BAFF THERAPY TO PROMOTE ANTI-TUMOR IMMUNITY

Abstract

Anti-tumor immune response are generated by induction of activated B cells to provide costimulatory signals necessary for T cell activation. Certain compositions are combined with anti-immune checkpoint inhibitors to generate a synergistic anti-tumor response.

Claims

1. A method of treating cancer comprising: administering to a subject in need thereof, a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF) wherein the BAFF activates an immune response to a tumor; thereby treating cancer.

2. The method of claim 1, wherein further comprising administering a tumor-specific antigen.

3. The method of claim 1, wherein the BAFF increases B-cell expression of CD40 and/or CD86 as compared to a control.

4. The method of claim 1, wherein the BAFF is protein or active fragment thereof.

5. A method of increasing an antigen specific immune response in vivo, comprising administering to a subject a composition comprising BAFF and a desired antigen for which a specific immune response is desired.

6. The method of claim 5, wherein the antigen comprises: a vaccine, a peptide, an irradiated cell, polynucleotide, oligonucleotide or combinations thereof.

7. The method of claim 5 wherein the antigen is a tumor antigen, a virus antigen or a combination thereof.

8. (canceled)

9. The method of claim 50, wherein the at least one checkpoint inhibitor comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

10. The method of claim 50, wherein the at least one checkpoint inhibitor is anti-PD-1 antibody.

11. The method of claim 50, wherein the BAFF increases B-cell expression of CD40 and/or CD86 as compared to a control.

12. The method of claim 50, wherein BAFF-treated B cells stimulate CD4.sup.+ T cell activity.

13. The method of claim 50, wherein the BAFF increases reduces T regulatory cell activity and increases TH17 activity.

14. The method of claim 50, wherein the BAFF is protein or active fragment thereof.

15. A method of treating cancer, comprising: co-culturing ex vivo, CD4.sup.+ T cells with B-cell activating factor (BAFF)-treated B cells obtained from a subject suffering from cancer; re-infusing the CD4.sup.+ T cells and BAFF-treated B cells into the subject; thereby treating the subject's cancer.

16. The method of claim 15, wherein the CD4.sup.+ T cells and BAFF-treated B cells are co-cultured with tumor cells obtained from the subject.

17. The method of claim 15, wherein the CD4.sup.+ T cells and BAFF-treated B cells are co-cultured with tumor cell antigens.

18. The method of claim 15, further comprising co-culturing the CD4.sup.+ T cells and BAFF-treated B cells with at least one checkpoint inhibitor.

19. The method of claim 15, wherein the at least one checkpoint inhibitor is co-administered to the patient, during re-infusion of the CD4.sup.+ T cells and BAFF-treated B cells.

20. The method of claim 18, wherein the at least one checkpoint inhibitor is administered to the patient prior to, during and/or after re-infusion of the CD4.sup.+ T cells and BAFF-treated B cells.

21. The method of claim 18, wherein the at least one checkpoint inhibitor comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

22-49. (canceled)

50. The method of claim 1 comprising administering to the subject a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF) and at least one checkpoint inhibitor;

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 is a series of graphs from flow cytometric analyses showing that B cells treated with BAFF express high levels of PD-L1.

[0045] FIG. 2 is a series of graphs from flow cytometric analyses showing B cells treated with BAFF have increased CD86 and CD40 expression.

[0046] FIG. 3 is a series of graphs from flow cytometric analyses showing B cells treated with BAFF have an improved ability to stimulate CD4.sup.+ T cells.

[0047] FIG. 4 is a series of graphs showing proliferation of CD4.sup.+ T cells in response to BAFF-treated B cells.

[0048] FIG. 5 is a graph showing recombinant BAFF synergies with anti-PD-1 therapy promotes ant-tumor immunity.

[0049] FIGS. 6A, 6B are graphs showing that BAFF is an effective therapeutic anti-cancer vaccine adjuvant.

[0050] FIG. 7A, 7B are graphs showing that BAFF as a vaccine adjuvant can increase the number of anti-tumor IgG antibodies.

[0051] FIGS. 8A and 8B are plots showing that B cells treated with BAFF show high levels of PD-L1 (FIG. 8A) and MHCII (FIG. 8B) expression. 1×10.sup.6 splenocytes from a B6 mouse were cultured for 72 hours in a flat bottom well of a 48-well plate containing 400 microliters of CLT medium (RPMI with 10% Fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol), with or without recombinant mouse BAFF (1 μg). Flow cytometry was performed on the cells after 72 hours. The BAFF-treated cells express high levels of PD-L1 in a dose-dependent fashion.

[0052] FIGS. 9A-9D are plots showing that B cells treated with BAFF show increased MHCII (FIGS. 9A and 9C) and CD40 (FIGS. 9B and 9D) expression.

