Although chimeric antigen receptor (CAR) T cells have demonstrated signs of antitumor activity against glioblastoma (GBM), tumor heterogeneity remains a critical challenge. To achieve broader and more effective GBM targeting, we developed a peptide-bearing CAR exploiting the GBM-binding potential of chlorotoxin (CLTX). We find that CLTX peptide binds a great proportion of tumors and constituent tumor cells. CAR T cells using CLTX as the targeting domain (CLTX-CAR T cells) mediate potent anti-GBM activity and efficiently target tumors lacking expression of other GBM-associated antigens. Treatment with CLTX-CAR T cells resulted in tumor regression in orthotopic xenograft GBM tumor models. CLTX-CAR T cells do not exhibit observable off-target effector activity against normal cells or after adoptive transfer into mice. Effective targeting by CLTX-CAR T cells requires cell surface expression of matrix metalloproteinase–2. Our results pioneer a peptide toxin in CAR design, expanding the repertoire of tumor-selective CAR T cells with the potential to reduce antigen escape.

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity, however, their indirect effects on the endogenous immune system is not well characterized. Remarkably, we demonstrate that CAR T cell treatment of mouse syngeneic glioblastoma activates intratumoral myeloid cells and induces endogenous T cell memory responses coupled with feed-forward propagation of CAR T responses. IFNγ production by CAR T cells and IFNγ-responsiveness of host immune cells is critical for tumor immune landscape remodeling to promote a more activated and less suppressive tumor microenvironment. The clinical relevance of these observations is supported by studies showing that human IL13Rα2-CAR T cells activate patient-derived endogenous T cells and monocyte/macrophages through IFN-signaling, as well as induce the generation of tumor-specific T cell responses in a responding patient with GBM. These studies establish that CAR T therapy has the potential to shape the tumor microenvironment, creating a context permissible for eliciting endogenous antitumor immunity. SIGNIFICANCE:Our findings highlight the critical role of IFNγ-signaling for a productive CAR T therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T cell immunity, emphasizing on the importance of host innate and adaptive immunity for CAR T therapy of solid tumors.Research.

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable clinical efficacy against B-cell malignancies, yet marked vulnerability to antigen escape and tumor relapse exists. Here we report the rational design and optimization of bispecific CART cells with robust activity against heterogeneous multiple myeloma (MM) that is resistant to conventional CART cell therapy targeting B-cell maturation antigen (BCMA). We demonstrate that BCMA/CS1 bispecific CART cells exhibit superior CAR expression and function compared to T cells that co-express individual BCMA and CS1 CARs. Combination therapy with anti-PD-1 antibody further accelerates the rate of initial tumor clearance in vivo, while CART cell treatment alone achieves durable tumor-free survival even upon tumor re-challenge. Taken together, the BCMA/CS1 bispecific CAR presents a promising treatment approach to prevent antigen escape in CART cell therapy against MM, and the vertically integrated optimization process can be used to develop robust cell-based therapy against novel disease targets.

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor–immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. Significance: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy. This article is highlighted in the In This Issue feature, p. 995

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