Qunrui Ye

Aldehyde dehydrogenase isoform 1 (ALDH1) has been proved useful for the identification of cancer stem cells. However, our knowledge of the expression and activity of ALDH1 in common epithelial cancers and their corresponding normal tissues is still largely absent. Therefore, we characterized ALDH1 expression in 24 types of normal tissues and a large collection of epithelial tumor specimens (six cancer types, n = 792) by immunohistochemical staining. Using the ALDEFUOR assay, ALDH1 activity was also examined in 16 primary tumor specimens and 43 established epithelial cancer cell lines. In addition, an ovarian cancer transgenic mouse model and 7 murine ovarian cancer cell lines were analyzed. We found that the expression levels and patterns of ALDH1 in epithelial cancers are remarkably distinct, and they correlate with their corresponding normal tissues. ALDH1 protein expression levels are positively correlated with ALDH1 enzymatic activity measured by ALDEFLUOR assay. Long-term in vitro culture doesn't significantly affect ALDH1 activity in epithelial tumor cells. Consistent with research on other cancers, we found that high ALDH1 expression is significantly associated with poor clinical outcomes in serous ovarian cancer patients (n = 439, p = 0.0036). Finally, ALDH(br) tumor cells exhibit cancer stem cell properties and are resistant to chemotherapy. As a novel cancer stem cell marker, ALDH1 can be used for tumors whose corresponding normal tissues express ALDH1 in relatively restricted or limited levels such as breast, lung, ovarian or colon cancer.

The costimulatory effects of CD27 on T lymphocyte effector function and memory formation has been confined to evaluations in mouse models, in vitro human cell culture systems, and clinical observations. Here, we tested whether CD27 costimulation actively enhances human T-cell function, expansion, and survival in vitro and in vivo. Human T cells transduced to express an antigen-specific chimeric antigen receptor (CAR-T) containing an intracellular CD3 zeta (CD3ζ) chain signaling module with the CD27 costimulatory motif in tandem exerted increased antigen-stimulated effector functions in vitro, including cytokine secretion and cytotoxicity, compared with CAR-T with CD3ζ alone. After antigen stimulation in vitro, CD27-bearing CAR-T cells also proliferated, up-regulated Bcl-X(L) protein expression, resisted apoptosis, and underwent increased numerical expansion. The greatest impact of CD27 was noted in vivo, where transferred CAR-T cells with CD27 demonstrated heightened persistence after infusion, facilitating improved regression of human cancer in a xenogeneic allograft model. This tumor regression was similar to that achieved with CD28- or 4-1BB-costimulated CARs, and heightened persistence was similar to 4-1BB but greater than CD28. Thus, CD27 costimulation enhances expansion, effector function, and survival of human CAR-T cells in vitro and augments human T-cell persistence and antitumor activity in vivo.

PURPOSE: Upregulation of CD137 (4-1BB) on recently activated CD8(+) T cells has been used to identify rare viral or tumor antigen-specific T cells from peripheral blood. Here, we evaluated the immunobiology of CD137 in human cancer and the utility of a CD137-positive separation methodology for the identification and enrichment of fresh tumor-reactive tumor-infiltrating lymphocytes (TIL) or tumor-associated lymphocytes (TAL) from ascites for use in adoptive immunotherapy. EXPERIMENTAL DESIGN: TILs from resected ovarian cancer or melanoma were measured for surface CD137 expression directly or after overnight incubation in the presence of tumor cells and homeostatic cytokines. CD137(pos) TILs were sorted and evaluated for antitumor activity in vitro and in vivo. RESULTS: Fresh ovarian TILs and TALs naturally expressed higher levels of CD137 than circulating T cells. An HLA-dependent increase in CD137 expression was observed following incubation of fresh enzyme-digested tumor or ascites in IL-7 and IL-15 cytokines, but not IL-2. Enriched CD137(pos) TILs, but not PD-1(pos) or PD-1(neg) CD137(neg) cells, possessed autologous tumor reactivity in vitro and in vivo. In melanoma studies, all MART-1-specific CD8(+) TILs upregulated CD137 expression after incubation with HLA-matched, MART-expressing cancer cells and antigen-specific effector function was restricted to the CD137(pos) subset in vitro. CD137(pos) TILs also mediated superior antitumor effects in vivo, compared with CD137(neg) TILs. CONCLUSIONS: Our findings reveal a role for the TNFR-family member CD137 in the immunobiology of human cancer where it is preferentially expressed on tumor-reactive subset of TILs, thus rationalizing its agonistic engagement in vivo and its use in TIL selection for adoptive immunotherapy trials.

Human T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-α (FRα) have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of their inability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FRα-specific scFv (MOv19) coupled to the T-cell receptor CD3ζ chain signaling module alone (MOv19-ζ) or in combination with the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BBζ). Primary human T cells transduced to express conventional MOv19-ζ or costimulated MOv19-BBζ CARs secreted various proinflammatory cytokines, and exerted cytotoxic function when cocultured with FRα(+) tumor cells in vitro. However, only transfer of human T cells expressing the costimulated MOv19-BBζ CAR mediated tumor regression in immunodeficient mice bearing large, established FRα(+) human cancer. MOv19-BBζ CAR T-cell infusion mediated tumor regression in models of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly, tumor response was associated with the selective survival and tumor localization of human T cells in vivo and was only observed in mice receiving costimulated MOv19-BBζ CAR T cells. T-cell persistence and antitumor activity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cell persistence but not antitumor activity in vivo. Our results show that anti-FRα CAR outfitted with CD137 costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limit the conventional FRα T-cell targeting strategy to provide potent antitumor activity in vivo.

BACKGROUND: The poor prognosis and the limited efficacy of targeted therapy in patients with triple-negative breast cancer (TNBC) have raised the need for alternative therapies. Recent studies have demonstrated that folate receptor-alpha (FRα) may represent an ideal tumor-associated marker for immunotherapy for TNBC. METHODS: The aim of the present study was to apply a chimeric antigen receptor (CAR) approach for the targeting of FRα expressed on TNBC cells and evaluate the antitumor activity of CAR-engineered T cells in vitro and in vivo. RESULTS: We found that human T cells expressing a FRα-specific CAR were potent and specific killers of TNBC cells that express moderate levels of FRα in vitro and significantly inhibited tumor outgrowth following infusion into immunodeficient mice bearing an MDA-MB-231 tumor xenograft. However, the antitumor activity of the FRα CAR T cells was modest when compared to the same CAR T cells applied in an ovarian tumor xenograft model where FRα expression is more abundant. Notably, FRα CAR T cells induced superior tumor regression in vivo against MDA-MB-231 that was engineered for overexpression of FRα. CONCLUSIONS: Taken together, our results show that FRα CAR T cells can mediate antitumor activity against established TNBC tumor, particularly when FRα is expressed at higher levels. These results have significant implications for the pre-selection of patients with high antigen expression levels when utilizing CAR-based adoptive T cell therapies of cancer in future clinical trials.