[0053] FIG. 10 is a series of plots showing that BAFF-treated B cells have an improved ability to stimulate CD4.sup.+ T cells. B cells were isolated from a B6 mouse using an EASYSEP™ Human B Cell Enrichment Kit. 0.5×10.sup.6 B cells per well were cultured in a flat bottom well of a 48-well plate containing 400 microliters of CLT medium (RPMI with 10% Fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol). Control or recombinant mouse BAFF (3 μg) were added to the well for 24 hours. For the last 8 hours, Ova peptide (sequence SLKISQAVHAAHAEINEAGR, SEQ ID NO: 1) were added. The plate was then spun and washed three times to remove any BAFF or excess peptide not processed by the B cells. B cells were resuspended in 400 microliters of fresh CLT medium. 1×10.sup.6 CD4 T cells from an OT2 mouse were added to each well containing B cells, allowing 48 hours of coculture with the B cells. CD4 T cells cocultured with BAFF-treated peptide pulsed B cells showed significant increase in CD44 and CD69 expression at the end of the 48 hour coculture experiment.

[0054] FIG. 11 is a series of plots showing that BAFF-treated B cells have an improved ability to stimulate CD4.sup.+ T cells. The experiments from FIG. 10 were repeated using CFSE-stained CD4.sup.+ T cells to demonstrate CD4.sup.+ T cell proliferation. These results show that BAFF-treated B cells have increased activation and an improved ability to stimulate and expand CD4.sup.+ T cells. However, they upregulate PD-L1 as a major compensatory mechanism.

[0055] FIG. 12 is a graph showing that systemic recombinant BAFF synergizes with anti-PD-1 therapy to promote anti-tumor immunity.

[0056] FIGS. 13A and 13B are plots showing that systemic recombinant BAFF reduces T regulatory cells (FIG. 13B) and increased TH17 (FIG. 13A) cells in tumor bearing mice.

[0057] FIGS. 14A and 14B are graphs showing that BAFF is an effective therapeutic anticancer vaccine adjuvant. FIG. 14A compares the tumor volume (mm.sup.3) between Mock-Vac and BAFF-Vac. FIG. 14B compares the percent survival between mice receiving the Mock-Vac versus the BAFF-Vac. A 3T3-derived cell line that secretes mouse BAFF. was created. FIGS. 14A and 14B show that use of this bystander BAFF-secreting cell line induces a more potent anti-tumor immune response when coadministered with irradiated tumor cells, as compared to a 3T3-mock cell line which does not secrete BAFF. Here, 20 NeuN mice were administered 5×10.sup.5 NT2.5 tumor cells (a mouse model of HER2.sup.+ breast cancer), injected subcutaneously into the R mammillary gland. On day 3 after tumor inoculation, mice received 3×10.sup.6 irradiated 3T3-BAFF+3×10.sup.6 irradiated NT2.5 tumor cells (BAFF-Vac, n=10) or 3×10.sup.6 irradiated 3T3-mock+3×10.sup.6 irradiated NT2.5 tumor cells (Mock-Vac, n=10), injected over 3 limbs. Survival for the group receiving irradiated 3T3-BAFF+NT2.5 tumor cells was greater than the group receiving the mock vaccine.

[0058] FIG. 15 is a series of plots showing that BAFF as a vaccine adjuvant can increase the number of anti-tumor IgG antibodies. Mice were treated as described for FIGS. 14A and 14B. At days 7 and 15 after vaccination with BAFF-Vac or Mock-Vac, 150 μl was collected using tail bleeds from each mouse using heparinized capillary tubes. The collected blood was spun down and serum was collected. Using FACs of the NeuN tumor cells, with the mouse serum used as a primary antibody, and using a secondary anti-IgG antibody, a marked increase in anti-tumor IgG was found at 15 days.

DETAILED DESCRIPTION

[0059] B-cell activating factor (BAFF) also known as tumor necrosis factor ligand superfamily member 13B as well as B Lymphocyte Stimulator (BLyS) is a protein that binds to three known receptors: TNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFF-R. BAFF is thought to contribute to the development of autoimmune disorders including lupus.

[0060] Recombinant BAFF was shown, in the examples section which follows, to induce activated B cells which provide costimulatory signals necessary for T cell activation, proliferation, and survival. It was also shown that BAFF leads to a compensatory increase of PD-L1 expression on B cells. The combination of BAFF with an anti-PD-1 pathway antibody leads to a synergistic anti-tumor response.

[0061] Accordingly, in certain embodiments, a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF). In other embodiments, a method of treating cancer comprises administering to a subject in need thereof, a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF).

[0062] The amino acid sequences of naturally occurring full-length human BAFF, BCMA, TACI, and BR3 are available under GenBank™ accession numbers AAD25356, BAB60895, AAP57629, and AAK91826, respectively.

