WO2017011444A1 - Methods and compositions for treating b cell cancers - Google Patents

Abstract

A method of directly reducing, inhibiting or suppressing the growth or metastasis of a B cell-derived cancer or tumor involves contacting the cancer cell or tumor cell with an activator or agonist of the endoplasmic reticulum-resident protein, STING. Another method involves administering to a subject with a B cell cancer or tumor an amount of a STING antagonist sufficient to directly kill the B cell tumor.

Description

METHODS AND COMPOSITIONS FOR TREATING B CELL CANCERS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the priority US Provisional Patent Application No. 62/191561 filed July 13, 2015, which application is incorporated by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. 5R01CA163910- 02 awarded by the National Institutes of Health and National Cancer Institute. The government has certain rights in this invention.

SEQUENCE LISTING

Applicant hereby incorporates by reference the Sequence Listing material filed in electronic form herewith. This file is labeled "WST153PCT_07112016_ST25.txt".

BACKGROUND OF THE INVENTION

Lymphoma is the most common blood cancer. The two main forms of lymphoma are Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Of the B-cell derived cancers, diffuse large B-cell lymphoma (DLBCL) is the most common form of NHL, accounting for up to 30 percent of newly diagnosed cases in the United States. DLBCL is an aggressive (fast- growing) lymphoma. It can arise in lymph nodes or outside of the lymphatic system, in the gastrointestinal tract, testes, thyroid, skin, breast, bone, or brain.

Current treatment for B cell lymphomas involves a combination of chemotherapy and the monoclonal antibody rituximab (Rituxan) with or without radiation therapy. Because DLBCL advances very quickly, rapid treatment is needed. The most widely used treatment for DLBCL is a mixture of rituximab and several chemotherapy drugs (cyclophosphamide, doxorubicin, vincristine, and prednisone). For many patients, DLBCL does not return after initial treatment; however, for some patients, the disease does return. For patients where the disease becomes refractory or relapses, secondary therapies may be necessary, including stem cell transplant and/or high-dose chemotherapy or other multi-agent chemotherapy.

There remains a need in the art for new and effective tools to facilitate treatment of metastatic B cell cancers and tumors.

SUMMARY OF THE INVENTION

In one aspect, a method of directly reducing, inhibiting or suppressing the growth or metastasis of a B cell-derived cancer or tumor comprises contacting the cancer cell or tumor cell with an activator or agonist of the endoplasmic reticulum-resident protein, STING. This method involves in one embodiment, administering to a mammalian subject having a B cell cancer or tumor an effective amount of a STING agonist. In another embodiment, the method involves administering the STING agonist in the absence of any other chemotherapeutic agent or therapy. In another embodiment, the method involves administering the STING agonist in an amount greater than that necessary to induce interferon production.

In another aspect, a pharmaceutical composition comprises a STING agonist in a pharmaceutically acceptable carrier or excipient. In one embodiment of this aspect, the composition further comprises an immune globulin.

In yet a further aspect, a therapeutic regimen comprises administering a STING agonist for suppression of the growth of a B cell cancer and administering immunoglobulin to restore normal B cell function following cancer cell suppression.

Other aspects and advantages of these compositions and methods are described further in the following detailed description of the preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A demonstrates that IRE-1 associates with STING. IRE-1 -/- MEFs, 5TGM1 cells, and A20 cells, were lysed in RIPA buffer or lysis buffer containing 1% NP-40, and immunoprecipitations were performed with anti-IRE-1 antibodies. Proteins

immunoprecipitated with anti-IRE-1 were immunoblotted with an anti-IRE-1 antibody. Data in this FIG. 1A are representative of three independent experiments. ** : immunoglobulin heavy chain; * : immunoglobulin light chain.

FIG. IB is similar to FIG. 1 A, except that the proteins immunoprecipitated with anti- IRE-1 were immunoblotted with an anti-STING (FIG. IB) antibody. Data replicates and symbols are the same as for FIG. 1A.

FIG. 1C is similar to FIG. 1A, except that immunoprecipitations were performed with anti-STING antibodies. Proteins immunoprecipitated with anti-STING antibodies were immunoblotted with an anti-STING antibody. Data replicates and symbols are the same as for FIG. 1A.

FIG. ID is similar to FIG. 1C, except that proteins immunoprecipitated with anti- STING antibodies were immunoblotted with an anti-IRE-1 antibody. Data replicates and symbols are the same as for FIG. 1A.

FIG. 2A shows the chemical structure of 2'2'-cGAMP.

FIG. 2B shows the chemical structure of c-di-UMP.

FIG. 2C shows the chemical structure of, 3'3'- cGAMP.

FIG. 2D shows the chemical structure of DMXAA.

FIG. 2E shows the chemical structure of 2'3'-cGAMP. FIG. 2F shows the chemical structure of CMA.

FIG. 2G is an immunoblot that shows that 3'3'-cGAMP is a potent STING agonist and activates STING more efficiently than DMXAA. WT MEFs were treated with 20 μΜ 3'3'- cGAMP or 20 μΜ DMXAA for indicated times and lysed for analysis of indicated proteins by immunoblots. AAA ATPase (p97) and actin serve as loading controls. Data is representative of three independent experiments.

FIG. 2H is an immunoblot that shows that 3'3'-cGAMP activates STING more efficiently than CMA. In the protocol of FIG. 2G, or 20 μΜ CMA replaced the DMXAA.

FIG. 21 is an immunoblot that shows the results of WT MEFs, untreated or treated with 20 μΜ 3'3'-cGAMP for 4 hour, and for which cell ly sates were immunoprecipitated by an anti-STING antibody. Bead-bound immunoprecipitated proteins were further treated with calf intestinal phosphatase (CIP) or λ phosphatase ( PPase) for 3 hours, and immunoblotted with an anti-STING antibody. Data in this FIG. 21 are representative of three independent experiments.

FIG. 2 J shows the results of WT MEFs radiolabeled for 4 h and chased for 4 h in the presence of 20 μΜ 3'3'-cGAMP, for which lysates were immunoprecipitated with an anti- STING antibody. Eluted proteins were treated with endo-H or PNGase F, and analyzed by SDS-PAGE and autoradiography. Data in this FIG. 2D are representative of three independent experiments.

FIG. 3A is an immunoblot showing that WT and IRE-1-/- MEFs were treated with 20 μΜ 3'3'-cGAMP for indicated times and then lysed for analysis of indicated proteins by immunoblots. Data are representative of three independent experiments. The IRE-l/XBP-1 pathway is critical for normal STING function.

FIGs. 3B and 3C are bar graphs showing that WT and IRE-1-/- MEFs were treated with 20 μΜ 3'3'-cGAMP for indicated times, and lysed for purification of total RNA and synthesis of cDNA. The mRNA expression levels of IFNa and IFN were measured by realtime quantitative PCR (RT-qPCR), performed in triplicate for each sample (n=3). Data from WT and IRE-1-/- MEFs were normalized to GAPDH (as 1) and shown as mean ± SD. Data are representative of three independent experiments.

FIG. 3D shows WT and IRE-1-/- MEFs that were radiolabeled for 4 h and chased for indicated times in the presence of 20 μΜ 3'3'-cGAMP. Lysates were immunoprecipitated with an anti-STING antibody, and analyzed by SDS-PAGE and autoradiography. The signal of STING was quantified by densitometry. This experiment was repeated for 3 times with similar results. FIG. 3E shows WT and XBP-1-/- MEFs that were treated with 20 μΜ 3'3'-cGAMP for indicated times and lysed for analysis of indicated proteins by immunoblots. Data are representative of three independent experiments.

FIG. 3F shows WT and XBP-1-/- MEFs that were treated with 20 μΜ 3'3'-cGAMP for indicated times were analyzed for the expression of ΓΡΝβ by RT-qPCR. Data are representative of three independent experiments.

FIG. 3G shows WT and IRE-l-/-MEFs that were untreated or treated with 3'3'- cGAMP (20 μΜ) for a course of 3 days, and subjected to XTT assays at the end of each day. Percentages of growth were determined by comparing treated groups with untreated control groups. Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. In another experiment (data not shown) but see Fig. 2G of Tang et al, Cane. Res., 76(8) at page 2140 (Apr 2016),WT and XBP-1-/- MEFs were treated with 20 mmol/L 3'3'-cGAMP for 24, 48, or 72 hours; some cells were treated with 20 mmol/L 3'3'-cGAMP for 24 hours, washed with fresh media twice, and incubated in fresh media for additional 24 or 48 hours. Cells were lysed for analysis of indicated proteins by immunoblots. Results are representative of three independent experiments.

FIG. 3H shows WT and XBP-1-/- MEFs that were untreated or treated with 3'3'- cGAMP (20 μΜ) for a course of 3 days, and subjected to XTT assays at the end of each day. Percentages of growth and data points were determined and derived as for FIG. 3G. Results are representative of three independent experiments.

FIG. 4A is an immunoblot that shows freshly purified B cells from XBP- 1 WT and XBP-IKO spleens that were treated with 20 μΜ 3'3'-cGAMP, and lysed for analysis by immunoblots for indicated proteins. Results shown in each immunoblot are representative of three independent experiments. For each experiment, naive XBP-1WT and XBP-IKO B cells were purified and pooled from at least two mouse spleens. STING agonists induce mitochondria-initiated apoptosis in B cells.

FIG. 4B show naive B cells purified from mouse spleens that were untreated or treated with c-di-UMP or 3'3'-cGAMP at indicated concentrations for 24 h, and subjected to XTT assays. Percentages of growth were determined by comparing treated with untreated groups. Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. Data are representative of three independent experiments.

FIG. 4C shows naive B cells that were cultured in the presence of LPS (20 μg/ml), CpG-1826 (0.5 μΜ) or poly(I;C) (10 μg/ml) for 2 days. At the end of each day, cells were subjected to XTT assays. Percentages of growth were determined by comparing colorimetric reading at Day 1 and Day 2 with that at Day 0. Each data point derived from four independent groups receiving the same treatment was plotted as mean ± SD. Results shown are representative of three independent experiments.

FIGs. 4D is a graph showing that naive B cells that were cultured for 3 days in the presence of LPS (20 μ^πιΐ) alone or LPS plus 15 μΜ 2'2'-cGAMP, 2'3'-cGAMP or 3'3'- cGAMP. At the end of each day, cells were subjected to XTT assays. Percentages of growth were determined by comparing cells treated with LPS plus cGAMP with those treated with LPS alone. Each data point derived from four independent groups receiving the same treatment was plotted as mean ± SD. Data are representative of three independent

experiments.

FIG. 4E shows naive B cells that were cultured in the presence of CpG-1826 (0.5 μΜ) alone or CpG- 1826 plus three types of cGAMP (15 μΜ). At the end of each day, cells were subjected to XTT assays. Percentages of growth were determined by comparing cells treated with CpG-1826 plus cGAMP with those treated with CpG-1826 alone. Each data point derived from four independent groups receiving the same treatment was plotted as mean ± SD. Data are representative of three independent experiments.

FIG. 4F show naive B cells stimulated for 2 days in the presence of LPS to allow for differentiation into plasmablasts. Plasmablasts were subsequently treated with indicated concentrations of 3'3'-cGAMP in the presence of LPS for additional 12 h, and lysed for analysis of indicated proteins by immunoblots. Data are representative of three independent experiments.

