Effective Targeting of TAG72+ Peritoneal Ovarian Tumors via Regional Delivery of CAR-Engineered T Cells.

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.

Novel strategies to target cancer stem cells by NK cells; studies in humanized mice.

We have previously shown that following selection, Natural Killer (NK) cells, differentiate Cancer stem cells (CSCs)/undifferentiated or poorly differentiated tumors via secreted and membrane bound IFN-gamma and TNF-alpha, leading to prevention of tumor growth and remodeling of the tumor microenvironment. Since conventional therapeutic strategies including chemotherapy and radiotherapy remain unsuccessful in treating stem-like tumors, there has been an increasing interest in NK cell based immunotherapy for the treatment of resistant tumors. In our recent studies, we used bone marrow, liver, thymus humanized (hu-BLT) mouse model to demonstrate the efficacy of adoptive transfer of ex vivo expanded super-charged NK cells in the selection and differentiation of stem-like tumors within the context of a fully reconstituted human immune system. We have also shown that CSCs differentiated with split anergized NK cells prior to implantation in humanized mice did not grow or metastasize. In this review, we present current advances in NK cell detection, expansion and therapeutic delivery, and discuss the utility of different humanized mouse models in studying NK cell based therapies in the preclinical settings.

Resistance to cytotoxicity and sustained release of interleukin-6 and interleukin-8 in the presence of decreased interferon-γ after differentiation of glioblastoma by human natural killer cells.

Natural killer (NK) cells are functionally suppressed in the glioblastoma multiforme (GBM) tumor microenvironment. We have recently shown that survival and differentiation of cancer stem-like cells (CSCs)/poorly differentiated tumors are controlled through two distinct phenotypes of cytotoxic and non-cytotoxic/split anergized NK cells, respectively. In this paper, we studied the function of NK cells against brain CSCs/poorly differentiated GBM and their NK cell-differentiated counterparts. Brain CSCs/poorly differentiated GBM, differentiated by split anergized NK supernatants (supernatants from NK cells treated with IL-2 + anti-CD16mAb) expressed higher levels of CD54, B7H1 and MHC-I and were killed less by the NK cells, whereas their CSCs/poorly differentiated counterparts were highly susceptible to NK cell lysis. Resistance to NK cells and differentiation of brain CSCs/poorly differentiated GBM by split anergized NK cells were mediated by interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Brain CSCs/poorly differentiated GBM expressed low levels of TNFRs and IFN-γRs, and when differentiated and cultured with IL-2-treated NK cells, they induced increased secretion of pro-inflammatory cytokine interleukin (IL)-6 and chemokine IL-8 in the presence of decreased IFN-γ secretion. NK-induced differentiation of brain CSCs/poorly differentiated GBM cells was independent of the function of IL-6 and/or IL-8. The inability of NK cells to lyse GBM tumors and the presence of a sustained release of pro-inflammatory cytokines IL-6 and chemokine IL-8 in the presence of a decreased IFN-γ secretion may lead to the inadequacy of NK cells to differentiate GBM CSCs/poorly differentiated tumors, thus failing to control tumor growth.

Adoptive transfer of osteoclast-expanded natural killer cells for immunotherapy targeting cancer stem-like cells in humanized mice.

Based on data obtained from oral, pancreatic and lung cancers, glioblastoma, and melanoma, we have established that natural killer (NK) cells target cancer stem-like cells (CSCs). CSCs displaying low MHC class I, CD54, and PD-L1 are killed by cytotoxic NK cells and are differentiated by split anergized NK cells through both membrane bound and secreted forms of TNF-α and IFN-γ. NK cells select and differentiate both healthy and transformed stem-like cells, resulting in target cell maturation and shaping of their microenvironment. In our recent studies, we have observed that oral, pancreatic, and melanoma CSCs were capable of forming large tumors in humanized bone marrow, liver, thymus (hu-BLT) mice with fully reconstituted human immune system. In addition, major human immune subsets including NK cells, T cells, B cells, and monocytes were present in the spleen, bone marrow, peripheral blood, and tumor microenvironment. Similar to our previously published in vitro data, CSCs differentiated with split anergized NK cells prior to implantation in mice formed smaller tumors. Intravenous injection of functionally potent osteoclast-expanded NK cells inhibited tumor growth through differentiation of CSCs in humanized mice. In this review, we present current approaches, advances, and existing limitations in studying interactions of the immune system with the tumor, in particular NK cells with CSCs, using in vivo preclinical hu-BLT mouse model. In addition, we discuss the use of osteoclast-expanded NK cells in targeting cancer stem-like tumors in humanized mice-a strategy that provides a much-needed platform to develop effective cancer immunotherapies.