[0063] In certain embodiments, a method of treating cancer comprises administering to a subject in need thereof, a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF) and at least one checkpoint inhibitor. In certain embodiments, a checkpoint inhibitor comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

[0064] Compositions and Methods of Treatment

[0065] Compositions can include cells expressing BAFF, vectors encoding BAFF molecules, a polynucleotide encoding BAFF or oligonucleotides thereof, a polypeptide of BAFF or peptides thereof, mutants, active fragments, orthologs, analogs or combinations thereof.

[0066] In certain embodiments, a composition comprises an isolated cell comprising a vector encoding B-cell activating factor (BAFF) or active fragments thereof. The cells are preferably of mammalian origin, however, the invention is not so limited. In certain embodiments, the cell is a B cell, a stem cell, a bone marrow cell, a tumor cell, or cell-line.

[0067] In certain embodiments, a method of treating cancer, comprises co-culturing ex vivo, CD4.sup.+ T cells with B-cell activating factor (BAFF)-treated B cells obtained from a subject suffering from cancer and re-infusing the CD4.sup.+ T cells and BAFF-treated B cells into the subject. The CD4.sup.+ T cells and BAFF-treated B cells can be co-cultured with tumor cells obtained from the subject or tumor antigens, so as to induce tumor cell specific T cells. The tumor cells can be irradiated.

[0068] In certain embodiments, a method of treating cancer comprises administering to a subject in need thereof, a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF). In certain embodiments, a method of treating cancer comprises administering to a subject in need thereof, a composition comprising a therapeutically effective amount of B-cell activating factor (BAFF) and an anti-tumor vaccine. Examples of anti-tumor vaccines include irradiated tumor cells, tumor antigens etc.

[0069] In other embodiments, a method of increasing an antigen specific immune response in vivo, comprises administering to a subject a composition comprising BAFF and a desired antigen for which a specific immune response is desired. The antigen can be a vaccine, a peptide, an irradiated cell, polynucleotide, oligonucleotide, etc.

[0070] In certain embodiments, a composition comprising BAFF is administered as an adjuvant.

[0071] In other embodiments, a method of increasing an antigen specific immune response in vivo, comprises isolating B cells from a subject; culturing the B cells with an antigen and B-cell activating factor (BAFF); co-culturing the BAFF- and antigen culture B cells with CD4.sup.+ T cells and reinfusing the T and B cells into the subject. The antigen can be any desired antigen, e.g. a tumor antigen, viral antigen and the like. The method may include administering a checkpoint inhibitor. Examples of checkpoint inhibitor include, without limitation an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

[0072] In other embodiments, a method of inducing a tumor response in vivo, comprises isolating B cells from a subject; transforming the B cells comprising a vector encoding B-cell activating factor (BAFF); pulsing or culturing the B cells with a tumor antigen; and re-infusing the cells into the patient. In certain embodiments, a checkpoint inhibitor is administered.

[0073] In certain embodiments, the B cells or cells comprising a vector encoding B-cell activating factor (BAFF) are cultured with tumor antigens, tumor cells and/or antigen presenting cells. In other embodiments, the CD4.sup.+ T cells, BAFF-treated B cells or cells comprising a vector encoding B-cell activating factor (BAFF) are cultured with tumor antigens, tumor cells and/or antigen presenting cells.

[0074] The compositions embodied herein can be used to increase an antigen specific immune response in vivo. In certain embodiments a method of increasing an antigen specific immune response in vivo, comprises isolating B cells from a subject; culturing the B cells with an antigen and B-cell activating factor (BAFF); co-culturing the BAFF- and antigen culture B cells with CD4.sup.+ T cells and reinfusing the T and B cells into the subject. The antigen can be any desired antigen for treating a disease, whether a tumor, virus etc.

[0075] In certain embodiments, an antigen specific immune response is directed to one or more tumor antigens. Accordingly, in some embodiments, a method of inducing a tumor response in vivo, comprises isolating B cells from a subject; transforming the B cells with a vector encoding B-cell activating factor (BAFF), or, transforming a cell with a vector encoding B-cell activating factor (BAFF); pulsing or culturing the B cells with a tumor antigen or tumor cells and re-infusing the cells into the patient. The tumor cells are irradiated or are non-irradiated. In certain embodiments, the cells are cultured with antigen presenting cells, e.g. macrophages, dendritic cells. In certain embodiments, a checkpoint inhibitor is administered. In certain embodiments, a checkpoint inhibitor comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

[0076] In certain embodiments, a composition comprises an isolated B lymphocyte, an isolated T lymphocyte, a B-cell activating factor (BAFF) or active fragments thereof. The composition optionally comprises tumor antigens or tumor cells. In certain embodiments, the composition further comprises a checkpoint inhibitor, wherein the checkpoint inhibitor comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

