Sons, New York). Additionally, large numbers of tissue samples are readily processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process disclosed in U.S. Pat. No. 4,843,155.

[00249] Thus, in some embodiments, the detection of a biomarker or other protein of interest is assayed at the nucleic acid level using nucleic acid probes. The term "nucleic acid probe" refers to any molecule that is capable of selectively binding to a specifically intended target nucleic acid molecule, for example, a nucleotide transcript. Probes are synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes are specifically designed to be labeled, for example, with a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or other labels or tags that are discussed above or that are known in the art. Examples of molecules that are utilized as probes include, but are not limited to, RNA and DNA.

[00250] For example, isolated mRNA are used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe comprises of, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a biomarker, biomarker described herein above.

Hybridization of an mRNA with the probe indicates that the biomarker or other target protein of interest is being expressed.

[00251] In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array. A skilled artisan readily adapts known mRNA detection methods for use in detecting the level of mRNA encoding the biomarkers or other proteins of interest.

[00252] An alternative method for determining the level of an mRNA of interest in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189 193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1 177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1197), rolling circle replication (U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, biomarker expression is assessed by quantitative fluorogenic RT-PCR (i.e., the TaqManO System).

[00253] Modifications or expression levels of an RNA of interest are monitored using a membrane blot (such as used in hybridization analysis such as Northern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The detection of expression also comprises using nucleic acid probes in solution.

[00254] In some embodiments, microarrays are used to determine expression or presence of one or more biomarkers. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, U.S. Pat. Nos.

6,040, 138, 5,800,992, 6,020,135, 6,033,860, 6,344,316, and U.S. Pat. Application

20120208706. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample. Exemplary microarray chips include FoundationOne and FoundationOne Heme from Foundation Medicine, Inc; GeneChip® Human Genome U133 Plus 2.0 array from Affymetrix; and Human

DiscoveryMAP® 250+ v. 2.0 from Myraid RBM. [00255] Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261. In some embodiments, an array is fabricated on a surface of virtually any shape or even a multiplicity of surfaces. In some embodiments, an array is a planar array surface. In some embodiments, arrays include peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040, 193 and 5,800,992, each of which is hereby incorporated in its entirety for all purposes. In some embodiments, arrays are packaged in such a manner as to allow for diagnostics or other manipulation of an all- inclusive device.

[00256] Any means for specifically identifying and quantifying a biomarker (for example, biomarker, a biomarker of cell survival or proliferation, a biomarker of apoptosis, a biomarker of a Btk-mediated signaling pathway) in the biological sample of a candidate subject is contemplated. Thus, in some embodiments, expression level of a biomarker protein of interest in a biological sample is detected by means of a binding protein capable of interacting specifically with that biomarker protein or a biologically active variant thereof. In some embodiments, labeled antibodies, binding portions thereof, or other binding partners are used. The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. In some embodiments, the label is detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, catalyzes chemical alteration of a substrate compound or composition that is detectable.

[00257] The antibodies for detection of a biomarker protein are either monoclonal or polyclonal in origin, or are synthetically or recombinantly produced. The amount of complexed protein, for example, the amount of biomarker protein associated with the binding protein, for example, an antibody that specifically binds to the biomarker protein, is determined using standard protein detection methodologies known to those of skill in the art. A detailed review of immunological assay design, theory and protocols are found in numerous texts in the art (see, for example, Ausubel et al, eds. (1995) Current Protocols in Molecular Biology) (Greene Publishing and Wiley- Interscience, NY)); Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, N.Y.).

[00258] The choice of marker used to label the antibodies will vary depending upon the application. However, the choice of the marker is readily determinable to one skilled in the art. These labeled antibodies are used in immunoassays as well as in histological applications to detect the presence of any biomarker or protein of interest. The labeled antibodies are either polyclonal or monoclonal. Further, the antibodies for use in detecting a protein of interest are labeled with a radioactive atom, an enzyme, a chromophoric or fluorescent moiety, or a colorimetric tag as described elsewhere herein. The choice of tagging label also will depend on the detection limitations desired. Enzyme assays (ELISAs) typically allow detection of a colored product formed by interaction of the enzyme -tagged complex with an enzyme substrate.

