Control of triple-negative breast cancer using ex vivo self-enriched, costimulated NKG2D CAR T cells.

Background: Triple-negative breast cancer (TNBC) is an aggressive disease that currently lacks effective targeted therapy. NKG2D ligands (NKG2DLs) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy.

Methods: We applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human TNBCs.

Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level.

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.

Multiparameter comparative analysis reveals differential impacts of various cytokines on CART cell phenotype and function ex vivo and in vivo.

Exogenous cytokines are widely applied to enhance the anti-tumor ability of immune cells. However, systematic comparative studies of their effects on chimeric antigen receptor (CAR)-engineered T (CART) cells are lacking. In this study, CART cells targeting folate receptor-alpha were generated and expanded ex vivo in the presence of different cytokines (IL-2, IL-7, IL-15, IL-18, and IL-21), and their expansion, phenotype and cytotoxic capacity were evaluated, in vitro and in vivo.

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity.

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate "on-target" toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains.

Finding a needle in a haystack: Activation-induced CD137 expression accurately identifies naturally occurring tumor-reactive T cells in cancer patients.

We have recently identified tumor necrosis factor receptor superfamily, member 9 (TNFRSF9, best known as CD137 or 4-1BB) as a biomarker of tumor-reactive T cells naturally occurring in cancer patients, and developed a rapid, accurate system to comprehensively isolate lymphocytes with tumor-rejecting properties from human biopsies. Our findings reveal a previously unappreciated role for CD137, a co-stimulatory TNFR family member, in the immunobiology of human cancer.

CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor.

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.

Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition.

NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested.

Primary human ovarian epithelial cancer cells broadly express HER2 at immunologically-detectable levels.

The breadth of HER2 expression by primary human ovarian cancers remains controversial, which questions its suitability as a universal antigen in this malignancy. To address these issues, we performed extensive HER2 expression analysis on a wide panel of primary tumors as well as established and short-term human ovarian cancer cell lines. Conventional immunohistochemical (IHC) analysis of multiple tumor sites in 50 cases of high-grade ovarian serous carcinomas revealed HER2 overexpression in 29% of evaluated sites.

Pro-survival signaling via CD27 costimulation drives effective CAR T-cell therapy.

The impact of CD27 costimulation on human T lymphocyte effector function and memory formation has been limited to evaluations using in vitro cell culture systems and observations from the clinic. We therefore investigated whether CD27 signaling augments antigen redirected human T-cell survival, expansion and function in vitro and in vivo.

CD27 costimulation augments the survival and antitumor activity of redirected human T cells in vivo.

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.

In vivo persistence, tumor localization, and antitumor activity of CAR-engineered T cells is enhanced by costimulatory signaling through CD137 (4-1BB).

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.