[0077] In certain embodiments, one or more tumor antigens include, but is not limited to, 5 alpha reductase, alpha-fetoprotein, AM-1, APC, April, BAGE, beta-catenin, Bcl12, bcr-abl, CA-125, CASP-8/FLICE, Cathepsins, CD19, CD20, CD21, CD23, CD22, CD33 CD35, CD44, CD45, CD46, CD5, CD52, CD55, CD59, CDCl27, CDK4, CEA, c-myc, Cox-2, DCC, DcR3, E6/E7, CGFR, EMBP, Dna78, farnesyl transferase, FGF8b, FGF8a, FLK-1/KDR, folic acid receptor, G250, GAGE-family, gastrin 17, gastrin-releasing hormone, GD2/GD3/GM2, GnRH, GnTV, GP1, gp100/Pme117, gp-100-in4, gp15, gp75/TRP-1, hCG, heparanse, Her2/neu, HMTV, Hsp70, hTERT, IGFR1, IL-13R, iNOS, Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT, MAGE-family, mammaglobin, MAP17, melan-A/MART-1, mesothelin, MIC A/B, MT-MMPs, mucin, MUC-1, NY-ESO-1, osteonectin, p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, PAP, PDGF, uPA, PRAME, probasin, progenipoientin, PSA, PSM, RAGE-1, Rb, RCAS1, SART-1, SSX-family, STAT3, STn, TAG-72, TGF-alpha, TGF-beta, Thymosin-beta-15, TNF-alpha, TYRP-, TYRP-2, tyrosinase, VEGF, ZAG, p16INK4, and glutathione-S-transferase.

[0078] Checkpoint Inhibitors

[0079] In certain embodiments, a method of treating cancer, comprises co-culturing ex vivo, CD4.sup.+ T cells with B-cell activating factor (BAFF)-treated B cells obtained from a subject suffering from cancer and re-infusing the CD4.sup.+ T cells and BAFF-treated B cells into the subject. The CD4.sup.+ T cells and BAFF-treated B cells can be co-cultured with tumor cells obtained from the subject or tumor antigens, so as to induce tumor cell specific T cells. The tumor cells can be irradiated. Further, at least one checkpoint inhibitor may be added to the culturing of cells and/or at least one checkpoint inhibitor is co-administered to the patient, during re-infusion of the CD4.sup.+ T cells and BAFF-treated B cells. In other embodiments, the at least one checkpoint inhibitor is administered to the patient prior to, during and/or after re-infusion of the CD4.sup.+ T cells and BAFF-treated B cells. Examples of checkpoint inhibitors include without limitation, an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β.

[0080] The duration and/or dose of treatment with checkpoint inhibitor therapies may vary according to the particular anti-immune checkpoint inhibitor agent or combination thereof (e.g., anti-ARG1 agents like small molecule inhibitors in combination with inhibitors of PD-1, PD-L1, PD-L2, CTLA4, and the like). An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. For example, dosage concentrations and dosing regimens are configured based upon one or more cancer related factors such as tumor size, tumor volume, cancer stage of a cancer patient or group of cancer patients (such as pre or post metastatic cancer). The invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.

[0081] The Programmed Death 1 (PD-1) protein is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Other members of the CD28 family include CD28, CTLA-4, ICOS and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified, Program Death Ligand 1 (PD-L1) and Program Death Ligand 2 (PD-L2). PD-L1 and PD-L2 have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53).

[0082] PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40 kDa type 1 transmembrane protein. PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1, but do not bind to CD28 or CTLA-4 (Blank et al. (2005) Cancer Immunol Immunother. 54:307-14). Binding of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3.zeta. (Sheppard et al. (2004) FEBS Lett. 574:37-41). PD-1 signaling attenuates PKC-0 activation loop phosphorylation resulting from TCR signaling, necessary for the activation of transcription factors NF-κB and AP-1, and for production of IL-2. PD-L1 also binds to the costimulatory molecule CD80 (B7-1), but not CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45:3567-72).

[0083] Expression of PD-L1 on the cell surface has been shown to be upregulated through IFN-γ stimulation. PD-L1 expression has been found in many cancers, including human lung, ovarian and colon carcinoma and various myelomas, and is often associated with poor prognosis (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53; Okazaki et al. (2007) Intern. Immun. 19:813-24; Thompson et al. (2006) Cancer Res. 66:3381-5). PD-L1 has been suggested to playa role in tumor immunity by increasing apoptosis of antigen-specific T-cell clones (Dong et al. (2002) Nat Med 8:793-800). It has also been suggested that PD-L1 might be involved in intestinal mucosal inflammation and inhibition of PD-L1 suppresses wasting disease associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63).