Radionuclides that serve as detectable labels include, for example, 1 -131 , 1-123, 1-125, Y-90, Re-188, Re-186, At-21 1 , Cu-67, Bi-212, and Pd-109. Examples of enzymes that serve as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophoric moieties include, but are not limited to, fluorescein and rhodamine. The antibodies are conjugated to these labels by methods known in the art. For example, enzymes and chromophoric molecules are conjugated to the antibodies by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Alternatively, conjugation occurs through a ligand-receptor pair. Examples of suitable ligand-receptor pairs are biotin-avidin or biotin-streptavidin, and antibody- antigen.

[00259] In certain embodiments, expression or presence of one or more biomarkers or other proteins of interest within a biological sample, for example, a sample of bodily fluid, is determined by radioimmunoassays or enzyme-linked immunoassays (ELISAs), competitive binding enzyme-linked immunoassays, dot blot (see, for example, Promega Protocols and Applications Guide, Promega Corporation (1991), Western blot (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Vol. 3, Chapter 18 (Cold Spring Harbor Laboratory Press, Plainview, N.Y.), chromatography such as high performance liquid chromatography (HPLC), or other assays known in the art. Thus, the detection assays involve steps such as, but not limited to, immunoblotting, immunodiffusion, Immunoelectrophoresis, or immunoprecipitation.

[00260] In certain other embodiments, the methods disclosed herein are useful for identifying and treating a hematological malignancy, including those listed herein, that are refractory to (i.e., resistant to, or have become resistant to) first-line oncotherapeutic treatments.

Samples

[00261] In some embodiments, the sample for use in the methods is obtained from dendritic cells such as murine bone marrow derived dendritic cells (BMDCs) and/or T cells.

[00262] In some embodiments, the sample for use in the methods is from any tissue or fluid from a patient. Samples include, but are not limited, to whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract. In some embodiments, the sample is a blood serum sample. In some embodiments, the sample is from a fluid or tissue that is part of, or associated with, the lymphatic system or circulatory system. In some embodiments, the sample is a blood sample that is a venous, arterial, peripheral, tissue, cord blood sample. In some embodiments, the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample contains one or more circulating tumor cells (CTCs). In some embodiments, the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).

[00263] In some embodiments, the samples are obtained from the individual by any suitable means of obtaining the sample using well-known and routine clinical methods. Procedures for obtaining fluid samples from an individual are well known. For example, procedures for drawing and processing whole blood and lymph are well-known and can be employed to obtain a sample for use in the methods provided. Typically, for collection of a blood sample, an anticoagulation agent (e.g., EDTA, or citrate and heparin or CPD (citrate, phosphate, dextrose) or comparable substances) is added to the sample to prevent coagulation of the blood. In some examples, the blood sample is collected in a collection tube that contains an amount of EDTA to prevent coagulation of the blood sample.

[00264] In some embodiments, the collection of a sample from the individual is performed at regular intervals, such as, for example, one day, two days, three days, four days, five days, six days, one week, two weeks, weeks, four weeks, one month, two months, three months, four months, five months, six months, one year, daily, weekly, bimonthly, quarterly, biyearly or yearly.

[00265] In some embodiments, the collection of a sample is performed at a predetermined time or at regular intervals relative to treatment with a TEC inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor, an ITK inhibitor, a TEC inhibitor, a RL inhibitor, or a BMX inhibitor. In some embodiments, the TEC inhibitor is an ITK inhibitor. In some embodiments, the TEC inhibitor is a BTK inhibitor.

[00266] In some embodiments, the collection of a sample is performed at a predetermined time or at regular intervals relative to treatment with an ITK inhibitor. For example, a sample is collected from a patient at a predetermined time or at regular intervals prior to, during, or following treatment or between successive treatments with an ITK inhibitor. In particular examples, a sample is obtained from a patient prior to administration of an ITK inhibitor, and then again at regular intervals after treatment with the ITK inhibitor has been effected. In some embodiments, the patient is administered an ITK inhibitor and one or more additional therapeutic agents. In some embodiments, the ITK inhibitor is an irreversible ITK inhibitor. In some embodiments, the ITK inhibitor is a reversible ITK inhibitor.