FIG. 4G shows naive B cells that were stimulated for 2 days in the presence of LPS to allow for differentiation into plasmablasts, which were subsequently treated with 15 μΜ 3'3'- cGAMP in the presence of LPS for indicated times. Lysates were analyzed for indicated proteins by immunoblots. Data are representative of three independent experiments.

FIG. 4H shows naive B cells that were stimulated for 2 days in the presence of LPS CpG-1826 to allow for differentiation into plasmablasts, which were subsequently treated with 15 μΜ 3 '3'- cGAMP in the presence of CpG-1826 for indicated times. Lysates and data were analyzed as described above.

FIG. 41 show B cells stimulated with LPS for 2 days that were untreated or treated with 15 μΜ 3'3'-cGAMP for additional 24 h, stained with Annexin V-PE and DAPI, and analyzed by flow cytofluorometry. Data are representative of three independent experiments. In another experiment (data not shown; but see Fig. 3 J of Tang et al, 2016, cited above) naive B cells were cultured in the presence of IL4 (100 ng/mL) plus CD40L (50 ng/mL) together with 3'3'-cGAMP at indicated concentrations for 3 days. At the end of each day, cells were subjected to XTT assays. Percentages of growth were determined by comparing colorimetric reading on each day with that on day 0. Each data point derived from four independent groups receiving the same treatment was plotted as meanSD. Results shown are representative of three independent experiments.

FIGs. 5 A shows that Εμ- TCL1 CLL, A20 B-cell lymphoma, and 5TGM1 multiple myeloma cells were untreated or treated with c-di-UMP (15 μΜ) or 3'3'-cGAMP (15 μΜ) for 3 days, and subjected to XTT assays at the end of each day. Percentages of growth were determined by comparing treated with untreated groups. Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. Results are representative of three independent experiments. Data shown in immunoblots in these FIGs. 5A-5D are representative of three independent experiments. STING agonists induce mitochondria-initiated apoptosis in B cell malignancies.

FIG. 5B shows 5TGM1 cells that were cultured for 24 h in the presence of 15 μΜ c- di-UMP or 3'3'-cGAMP, and lysed for analysis of indicated proteins by immunoblots.

FIG. 5C shows 5TGM1 cells that were cultured for 12 h in the presence of 3'3'- cGAMP at indicated concentrations, and lysed for analysis of indicated proteins by immunoblots.

FIG. 5D shows 5TGM1 cells that were cultured in the presence of 15 μΜ 3'3'- cGAMP for indicated times, and lysed for analysis of indicated proteins by immunoblots.

FIG. 6A is an immunoblot that shows A20, A20 STING-ZFN, 5TGM1 and 5TGM1 STING-ZFN cells that were lysed for analysis of the expression of STING and p97. Data are representative of three independent experiments. STING-null A20 and 5TGM1 cells are resistant to 3'3'-cGAMP-induced apoptosis.

FIG. 6B shows A20, A20 STING-ZFN, 5TGM1 and 5TGM1 STING-ZFN cells that were untreated or treated for 72 h with 3 '3'- cGAMP at indicated concentrations, and subjected to XTT assays. Percentages of growth were determined by comparing treated with untreated groups. Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. Results are representative of three independent experiments.

FIG. 6C is an immunoblot that shows 5TGM1 and 5TGM1 STING-ZFN cells that were treated with 20 μΜ 3'3'-cGAMP for indicated times and lysed for analysis of indicated proteins by immunoblots. Data are representative of three independent experiments. FIG. 6D is an immunoblot that shows A20 and A20 STING-ZFN cells that were treated with 20 μΜ 3'3'-cGAMP for indicated times and lysed for analysis of indicated proteins by immunoblots. Data are representative of three independent experiments. In other experiments (see Figs. 5D and 5E of Tang et al, 2016 cited above), wild-type MEFs, IRE-1-/- MEFs, A20 lymphoma, and 5TGM1 myeloma cells were radiolabeled for 12 hours and chased for indicated times in the presence of 20 mmol/L 3'3'-cGAMP. Lysates of equal radioactive counts from each sample were immunoprecipitated with an anti- STING antibody and analyzed by SDS-PAGE and autoradiography. The signal of STING was quantified by densitometry, and data were plotted. This experiment was repeated three times with similar results. 5TGM1 cells were untreated or treated with 20 mmol/L 3'3'-cGAMP for 8 hours, coimmunostained with anti-STING and anti-Man2Al antibodies, and analyzed by confocal microscopy. Scale bar, 10 mm.

FIG. 7A is a bar graph showing that 5TGM1 or 5TGM1 STING-ZFN cells were untreated or treated with 20 μΜ 3'3'-cGAMP for 4 h. Cells were lysed for RNA extraction and analyzed by RT-qPCR for the expression of IFNa. Data were normalized to GAPDH and shown as mean ± SD. Data are representative of three independent experiments.

FIG. 7B is a bar graph showing the same experiment of FIG. 7A but cells were analyzed by RT-qPCR for the expression of ΓΡΝβ.

FIG. 7C is a bar graph showing treatment of A20 and A20 STING-ZFN cells were untreated or treated with 20 μΜ 3'3'-cGAMP for 4 h, then extracted and analyzed for IFNa as in FIG. 7A.

FIG. 7D is a bar graph showing the same experiment of FIG. 7C but cells were analyzed by RT-qPCR for the expression of IFN . The IRE- 1/XBP- 1 pathway but not type- 1 interferon is responsible for 3'3'-cGAMPinduced apoptosis.

FIG. 7E is a graph showing that 5TGM1 and A20 cells were untreated or treated with

IFN of indicated concentrations for 24 h, and subjected to XTT assays. Percentages of growth were determined by comparing treated with untreated groups. Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. Data are representative of three independent experiments.

FIG. 7F is a series of immunoblots showing that 5TGM1 and A20 cells were treated with 20 μΜ 3'3'-cGAMP or 100 ng/mL IFN for 24 h, and lysed for analysis of indicated proteins by immunoblots.

FIG. 7G is an immunoblot showing that 5TGM1 cells were treated with 20 μΜ 3'3'- cGAMP or 20 μΜ 3'3'-cGAMP plus 3.5 μΜ BFA for indicated times, and lysed for analysis of indicated proteins by immunoblots. Immunoblot data are representative of three independent experiments.

FIG. 7H is a bar graph showing that 5TGM1 cells were untreated or treated with 20 μΜ 3'3'-cGAMP or 20 μΜ 3'3'-cGAMP plus BFA of increasing concentrations (0, 0.5, 1 and 3 μΜ) for 24 h, and subjected to XTT assays. Percentages of growth were determined by comparing treated with untreated groups. Fold increase in survival was determined by comparing cells treated with 3'3'-cGAMP plus 0.5, 1 or 3 μΜ BFA with those treated with 3'3'-cGAMP plus 0 μΜ BFA . Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. Data are representative of three independent experiments. See also, Fig. 61 in Tang et al 2016 cited above.

FIGs. 8A-8F show that STING agonists do not induce apoptosis in melanoma, hepatoma, and Lewis lung cancer cells. In FIGs. 8A-8C, B16 melanoma (8A), Hepa 1-6 hepatoma (8B), or LL/2 Lewis lung cancer (8C) cells were untreated or treated with 3'3'- cGAMP (20 μΜ), DMXAA (20 μΜ) or CMA (20 μΜ) for 3 days, and subjected to XTT assays at the end of each day. Percentages of growth were determined by comparing treated groups with untreated groups. Each data point derived from four independent groups receiving exactly the same treatment was plotted as mean ± SD. Data are representative of three independent experiments.

FIG. 8D is a graph showing that 5TGM1 and Hepa 1-6 cells were treated with 20 μΜ 3'3'-cGAMP for indicated times, and lysed for analysis of indicated proteins by immunoblots.

FIGs. 8E-8G show that B 16 (8E), Hepa 1-6 (8F) and LL/2 (8G) cells were treated with 20 μΜ 3'3'-cGAMP or 20 μΜ DMXAA for indicated times and lysed for analysis of indicated proteins by immunoblots. All immunoblot data in these FIGs. 8A-8F are representative of three independent experiments.

FIGs. 9A-9D show that STING-deficient 5TGM1 and A20 cells respond normally to

ER stress inducers, and exhibit normal intracellular transport of class I MHC molecules. In FIG. 9A and 9B, 5TGM1 and 5TGM1 STINGZFN cells (9A) or A20 and A20 STING-ZFN cells (9B) were treated with 5 mM DTT, 2.5 μΜ thapsigargin (Tg), 5 μg/mL tunicamycin (Tu), 100 ng/mL subtilase cytotoxin (SubAB), 20 μΜ B-I09, 3.5 μΜ Brefeldin A (BFA), or 50 μΜ MG132 for 3 h. Cells were lysed for analysis of indicated proteins by immunoblots. Data are representative of three independent experiments. In FIGs. 9C-9D, 5TGM1 and 5TGM1 STING-ZFN cells (9C) or A20 and A20 STING-ZFN cells (9D) were radiolabeled for 15 min, chased for indicated time and lysed. Ly sates were immunoprecipitated using an anti- class I MHC heavy chain (HC) antibody, and analyzed by SDS-PAGE followed by autoradiography. CHO and CHO* represent high mannose-type glycans and complex-type glycans, respectively. Data are representative of three independent experiments.

FIG. 10A shows PBMCs isolated from 2 representative CLL-bearing Εμ-TCLl mice were stained with CD3-APC-Cy7, IgM-PE-Cy7, B220-FITC, CD5-APC and DAPI. Gated live CD3- IgM+ B cell populations were analyzed for the expression of B220 and CD5.

FIG. 10B shows that CLL-bearing Εμ-TCLl mice were injected with vehicle (20% DMSO in PBS, n=9) or 3'3'-cGAMP (10 mg/kg, n=10) daily for the first 5 days in a week for a period of three weeks. Each week, blood was collected by submandibular bleeding to measure lymphocyte numbers using a HemaTrue Hematology Analyzer (HESKA), and the data were plotted as mean ± SEM. * P value < 0.05. NS: non-significant.

FIG. IOC shows that 5TGM1 or 5TGM1 STING-ZFN cells (5 χ 10⁶) were intravenously injected into KaLwRij mice (8 mice each group). Half of the 5TGM1 -grafted and 5TGM1 STING-ZFN-grafted mice were intraperitoneally injected with 3'3'-cGAMP (10 mg/kg) daily for the first 5 days of each of the first three weeks, and subjected to Kaplan- Meier survival analysis. * P value < 0.05. NS: non-significant. Intraperitoneal injections of 3'3'-cGAMP lead to leukemic regression in CLL-bearing Εμ-TCLl mice and prolong the survival of KaLwRij mice grafted with multiple myeloma.

FIG. 10E a show that 5TGM1 cells (5x 10⁶) were subcutaneously injected into immunodeficient NSG mice (n = 10) on day 0. Five 5 TGM1 -grafted NSG mice were intraperitoneally injected with the vehicle (20% DMSO in PBS) and the other five with 3'3'- cGAMP (10 mg/kg). A single injection occurred daily on the first 5 days of each of the first three weeks. No injection, but data recording was performed on day 22 and day 23. Tumor volume was plotted as mean ^SD.

