Impressive clinical efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy for hematological malignancies have prompted significant efforts in achieving similar responses in solid tumors. The lack of truly restricted and uniform expression of tumor-associated antigens, as well as limited T cell persistence and/or tumor trafficking pose major challenges for successful translation of CAR T cell therapy in solid tumors. Recent studies have demonstrated that aberrantly glycosylated cell surface proteins on tumor cells are amenable CAR targets. Tumor-associated glycoprotein 72 (TAG72) antigen is the sialyl-Tn found on multiple O-glycoproteins expressed at high levels on the surface of several cancer types, including ovarian cancer. Here, we developed a humanized TAG72-specific CAR containing a 4-1BB intracellular co-stimulatory signaling domain (TAG72-BBζ). TAG72-BBζ CAR T cells showed potent antigen-dependent cytotoxicity and cytokine production against multiple TAG72⁺ ovarian cancer cell lines and patient-derived ovarian cancer ascites. Using in vivo xenograft models of peritoneal ovarian tumors, regional intraperitoneal delivery of TAG72-BBζ CAR T cells significantly reduced tumor growth, extended overall survival of mice, and was further improved with repeat infusions of CAR T cells. However, reduced TAG72 expression was observed in early recurring tumors, which coincided with a lack of T cell persistence. Taken together, we demonstrate efficacy with TAG72-CAR T cells in ovarian cancer, warranting further investigations as a CAR T cell therapeutic strategy for this disease.

Key Points: CpG(A)-siRNA oligonucleotides allow for targeting genes specifically in human TLR9+ immune cells and blood cancer cells. Tumoricidal and immunostimulatory properties of CpG(A)-STAT3 siRNA provide a novel therapeutic opportunity for hematologic malignancies.

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 silencing in Toll-like receptor 9 (TLR9)-positive hematopoietic cells using CpG-small interfering RNA (siRNA) conjugates. Here, we assessed the therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used mouse Cbfb-MYH11/Mpl-induced leukemia model, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require host’s effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo upregulating major histocompatibility complex class-II, costimulatory and proinflammatory mediators, such as interleukin-12, while downregulating coinhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen–specific immune responses, increase the ratio of tumor-infiltrating CD8⁺ T cells to regulatory T cells in various organs, and result in CD8⁺ T-cell–dependent regression of leukemia. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce immunity against AML and potentially other TLR9-positive blood cancers.

Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo : Role in osteoclast-mediated NK cell activation Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: Role in osteoclast-mediated NK cell activation

Our previous reports demonstrated that the magnitude of NK cell mediated cytotoxicity correlated directly with the stage and levels of differentiation of tumor cells. In addition, we have shown previously that activated NK cells induce growth inhibition of cancer cells through induction of differentiation resulting in the resistance of tumor cells to NK cell mediated cytotoxicity through secreted cytokines, as well as direct NK-tumor cell contact. In this report we show that in comparison to IL-2+anti-CD16mAb treated NK cells, activation of NK cells by probiotic bacteria sAJ2 in combination with IL-2 and anti-CD16mAb substantially decreases tumor growth and induces maturation, differentiation, and resistance of Oral Squamous Cancer Stem Cells (OSCSCs), Mia-Paca (MP2) stem-like/poorly differentiated pancreatic tumors, and healthy Stem Cells of Apical Papillae (SCAPs) through increased secretion of IFN-γ and TNF-α, as well as direct NK-tumor cell contact. Tumor resistance to NK cell mediated killing induced by IL-2+anti-CD16mAb+sAJ2 treated NK cells is mediated by both IFN-γ and TNF-α since antibodies to both, and not each cytokine alone, was able to restore tumor sensitivity to NK cells. Increased surface expression of CD54, B7H1, and MHC class I on NK differentiated tumors was mediated by IFN-γ since the addition of anti-IFN-γ abolished their increase and restored the ability of NK cells to trigger cytokine and chemokine release; whereas, differentiated tumors inhibited cytokine release by the NK cells. Monocytes synergized with NK cells in the presence of bacteria to induce regulated differentiation of stem cells through secretion of IL-10 resulting in resistance to NK cell mediated cytotoxicity and inhibition of cytokine release. Therefore, probiotic bacteria condition activated NK cells to provide augmented differentiation of cancer stem cells resulting in inhibition of tumor growth, increased tumor differentiation and decreased inflammatory cytokine release.

Cancer radiotherapy may be immunogenic, but it is unclear why its immunogenic effects are rarely sufficient to prevent tumor recurrence. Here, we report a novel Toll-like receptor 9 (TLR9)–dependent mechanism that initiates tumor regrowth after local radiotherapy. Systemic inhibition of TLR9, but not TLR4, delayed tumor recurrence in mouse models of B16 melanoma, MB49 bladder cancer, and CT26 colon cancer after localized high-dose tumor irradiation. Soluble factors in the microenvironment of regressing tumors triggered TLR9 signaling in freshly recruited myeloid cells appearing within four days of radiotherapy. The tumorigenic effects of TLR9 depended on MyD88/NF-κB–mediated upregulation of interleukin (IL)-6 expression, which in turn resulted in downstream activation of Jak/STAT3 signaling in myeloid cells. In comparing global gene expression in wild-type, TLR9-, or STAT3-deficient myeloid cells derived from irradiated tumors, we identified a unique set of TLR9/STAT3–regulated genes involved in tumor-promoting inflammation and revascularization. Blocking STAT3 function by two myeloid-specific genetic strategies corrected TLR9-mediated cancer recurrence after radiotherapy. Our results suggest that combining localized tumor irradiation with myeloid cell–specific inhibition of TLR9/STAT3 signaling may help eliminate radioresistant cancers. Cancer Res; 73(24); 7211–21. ©2013 AACR.