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Preclinical and early clinical studies have demonstrated that chimeric antigen receptor (CAR)-redirected T cells are highly promising in cancer therapy. We observed that targeting HER2 in a glioblastoma (GBM) cell line results in the emergence of HER2-null tumor cells that maintain the expression of nontargeted tumor-associated antigens. Combinational targeting of these tumor-associated antigens could therefore offset this escape mechanism. We studied the single-cell coexpression patterns of HER2, IL-13Rα2, and EphA2 in primary GBM samples using multicolor flow cytometry and immunofluorescence, and applied a binomial routine to the permutations of antigen expression and the related odds of complete tumor elimination. This mathematical model demonstrated that cotargeting HER2 and IL-13Rα2 could maximally expand the therapeutic reach of the T cell product in all primary tumors studied. Targeting a third antigen did not predict an added advantage in the tumor cohort studied. We therefore generated bispecific T cell products from healthy donors and from GBM patients by pooling T cells individually expressing HER2 and IL-13Rα2-specific CARs and by making individual T cells to coexpress both molecules. Both HER2/IL-13Rα2-bispecific T cell products offset antigen escape, producing enhanced effector activity in vitro immunoassays (against autologous glioma cells in the case of GBM patient products) and in an orthotopic xenogeneic murine model. Further, T cells coexpressing HER2 and IL-13Rα2-CARs exhibited accentuated yet antigen-dependent downstream signaling and a particularly enhanced antitumor activity.

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.

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.

IntroductionImmunotherapy with antigen-specific T cells has shown promise in the treatment of hematologic malignancies in preclinical models and in phase 1/2 clinical studies. [1][2][3] One attractive strategy to generate tumor-specific T cells is by genetic modification with chimeric antigen receptors (CARs), which consist of an extracellular antigen-recognition domain, a transmembrane domain, and an intracellular signaling domain derived from the TCR CD3-chain often linked to costimulatory molecule endodomains. 4,5 CARs targeting CD19 and CD20 antigens for the treatment of hematologic malignancies have been explored extensively, but this approach is limited to B cell-derived malignancies and may produce prolonged impairment of humoral immunity because of the potentially long life span of T cells. 6,7 It is therefore desirable to prepare CARs directed against alternative antigens that could broaden the spectrum of potentially treatable tumors and/or potentially reduce damage to normal cells.CD70 is the membrane-bound ligand of the CD27 receptor, which belongs to the tumor necrosis factor receptor superfamily. 8,9 CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkin lymphoma, Waldenström macroglobulinemia, and multiple myeloma, and by human T-lymphotropic virus type 1-and EBV-associated malignancies. [10][11][12][13][14] In addition, CD70 is expressed by nonhematologic malignancies such as renal cell carcinoma and glioblastoma. 15,16 Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells. Whereas CD70/CD27 costimulation plays a role in T-cell activation, CD70/ CD27 signaling is not essential for the development and maintenance of a functional immune system, because CD27-knockout mice have no overt immunodeficiency and recover from influenza virus infection within the same time frame as wild-type mice. 17,18 Targeting CD70-positive malignancies with CD70-specific monoclonal antibodies has shown promise in preclinical animal models, 14,19,20 and we have now evaluated whether T cells can be redirected to CD70 by forced expression of the appropriate CAR. Because CARs consist of an extracellular antigen-recognition domain derived from murine monoclonal antibodies, they may induce human anti-mouse antibody on infusion unless fully humanized. 21,22 One potential strategy to overcome this limitation is to engineer the antigen-recognition domain using endogenous protein ligands or receptors rather than monoclonal antibodies. 23,24 To target CD70 with T cells, we took advantage of the physiologic CD70/CD27 interaction and generated a CD70-specific CAR, which consists of full-length CD27 as the antigen-recognition domain fused to the intracellular domain of the CD3-chain. Engagement of chimeric CD27-by tumor targets expressing the CD70 ligand resulted in T-cell activation and CD27 costimulation, which was dependent on the presence of the TRAF2-binding site within the cytoplasmic tail of CD27. CD70-specifi...

Outcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting IL13R[.alpha]2, HER2, EGFRvIII, or EphA2 has shown promise for the treatment of glioma in preclinical models. Based on IL13Rα2-targeted immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARs with IL13 muteins as antigen binding domains. While the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells. We constructed four 2nd generation CARs with IL13 muteins with one or two amino acid substitutions. T cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by IFNα production. IL13Rα2 protein induced significantly more IL2, indicating that IL13 mutein-CAR T cells have a higher affinity to IL13Rα2 than IL13Rα1. In cytotoxcity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. While we observed no significant differences between IL13 mutein CAR T cells in vitro, only T cells expressing IL13 mutein CARs with an E13K amino acid substitution had antitumor activity in vivo that resulted in a survival advantage of treated animals. Our study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.

Glioblastoma (GBM) is the most common primary brain tumor, and despite aggressive therapy with surgery, radiation, and chemotherapy, average survival remains at about 1.5 years. The highly infiltrative and invasive nature of GBM requires that alternative treatments for this disease be widespread and targeted to tumor cells. Immunotherapy in the form of tumor vaccines has the potential to meet this need. Vaccines against GBM hold the promise of triggering specific and systemic antitumor immune responses that may be the key to eradicating this unrelenting cancer. In this review, we will discuss past and present clinical trials of various GBM vaccines and their potential impact on the future care of GBM patients. There have been many promising phase I and phase II GBM vaccine studies that have led to ongoing and upcoming phase III trials. If the results of these randomized trials show a survival benefit, immunotherapy will become a standard part of the treatment of this devastating disease.

Cancer cells can live and grow if they succeed in creating a favorable niche that often includes elements from the immune system. While T lymphocytes play an important role in the host response to tumor growth, the mechanism of their trafficking to the tumor remains poorly understood. We show here that T lymphocytes consistently infiltrate the primary brain cancer, medulloblastoma. We demonstrate, both in vitro and in vivo, that these T lymphocytes are attracted to tumor deposits only after the tumor cells have interacted with tumor vascular endothelium. Macrophage Migration Inhibitory Factor (MIF)” is the key chemokine molecule secreted by tumor cells which induces the tumor vascular endothelial cells to secrete the potent T lymphocyte attractant “Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES).” This in turn creates a chemotactic gradient for RANTES-receptor bearing T lymphocytes. Manipulation of this pathway could have important therapeutic implications.