FIG. 10F was a graph of the results of the experiment of FIG. 10E, in which body weight was compared with the weight recorded on day 1 (100%) and plotted as mean + SD.

FIG. 11 is a graph showing deletion of mouse STING gene by ZFN. Zinc finger nuclease (ZFN) mRNA reagents specific to STING were designed, assembled and tested for functionality using CompoZr fluorescent protein (FP)-linked ZFN technology. Successful delivery of mouse STING-specific ZFN mRNA was confirmed by Surveyor Mutation Detection Assay. Digested heteroduplexed DNA was resolved on a 10% TBE-PAGE gel to verify the cleavage of the 326 bp product into 181 bp and 145 bp fragments.

DETAILED DESCRIPTION

Methods and compositions are provided for use in directly reducing, inhibiting or suppressing the growth or metastasis of a B cell-derived cancer or tumor. The inventors determined that STING agonists induce mitochondria mediated apoptosis potently in B cells and B cell malignancies while inducing production of interferons in fibroblasts, melanoma, hepatoma, and Lewis lung cancer cells without suppressing cell growth. STING agonists induce apoptosis in B cell malignancies via binding to STING because no cytotoxicity was observed in B cell lymphoma and multiple myeloma cell lines in which the STING gene was deleted with zinc finger nucleases.

The IRE- 1/XBP-l pathway of the endoplasmic reticulum (ER) stress response is required for the function of STING, an ER-resident transmembrane protein critical for cytoplasmic DNA sensing and production of type I interferons. The IRE- 1/XBP-l pathway is downstream of STING because IRE-1- or XBP-1 -deficient cells fail to respond to STING agonists by producing interferons, while the IRE- 1/XBP-l pathway can be activated normally in cells missing STING. Further, it was determined that transient activation of the IRE- 1/XBP-l pathway protects agonist-stimulated B cell malignancies from apoptosis, suggesting a survival role of the IRE- 1/XBP-l pathway in B cell malignancies. When mice carrying leukemia or multiple myeloma were injected with the STING agonist, 3'3'-cGAMP, dramatic tumor regression and prolonged survival of mice were observed. STING agonists thus directly target B cell malignancies, other than their role in boosting an anti-tumor immune response.

COMPONENTS OF THE METHODS AND COMPOSITIONS

By "STING" (TMEM173) is meant the stimulator of interferon gene which encodes an endoplasmic reticulum (ER)-resident protein, which when activated, can lead to the phosphorylation of interferon regulatory factor 3 (IRF3) to allow for the production of type I interferons (IFNa and IFN ) to stimulate the immune system^{1"5 15"18}. In mammalian cells, a cytoplasmic DNA sensor, cyclic GMP-AMP synthase (cGAS) generates 2'3'-cGAMP as an endogenous ligand to activate STING when the sensor binds to double-stranded DNA of bacterial, viral or self origin that is present in the cytoplasm. Such double-stranded DNA in the cytoplasm is a danger signal of infections or cell anomalies. The genomic sequence and isoforms for murine STING are identified at NCBI database, Gene ID72512. The protein sequences for these isoforms are identified in NCBI by Accession Nos. AMD16372.1, NP_001276521.1, NP_001276520.1, NP_082537.1, XP_017173483.1 and Q3TBT3.2.

The genomic sequence and isoforms for human STING are identified at NCBI database, Gene ID No. 340061. The protein sequences for these sequences and isoforms are identified by Accession Nos. XP_011535042.1, XP_0011535941.1, XP001288667.1, XP005268502.1 and NP 938023.1. STING resides in the ER ' . The lumen of the ER contains chaperone proteins such as HSP70-like GRP78/BiP, HSP90-like GRP94, protein disulfide isomerase (PDI), and lectin- binding calreticulin and calnexin to facilitate the folding, assembly and quality control of integral membrane proteins and secretory proteins. The ER is also equipped with sensor molecules that have the capability in activating the ER stress response upon stress conditions³¹'³². One of these sensor molecules is IRE-1. IRE-1 is an ER resident

transmembrane protein that contains an ER stress sensor domain facing the lumen of the ER, and this luminal domain is linked to its cytoplasmic kinase/RNase domain by a

transmembrane domain. Upon activation, IRE- 1 undergoes oligomerization and

autophosphorylation to assemble a functional RNase that specifically splices 26 nucleotides from the mature XBP-1 mRNA in mammalian cells^33"37. Such an excision followed by the subsequent ligation of the mRNA leads to a reading frame shift in translation, and the spliced XBP-1 mRNA encodes a larger 54-kDa transcription factor, XBP-ls, in mammalian cells³⁶. XBP-ls is responsible for upregulating the synthesis of lipids and chaperones, contributing to the restoration of a homeostatic ER^38"40.

Stimulations of B cells with the TLR4 ligand (lipopolysaccharides, LPS) or TLR9 ligand (CpG) activate the IRE- 1/XBP- 1 pathway to support B cell growth and differentiation, as evidenced by robust B cell proliferation and antibody production^41"44. The lack of IRE-1 or XBP-1 blocks the antibody -producing function of B cells⁴¹'⁴²' ⁴⁴' ⁴⁵. Although STING-/- mice have been shown to be incapable of mounting antibody responses after immunization with a DNA vaccine encoding ovalbumin¹⁸, the response of B cells to STING agonists is still unknown. In addition, it is unclear whether STING interacts with other ER-resident proteins and plays a role in responding to stresses in the ER.

"Agonists or Activators of STING" include without limitation, any molecule, naturally occurring or synthetic that binds to the STING molecule and activates STING to stimulate its activity and related pathways. In one embodiment, the STING agonist is a cyclic dinucleotide or a chemical molecule that binds to and activates STING. The bacteria-produced cyclic dinucleotides, c-di-AMP, c-di-GMP and 3'3'-cGAMP, are agonists of STING. See Tang et al 2016, cited herein for supplemental figure 3, or provisional application Figs. 11 and 12, which show a graph showing the analysis of 3'3'-cGAMP that was chemically synthesized and analyzed by RP-HPLC or by NMR.

In another embodiment, the unnatural or synthetic dinucleotide 2'2'-cGAMP is an agonist of STING. In another embodiment, the dinucleotide 2'3'-GAMP is an agonist of STING. Some of these molecules bind to and activate mammalian STING, including mouse STING^5"14. c-di-GMP and c-di-AMP have been proposed as adjuvants to elicit potent immune response^19"21. In addition, 2'3'-cGAMP was shown to aid in radiation-based cancer therapy²². In another embodiment, the STING agonist is the chemical molecule is 5, 6-dimethyl- xanthenone-4-acetic acid (DMXAA). DMXAA has been reported to disrupt tumor vasculature and boost the immune system by producing cytokines^23"26 . In another embodiment, the STING agonist is the chemical molecule is lO-carboxymethyl-9-acridanone (CMA), a known anti-viral compound that can induce type I interferons²⁷'²⁸. Both DMXAA and CMA were recently co-crystallized with STING¹⁴' ²⁹' ³⁰.

By "B-cell derived cancer" is meant any of the following diseases or disorders, i.e., B cell lymphoma, chronic lymphocytic leukemia, small cell lymphocytic lymphoma, non-

Hodgkin lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma (MZL), mucosa-associated lymphatic tissue lymphoma (MALT), mantle cell lymphoma (MCL), Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacyticlymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell/histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma, leg type, EBV positive diffuse large B-cell lymphoma, diffuse large B-cell lymphoma associated with inflammation, intravascular large B-cell lymphoma, ALK- positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, B-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, and B- cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma.

By the term or phrase "directly reducing, inhibiting or suppressing the growth or metastasis" as used herein is meant that the STING agonist or activator operates to retard growth of, reduce the rate of growth of, or kill, a B cell-derived cancer cell while the B cell- derived cancer cell is in contact with the agonist or activator, without the need for cytokine induction of activity.

The terms "subject", "patient", or "mammalian subject", as used herein includes primarily humans, but can also be extended to include a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. In one embodiment, the subject of these methods and compositions is a human. Still other suitable mammalian subjects include, without limitation, murine, rat, canine, feline, porcine, bovine, ovine, and others.

By "chemotherapeutic agent or therapy" is meant a drug or therapy designed for using in treating cancers. One of skill in the art would readily be able to select a chemotherapeutic for formulations with or for administration with STING agonists or activators based on consideration of such factors as the B cell-derived cancer being treated and stage of the cancer, the subject's age and physical condition, among others factors. Examples of

chemotherapeutics which may be utilized as described herein include, without

limitation,cisplatin, carboplatin, 5-fluorouracil, cyclophosphamide, Oncovin, vincristine, prednisone, rituximab, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, carmustine, lomustine, semustine, thriethylenemelamine, triethylene thiophosphoramide, hexamethylmelamine altretamine, busulfan, triazines dacarbazine, methotrexate, trimetrexate, fluorodeoxyuridine, gemcitabine, cytosine arabinoside, 5- azacytidine, 2,2'-difluorodeoxycytidine, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'- deoxycoformycin, erythrohydroxynonyladenine, fludarabine phosphate, 2- chlorodeoxyadenosine, camptothecin, topotecan, irinotecan, paclitaxel, vinblastine, vincristine, vinorelbine, docetaxel, estramustine, estramustine phosphate, etoposide, teniposide, mitoxantrone, mitotane, or aminoglutethimide. Other therapies for use with the methods and compositions using STING agonist or activators as described herein include non- chemical therapies. In one embodiment, the additional or adjunctive therapy includes, without limitation, radiation, acupuncture, surgery, chiropractic care, passive or active

immunotherapy, X-ray therapy, ultrasound, diagnostic measurements, e.g., blood testing. In one embodiment, these therapies are be utilized to treat the patient. In another embodiment, these therapies are utilized to determine or monitor the progress of the disease, the course or status of the disease, relapse or any need for booster administrations of the compounds discussed herein.

By "administering" or "route of administration" is delivery of the STING agonist or activator, with or without a pharmaceutical carrier or excipient, or with or without another chemotherapeutic agent into the environment of the B cell-derived cancer cell or tumor microenvironment of the subject. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, systemic routes, such as intraperitoneal, intravenous, intranasal, intravenous, intramuscular, intratracheal, subcutaneous, and other parenteral routes of administration or intratumoral or intranodal administration. In one embodiment, the route of administration is oral. In another embodiment, the route of administration is intraperitoneal. In another embodiment, the route of administration is intravascular. Routes of administration may be combined, if desired. In some embodiments, the administration is repeated periodically.

By "B cell stimulator" is meant an immunoglobulin or immunoglobulin formulation that encourages the development of B cells or a pharmaceutical agent capable of protecting the subject from infections as a result of the absence of B cells, e.g., IVIG.