[00267] In some embodiments, the collection of a sample is performed at a predetermined time or at regular intervals relative to treatment with a BTK inhibitor. For example, a sample is collected from a patient at a predetermined time or at regular intervals prior to, during, or following treatment or between successive treatments with a BTK inhibitor. In particular examples, a sample is obtained from a patient prior to administration of a BTK inhibitor, and then again at regular intervals after treatment with the BTK inhibitor has been effected. In some embodiments, the patient is administered a BTK inhibitor and one or more additional therapeutic agents. In some embodiments, the BTK inhibitor is an irreversible BTK inhibitor. In some embodiments, the BTK inhibitor is a reversible BTK inhibitor. In some embodiments, the BTK inhibitor is ibrutinib. In some embodiments, the BTK inhibitor is selected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-lOl/CC-101 (Avila Therapeutics/Celgene

Corporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics), BMS-488516 (Bristol-Myers Squibb), BMS- 509744 (Bristol-Myers Squibb), CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891, HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS- 123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) and LFM-A13.

[00268] In some embodiments, the collection of a sample is performed at a predetermined time or at regular intervals relative to treatment with ibrutinib. For example, a sample is collected from a patient at a predetermined time or at regular intervals prior to, during, or following treatment or between successive treatments with ibrutinib. In particular examples, a sample is obtained from a patient prior to administration of ibrutinib, and then again at regular intervals after treatment with ibrutinib has been effected. In some embodiments, the patient is

administered ibrutinib and one or more additional therapeutic agents.

Kits/Article of Manufacture

[00269] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.

[00270] The articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.

[00271] For example, the container(s) include ibrutinib, optionally in a composition or in combination with an additional agent as disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.

[00272] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.

[00273] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.

[00274] In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for

administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

EXAMPLES

[00275] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. Example 1

Mice strains

[00276] Female C57BL/6 mice (age 8-10 weeks) and OT-II TCR transgenic mice (age 8-10 weeks) were purchased from Harlan and Jackson Laboratories respectively. All animals were housed in a pathogen-free animal facility in The Ohio State University in accordance with National Institutes of Health and institutional guidelines.

In vitro DC activation studies

[00277] BMDCs from C57BL/6 mice were cultivated. Cells were treated with DMSO (control) or ΙμΜ ibrutinib (Pharmacyclics, Inc., Sunnyvale, CA) for 30 minutes, washed and treated with ^g/ml LPS (Sigma- Aldrich, St. Louis, MO) at 37°C and 5% CO ₂ . Culture supernatants were collected at 24 and 48 hours and analyzed for the production of cytokines and nitric oxide (NO). Cells were analyzed for expression of CD40, CD80, CD86 and MHC-II (BD Biosciences, San Jose, CA) by flow cytometry. For real-time PCR analysis, RNA was extracted, reverse transcribed and PCR amplified using SYBR Green (BioRad, Hercules, CA). Primers were obtained from PRIMER BANK. Data were normalized to β-actin and represented as fold induction over unstimulated cells by AACT method.

T cell co-culture and proliferation studies

[00278] BMDCs were treated with control or Ibrutinib as described above, pulsed with 2μg/ml (323-339) OVA-peptide (Anaspec, Fremont, CA) for 2 hours followed by overnight stimulation with LPS. DCs were co-cultured with nylon- wool enriched T cells from spleens of OT-II mice (ratio 1 :4) for 5 days. T cells were stained with 5μΜ CFSE to evaluate T cell proliferation. Cytokine production was determined in culture supernatants.

Statistical analysis

[00279] All statistical analyses were done using Prism 5 (GraphPad Software, San Diego, CA). Student's unpaired t test was employed to determine statistical significance of values obtained. The p values <0.05 were considered statistically significant.

Ibrutinib treatment alters cytokine and NO response in DCs

[00280] Since Btk is important for TLR-4 signaling in DCs, NO and cytokines were examined to determine whether ibrutinib modulated their production in DCs upon stimulation with TLR-4 agonist, LPS. Treatment with ibrutinib dampened TNF-a, IL-10 and NO production compared to controls, while IL-12 production was comparable between both groups (Figure 1 A). Further, IL- 6, TGF-β and IL-18 expression was significantly enhanced upon Ibrutinib treatment compared to control treatment. There was a trend towards higher IL- 1 β expression in Ibrutinib-treated DCs compared to controls but this difference was not statistically significant (Figure IB). Diminished IL- 10 production in Btk ' macrophages is associated with a concomitant increase in IL-6 production. These results indicate that Ibrutinib alters TLR-4 mediated cytokine and NO production in DCs which could subsequently reprogram T cell responses.