[0084] Studies with checkpoint inhibitor antibodies for cancer therapy have generated unprecedented response rates in cancers previously thought to be resistant to cancer treatment (see, e.g., Ott & Bhardwaj, 2013, Frontiers in Immunology 4:346; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Pardoll, 2012, Nature Reviews 12:252-264). Therapy with antagonistic checkpoint blocking antibodies against CTLA-4, PD-1 and PD-L1 are one of the most promising new avenues of immunotherapy for cancer and other diseases. In contrast to the majority of anti-cancer agents, checkpoint inhibitor do not target tumor cells directly, but rather target lymphocyte receptors or their ligands in order to enhance the endogenous antitumor activity of the immune system. (Pardoll, 2012, Nature Reviews 12:252-264) Because such antibodies act primarily by regulating the immune response to diseased cells, tissues or pathogens, they may be used in combination with other therapeutic modalities, such as antibody-drug conjugates (ADCs), to enhance the anti-tumor effect of the ADCs.

[0085] Programmed cell death protein 1 (PD-1, also known as CD279) encodes a cell surface membrane protein of the immunoglobulin superfamily, which is expressed in B cells and NK cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll, 2012, Nature Reviews 12:252-264). Anti-PD1 antibodies have been used for treatment of melanoma, non-small-cell lung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triple-negative breast cancer, leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85).

[0086] Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011, Curetech LTD.). Anti-PD1 antibodies are commercially available, for example from ABCAM™ (AB137132), BIOLEGEND™ (EH12.2H7, RMP1-14) and Affymetrix Ebioscience (J105, J116, MIH4).

[0087] Programmed cell death 1 ligand 1 (PD-L1, also known as CD274) is a ligand for PD-1, found on activated T cells, B cells, myeloid cells and macrophages. The complex of PD-1 and PD-L1 inhibits proliferation of CD8.sup.+ T cells and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65). Anti-PDL1 antibodies have been used for treatment of non-small cell lung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematologic malignancies (Brahmer et al., 2012, N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14:13044-51).

[0088] Exemplary anti-PDL1 antibodies include MDX-1105 (MEDAREX), durvalumab (MEDI4736, MEDIMMUNE) atezolizumab (TECENTRIQ™, MPDL3280A, GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB). Anti-PDL1 antibodies are also commercially available, for example from AFFYMETRIX EBIOSCIENCE (MIH1).

[0089] In certain embodiments, a checkpoint inhibitor is an RNA interfering agent.

[0090] Combination Therapies.

[0091] The compositions of the invention embodied herein, can be administered with one or more alternative treatment regimens, such as targeted and/or untargeted anti-cancer therapies can be administered. Combination therapies are also contemplated and can comprise, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation and chemotherapy, each combination of which can be with or without anti-immune checkpoint inhibitor therapy.

[0092] Immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). For example, anti-VEGF and mTOR inhibitors are known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.

[0093] In one embodiment, chemotherapy is used. Chemotherapy includes the administration of a chemotherapeutic agent. Such a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs: mercaptopurine and thioguanine: DNA antimetabolites: 2′-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-L and/or PARP-2) inhibitors are used and such inhibitors are well known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34; 3-aminobenzamide (Trevigen); 4-amino-1,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025. The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta.-nicotinamide adenine dinucleotide (NAD.sup.+) into nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis (Bouchard V. J. et al., Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)). Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single-strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q. et al. (1997) Genes Dev 11:2347-2358). Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The foregoing examples of chemotherapeutic agents are illustrative, and are not intended to be limiting.

[0094] In another embodiment, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (I-125 palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al., eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

[0095] In another embodiment, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).

[0096] In another embodiment, hyperthermia, a procedure in which body tissue is exposed to high temperatures (up to 106° F.) is used. Heat may help shrink tumors by damaging cells or depriving them of substances they need to live. Hyperthermia therapy can be local, regional, and whole-body hyperthermia, using external and internal heating devices. Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) to try to increase their effectiveness. Local hyperthermia refers to heat that is applied to a very small area, such as a tumor. The area may be heated externally with high-frequency waves aimed at a tumor from a device outside the body. To achieve internal heating, one of several types of sterile probes may be used, including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae; and radiofrequency electrodes. In regional hyperthermia, an organ or a limb is heated. Magnets and devices that produce high energy are placed over the region to be heated. In another approach, called perfusion, some of the patient's blood is removed, heated, and then pumped (perfused) into the region that is to be heated internally. Whole-body heating is used to treat metastatic cancer that has spread throughout the body. It can be accomplished using warm-water blankets, hot wax, inductive coils (like those in electric blankets), or thermal chambers (similar to large incubators). Hyperthermia does not cause any marked increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause discomfort or even significant local pain in about half the patients treated. It can also cause blisters, which generally heal rapidly.