By "pharmaceutically acceptable carrier or excipient" is meant a solid and/or liquid carrier, in in dry or liquid form and pharmaceutically acceptable. The compositions are typically sterile solutions or suspensions. Examples of excipients which may be combined with the STING agonist or activator include, without limitation, solid carriers, liquid carriers, adjuvants, amino acids (glycine, glutamine, asparagine, arginine, lysine), antioxidants (ascorbic acid, sodium sulfite or sodium hydrogen-sulfite), binders (gum tragacanth, acacia, starch, gelatin, polyglycolic acid, polylactic acid, poly-d,l-lactide/glycolide, polyoxaethylene, polyoxapropylene, polyacrylamides, polymaleic acid, polymaleic esters, polymaleic amides, poly acrylic acid, poly acrylic esters, polyvinylalcohols, polyvinylesters, polyvinylethers, polyvinylimidazole, polyvinylpyrrolidon, or chitosan), buffers (borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids), bulking agents (mannitol or glycine),

carbohydrates (such as glucose, mannose, or dextrins), clarifiers, coatings (gelatin, wax, shellac, sugar or other biological degradable polymers), coloring agents, complexing agents (caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl- -cyclodextrin), compression aids, diluents, disintegrants, dyes, emulsifiers, emollients, encapsulating materials, fillers, flavoring agents (peppermint or oil of wintergreen or fruit flavor), glidants, granulating agents, lubricants, metal chelators (ethylenediamine tetraacetic acid (EDTA)), osmo-regulators, pH adjusters, preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, hydrogen peroxide, chlorobutanol, phenol or thimerosal), solubilizers, sorbents, stabilizers, sterilizer, suspending agent, sweeteners (mannitol, sorbitol, sucrose, glucose, mannose, dextrins, lactose or aspartame), surfactants, syrup, thickening agents, tonicity enhancing agents (sodium or potassium chloride) or viscosity regulators. See, the excipients in

"Handbook of Pharmaceutical Excipients", 5^th Edition, Eds.: Rowe, Sheskey, and Owen, APhA Publications (Washington, DC), 2005 and US Patent No. 7,078,053, which are incorporated herein by reference. The selection of the particular excipient is dependent on the nature of the compound selected and the particular form of administration desired. Solid carriers include, without limitation, starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, calcium carbonate, sodium carbonate, bicarbonate, lactose, calcium phosphate, gelatin, magnesium stearate, stearic acid, or talc. Fluid carriers without limitation, water, e.g., sterile water, Ringer's solution, isotonic sodium chloride solution, neutral buffered saline, saline mixed with serum albumin, organic solvents (such as ethanol, glycerol, propylene glycol, liquid polyethylene glycol, dimethylsulfoxide (DMSO)), oils (vegetable oils such as fractionated coconut oil, arachis oil, corn oil, peanut oil, and sesame oil; oily esters such as ethyl oleate and isopropyl myristate; and any bland fixed oil including synthetic mono- or diglycerides), fats, fatty acids (include, without limitation, oleic acid find use in the preparation of injectables), cellulose derivatives such as sodium carboxymethyl cellulose, and/or surfactants.

By "effective amount" is meant the amount or concentration ( by single dose or in a dosage regimen delivered per day) of the STING agonist or activator sufficient to retard, suppress or kill the B-cell derived cancer or tumor, while providing the least negative side effects to the treated subject. One of skill in the art would be able to determine the amount of these STING agonists to administer alone or in combination with an additional reagent, e.g., chemotherapeutic, antibiotic or the like. In one embodiment, the effective amount is an amount larger than that required when a STING agonist is administered to induce cytokine production in a subject. In another embodiment, the effective amount of the STING agonist is the same as that required to induce interferon production. In still another embodiment, the effective amount is that required to kill the B cell-derived cancer or tumor when administered in combination with a B cell stimulatory agent, such as an immune globulin. In a further embodiment, the combination of the STING agonist or activator with another pharmacological agent or treatment protocol permits lower than usual amounts of the STING agonist and additional chemotherapeutic agent to achieve the desired therapeutic effect. In another embodiment, the combination of the STING agonist with another chemotherapy treatment protocol permits adjustment of the additional protocol regimen to achieve the desired therapeutic effect. In one embodiment, the effective amount of the STING agonist is within the range of 1 mg/kg body weight to 100 mg/kg body weight in humans including all integers or fractional amounts within the range. In certain embodiments, the effective amount is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/kg body weight, including all integers or fractional amounts within the range. In one embodiment, the above amounts represent a single dose. In another embodiment, the above amounts define an amount delivered to the subject per day. In another embodiment, the above amounts define an amount delivered to the subject per day in multiple doses. In still other embodiments, these amounts represent the amount delivered to the subject over more than a single day.

It is to be noted that the term "a" or "an" refers to one or more. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively, i.e., to include other unspecified components or process steps. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively, i.e., to exclude components or steps not specifically recited.

As used herein, the term "about" means a variability of 10 % from the reference given, unless otherwise specified.

Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to published texts, which provide one skilled in the art with a general guide to many of the terms used in the present application.

METHODS

In one embodiment, a method of directly reducing, inhibiting or suppressing the growth or metastasis of a B-cell-derived cancer or tumor involves contacting the cancer cell or tumor cell with an activator or agonist of the endoplasmic reticulum-resident protein, STING. Such a method includes treating any one or more of the B-cell derived cancers described above in a subject in need of such treatment.

In one such embodiment, this method involves administering to a mammalian subject having a B cell cancer or tumor an effective amount of a STING agonist. In certain embodiments, the STING agonist is administered in the absence of any other

chemotherapeutic agent or other chemical or non-chemical therapy designed for treatment of the cancer of a symptom thereof. In such a method, the subject is administered the STING agonist/activator alone or in a suitable pharmaceutical formulation.

In another embodiment, the method involves administering to the subject a STING agonist in combination with a pharmaceutical agent capable of stimulating production of new healthy B cells. In one method, the B cell stimulating agent is administered during treatment with the STING activator or agonist. In another method, the B cell stimulating agent is administered following administration of the STING activator or agonist after a determination that the B cell cancer or tumor has been sufficiently inhibited. In another embodiment, the STING agonist is also administered in combination with another chemotherapeutic or an antibiotic or other pharmaceutical component designed to treat other aspects of the B cell cancer. In yet another embodiment, the STING agonist or a pharmaceutical composition containing it, is administered in concert with an anti-cancer or chemotherapy regimen. In another embodiment, the STING agonist can be administered alone or in combination with another agent in a pharmaceutically acceptable carrier or excipient.

The method of treatment of a B cell-derived cancer further includes administering the STING agonist/activator and any other therapeutic agent via any conventional route, including the routes described in detail above. In certain embodiments, the route of administration is the oral route, intraperitoneal route, or intravascular route. In other embodiments, the STING agonist/activator is administered using one route and any additional agent, such as the immune globulin or other chemotherapeutic or antibiotic is administered via a different route. In yet another embodiment, all therapeutic agents are administered via the same route.

A specific therapeutic regimen for treatment of a B cell derived cancer comprises administering a STING agonist for suppression of the growth of a B cell cancer and administering immunoglobulin to restore normal B cell function following cancer cell suppression. Intravenous immunoglobulin (IVIG) can be administered during the treatment period to maintain adequate levels of antibodies to prevent infections.

In one embodiment, the STING agonist or activator is combined with one or more of these other pharmaceutical agents, such as the immune globulin or other chemotherapeutic agent or therapy, i.e., delivered to the patient concurrently. In another embodiment, the STING agonist or activator is administered to the patient concurrently with one or more of these pharmaceutical agents or therapies. In a further embodiment, the compound is administered prior to another chemotherapeutic agent or therapy, e.g., another

chemotherapeutic agent, such as cisplatin. In still another embodiment, the compound is administered subsequent to one or more of these pharmaceutical agents or therapies, e.g., after the subject is treated with radiation to reduce tumor size. Still other embodiments involves administering the STING agonist/antagonist in a specifically ordered therapeutic regimen or substantially simultaneously with other therapeutic agents or therapies.

In yet another embodiment, the method of treating a B cell cancer involves administering the STING agonist/activator in an effective amount larger than that necessary to induce interferon production. In still further embodiments, the amount of STING agonist to kill a B cell cancer is larger than that to induce the production of interferons. In other embodiments, when the STING agonist/activator is administered with other chemotherapeutics, the amount of STING required can be more, equal to, or less than the amount necessary to induced the production of interferons. According to any of these methods the B cell cancer or tumor cell is killed directly by the STING agonist/antagonist without the need for additional use of, or induction of cytokines.

PHARMACEUTICAL FORMULATIONS

As another aspect, a novel pharmaceutical composition comprises a STING agonist in a pharmaceutically acceptable carrier or excipient in an effective amount to reduce, inhibit, retain or suppress growth of the B cell derived cancer or tumor. In one aspect, the pharmaceutical composition contains, e.g., about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, to about 90% of the STING agonist or activator in combination with a pharmaceutical carrier or excipient. The pharmaceutical excipients may be one of more of those identified above. In another aspect, the pharmaceutical composition further comprises an agent that increases healthy B cell production, such as an immune globulin.

In another embodiment, the pharmaceutical composition contains a STING agonist and a compound described herein and a chemotherapeutic. Alternatively, the STING agonist is formulated with a chemotherapeutic for treatment of the B cell derived cancers described herein. In one embodiment, the chemotherapeutic is selected from among those described above. Alternatively, the STING agonist is formulated with another effective compound or reagent for treatment of the B cell derived cancers described herein, such as a antibiotic or bactericide, a surfactant, or other reagent commonly used in formulation anti-cancer compositions.

The forms of the pharmaceutical compositions may be liquid, solid or a suspension or semi-solid and designed for use with a desired administrative route, such as those described herein. In one embodiment, the compositions contain a single dose of the STING agonist that is greater than that required to stimulate the production of interferon. In one embodiment, the compositions contain a single dose of the STING agonist that is less than that required to stimulate the production of interferon. In one embodiment, the compositions contain a single dose of the STING agonist that is less than that required to stimulate the production of interferon. In still other embodiments, the doses and dosage regimens are adjusted for the particular B cell-derived cancer, and the stage of the cancer, physical status of the subject. Such doses may range from about 1 to about 100 mg/kg subject body weight, as discussed above and include dosage regimens designed to administer the effective amount in smaller repeated doses.

Other aspects of the methods and compositions are described in the examples below. In summary, Examples 1-11 below demonstrate that STING agonists induce phosphorylation of STING and IRF3, leading to the production of type-I interferons and phosphorylation of STAT1 in MEFs, melanoma, hepatoma and Lewis lung cancer cells (FIGs. 2B, 2C, 2D, 3A, 3B, 3C, 3E, 3F, 8E, 8F and 8G). Continuous incubation with these agonists exerts little impact on the growth of these cells (FIGs. 3G, 3H and 8A-8C). Although STING agonists can also trigger malignant B cells to produce type-I interferons shortly after stimulation (FIG. 7A-7D), continuous incubation induces normal and malignant B cells to undergo rapid apoptosis (FIGs. 4A-4I, 5A-5D, 6C and 6D). STING agonist-induced apoptosis is clearly mediated by STING because STING-ZFN cells do not undergo such apoptosis (FIGs. 6A-6D). STING mediates the production of type I interferons in MEFs, melanoma, hepatoma and Lewis lung cancer cells, but apoptosis in normal and malignant B cells.

Different from MEFs, melanoma, hepatoma and Lewis lung cancer cells, normal and malignant B cells are incapable of degrading STING efficiently after stimulation by STING agonists (FIGs. 4A, 4G, 4H, 4F, 5C, 6C, 6D, and 7G). The prolonged existence of agonist- bound STING engages activation of apoptotic machineries through protein complex formation in the ER or Golgi apparatus. Upon 3'3'-cGAMP stimulations, IRE-1-/- MEFs are also less capable in degrading STING, but they do not undergo apoptosis like B cells even after prolonged treatment. Such a difference may be attributed to (i) the intrinsic lower expression levels of STING in MEFs, (ii) the different phosphorylation status of STING in MEFs, and (iii) the lack of B-cell-specific partner proteins in MEFs to allow for the formation of protein complexes that can initiate apoptosis.