Treatment with Ibrutinib modifies the expression ofMHC-II and co-stimulatory molecules

[00281] Since LPS stimulation affects the expression of MHC-II and co-stimulatory molecules in DCs, the effect of Ibrutinib on the expression of these molecules were examined. Ibrutinib treatment reduced the percentage of MHC-II ⁺ and CD86 ⁺ cells and increased the percentage of CD80 ⁺ cells compared to controls (Figure 1C). Further, Ibrutinib -treated cells displayed lower levels of MHC-II and CD86 expression and higher CD80 expression compared to controls (Figure ID). However, expression levels and percentages of CD40 ⁺ cells remained similar between Ibrutinib and control-treated DCs (Figure 1C-1D). It has been previously shown that IL-6 and TGF-β dampen the surface expression of MHC-II and co-stimulatory molecules. Hence, it is possible that the increased production of IL-6 and TGF-β in Ibrutinib-treated DCs contributes to the reduction of MHC-II and CD86 expression in these cells. The results of this study contrast with a human DC study on human DCs which showed no effect of Ibrutinib treatment on cytokine responses or co -stimulatory molecule expression after LPS stimulation. One possible explanation to this discrepancy is in the concentration of Ibrutinib used (ΙμΜ) which is consistent with other studies and within pharmacologic range obtained in patients but lower than the concentration (ΙΟμΜ) used in the human DC study. In vitro assays indicate that Ibrutinib could bind to other kinases at varied concentrations and exposure times which may differentially activate cells. Hence, the use of lower concentrations of Ibrutinib ensures target specificity in in vitro studies.

Ibrutinib-treated DCs promote Thl 7 differentiation

[00282] DC-derived cytokines such as IL-6 and TGF-β are critical in the initiation of a Thl 7 immune response. Based on the cytokine profile generated upon Ibrutinib treatment, Ibrutinib- treated DCs would promote Thl 7 cell differentiation. This was investigated by using an in vitro antigen-specific DC-T cell co-culture model. T cells co-cultured with LPS/Ibrutinib-treated DCs displayed higher proliferation rates compared to T cells co-cultured with LPS only-treated DCs (Figure 2A). The production of T cell specific cytokines in the co-culture supernatants was also evaluated. LPS/Ibrutinib-treated DCs enhanced the production of IL-17, but not IFN-γ or IL-13, by T cells, compared to LPS only-treated DCs (Figure 2B-2D). The presence of IL-4 in either treatment was not detected. The balance of cytokines and co -stimulatory molecules expressed by DCs determines the T-helper subset generated during T cell differentiation. The production of both IL-6 and TGF-β by DCs promotes Thl 7 differentiation. Further, Thl 7 polarization can also be mediated by other cytokines such as IL-Ι β and IL-18, which were observed to be increased in LPS/Ibrutinib-treated DCs (Figure IB). Although CD80-CD86/CD28-CTLA4 mediated co- stimulation has been suggested to suppress Thl7 cell differentiation, this study suggests that differential regulation of CD80 and CD86 expression may complement the cytokine signals driving Thl7 cell polarization. Taken together, the results indicate that Ibrutinib modulates DC activation which subsequently promotes Thl7 cell differentiation.

Example 2

Mice Strains

[00283] Female C57BL/6 mice (age 8-10 weeks) and OT-II TCR transgenic mice (age 8-10 weeks) were purchased from Harlan and Jackson Laboratories respectively. All animals were housed in a pathogen-free animal facility in The Ohio State University in accordance with National Institutes of Health and institutional guidelines.

Cultivation and in vitro studies with bone marrow derived dendritic cells

[00284] Bone marrow derived DCs from C57BL/6 mice were cultivated as described herein. Briefly, bone marrow cells were isolated from femurs and tibias of mice, treated with ACK lysis buffer and plated in complete RPMI medium supplemented with 10% fetal bovine serum (Atlanta Biologicals), 1 % penicillin (20 Units/ml)/streptomycin (20 μg/ml) (Life Technologies) and 20 ng/ml GMCSF (Peprotech) for 7 days. At Day 1, PBS or 1 μΜ ibrutinib (Pharmacyclics LLC) was added to the culture and remained in the supernatant until the end of the culture period to generate untreated- and ibrutinib-treated DCs respectively. At Day 7, untreated and ibrutinib-treated DCs were collected by gently harvesting the cells in the floating fraction to obtain > 60%> purity of CD 11 c ⁺ DCs in the floating fraction of the culture medium and analyzed for expression of surface markers by flow cytometry. Cells were rested overnight prior to subsequent LPS activation studies.

