Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor—the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment.

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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.

UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.

Triple-negative breast cancer (TNBC) is an aggressive disease lacking targeted therapy. In this study, we developed a CAR T cell-based immunotherapeutic strategy to target TEM8, a marker initially defined on endothelial cells in colon tumors that was discovered recently to be upregulated in TNBC. CAR T cells were developed that upon specific recognition of TEM8 secreted immunostimulatory cytokines and killed tumor endothelial cells as well as TEM8-positive TNBC cells. Notably, the TEM8 CAR T cells targeted breast cancer stem-like cells, offsetting the formation of mammospheres relative to nontransduced T cells. Adoptive transfer of TEM8 CAR T cells induced regression of established, localized patient-derived xenograft tumors, as well as lung metastatic TNBC cell line-derived xenograft tumors, by both killing TEM8 TNBC tumor cells and targeting the tumor endothelium to block tumor neovascularization. Our findings offer a preclinical proof of concept for immunotherapeutic targeting of TEM8 as a strategy to treat TNBC. These findings offer a preclinical proof of concept for immunotherapeutic targeting of an endothelial antigen that is overexpressed in triple-negative breast cancer and the associated tumor vasculature. .

Atopic asthma is a chronic inflammatory disease of the lungs generally marked by excessive Th2 inflammation. The role of allergen-specific IgG in asthma is still controversial; however, a receptor of IgG-immune complexes (IgG-ICs), FcγRIII, has been shown to promote Th2 responses through an unknown mechanism. Herein, we demonstrate that allergen-specific IgG-ICs, formed upon reexposure to allergen, promoted Th2 responses in two different models of IC-mediated inflammation that were independent of a preformed T cell memory response. Development of Th2-type airway inflammation was shown to be both FcγRIII and TLR4 dependent, and T cells were necessary and sufficient for this process to occur, even in the absence of type 2 innate lymphoid cells. We sought to identify downstream targets of FcγRIII signaling that could contribute to this process and demonstrated that bone marrow-derived DCs, alveolar macrophages, and respiratory DCs significantly upregulated IL-33 when activated through FcγRIII and TLR4. Importantly, IC-induced Th2 inflammation was dependent on the ST2/IL-33 pathway. Our results suggest that allergen-specific IgG can enhance secondary responses by ligating FcγRIII on antigen-presenting cells to augment development of Th2-mediated responses in the lungs via an IL-33-dependent mechanism.

Refractory metastatic rhabdomyosarcoma is largely incurable. Here we analyze the response of a child with refractory bone marrow metastatic rhabdomyosarcoma to autologous HER2 CAR T cells. Three cycles of HER2 CAR T cells given after lymphodepleting chemotherapy induces remission which is consolidated with four more CAR T-cell infusions without lymphodepletion. Longitudinal immune-monitoring reveals remodeling of the T-cell receptor repertoire with immunodominant clones and serum autoantibodies reactive to oncogenic signaling pathway proteins. The disease relapses in the bone marrow at six months offtherapy. A second remission is achieved after one cycle of lymphodepletion and HER2 CAR T cells. Response consolidation with additional CAR T-cell infusions includes pembrolizumab to improve their efficacy. The patient described here is a participant in an ongoing phase I trial (NCT00902044; active, not recruiting), and is 20 months off T-cell infusions with no detectable disease at the time of this report.

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. Supplementary informationThe online version of this article (https://