Recently, in vitro treatment of 2'3'-cGAMP was shown to upregulate the surface expression of CD86 and increase proliferative activity in B cells purified from the mouse spleen⁵⁵. In this experiment, B cells were pulse-treated for 30 min with 2'3'-cGAMP (30 μΜ) dissolved in the permeabilization solution containing digitonin, washed twice with RPMI- 1640 complete medium, and cultured in the presence of 0.6 μΜ 2'3'-cGAMP for 2 days before analysis. Our data and examples below demonstrate that STING agonists exert distinct effects on different cell types, and that continuous incubation with STING agonists induces normal and malignant B cells to die rapidly. While the expression levels of IRE-1 and XBP-1 stay constant in response to STING agonists in nonhematopoietic cells, STING agonist- induced apoptosis leads to the significant degradation of IRE-1 and XBP-ls in normal and malignant B cells. BFA blocks vesicular transport between the ER and the Golgi apparatus, causes the ER stress, and activates the IRE- 1/XBP- 1 pathway. Transient activation of the IRE- 1/XBP- 1 pathway using BFA attenuates activation of apoptosis and increases the survival of STING agonist-treated malignant B cells. Upon activation by the agonists, STING needs to be transported from the ER to the Golgi apparatus for phosphorylation. Thus, we observed decreased phosphorylation of STING in malignant B cells treated with BFA.

STING agonist-induced apoptosis leads to the significantly reduced expression of IRE-1 and XBP-ls (FIGs. 5C-5D). Transient activation of this pathway attenuates activation of apoptosis and increases the survival of STING agonist-treated malignant B cells (FIGs. 7G- 7H).

STING agonists have been proposed to be used as adjuvants for vaccinations and cancer therapy^19"22' ⁵⁶. Such applications rely on the capability of STING agonists in triggering the production of type-I interferons. Type-I interferons subsequently bind to IFNAR and activate the JAK-STAT signaling pathway to allow for the increased expression of cytokines such as TNFa, IL-Ιβ, IL6 and CXCL10. Type-I interferons together with these cytokines boost the immune system by promoting proliferation, differentiation, survival and

mobilization of a number of immune cells. Our data and the examples below show that STING agonists are cytotoxic to mouse B cells (FIGs. 4A-4I), thus indicating their use as adjuvants to boost antibody production may not be feasible. Nevertheless, the specific cytotoxicity of STING agonists to malignant mouse B cells (FIGs. 5A-5D and 1 OA- IOC) provides evidence for therapeutic use of STING agonists in treating B cell malignancies in addition to their immunomodulatory activity, which is also against cancer.

Unmet medical needs still exist for the treatment of relapsed and refractory B-cell- derived malignancies such as chronic lymphocytic leukemia, mantle cell lymphoma, and multiple myeloma. The disadvantage of using STING agonists in treating B cell malignancies at least includes the collateral damage to normal B cells. However, intravenous

immunoglobulin (IVIG) can be administered during the treatment period to maintain adequate levels of antibodies to prevent infections. Such treatment has been shown useful in concert with administration of Rituximab (an anti-CD20 monoclonal antibody) prescribed for the treatment of various B cell malignancies that destroys both normal and malignant B cells.

Additionally the examples below demonstrate that IRE-1-/- and XBP-1-/- MEFs, STING agonists elicit compromised phosphorylation of STING and IRF3, reduced production of type-I interferons, and decreased phosphorylation of STAT1 (FIGs. 3A-3H), suggesting that the normal function of STING depends on the IRE-l/XBP-1 pathway of the ER stress response. Together with the data showing that the IRE-l/XBP-1 pathway can be activated normally in STING-ZFN cells by ER stress inducers (FIGs. 9A-9B), this provides evidence that the IRE-l/XBP-1 pathway is downstream of STING. These examples are provided for the purpose of illustration only. The invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein.

EXAMPLE 1: MATERIALS AND METHODS

Mice - The XBP-l^f/f, CD19Cre/XBP-l^f/f, Εμ-TCLl and KaLwRij mice were maintained at our animal facility strictly following the guidelines provided by the Wistar Institute Committee on Animal Care.

Purification of mouse B cells and Εμ-TCLl CLL cells - Splenocytes were obtained from mice by mashing the spleens through cell strainers followed by RBC lysis (Qiagen). Mouse B cells and Εμ-TCLl CLL cells were purified from mouse spleens by negative selection using CD43 (Ly48) and Pan-B magnetic beads (Miltenyi Biotech), respectively, according to the manufacturer's instructions.

Flow cytometric analysis - Peripheral blood mononuclear cells (PBMCs) were blocked for 30 minutes using FBS. Cell surface staining was achieved by incubating cells at 4°C for 30 minutes with the following anti-mouse antibodies: CD3 (145-2C11; Biolegend), IgM (e- Bioscience), B220 (RA3-6B2; BD Pharmingen) and CD5 (53-7.3; eBioscience). Viability staining was accomplished using DAPI exclusion during acquisition. Apoptotic cells were detected by Annexin V-PE/DAPI staining (BD Pharmingen). Acquisition of B-cell and CLL cell populations was performed on a LSRII cytometer (BD Biosciences) harboring a custom configuration for the Wistar Institute. Cytometry data was analyzed using Flow Jo software version 7.6.1 (Tree Star Inc.).

Antibodies and reagents - Polyhistidine-tagged mouse IRE-1 (a. a. 21-445) SEQ ID NO: 10 and mouse STING (a.a. 139-379) SEQ ID NO: 11 proteins were expressed and purified from BL21(DE3) bacterial cells by Ni-NTA affinity column chromatography

(Qiagen) followed by size exclusion column chromatography (GE Healthcare). Polyclonal antibodies against mouse IRE-1 or mouse STING were generated in rabbits, and affinity- purified against recombinant proteins. Antibodies to phospho-IRF3 (Cell Signaling), IRF3 (Cell Signaling), phospho- STAT1 Y701 (Cell Signaling), IRE-1 (Cell Signaling), XBP-1 (Cell Signaling), GRP94 (Stressgen), calnexin (Stressgen), caspase 9 (Cell Signaling), cleaved caspase 9 (Cell Signaling), caspase 3 (Cell Signaling), cleaved caspase 3 (Cell Signaling), caspase 7 (Cell Signaling), cleaved caspase 7 (Cell Signaling), PARP (Cell Signaling), MCLl (Cell Signaling), AIP1 (Cell Signaling) cleaved PARP (Cell Signaling), p97 (Fitzgerald) and actin (Sigma) were obtained commercially. Polyclonal antibodies against BiP/GRP78, PDI and class I MHC molecules were generated in rabbits. LPS (Sigma), 9-Oxo- 10(9H)- acridineacetic acid (10-carboxymethyl-9-acridanone, CMA) (Sigma), 5,6- dimethylxanthenone-4- acetic acid (DMXAA) (Sigma), dithiothreitol (DTT) (Sigma), brefeldin A (BFA) (Cell Signaling), recombinant IFN (Biolegend), CpG-1826

oligodeoxynucleotides (TIB-Molbiol), tunicamycin (Enzo Life Sciences), thapsigargin (Enzo Life Sciences), MG-132 (Enzo Life Sciences), 2'2'-cGAMP (InvivoGen), 2'3'-cGAMP (InvivoGen), c-di-UMP (InvivoGen) and poly(LC) (InvivoGen) were purchased

commercially. Subtilase cytotoxin (SubAB) was provided by Dr. James C. Paton at the University of Adelaide. We developed and synthesized the IRE-1 R ase inhibitor, B-I09 (50). 3'3'-cGAMP were chemically synthesized in house according to the known synthetic route ^l' ⁵¹ ' ⁵⁸, and its purity and identity are demonstrated by reversed-phase high-performance liquid chromatography and nuclear magnetic resonance, respectively (FIGs.11 and 12).

Cell culture - Mouse B cells and Εμ-TCLl CLL, A20 B-cell lymphoma and 5TGM1 multiple myeloma cells were cultured in the RPMI 1640 media (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 0.1 mM β-mercaptoethanol (β-ΜΕ). M5TGM1 cells were tested for the secretion of immunoglobulin and the surface expression of plasma cell marker, CD138, every 6 months. Mouse embryonic fibroblasts (MEFs), IRE-1-/- MEFs, XBP-1-/- MEFs, B16 melanoma, Hepa 1-6 hepatoma, LL/2 Lewis lung carcinoma were cultured in Dulbecco's Modified Eagle's Medium (Gibco) with the same supplemental nutrients. All the cell lines were negative for mycoplasma contamination.

Deletion of the STING gene in 5TGM1 and A20 cells with zinc finger nucleases (ZFN) and establishment of STING-null 5TGM1 and A20 cells - ZFN mRNA reagents specific to mouse STING (TMEM173) were designed, assembled and tested for functionality using CompoZr® fluorescent protein (FP)-linked ZFN technology (Sigma-Aldrich, CSTZFN). The ZFN target sequence (cut site in lowercase) is:

GGCCTGGTCATACTACATtgggtaCTTGCGGTTGATCTT SEQ ID NO: l. One million of A20 or 5TGM1 cells were combined with ZFNs and Nucleofector Solution V (Lonza, VACA- 1003) and nucleofected using the Amaxa Nucleofector I device (Lonza) with program L-013. The nucleofected cells were allowed to recover for 48 h. FP-expressing cells were collected in a pool via FACS and a portion was used for genomic DNA purification. STING (TMEM173) was amplified with JumpStart™ REDTaq® Ready Mix™ Reaction Mix (Sigma-Aldrich, P0982) with the primer pair FWD 5'- CAAGAGAAGGGCTTTGGACA-3 ' SEQ ID NO: 2 and REV 5'- GCTCCTGCCTCAAAGATCAC-3' SEQ ID NO: 3 using the thermal cycle program: 95°C for 5 min; 35 cycles: 95°C for 30 sec, 60°C for 30 sec, 72°C for 45 sec; 72°C for 5 min; and 4°C hold. The resulting PCR product was denatured, re-annealed, and digested using Surveyor® Mutation Detection Kit (IDT, 706025) according to manufacturer instructions. Digested heteroduplexed DNA was resolved on a 10% TBE gel to verify the cleavage of the 326 bp product into 181 bp and 145 bp fragments (FIG. 13). 5TGM1 STING- ZFN and A20 STING-ZFN cells were cloned via limited dilution and cultured in the RPMI 1640 media (Gibco) with supplements described above.