[0097] In still another embodiment, photodynamic therapy (also called PDT, photoradiation therapy, phototherapy, or photochemotherapy) is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light PDT destroys cancer cells through the use of a fixed-frequency laser light in combination with a photosensitizing agent. In PDT, the photosensitizing agent is injected into the bloodstream and absorbed by cells all over the body. The agent remains in cancer cells for a longer time than it does in normal cells. When the treated cancer cells are exposed to laser light, the photosensitizing agent absorbs the light and produces an active form of oxygen that destroys the treated cancer cells. Light exposure must be timed carefully so that it occurs when most of the photosensitizing agent has left healthy cells but is still present in the cancer cells. The laser light used in PDT can be directed through a fiber-optic (a very thin glass strand). The fiber-optic is placed close to the cancer to deliver the proper amount of light. The fiber-optic can be directed through a bronchoscope into the lungs for the treatment of lung cancer or through an endoscope into the esophagus for the treatment of esophageal cancer. An advantage of PDT is that it causes minimal damage to healthy tissue. However, because the laser light currently in use cannot pass through more than about 3 centimeters of tissue (a little more than one and an eighth inch), PDT is mainly used to treat tumors on or just under the skin or on the lining of internal organs. Photodynamic therapy makes the skin and eyes sensitive to light for 6 weeks or more after treatment. Patients are advised to avoid direct sunlight and bright indoor light for at least 6 weeks. If patients must go outdoors, they need to wear protective clothing, including sunglasses. Other temporary side effects of PDT are related to the treatment of specific areas and can include coughing, trouble swallowing, abdominal pain, and painful breathing or shortness of breath.

[0098] In yet another embodiment, laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors. The term “laser” stands for light amplification by stimulated emission of radiation. Ordinary light, such as that from a light bulb, has many wavelengths and spreads in all directions. Laser light, on the other hand, has a specific wavelength and is focused in a narrow beam. This type of high-intensity light contains a lot of energy. Lasers are very powerful and may be used to cut through steel or to shape diamonds. Lasers also can be used for very precise surgical work, such as repairing a damaged retina in the eye or cutting through tissue (in place of a scalpel). Although there are several different kinds of lasers, only three kinds have gained wide use in medicine: Carbon dioxide (CO.sub.2) laser—This type of laser can remove thin layers from the skin's surface without penetrating the deeper layers. This technique is particularly useful in treating tumors that have not spread deep into the skin and certain precancerous conditions. As an alternative to traditional scalpel surgery, the CO.sub.2 laser is also able to cut the skin. The laser is used in this way to remove skin cancers. Neodymium:yttrium-aluminum-garnet (Nd:YAG) laser—Light from this laser can penetrate deeper into tissue than light from the other types of lasers, and it can cause blood to clot quickly. It can be carried through optical fibers to less accessible parts of the body. This type of laser is sometimes used to treat throat cancers. Argon laser—This laser can pass through only superficial layers of tissue and is therefore useful in dermatology and in eye surgery. It also is used with light-sensitive dyes to treat tumors in a procedure known as photodynamic therapy (PDT). Lasers have several advantages over standard surgical tools, including: Lasers are more precise than scalpels. Tissue near an incision is protected, since there is little contact with surrounding skin or other tissue. The heat produced by lasers sterilizes the surgery site, thus reducing the risk of infection. Less operating time may be needed because the precision of the laser allows for a smaller incision. Healing time is often shortened; since laser heat seals blood vessels, there is less bleeding, swelling, or scarring. Laser surgery may be less complicated. For example, with fiber optics, laser light can be directed to parts of the body without making a large incision. More procedures may be done on an outpatient basis. Lasers can be used in two ways to treat cancer; by shrinking or destroying a tumor with heat, or by activating a chemical—known as a photosensitizing agent—that destroys cancer cells. In PDT, a photosensitizing agent is retained in cancer cells and can be stimulated by light to cause a reaction that kills cancer cells. CO.sub.2 and Nd:YAG lasers are used to shrink or destroy tumors. They may be used with endoscopes, tubes that allow physicians to see into certain areas of the body, such as the bladder. The light from some lasers can be transmitted through a flexible endoscope fitted with fiber optics. This allows physicians to see and work in parts of the body that could not otherwise be reached except by surgery and therefore allows very precise aiming of the laser beam. Lasers also may be used with low-power microscopes, giving the doctor a clear view of the site being treated. Used with other instruments, laser systems can produce a cutting area as small as 200 microns in diameter—less than the width of a very fine thread. Lasers are used to treat many types of cancer. Laser surgery is a standard treatment for certain stages of glottis (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancers. In addition to its use to destroy the cancer, laser surgery is also used to help relieve symptoms caused by cancer (palliative care). For example, lasers may be used to shrink or destroy a tumor that is blocking a patient's trachea (windpipe), making it easier to breathe. It is also sometimes used for palliation in colorectal and anal cancer. Laser-induced interstitial thermotherapy (LITT) is one of the most recent developments in laser therapy. LITT uses the same idea as a cancer treatment called hyperthermia; that heat may help shrink tumors by damaging cells or depriving them of substances they need to live. In this treatment, lasers are directed to interstitial areas (areas between organs) in the body. The laser light then raises the temperature of the tumor, which damages or destroys cancer cells.

