Qunrui Ye

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. 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.

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. 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.

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. CD137pos 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 CD137pos TILs, but not PD-1pos or PD-1neg CD137neg 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 CD137pos subset in vitro. CD137pos TILs also mediated superior antitumor effects in vivo, compared with CD137neg 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. Clin Cancer Res; 20(1); 44–55. ©2013 AACR.

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. To redirect human T cells against NKG2DLs, an NKG2DL-specific CAR was generated by fusing the extracellular domain of the NKG2D receptor to the 4-1BB costimulatory and CD3-ζ chain signaling domains. In vitro expansion of chimeric NKG2D CAR T cells was delayed compared with untransduced T cells and control CAR T cells; the likely result of fratricide among activated T cells expressing NKG2DLs. However, NKG2D CAR T cells did expand and were selectively enriched during prolonged culture. In coculture, CD4(+) and CD8(+) NKG2D CAR T cells specifically recognized and killed NKG2DL-expressing ovarian cancer cell lines but not NKG2DL-negative cells. Notably, pretreatment of ovarian cancer cells expressing moderate to low levels of NKG2DLs with the histone deacetylase inhibitor sodium valproate (VPA) upregulated NKG2DL cell surface expression and consequently enhanced their immune recognition by chimeric NKG2D CAR T cells. Our results demonstrate that VPA-induced upregulation of NKG2DL expression enhances the immune recognition of ovarian cancer cells by engineered NKG2D CAR T cells, and rationalizes the use of VPA in combination with NKG2DL-targeted immunotherapy in ovarian cancer.

Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

BackgroundDendritic cells (DCs) are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines.MethodsMonocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1) culture media; 2) culture surface; 3) duration of activating DCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma; 4) method of DC harvest; and 5) cryomedia and final DC product formulation.ResultsDCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC) Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs), and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and replated in fresh media.ConclusionsThis study examined criteria including DC phenotype, viability, IL-12p70 production and the ability to stimulate MLR as metrics of whole oxidized tumor lysate-pulsed DC immunogenicity and functionality. Development and optimization of this unique method is now being tested in a clinical trial of autologous oxidized tumor lysate-pulsed DC in clinical-scale in recurrent ovarian, primary peritoneal or fallopian tube cancer (NCT01132014).

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

BackgroundThe 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.MethodsThe 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.ResultsWe 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.ConclusionsTaken 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.

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. Moreover, the effect of the administration of these cytokines along with CART cells in vivo was also studied. IL-2, IL-7, and IL-15 favored the ex vivo expansion of CART cells compared to other cytokines or no cytokine treatment. IL-7 induced the highest proportion of memory stem cell-like CART cells in the final product, and IL-21 supported the expansion of CART cells with a younger phenotype, while IL-2 induced more differentiated CART cells. IL-2 and IL-15-exposed CART cells secreted more proinflammatory cytokines and presented stronger tumor-lysis ability in vitro. However, when tested in vivo, CART cells exposed to IL-2 ex vivo showed the least anti-tumor effect. In contrast, the administration of IL-15 and IL-21 in combination with CART cells in vivo increased their tumor killing capacity. According to our results, IL-7 and IL-15 show promise to promote ex vivo expansion of CART cells, while IL-15 and IL-21 seem better suited for in vivo administration after CART cell infusion. Collectively, these results may have a profound impact on the efficacy of CART cells in both hematologic and solid cancers.

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.

Abstract A49: Investigating the expression of inhibitory surface molecules in ovarian cancer tumor-infiltrating lymphocytes.

Ovarian cancer causes more deaths than any other cancer of the female reproductive system. However, novel therapies, including immunotherapy are now being utilized to treat this cancer. In addition, efforts have been made to identify and evaluate the subsets of cells within the tumor environment that correspond with patient overall survival. Although the presence of intraepithelial CD3+ T cells and CD8+ T cells have been correlated with patient survival and CD137, a co-stimulatory molecule, has been identified as a biomarker for tumor-reactive cells, studies have not thoroughly investigated the phenotype of the T cells, specifically the CD4+, CD8+, and CD137+ subsets. In our study, we used a multi-parameter flow cytometry to measure inhibitory molecules, Th1 cytokines, and the effector phenotype in the T-cell subsets. We observed that the CD4+ and CD8+ T cells found within the tumor environment express inhibitory receptors, very limited Th1 cytokine producing cells and a decreased expression of Granzyme B within the CD8+ subset. The CD137+ population also expressed inhibitory molecules, but was still able to exhibit an effector phenotype and comprise Th1 cytokine producing cells. In conclusion, using multi-parameter flow cytometry enabled us to investigate the expression of inhibitory molecules on the CD4+, CD8+, and CD137+ subsets and measure their effector phenotype. We found that the T cells express many inhibitory receptors and this may hinder their ability to become optimal effector cells that maintain tumor control. However, future studies utilizing antibodies that block these inhibitory receptors may increase the T cells9 ability to exhibit an increased effector phenotype and contain more Th1 cytokine producing cells. Citation Format: Jessica Chacon, Tori Yamamoto, Thomas Garrabrant, Gabor Kari, Andrew Best, Qunrui Ye, Daniel Powell, Jr. Investigating the expression of inhibitory surface molecules in ovarian cancer tumor-infiltrating lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A49.