Mass Spectrometry - Protein bands were stained with Coomassie Brilliant Blue G-250, excised, reduced, alkylated, and digested with trypsin (Promega). Reverse-phase liquid chromatography tandem mass spectrometry (LC/MS-MS) analysis was performed by the Wistar Proteomics Facility using a Q Exactive HF mass spectrometer (Thermo Scientific) coupled with a Nano-ACQUITY UPLC system (Waters). Eluted peptides were analyzed by the mass spectrometer set to repetitively scan m/z from 400 to 2,000 in positive ion mode. The full mass spectrometry (MS) scan was collected at 70,000 resolution followed by data- dependent MS/MS scans at 17,500 resolution on the 20 most abundant ions exceeding a minimum threshold of 10,000. Peptide match was set as preferred, and exclude isotopes option and charge-state screening were enabled to reject singly and unassigned charged ions. MS data were analyzed with MaxQuant 1.5.2.8 (Ref: PMID 19029910). MS/MS spectra were searched against the mouse UniProt protein database using full tryptic specificity with up to two missed cleavages, static carboxamidomethylation of Cys, and variable oxidation of Met, protein N- terminal acetylation, and phosphorylation on Ser, Thr, and Tyr. Modified peptides were required to have a minimum score of 40. Consensus identification lists were generated with false discovery rates of 1% at protein, peptide, and site levels. MS/MS assignment of phosphorylated peptides was manually inspected and peaks were labeled using pLabel (pfind.ict.ac.cn).

Protein isolation, immunoprecipitation, dephosphorylation and immunoblotting - Cells were lysed in RIPA buffer (10 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 1 mM EDTA) supplemented with protease inhibitors (Roche) and phosphatase inhibitors. Protein concentrations were determined by BCA assays (Pierce). In some experiments, target proteins were immunoprecipitated with antibodies together with Protein G-agarose beads (Sigma), and bead-bound proteins were dephosphorylated using calf intestinal alkaline phosphatase (CIP, New England Biolabs) or lambda protein phosphatase ( PPase, New England Biolabs). Samples were boiled in SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8; 2% SDS; 10% glycerol; 0.1% bromophenol blue) with β-ΜΕ and analyzed by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, blocked in 5% non-fat milk (wt/vol in PBS), and immunoblotted with indicated primary antibodies and appropriate horseradish peroxidase-conjugated secondary antibodies. Immunoblots were developed using Western Lighting Chemiluminescence Reagent (Perkin-Elmer).

Pulse chase experiments, immunoprecipitation and protein deglycosylation - Cells were starved in methionine- and cysteine-free media containing dialyzed serum for 1 h, then pulse-labeled with 250 μθί/ιηΐ [35S]-methionine and [35 S] -cysteine (Perkin-Elmer) for indicated times. After labeling, cells were incubated in the chase medium containing unlabeled methionine (2.5 mM) and cysteine (0.5 mM). In some experiments, 3'3'-cGAMP was added in the chase medium. At the end of each chase interval, cells were lysed in RIPA buffer containing protease inhibitors. Pre-cleared lysates were incubated with an anti-mouse STING or anti-class I MHC antibody, together with Protein Gagarose beads. Bead-bound proteins were eluted using glycoprotein denaturing buffer (0.5% SDS, 1% β-ΜΕ) or reducing Laemmli SDS-PAGE sample buffer. Enzymatic deglycosylation of proteins was achieved by denaturation of the immunoprecipitates in glycoprotein denaturing buffer at 95°C for 10 min, followed by addition of sodium citrate (pH 5.5) to a final concentration of 50 mM, and incubated with Endo H (New England Biolabs) at 37°C for 3 h. Alternatively, sodium phosphate (pH 7.5) and NP- 40 were added to the denatured cell lysates to a final concentration of 50 mM and 1%, respectively, and the mixture was incubated with PNGase F (New England Biolabs) at 37°C for 3 h. Protein samples were then analyzed by SDS-PAGE and visualized by autoradiography. Densitometric quantification of radioactivity was performed on a Phosphorlmager (Fujifilm BAS-2500) using Image Reader BAS-2500 VI.8 software (Fujifilm) and Multi Gauge V2.2 (Fujifilm) software for analysis.

Reverse transcription and polymerase chain reaction (PCR) - Total RNA was isolated using TRIzol reagent (Invitrogen). Complementary DNA was synthesized from RNA using Superscript II reverse transcriptase (Invitrogen). The following sets of primers were used together with Platinum Taq DNA polymerase (Invitrogen) in PCR to detect the expression of mouse IFNa (CCA CAG GAT CAC TGT GTA CCT GAG A) SEQ ID NO: 4 and (CTG ATC ACC TCC CAG GCA CAG) SEQ ID NO: 5; mouse IFN (CAT CAA CTA TAA GCA GCT CCA) SEQ ID NO: 6 and (TTC AAG TGG AGA GCA GTT GAG) SEQ ID NO: 7 and mouse GAPDH (CTC ATG ACC ACA GTC CAT GC) SEQ ID NO: 8 and (CAC ATT GGG GGT AGG AAC AC) SEQ ID NO: 9. Immunofluorescence staining and confocal microscopy- A total of 2 x 10⁴ to 5 X10⁴ 5TGM1 or 5TGM1 STING-ZFN cells were seeded on a coverglass, treated with 3'3'-cGAMP for indicated times, spun down onto the coverglass (1,200 rpm; 10 minutes), fixed in the fixation solution (acetone : methanol =4:6) at -20 °C for 15 minutes, and air-dried. Cells were rehydrated with PBS, blocked in 3% BSA (in PBS), and incubated with rabbit anti-STING and mouse anti-Man2Al (Mannosidase II; Biolegend) primary antibodies and subsequently with Alexa 488-conjugated goat anti-rabbit (Invitrogen Molecular Probes) and Alexa 594- conjugated goat anti-mouse (Life Technologies) secondary antibodies. The coverglass was mounted on a glass slide and cells were observed using a Leica TCS SP5 II confocal microscope.

Cell proliferation XTT assays - Appropriate numbers of cells were suspended in phenol red-free culture media, seeded in 96-well cell culture plates, and treated with STING agonists, TLR ligands or other chemical compounds. Every 24 hours after the treatment, cells were spun down and proliferation was assessed by XTT assays (Roche) according to the manufacturer's instructions. Briefly, 50 μΐ XTT labeling reagent, 1 μΐ electron-coupling reagent and 100 μΐ phenol red-free culture media were combined and applied to each well of the 96-well plates. Cells were then incubated for 4 h in a CO₂ incubator to allow for the yellow tetrazolium salt XTT to be cleaved by mitochondrial dehydrogenases of metabolic active cells to form the orange formazan dye, which can be quantified at 492 nm using a BioTek Synergy HI MicroPlate Reader.

In vivo treatment of mice with 3'3'-cGAMP - The peripheral blood of Εμ-TCLl mice was collected by submandibular bleeding. Εμ-TCLl mice with high CLL burden were identified by measuring lymphocyte numbers using a HemaTrue Hematology Analyzer (HESKA) and examining the percentage of CLL cells in PBMCs. These mice received intraperitoneal injections with 3'3'-cGAMP (10 mg/kg) dissolved in 20% DMSO in PBS on Days 1, 2, 3, 4, 5, 8, 9, 10, 11, 12, 15, 16, 17, 18 and 19. Lymphocyte numbers in their peripheral blood were measured on Days 7, 14 and 21. KaLwRij mice were intravenously injected with 5 ^χ 10⁶ 5TGM1 or 5TGM1 STING-ZFN multiple myeloma cells on DO;

intraperitoneally injected with 3'3'-cGAMP (10 mg/kg) on Days 3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 17, 18, 19, 20 and 21; and monitored for survival.

Statistics - The Kaplan-Meier analysis was used to evaluate mouse survival data. A P value of less than 0.05 was considered significant.

EXAMPLE 2: IRE-1 associates with STLNG. To investigate the interaction proteins of IRE- 1 on the ER membrane, we generated several rabbit polyclonal antibodies against the luminal domain of murine IRE-1 (a.a. 21-445; SEQ ID NO: 10). Affinity-purified IRE-1 antibodies were used to immunoprecipitate IRE-1 together with its interaction partners in lipopolysaccharide (LPS)-stimulated wild-type mouse B cells. The immunoprecipitated protein complex was analyzed on an SDS-PAGE gel. A prominent ~35 kDa protein band co-immunoprecipitated with IRE-1 was excised from the gel. After in-gel proteolytic digestion, the samples were subjected to peptide sequencing with LC- MS/MS. STING was identified as an associated protein of IRE-1. We generated rabbit polyclonal antibodies against the cytoplasmic domain of mouse STING (a.a. 139-379; SEQ ID NO: 11).

To confirm that IRE-1 interacts with STING, we performed immunoprecipitations using anti-IRE-1 or anti-STING antibodies in IRE-1-/- MEFs (mouse embryonic fibroblasts), 5TGM1 cells (mouse multiple myeloma line expressing high levels of IRE-1), and A20 cells (mouse B cell lymphoma line expressing low levels of IRE-1). Proteins immunoprecipitated with the anti-IRE-1 antibody were immunoblotted with anti-IRE-1 or anti-STING antibodies (FIGs. 1A, IB and 1C), and those immunoprecipitated with the anti- STING antibody were also immunoblotted with anti-STING or anti-IRE- 1 antibodies (FIGs 1C and ID). The association of IRE-1 and STING was preserved not only in 1% NP-40 buffer but also in stringent RIPA buffer containing 0.1% SDS, 0.5% sodium deoxycholate, and 1% NP-40.

In still another experiment showing that STING co-immunoprecipitates with IRE-1, three-day LPS -stimulated wild-type B cell lysates were immunoprecipitated using an anti- IRE-1 antibody. Immunoprecipitates were analyzed by SDS-PAGE, and stained with Coomassie Brilliant Blue G-250. IRE-1 was confirmed by LC-MS/MS sequencing. IRE-l 's interactor was identified as STING (TMEM173) by LC-MS/MS sequencing.

STING peptides identified by LC-MS/MS included the following peptides in Table I

(amino acid coverage: 128/378 = 33.9%)

TABLE I

LLISGMDQPLPLR 362-374 17

MFNQLHNNMLSGAGSR 180-195 18

PYSNLHPAIPRPR 2-14 19

QEEKEEVTMNAPMTSVAPPPSVLSQEPR 334-361 20

TLEEILEDVPESR 293-305 21

Tandem MS spectra of phosphorylated S357 and S365 peptides of mouse STING were observed. Representative MS/MS spectra of the triply charged S357 phosphorylated peptide 334QEEKEEVTMNAPMTSVAPPPSVLSQEPR361 (m/z 1065.1510, 1.39 ppm error) SEQ ID NO: 20, and the double charged S365 phosphorylated peptides

362LLI S GMDQPLPLR374 (m/z 774.8945, 2.46 ppm error) SEQ ID NO: 17 were reported (not shown, but see Supplementary Fig. 7 in Tang et al 2016, cited herein.

EXAMPLE 3: 3 '3 '-cGAMP is a potent STING agonist.

Cyclic dinucleotides 2'2'-cGAMP, 3'3'-cGAMP and 2'3'-cGAMP but not c-di-UMP can bind to STING (FIG. 2A)⁹' ¹⁴. DMXAA and CMA are chemical compounds that bind to mouse but not human STING (FIG. 2A)¹⁴' ²⁹' ³⁰' ⁴⁶' ⁴⁷. We treated MEFs with 3'3'-cGAMP, DMXAA and CMA, and determined the capability of these compounds in activating phosphorylation of IRF3, which leads to the production of type I interferons (IFNa and IFN ) and the subsequent phosphorylation of STATl as a result of interferon-α/β receptor (IFNAR) activation by IFNa and IFN in an autocrine fashion (FIGs. 2B and 2C).