[0099] Pharmaceutical Compositions

[0100] The compositions of the invention can be administered as pharmaceutical compositions. In certain embodiments, a pharmaceutical composition comprises an isolated cell wherein the isolated cell comprises a vector encoding a B-cell activating factor (BAFF) or active fragments thereof. In certain embodiments, the pharmaceutical composition further comprises a checkpoint inhibitor. In certain embodiments, the checkpoint inhibitor comprises an inhibitor of: PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 or TGFR-β. The isolated cell is a B cell, a stem cell, a bone marrow cell, a tumor cell, or cell-line.

[0101] The pharmaceutical compositions of the present invention may be specially formulated, in pharmaceutically acceptable carriers or salts, for parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; intravaginally or intrarectally, for example, as a pessary, cream or foam; or aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

[0102] The phrase “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0103] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose, (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar, (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water, (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0104] The term “pharmaceutically-acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex encompassed by the invention. These salts can be prepared in situ during the final isolation and purification of the agents, or by separately reacting a purified agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.

[0105] In other cases, the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex. These salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

[0106] All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, applicants do not admit any particular reference is “prior art” to their invention.

EXAMPLES

[0107] The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

Example 1: B Cells Treated with BAFF Show High Levels of PD-L1 Expression

[0108] 1×10.sup.6 splenocytes from a B6 mouse were cultured for 72 hours in a flat bottom well of a 48-well plate containing 400 microliters of CLT medium (RPMI with 10% Fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol). Recombinant mouse BAFF (0 μg, 1 μg, or 2 μg) was added to each well.

[0109] Flow cytometry was performed on the cells after 72 hours. The BAFF-treated cells express high levels of PD-L1 in a dose-dependent fashion.

Example 2: B Cells Treated with BAFF Show Increased CD86 and CD40 Expression

[0110] As compared to control, BAFF-cultured B cells show increased expression of CD86 and CD40. As compared to B cells activated by ant-IgM antibody, B cells activated by BAFF show increased expression of PD-L1 and decreased expression of PD-1.

Example 3: BAFF-Treated B Cells have an Improved Ability to Stimulate CD4.SUP.+ T Cells

[0111] B cells were isolated from a B6 mouse using a EASYSEP™ Human B Cell Enrichment Kit. 0.5×10.sup.6 B cells per well were cultured in a flat bottom well of a 48-well plate containing 400 microliters of CLT medium (RPMI with 10% Fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol). Control or recombinant mouse BAFF (3 μg) were added to the well for 24 hours. For the last 8 hours, Ova peptide (sequence SLKISQAVHAAHAEINEAGR (SEQ ID NO: 1)) were added. The plate was then spun and washed three times to remove any BAFF or excess peptide not processed by the B cells. B cells were resuspended in 400 microliters of fresh CLT medium. 1×10.sup.6 CD4 T cells from an OT2 mouse were added to each well containing B cells, allowing 48 hours of co-culture with the B cells.

[0112] CD4 T cells co-cultured with BAFF-treated peptide pulsed B cells showed significant increase in CD44 and CD69 expression at the end of the 48 hour co-culture experiment.

[0113] This experiment was repeated with CFSE-stained CD4 T cells to demonstrate CD4 T cell proliferation.

Example 4: BAFF is an Effective Therapeutic Anticancer Vaccine Adjuvant

[0114] A 3T3-derived cell line was created that secretes mouse BAFF. Here it was shown that use of this bystander BAFF-secreting cell line induced a more potent anti-tumor immune response when co-administered with irradiated tumor cells, as compared to a 3T3-mock cell line which does not secrete BAFF. 20 NeuN mice were administered 5×10.sup.5 NT2.5 tumor cells (a mouse model of HER2.sup.+ breast cancer), injected subcutaneously into the R mammillary gland. On day 3 after tumor inoculation, mice received 3×10.sup.6 irradiated 3T3-BAFF+3×10.sup.6 irradiated NT2.5 tumor cells (BAFF-Vac, n=10) or 3×10.sup.6 irradiated 3T3-mock+3×10.sup.6 irradiated NT2.5 tumor cells (Mock-Vac, n=10), injected over 3 limbs. Survival for the group receiving irradiated 3T3-BAFF+NT2.5 tumor cells was greater than the group receiving the mock vaccine.

Example 5: BAFF as a Vaccine Adjuvant can Increase the Number of Anti-Tumor IgG Antibodies

[0115] Mice were treated as described above in Example 4. At days 7 and 15 after vaccination with BAFF-Vac or Mock-Vac, 150 μl were collected using tail bleeds from each mouse using heparinized capillary tubes. The collected blood was spun down and serum was collected. Using FACs of the NeuN tumor cells, with the mouse serum used as a primary antibody, and using a secondary anti-IgG antibody, a marked increase in anti-tumor IgG was evident at 15 days.

