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Dendritic cell (DC) vaccines targeting only cancer cells have produced limited antitumor activity in most clinical studies. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To co-target CAFs and tumor cells we developed a new compound DC vaccine that encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2). DC-shA20-FAP-TRP2 vaccination induced robust FAP- and TRP2-specific T-cell responses, resulting in greater antitumor activity in the B16 melanoma model in comparison to monovalent vaccines or a vaccine encoding antigens and a control shRNA. DC-shA20-FAP-TRP2 vaccination enhanced tumor infiltration of CD8-positive T cells, and induced antigen-spreading resulting in potent antitumor activity. Thus, co-targeting of tumor cells and CAFs results in the induction of broad-based tumor-specific T-cell responses and has the potential to improve current vaccine approaches for cancer.

Immunotherapy for solid tumors has shown promise in preclinical as well as early clinical studies. However, its efficacy remains limited. The hindrance to achieving objective, long-lasting therapeutic responses in solid tumors is, in part, mediated by the dynamic nature of the tumor and its complex microenvironment. Tumor-directed therapies fail to eliminate components of the microenvironment, which can reinstate a tumorigenic milieu and contribute to recurrence. Cancer-associated fibroblasts (CAFs) form the most preponderant cell type in the solid tumor microenvironment. Given their pervasive role in facilitating tumor growth and metastatic dissemination, CAFs have emerged as attractive therapeutic targets in the tumor microenvironment. In this article, we highlight the cross-talk between CAFs and cancer cells, and discuss how targeting CAFs has the potential to improve current immunotherapy approaches for cancer.

BackgroundT cell immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) has shown promise in preclinical models as well as early clinical studies. However, patients with solid tumors often do not respond as well as patients with hematological malignancies. This lack of efficacy for solid tumors is most likely due to several factors including a) emergence of immune escape mutants, and b) inability of tumor-specific T cells to recognize and destroy the vascular bed of solid tumors, which is critical for their malignant growth. The aim of this project is to generate CARs specific for tumor endothelial marker (TEM) 8, and evaluate their anti-vasculature and anti-tumor activity in preclinical tumor models.Methods/resultsWe generated a retroviral encoding a TEM8-specific CAR consisting of the TEM8-specific single chain variable fragment AF344, a hinge/transmembrane domain, and a CD28.41BB.z endodomain. CD3/CD28-activated T cells were transduced with RD114-pseudotyped retroviral particles to generate TEM8-specific T cells and CAR expression was confirmed by FACS analysis. To evaluate the functionality of TEM8-specific T cells we used TEM8-negative cell lines (U373, A549, LM7, 293T) and 293T cells that were genetically modified to either express human TEM8 (293T.hTEM8) or murine TEM8 (293T.mTEM8). TEM8-specific T cells recognized target cells in an antigen-dependent fashion as judged by their ability to secrete pro-inflammatory cytokines (IFN-g and IL-2) in coculture assays, and kill TEM8-positive target cells. Importantly, TEM8-specific T cells readily recognized mTEM8-positive target cells, which will allow us to evaluate the safety and efficacy of TEM8-specific T cells in xenograft and immune competent murine tumor models.ConclusionWe have constructed a TEM8-specific CAR and have shown that T cells expressing this CAR recognize and kill hTEM8- or mTEM8-positive target cells. Animal studies are in progress to determine their safety and efficacy. Targeting the tumor vasculature with TEM8-specific T cells may improve current T cell immunotherapies for solid tumors.

BackgroundImmunotherapy with T cells or bispecific T cell engagers is one promising approach to improve outcomes for patients with CD19-positive hematological malignancies. We had previously shown that T cells expressing bispecific T cell engagers that recognize CD19 and CD3 (CD19-ENG T cells) are activated in an antigen dependent manner, recruit resident T cells to tumors, and have anti-tumor activity in preclinical models. In this project we now wanted to evaluate if provision of co-stimulation enhances the effector function of CD19-ENG T cells by expressing CD80 and 41BBL on their cell surface (CD19 ENG/costim T cells).MethodsCD19-ENG T cells were generated by transducing T cells with a retroviral vector encoding a CD19-specific T cell engager, and CD19-ENG/Costim T cells were generated by double transducing T cells with the previous construct and a 2nd retroviral vector encoding the costimulatory molecules 41BBL and CD80. The effector function of the generated T cells was evaluated in vitro and in a xenograft model.ResultsCD19-ENG and CD19-ENG/Costim T cells recognized CD19+ lymphoma (Daudi, Raji) and acute leukemia (BV173) cells as judged by IFN-g secretion. Both ENG T cell populations produced IL-2 in the presence of CD19-positive targets expressing CD80 and CD86 (Daudi and Raji). However, CD19-ENG/Costim T cells produced higher levels of IL-2 in comparison to CD19-ENG T cells after stimulation with BV173 (CD19+CD80-CD86-). ENG and ENG/Costim T cells specific for an irrelevant antigen (EphA2) did not produce cytokines, confirming antigen dependence. Specificity was confirmed in cytotoxicity assays. In vivo anti-tumor activity of CD19-ENGand CD19-ENG/Costim T cells was assessed in a BV173/NSG xenograft model. First we determined the minimal required CD19-ENG T cell dose to observe anti-tumor effects. Three injections of 1x107 CD19-ENG T cells cured 5/5 mice, one injection 2/5 mice, and one dose of 1x106 CD19-ENG T cells had no anti-tumor effects. In contrast one dose of 1x106 or 1x107 CD19-ENG/Costim T cells cured 5/5 mice. ENG T cells or ENG/Costim T cells recognizing EphA2 had no anti-tumor effects.ConclusionsProvision of co-stimulation increases the effector function of CD19-ENG T cells resulting in enhanced anti-tumor activity in vivo. Genetically modifying T cells to express engagers and additional molecules to enhance their effector function may present a promising alternative to current CD19-targeted immunotherapies.