We found that in intact cells, 3'3'-cGAMP is more efficient than DMXAA in activating STING, as judged by phosphorylation of STING, IRF3 and STATl (FIG. 2B). The phosphorylation of STING was confirmed by treatments of immunoprecipitated STING with calf intestinal phosphatase (CIP) or λ phosphatase ( PPase) and disappearance of the phosphorylated STING protein band (FIG. 2D).

Activation of STING by 3'3'-cGAMP or DMXAA also causes STING to degrade (FIGs. 2B-2C). Although CMA was shown to bind to STING in a protein crystal structure²⁹, it does not activate STING in cells (FIG. 2C). Mouse STING contains one potential N-linked glycosylation site (Asn) in its luminal domain, but no glycosylation was detected in our deglycosylation experiments using endo-H or PNGase F (FIG. 2E).

EXAMPLE 4: The IRE-l/XBP-1 pathway is required for normal STING function.

To assess whether IRE-1 is required for activation of STING, we treated wild-type and IRE-1-/- MEFs with 3'3'-cGAMP for 0, 2, 4, 8, 12, or 24 h, and detected a significantly delayed and weaker phosphorylation of STING and IRF3 in IRE-1-/- MEFs (FIG. 3A). Correspondingly, the production of IFNa and IFN decreased significantly (FIGs.3B-3C), leading to inefficient phosphorylation of STATl in IRE-1-/- MEFs (FIG. 3A). Upon 3'3'- cGAMP stimulation, STING undergoes phosphorylation and degradation (FIGs. 2B-2C and 3A).

Thus, we measured the half-life of STING in 3 '3-cGAMP-treated cells by pulse chase experiments (FIG. 3D). STING in 3'3'-cGAMP-stimulated wild-type MEFs has a half-life of approximately 5 h, but it acquires an approximately 10-h half-life in 3'3'-cGAMP-stimulated IRE-1-/- MEFs (FIG. 3D), suggesting association with IRE-1 is critical for STING function and degradation. We examined XBP-1, a transcription factor regulated by the RNase activity of IRE-1. 3'3'-cGAMP-induced phosphorylation of STING, IRF3 and STATl as well as IFN production were similarly compromised in XBP-1-/- MEFs (FIGs. 3E-3F). Despite the significant reduction of 3'3'-cGAMP-induced activation of STING in IRE-1-/- and XBP-1 -/- MEFs, 3'3'-cGAMP does not impact the growth of these cells as well as wild-type MEFs (FIGs. 3G-3H).

EXAMPLE 5: B cells respond to STING agonists by undergoing mitochondria-mediated apoptosis.

To recapitulate compromised STING activation in XBP-1KO B cells, we treated naive B cells purified from XBP-lf/f and CD19Cre/XBP-lf/f mice with 3'3'-cGAMP. Contrary to our results from MEFs, STING in XBP-1 WT and XBP-1KO B cells does not undergo degradation upon stimulations by 3'3'-cGAMP (FIG. 4A). B cells initiate rapid apoptosis shortly after 3'3'-cGAMP stimulations, as judged by cleavage of a caspase substrate, PARP (FIG. 4A). 3'3'-cGAMP but not c-di-UMP induces cytotoxicity in B cells in a dose- dependent fashion (FIG. 4B), suggesting that cytotoxicity is a result of STING binding.

LPS and CpG can induce B cell proliferation and differentiation by engaging Toll-like receptor (TLR) 4 and TLR9, respectively (FIG. 4C). We investigated whether STING agonists have an impact on LPS- or CpG-induced cell growth. Co-incubation of 2'2'- cGAMP, 2'3'-cGAMP or 3'3'-cGAMP retards LPS- or CpG-induced B cell proliferation (FIGs. 4D-4E).

To examine the impact of STING agonists on antibody-secreting plasma cells, we first incubated purified naive B cells in LPS or CpG for 48 h to trigger their differentiation into plasmablasts, and subsequently treated these cells with 3'3'-cGAMP in a dose-dependent (FIG. 4F) and time-dependent (FIGs. 4G-4H) manner. Similarly, 3'3'-cGAMP does not induce the degradation of STING in these plasmablasts, but it triggers mitochondria-dependent apoptosis as evidenced by the cleavage of caspase 9, caspase 3, caspase 7 and PARP (FIGs. 4F-4H). Treatment of plasmablasts with 3'3'-cGAMP for 24 h turns almost all plasmablasts into Annexin V+/DAPI+ apoptotic cells (FIG. 41). 3'3'-cGAMP also potently suppresses IL4- and CD40L-induced B-cell growth (FIG. 4J).

EXAMPLE 6: STING agonists are cytotoxic to B cell leukemia, lymphoma and multiple myeloma.

Because STING agonists induced apoptosis in primary B cells and plasmablasts, we investigated whether these agonists would also be cytotoxic to primary B-cell chronic lymphocytic leukemia freshly purified from Εμ-TCLl mice^48"50, A20 B-cell lymphoma and 5TGM1 multiple myeloma. We treated these malignant B cells with 3'3'-cGAMP and c-di- UMP. 3'3'-cGAMP but not c-di-UMP was able to induce apoptosis in malignant B cells (FIGs. 5A-5B). When 5TGM1 multiple myeloma cells were treated with increasing concentrations of 3'3'-cGAMP for 12 h, we clearly detected a dose-dependent activation of mitochondria-mediated apoptosis, as shown by the decreased expression of MCL 1 and increased cleavage of caspase 9, caspase 3 and PARP (FIG. 5C). Increased apoptosis of 5TGM1 cells is associated with the decreased expression of IRE- 1 and XBP-1 in a dose- dependent manner (FIG. 5C).

In time-course experiments where 5TGM1 cells were treated with 3'3'-cGAMP, we observed time dependent activation of mitochondria-mediated apoptosis, accompanied by the time-dependent decreased expression of IRE- 1 and increased expression of AIP1, an IRE-1- associated proapoptotic protein⁵¹ (FIG. 5D).

EXAMPLE 7: STING agonists induce apoptosis in malignant B cells through binding to STING

To test whether 3'3'-cGAMP-induced apoptosis in malignant B cells is through binding to STING, we designed specific zinc finger nucleases to disrupt the mouse STING gene in A20 and 5TGM1 cell lines. Our ZFN design incorporates a GFP reporter to facilitate enrichment of STING-ZFN-positive cells after Nucleofection by FACS. By limited dilution cloning, we established STING-null A20 (A20 STING-ZFN) and 5TGM1 (5TGM1 STING- ZFN) clones (FIG. 6A). We treated 5TGM1 STING-ZFN and A20 STINGZFN cells with increasing concentration of 3'3'-cGAMP for a course of 72 h. Both STING-ZFN cell lines resist 3'3'-cGAMP-induced apoptosis (FIGs. 6B-6D).

EXAMPLE 8: STING does not degrade efficiently in malignant B cells, but undergoes phosphorylation and forms aggregates upon stimulation with 3 '3 '-cGAMP

To illustrate the difference between MEFs and malignant B cells in the degradation of STING upon 3'3'-cGAMP stimulations, we performed pulse chase experiments using wild- type MEFs, IRE- 17^" MEFs, A20 and 5TGM1 cells, and immunoprecipitated STING using anti-STING antibodies. When compared with wildtype and IRE-IT MEFs, A20 and 5TGM1 cells synthesize more STING and are less efficient in degrading it upon 3'3'-cGAMP stimulations (FIG. 6E). To compare the phosphorylation status of STING upon 3'3'-cGAMP stimulations, we chemically crosslinked anti-mouse STING antibodies to protein G-Sepharose beads by dimethyl pimelimidate (DMP), used these beads to immuno-precipitate STING from unstimulated and 3'3'-cGAMP-stimulated wild-type MEFs and 5TGM1 cells, and analyzed the immunoprecipitates by LC/MS-MS after tryptic digestion. By extracted ion

chromatograms (XIC) analyses, we detected in two independent experiments that S357 and S365 of STING were phosphorylated in 3'3' -cGAMP-treated 5TGM1 samples, and that S365 of STING was phosphorylated in 3'3'-cGAMP-treated MEFs (FIGs 14A and 14B). We did not detect phosphorylation of STING in untreated samples or obtain evidence showing that S357 of STING was phosphorylated in 3'3'-cGAMP-treated MEFs. To investigate the intracellular localization of STING in malignant B cells after stimulations with 3'3'-cGAMP, we demonstrated that our affinity -purified anti-mouse STING antibody was suitable for immunofluorescence staining because the immunofluorescence signal of STING was observed only in wild-type 5TGM1 but not 5TGM1 STING-ZFN cells (FIG. 15). STING forms aggregates in 3'3'-cGAMP-stimulated 5TGM1 cells undergoing rapid apoptosis (FIGs. 6C and 6F and 5D). These aggregates colocalized with alpha-mannosidase II (Man2Al) in the ER and Golgi apparatus (FIG. 6F).

EXAMPLE 9: The IRE-l/XBP-1 pathway but not the production of type-1 interferons is responsible for 3 '3 ' -cG AMP -induced apoptosis in malignant B cells.

While 5TGM1 STING-ZFN and A20 STING-ZFN cells do not produce IFNa and IFN in response to 3'3'-cGAMP stimulations, both STING-proficient 5TGM1 and A20 cells can produce IFNa and IFN in the first few hours of 3'3'-cGAMP stimulations before they succumb to death (FIGs. 7A-7D). To examine whether type I interferons can account for 3'3'- cGAMP-induced apoptosis, we treated 5TGM1 and A20 cells with increasing concentrations of recombinant IFN for 24 h. Even at the non-physiologically high concentration of 200 ng/mL, the 24-h IFN treatment accounts for approximately 30% growth inhibition (FIG. 7E) but not apoptosis, as confirmed by no evidence of caspase 9, caspase 3 and PARP cleavage

(FIG. 7F). This does not account for more than 50% apoptosis in A20 cells and 80% apoptosis in 5TGM1 cells after treatments with 3'3'-cGAMP for 24 h (FIG. 5A).

In light of that the IRE-l/XBP-1 pathway is suppressed in response to 3'3'-cGAMP- induced apoptosis (FIGs. 5C-5D), and that B-cell leukemia, lymphoma and myeloma requires the IRE- 1/XBP- 1 pathway for survival ' ' ' , we hypothesized that transient activation of the IRE-l/XBP-1 pathway might be able to rescue, or counter, 3'3'- cGAMP-induced apoptosis. We chose to use Brefeldin A (BFA) to activate the IRE-l/XBP-1 pathway, as it induces phosphorylation of IRE-1 and splicing of XBP-1 (FIG. 7G). When comparing 5TGM1 cells treated with 3'3'-cGAMP and 3'3'-cGAMP plus BFA, we observed that mitochondria- initiated apoptosis is significantly abated in 5TGM1 cells treated with 3'3'-cGAMP in combination with BFA (FIG 7G). Increased concentrations of BFA is positively correlated with the survival of 3'3'-cGAMP-treated 5TGM1 cells (FIG. 7H). To further investigate the role of XBP-ls in 3'3'-cGAMP-induced B-cell death, we treated LPS-stimulated XBP-1- proficient and XBP- 1-deficient B cells with 3'3'-cGAMP, and observed that XBP- 1-deficient B cells are more susceptible to 3'3'-cGAMP-induced apoptosis (Fig. 61). B-109 is an inhibitor that potently suppresses the expression of XBP-ls³⁸. B-109 enhances 3'3'-cGAMP-induced apoptosis in LPS-stimulated wild-type B cells and A20 cells (Figs not shown).