In vitro LPS activation studies

[00285] Overnight rested untreated and ibrutinib-treated DCs were treated with 1 μg/ml LPS (Sigma Aldrich) at 37°C and 5% C0 ₂ for 24 or 48 hours. After 24 hours, supernatants were harvested for cytokine analyses by ELISA and cells were isolated for flow cytometric analysis. After 48 hours, supernatants were harvested to determine nitric oxide production by Griess assay.

T-cell co-culture and proliferation studies

[00286] Overnight rested untreated- and ibrutinib-treated DCs were pulsed with 10 μg/ml OVA-peptide (323-339) (Anaspec) for 2 hours and treated with 1 μg/ml LPS (Sigma Aldrich) for 22 hours. After OVA/LPS stimulation, DCs were cultured in 1 :4 ratio for 6 days with CFSE- stained, nylon- wool enriched T cells from OT-II mice. After 6 days of DC-T cell co-culture, T cell proliferation was evaluated by flow cytometry based on the reduction of CFSE fluorescence. Culture supernatants were harvested to for cytokine analyses by ELISA. Flow cytometry

[00287] Cells were prepared for flow cytometry analysis as described herein. In order to study the expression of surface markers on DCs, cells blocked using normal mouse serum and incubated with conjugated antibodies against various cell surface markers including CD1 lc, Ly6C, MHC-II, CD80, CD86 and CC 7 (Biolegend). Samples were acquired on a BD FACS Calibur (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Inc.). Analysis was conducted by gating on CD1 lc ⁺ cells using isotype controls for the corresponding conjugated antibody. In order to measure proliferation in T cell co-culture assay, cells from co- cultures assays were blocked as mentioned above and incubated with conjugated antibody against CD4 (Biolegend). CD4 ⁺ T cells were gated based on isotype controls and percentage of proliferating cells was measured by evaluating reduction of CFSE using the Proliferation platform in the FlowJo software.

Statistical analysis

[00288] All statistical analyses were done using Prism 5 (GraphPad Software). Student's unpaired t test was employed to determine statistical significance of values obtained. The p values less than 0.05 were considered statistically significant.

Ibrutinib treatment enhances the development and maturation of DCs

[00289] In order to study whether inhibition of Btk using ibrutinib treatment affects the development of DCs, the proportion of CD1 lc ⁺ DCs generated from untreated- and ibrutinib- treated DC cultures was examined. Ibrutinib-treated DC cultures had a significantly higher percentage of CD1 lc ⁺ DCs compared to untreated DC cultures (Figure 3 A). The maturation status of untreated- and ibrutinib-treated DCs was also analyzed by measuring the expression of surface markers such as Ly6C, MHC-II and co-stimulatory molecule, CD80. Ibrutinib treatment increased the percentage of MHC-II ⁺ and CD80 ⁺ DCs and decreased the percentage of Ly6C ⁺ DCs (Figure 3B). Further, ibrutinib-treated DCs displayed higher levels of MHC-II and CD80 and lower levels of Ly6C compared to untreated DCs (Figure 3C).

[00290] When DCs undergo maturation, they downregulate Ly6C, upregulate CD1 lc and also enhance the expression of MHC-II and CD80. Ibrutinib treatment reduced the expression of Ly6C and increased the expression of MHC-II and CD80 on DCs suggesting that it promotes the maturation of DCs. Taken together, the results indicate that inhibition of Btk using ibrutinib enhances the development and maturation of bone marrow-derived DCs.