Example 6: B Cells Treated with BAFF Show High Levels of PD-L1 and MHCII Expression

[0116] 1×10.sup.6 splenocytes from a B6 mouse were cultured for 72 hours in a flat bottom well of a 48-well plate containing 400 microliters of CLT medium (RPMI with 10% Fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol), with or without recombinant mouse BAFF (1 μg). Flow cytometry was performed on the cells after 72 hours. The BAFF-treated cells express high levels of PD-L1 (FIG. 8A) in a dose-dependent fashion.

[0117] B cells treated with BAFF show increased MHCII (FIGS. 9A and 9C) and CD40 (FIGS. 9B and 9D) expression.

Example 7: BAFF-Treated B Cells have an Improved Ability to Stimulate CD4.SUP.+ T Cells

[0118] B cells were isolated from a B6 mouse using an EASYSEP™ Human B Cell Enrichment Kit. 0.5×10.sup.6 B cells per well were cultured in a flat bottom well of a 48-well plate containing 400 microliters of CLT medium (RPMI with 10% Fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin, and 50 μM β-mercaptoethanol). Control or recombinant mouse BAFF (3 μg) were added to the well for 24 hours. For the last 8 hours, Ova peptide (sequence SLKISQAVHAAHAEINEAGR, SEQ ID NO: 1) were added. The plate was then spun and washed three times to remove any BAFF or excess peptide not processed by the B cells. B cells were resuspended in 400 microliters of fresh CLT medium. 1×10.sup.6 CD4 T cells from an OT2 mouse were added to each well containing B cells, allowing 48 hours of coculture with the B cells. CD4 T cells cocultured with BAFF-treated peptide pulsed B cells showed significant increase in CD44 and CD69 expression at the end of the 48 hour coculture experiment (FIG. 10).

Example 8: BAFF-Treated B Cells have an Improved Ability to Stimulate CD4.SUP.+ T Cells

[0119] The experiments from FIG. 10 were repeated using CFSE-stained CD4.sup.+ T cells to demonstrate CD4.sup.+ T cell proliferation. These results show that BAFF-treated B cells have increased activation and an improved ability to stimulate and expand CD4.sup.+ T cells. However, they upregulate PD-L1 as a major compensatory mechanism.

Example 9: Systemic Recombinant BAFF Administration

[0120] Systemic recombinant BAFF administration synergizes with anti-PD-1 therapy to promote anti-tumor immunity (FIG. 12). FIGS. 13A and 13B demonstrate that systemic recombinant BAFF reduces T regulatory cells (FIG. 13B) and increased TH17 (FIG. 13A) cells in tumor bearing mice.

Example 10: BAFF is an Effective Therapeutic Anticancer Vaccine Adjuvant

[0121] Results shown in FIGS. 14A and 14B demonstrate that BAFF is an effective therapeutic anticancer vaccine adjuvant. FIG. 14A compares the tumor volume (mm.sup.3) between Mock-Vac and BAFF-Vac. FIG. 14B compares the percent survival between mice receiving the Mock-Vac versus the BAFF-Vac. A 3T3-derived cell line that secretes mouse BAFF was created. Use of this bystander BAFF-secreting cell line induces a more potent anti-tumor immune response when coadministered with irradiated tumor cells, as compared to a 3T3-mock cell line which does not secrete BAFF. Here, 20 NeuN mice were administered 5×10.sup.5 NT2.5 tumor cells (a mouse model of HER2.sup.+ breast cancer), injected subcutaneously into the R mammillary gland. On day 3 after tumor inoculation, mice received 3×10.sup.6 irradiated 3T3-BAFF+3×10.sup.6 irradiated NT2.5 tumor cells (BAFF-Vac, n=10) or 3×10.sup.6 irradiated 3T3-mock+3×10.sup.6 irradiated NT2.5 tumor cells (Mock-Vac, n=10), injected over 3 limbs. Survival for the group receiving irradiated 3T3-BAFF+NT2.5 tumor cells was greater than the group receiving the mock vaccine.

[0122] BAFF as a vaccine adjuvant can also increase the number of anti-tumor IgG antibodies (FIG. 15). Mice were treated as described above. At days 7 and 15 after vaccination with BAFF-Vac or Mock-Vac, 150 μl was collected using tail bleeds from each mouse using heparinized capillary tubes. The collected blood was spun down and serum was collected. Using FACs of the NeuN tumor cells, with the mouse serum used as a primary antibody, and using a secondary anti-IgG antibody, a marked increase in anti-tumor IgG was found at 15 days.

OTHER EMBODIMENTS

[0123] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0124] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.