EXAMPLE 10: STING agonists do not induce apoptosis in melanoma, hepatoma and Lewis lung cancer cells.

To investigate whether 3'3'-cGAMP exerts apoptosis in other types of cancer, we treated B16 (melanoma), Hepa 1-6 (hepatoma) and LL/2 (Lewis lung cancer) cells with 3'3'- cGAMP, DMXAA and CMA. None of the STING agonists influence the growth of these cells (FIGs. 8A-8C), except that there is an approximately 20% growth inhibition in Hepa 1-6 cells after treatment with 3'3'-cGAMP for 24 h (FIG. 8B). To determine whether this was a result of cytostasis or cytotoxicity, we compared 3'3'-cGAMPtreated 5TGM1 myeloma cells with 3'3'-cGAMP -treated Hepa 1-6 cells. There is no mitochondria mediated apoptosis observed in 3'3'-cGAMP-treated Hepa 1-6 cells (FIG. 8D). Similar to MEFs (FIGs. 2B-2C), B16, Hepa 1-6 and LL/2 cells can respond to 3'3'-cGAMP by phosphorylating and degrading STING, and inducing phosphorylation of IRF3 and STAT1 (FIGs. 8E-8G). There is also no significant change in the expression levels of IRE-1 and XBP-1 during the course of 3'3'- cGAMP stimulations (data not shown). As 3'3'-cGAMP can induce apoptosis in normal B cells, we tested whether it is also cytotoxic to normal T cells. We purified T cells from wild- type mice, treated them with 3'3'-cGAMP in the presence of IL2 (to maintain T-cell survival in culture), and observed no apoptosis in 3'3'-cGAMP-treated CD4p or CD8p T cells (data not shown; see Supplementary Fig 11 in Tang 2016 cited herein).

EXAMPLE 11: STING-deflcient cells respond to ER stress inducers by activating the IRE- l/XBP-1 pathway and exhibit normal intracellular transport of class IMHC molecules. The association of STING with IRE-1 intrigued us to investigate whether STING is involved in activation of the IRE- 1/XBP- 1 pathway in response to various ER stress inducers. Each ER-stress inducer requires a distinct time duration to achieve maximal activation of the ER stress response. We stimulated 5TGM1 and 5TGM1 STING-ZFN cells for 3 h with dithiothreitol (DTT, 5 mM), thapsigargin (Tg, 2.5 μΜ), tunicamycin (Tu, 5 μg/mL), subtilase cytotoxin (SubAB which cleaves BiP and activates the IRE- 1/XBP- 1 pathway⁵⁴, 100 ng/mL), B-I09 (an IRE- 1/XBP- 1 pathway inhibitor⁵⁰, 20 μΜ), Brefeldin A (BFA, 3.5 μΜ) and proteasomal inhibitor (MG132, 50 μΜ).

We observed no striking difference in activation of the IRE- 1/XBP- 1 pathway and the expression of BiP/GRP78, GRP94, PDI and calnexin between 5TGM1 and 5TGM1 STING- ZFN cells, except the increased expression of XBP- 1 in thapsigargin- and SubAB-treated 5TGM1 STING-ZFN cells (FIG. 9A). However, this difference in the expression of XBP- Is in response to thapsigargin and SubAB was not observed between A20 and A20 STING-ZFN (FIG. 9B). In addition, the 3-h treatments with BFA or MG132 do not induce robust activation of the IRE- 1/XBP- 1 pathway in A20 and A20 STING-ZFN cells (FIG. 9B). The quality control of the ER allows only correctly folded and assembled client proteins to exit the ER and be transported to their final destinations. The class I MHC molecule is one of such proteins. BI09 enhanced the cytotoxicity of 3'3'-cGAMP in normal and malignant B cells. See Suppl. Fig. 9A and 9B in Tang et al, 2016, cited herein.

To examine whether the lack of STING on the ER membrane can disrupt the quality control function of the ER, we examined the transport of class I MHC molecules in 5TGM1, 5TGM1 STING-ZFN, A20 and A20 STING-ZFN cells by pulse chase experiments (FIGs. 9C- 9D). Class I MHC molecules in 5TGM1 STING-ZFN and A20 STING-ZFN acquired complex glycans in the Golgi apparatus just like those in the respective STING proficient counterparts (FIGs. 9C-9D), suggesting a normal ER quality control function in STING- deficient cells.

EXAMPLE 12: Intraperitoneal injections of 3 '3 '-cGAMP induce leukemic regression in Εμ- TCL1 mice and prolong the survival of 5TGM1 -grafted KaLwRij mice and suppress myeloma growth in NSG mice

Since 3'3'-cGAMP is potent in inducing apoptosis in malignant B cells in culture

(FIGs. 5A-5D), we investigated whether it can similarly elicit apoptosis in B cell malignancies in mice. We identified CLL bearing Εμ-TCLl mice by a complete blood count (CBC). We also analyzed the ratio of B220+/CD5+ CLL cells to B220+/CD5- precancerous B cells in the gated CD3-/CD19+/IgM+ population in the peripheral blood of the Εμ-TCLl mice by flow cytofluorometry. Only mice that carry >8000 lymphocytes per μΐ_^ blood and >90%

B220+/CD5+ CLL cells in the CD3-/CD19+/IgM+ population are selected for injection studies (FIGs. 10A- 10B). We observed a dramatic leukemic regression in CLL-bearing Εμ- TCL 1 mice intraperitoneally injected with 3'3 '-cGAMP (10 mg/kg) solubilized in 20% DMSO in PBS, but not in those mice injected with only the vehicle (FIG. 10B). By performing immunohistochemcial staining of cleaved caspase-3, we showed that 3'3 '-cGAMP induces apoptosis in the spleens of 3 '3 '-cGAMP-injected Εμ-TCLl mice (FIG. 10D).

To investigate whether the lack of STING can alter malignant phenotypes of 5TGM1 cells in vivo, we injected intravenously 5 ^χ 10⁶ 5TGM1 or 5TGM1 STING-ZFN cells back to KaLwRij mice (FIG. IOC). No significant difference in survival was observed between mice injected with 5TGM1 and 5TGM 1 STING-ZFN cells (FIG. IOC). Some 5TGMl-grafted and 5TGM1 STING-ZFN-grafted mice were intraperitoneally injected with 3'3'-cGAMP (10 mg/kg) daily for the first 5 days of each of the first three week. Injections with 3'3 '-cGAMP significantly prolong the survival of 5 TGM1 -grafted mice (FIG. IOC).

We also observed increased survival of 5TGM1 STING-ZFN-grafted mice (FIG.

IOC), suggesting a role of 3'3 '-GAMP in boosting an anti-tumor immune response. To highlight the direct effect of 3'3 '-cGAMP in targeting malignant B cells in vivo without the help of a functional immune system, we grafted immunodeficient NSG mice with 5TGM1 cells subcutaneously, and showed that injections with 3'3 '-cGAMP can suppress the growth of multiple myeloma without the presence of T, B, or natural killer cells (data not shown; see suppl figures of Tang et al 2016, cited herein). We confirmed that myeloma cells remain in the tumor injection site, and do not migrate to bone marrow, peripheral blood, and spleens after 3 '3'-cGAMP injections (data not shown). Injections with 3'3'-cGAMP also do not cause NSG mice to lose weight (data not shown). Such an immune-boosting function of 3 '3 '- cGAMP in suppressing tumor growth is independent of the expression of STING in cancer cells.

While the expression levels of IRE- 1 and XBP- 1 stay constant in response to STING agonists in nonhematopoietic cells, STING agonist-induced apoptosis leads to the significant degradation of IRE- 1 and XBP- ls in normal and malignant B cells. BFA blocks vesicular transport between the ER and the Golgi apparatus, causes the ER stress, and activates the IRE- 1/XBP- l pathway. Transient activation of the IRE- l/XBP- 1 pathway using BFA attenuates activation of apoptosis and increases the survival of STING agonist-treated malignant B cells. Upon activation by the agonists, STING needs to be transported from the ER to the Golgi apparatus for phosphorylation. Thus, we observed decreased phosphorylation of STING in malignant B cells treated with BFA.

To further support our hypothesis that activation of the prosurvival IRE-l/XBP-1 pathway can protect B cells from STING agonist induced apoptosis, we showed that deletion of the XBP-1 gene and chemical inhibition of XBP-ls can aggrandize the growth suppression effect of STING agonists in normal and malignant B cells.

Each and every patent, patent application, and publication, including US provisional application No. 62/191561 and Tang, C-H A. et al, Cancer Res. 76(8):2137-2152 (April 15, 2016), cited throughout the disclosure, is expressly incorporated herein by reference in its entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention are devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include such embodiments and equivalent variations.

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TABLE II

(Sequence Listing Free Text)

The following information is provided for sequences containing free text under numeric identifier <223>.

Claims

CLAIMS:

1. A method of directly reducing, inhibiting or suppressing the growth or metastasis of a B cell-derived cancer or tumor comprising contacting the cancer cell or tumor cell with an activator or agonist of the endoplasmic reticulum-resident protein, STING.

2. The method according to claim 1, comprising administering to a mammalian subject having a B cell cancer or tumor an effective amount of a STING agonist.

3. The method according to claim 1 or 2, wherein the STING agonist is a cyclic dinucleotide or a chemical molecule that binds to and activates STING.

4. The method according to claim 3, wherein the cyclic dinucleotide is c-di-AMP, c-di- GMP, 3'3'-GAMP, 2'2-GAMP, 2'3'-GAMP.

5. The method according to claim 3, wherein the chemical molecule is 5, 6-dimethyl- xanthenone-4-acetic acid or 10-carboxymethyl-9-acridanone.

6. The method according to claim 1, wherein the STING agonist is administered in the absence of any other chemotherapeutic agent or therapy.

7. The method according to any of claim 1, wherein the subject is administered a pharmaceutical agent capable of protecting the subject from infections as a result of the absence of B cells.

8. The method according to claim 7, wherein the pharmaceutical agent is an intravenous immunoglobulin (IVIG) composition.

9. The method according to claim 8, wherein the IVIG is administered during treatment with the STING activator or agonist or following administration of the STING activator or agonist after a determination that the B cell cancer or tumor has been sufficiently inhibited.

10. The method according to claim 1, wherein the cancer is killed directly by the STING agonist or activator.

11. The method according to claim 1, wherein the B-cell cancer is a B cell lymphoma, B cell lymphoma or multiple myeloma.

12. The method according to claim 2, comprising administering the STING agonist in a pharmaceutically acceptable carrier or excipient.

13. The method according to claim 12, comprising administering the STING agonist via the oral route, intraperitoneal route, intramuscular route or intravascular or route.

14. The method according to claim 1, wherein the STING agonist is administered in an amount more than that necessary to induce interferon production.

15. A pharmaceutical composition comprising a STING agonist in a pharmaceutically acceptable carrier or excipient.

16. The composition according to claim 15, further composition an immune globulin.

17. A therapeutic regimen comprising administering a STING agonist for suppression of the growth of a B cell cancer and administering IVIG to restore immunoglobulin levels in the plasma following suppression of B cell function in producing immunoglobulin.