Ibrutinib modulates activation of DCs in response to LPS stimulation

[00291] Since ibrutinib-treated DCs are more mature compared to untreated DCs, whether ibrutinib-treated DCs would respond more robustly to inflammatory stimuli was analyzed. Using a TLR-4 ligand, LPS, as an immunogen, the expression of MHC-II and co-stimulatory molecules such as CD80, CD86 and CD40 in untreated- and ibrutinib-treated DCs was compared. It was observed that ibrutinib treatment increases the percentage of MHC-II ⁺ and CD80 ⁺ DCs and decreases the percentage of CD86 ⁺ DCs upon LPS stimulation (Figure 4A). Additionally, LPS/ibrutinib-treated DCs displayed higher levels of MHC-II and CD80 and lower levels of CD86 compared to LPS/untreated DCs (Figure 4B). There was no significant difference in the percentage of CD40 ⁺ DCs or levels of CD40 between LPS/untreated- and LPS/ibrutinib-treated DCs (Figure 4A - 4B).

[00292] After maturation and activation, DCs migrate from peripheral sites into draining lymph nodes in order to activate T cell responses. They migrate using a chemokine receptor, CCR7, which directs DCs into T cell specific sites within the draining lymph node. Since ibrutinib treatment enhances the maturation and activation status of DCs, it was determined whether this would also enhance CCR7 expression on DCs. A higher percentage of CCR7 ⁺ DCs in

LPS/ibrutinib-treated DC cultures compared to LPS/untreated DC cultures (Figure 4C), was observed.

[00293] When LPS-induced cytokine and nitric oxide responses in untreated- and ibrutinib- treated DCs was compared, it was observed that LPS/ibrutinib-treated DCs produced higher levels of IL-10 and IFN-β (Figure 5A - 5B) and lower levels of IL-6, IL-12 and NO (Figure 5C - 5E). There was no significant difference in TNF-a production between LPS/untreated- and LPS/ibrutinib-treated DCs (Figure 5F).

[00294] Since ibrutinib differentially regulates surface marker expression and cytokine production (Figure 4 and 5), it was studied whether this would impact the ability of DCs to activate CD4 ⁺ T cell responses. In this regard, an OVA antigen specific DC: T cell co-culture model was used. It was observed that LPS/ibrutinib-treated DCs promoted higher rates of T cell proliferation compared to LPS/untreated DCs (Figure 6A). The levels of cytokines in the co- culture supernatants were also measured. While LPS/ibrutinib-treated DCs enhanced IFN-γ and IL-13 production at earlier and later time points compared to LPS/untreated DCs, IL-17 production was significantly higher at a later time point (Figure 6B - 6D). Taken together, the results indicate that ibrutinib differentially regulates LPS-mediated surface marker expression and cytokine production in DCs to enhance CD4 ⁺ T cell activation.

[00295] As shown, LPS-mediated responses upon ibrutinib treatment of differentiated DCs demonstrated that ibrutinib-treated DCs decrease CD86 and NO production and increase CD80 and IL-10 production (Figure 4A, 4B, 5A and 5E). Indeed, Btk plays an inhibitory role during in vitro development, and Btk deficiency promotes the development of DCs which are more activated compared to wildtype DCs. Btk ^7" DCs express higher levels of MHC-II and CD80 and promote higher CD4 ⁺ T cell responses compared to wildtype DCs (Figure 4A, 4B and 6A). As shown, Btk ^{" "} DCs produce lower amounts of IL-10 compared to WT DCs. Reduced IL-10 production by Btk ^7" DCs enhances DC-mediated T cell proliferation. As shown, higher IL-10 production by DCs upon Btk inhibition may not necessarily dampen DC-mediated T cell proliferation (Figure 5A and 6A). The higher expression of surface markers such as MHC-II and CD80 and (Figure 4A and 4B) production of cytokines such as IFN-β (Figure 5B) upon ibrutinib treatment counterbalances the inhibitory effects of IL-10 on T cell activation and differentiation. Additionally, the lower NO production by DCs upon ibrutinib treatment could also be responsible for the enhanced DC-mediated T cell proliferation.

[00296] Dendritic cell-based immunotherapies have been employed to promote the maturation and activation of non- functional DCs in cancer patients in order to elicit higher anti-cancer T cell responses. The success of these therapies is dependent on the culture of mature DCs which display high T cell-stimulatory capacity and express high levels of CCR7 to home to lymph nodes. However, many DC based therapies do not have high clinical efficacy because the DC culture strategies generate immature or partially mature DCs. Since ibrutinib enhances the expression of MHC-II, co-stimulatory molecules, and CCR7, and it promotes DC-mediated T cell activation, it may be used for the generation of mature DCs for cancer therapy.

[00297] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.