Cancer-associated fibroblasts (CAFs), the principle component of the tumor-associated stroma, form a highly protumorigenic and immunosuppressive microenvironment that mediates therapeutic resistance. Co-targeting CAFs in addition to cancer cells may therefore augment the antitumor response. Fibroblast activation protein-α (FAP), a type 2 dipeptidyl peptidase, is expressed on CAFs in a majority of solid tumors making it an attractive immunotherapeutic target. To target FAP-positive CAFs in the tumor-associated stroma, we genetically modified T cells to express a FAP-specific chimeric antigen receptor (CAR). The resulting FAP-specific T cells recognized and killed FAP-positive target cells as determined by proinflammatory cytokine release and target cell lysis. In an established A549 lung cancer model, adoptive transfer of FAP-specific T cells significantly reduced FAP-positive stromal cells, with a concomitant decrease in tumor growth. Combining these FAP-specific T cells with T cells that targeted the EphA2 antigen on the A549 cancer cells themselves significantly enhanced overall antitumor activity and conferred a survival advantage compared to either alone. Our study underscores the value of co-targeting both CAFs and cancer cells to increase the benefits of T-cell immunotherapy for solid tumors.

Adoptive immunotherapy with antigen-specific T cells has shown promise for the treatment of malignancies. However, infused T cells are unable to redirect resident T cells, limiting potential benefit. While the infusion of bispecific T-cell engagers can redirect resident T cells to tumors, these molecules have a short half-life, and do not self amplify. To overcome these limitations, we generated T cells expressing a secretable T-cell engager specific for CD3 and EphA2, an antigen expressed on a broad range of human tumors (EphA2-ENG T cells). EphA2-ENG T cells were activated and recognized tumor cells in an antigen-dependent manner, redirected bystander T cells to tumor cells, and had potent antitumor activity in glioma and lung cancer severe combined immunodeficiency (SCID) xenograft models associated with a significant survival benefit. This new class of tumor-specific T cells, with the unique ability to redirect bystander T cells, may be a promising alternative to current immunotherapies for cancer.

Outcomes for patients with glioblastoma (GBM) remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)-13Rα2, epidermal growth factor receptor variant III (EGFRvIII), or human epidermal growth factor receptor 2 (HER2) has shown promise for the treatment of gliomas in preclinical models and in a clinical study (IL-13Rα2). However, targeting IL-13Rα2 and EGFRvIII is associated with the development of antigen loss variants, and there are safety concerns with targeting HER2. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) has emerged as an attractive target for the immunotherapy of GBM as it is overexpressed in glioma and promotes its malignant phenotype. To generate EphA2-specific T cells, we constructed an EphA2-specific CAR with a CD28-ζ endodomain. EphA2-specific T cells recognized EphA2-positive glioma cells as judged by interferon-γ (IFN-γ) and IL-2 production and tumor cell killing. In addition, EphA2-specific T cells had potent activity against human glioma-initiating cells preventing neurosphere formation and destroying intact neurospheres in coculture assays. Adoptive transfer of EphA2-specific T cells resulted in the regression of glioma xenografts in severe combined immunodeficiency (SCID) mice and a significant survival advantage in comparison to untreated mice and mice treated with nontransduced T cells. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive GBM.

T cells expressing chimeric antigen receptors (CARs) or the infusion of bispecific T-cell engagers (BITEs) have shown antitumor activity in humans for CD19-positive malignancies. While BITEs redirect the large reservoir of resident T cells to tumors, CAR T cells rely on significant in vivo expansion to exert antitumor activity. We have shown that it is feasible to modify T cells to secrete solid tumor antigen-specific BITEs, enabling T cells to redirect resident T cells to tumor cells. To adapt this approach to CD19-positive malignancies we now generated T cells expressing secretable, CD19-specific BITEs (CD19-ENG T cells). CD19-ENG T cells recognized tumor cells in an antigen-dependent manner as judged by cytokine production and tumor killing and redirected bystander T cells to tumor cells. Infusion of CD19-ENG T cells resulted in regression of leukemia or lymphoma in xenograft models and a survival advantage in comparison to control mice. Genetically modified T cells expressing engager molecules may present a promising addition to current CD19-targeted immunotherapies.

Dendritic cell (DC) vaccines targeting only cancer cells have produced limited antitumor activity in most clinical studies. Targeting cancer-associated fibroblasts (CAFs) in addition to cancer cells may enhance antitumor effects, since CAFs, the central component of the tumor stroma, directly support tumor growth and contribute to the immunosuppressive tumor microenvironment. To co-target CAFs and tumor cells we developed a new compound DC vaccine that encodes an A20-specific shRNA to enhance DC function, and targets fibroblast activation protein (FAP) expressed in CAFs and the tumor antigen tyrosine-related protein (TRP)2 (DC-shA20-FAP-TRP2). DC-shA20-FAP-TRP2 vaccination induced robust FAP- and TRP2-specific T-cell responses, resulting in greater antitumor activity in the B16 melanoma model in comparison to monovalent vaccines or a vaccine encoding antigens and a control shRNA. DC-shA20-FAP-TRP2 vaccination enhanced tumor infiltration of CD8-positive T cells, and induced antigen-spreading resulting in potent antitumor activity. Thus, co-targeting of tumor cells and CAFs results in the induction of broad-based tumor-specific T-cell responses and has the potential to improve current vaccine approaches for cancer.