Armored bicistronic CAR T cells with dominant-negative TGF-ß receptor II to overcome resistance in glioblastoma.

Chimeric antigen receptor (CAR) T cells have shown significant efficacy in hematological diseases. However, CAR T therapy has demonstrated limited efficacy in solid tumors, including glioblastoma (GBM). One of the most important reasons is the immunosuppressive tumor microenvironment (TME), which promotes tumor growth and suppresses immune cells used to eliminate tumor cells. The human transforming growth factor ß (TGF-ß) plays a crucial role in forming the suppressive GBM TME and driving the suppression of the anti-GBM response. To mitigate TGF-ß-mediated suppressive activity, we combined a dominant-negative TGF-ß receptor II (dnTGFßRII) with our previous bicistronic CART-EGFR-IL13Rα2 construct, currently being evaluated in a clinical trial, to generate CART-EGFR-IL13Rα2-dnTGFßRII, a tri-modular construct we are developing for clinical application. We hypothesized that this approach would more effectively subvert resistance mechanisms observed with GBM. Our data suggest that CART-EGFR-IL13Rα2-dnTGFßRII significantly augments T cell proliferation, enhances functional responses, and improves the fitness of bystander cells, particularly by decreasing the TGF-ß concentration in a TGF-ß-rich TME. In addition, in vivo studies validate the safety and efficacy of the dnTGFßRII cooperating with CARs in targeting and eradicating GBM in an NSG mouse model.

The IAP antagonist birinapant enhances chimeric antigen receptor T cell therapy for glioblastoma by overcoming antigen heterogeneity.

Antigen heterogeneity that results in tumor antigenic escape is one of the major obstacles to successful chimeric antigen receptor (CAR) T cell therapies in solid tumors including glioblastoma multiforme (GBM). To address this issue and improve the efficacy of CAR T cell therapy for GBM, we developed an approach that combines CAR T cells with inhibitor of apoptosis protein (IAP) antagonists, a new class of small molecules that mediate the degradation of IAPs, to treat GBM. Here, we demonstrated that the IAP antagonist birinapant could sensitize GBM cell lines and patient-derived primary GBM organoids to apoptosis induced by CAR T cell-derived cytokines, such as tumor necrosis factor. Therefore, birinapant could enhance CAR T cell-mediated bystander death of antigen-negative GBM cells, thus preventing tumor antigenic escape in antigen-heterogeneous tumor models in vitro and in vivo. In addition, birinapant could promote the activation of NF-κB signaling pathways in antigen-stimulated CAR T cells, and with a birinapant-resistant tumor model we showed that birinapant had no deleterious effect on CAR T cell functions in vitro and in vivo. Overall, we demonstrated the potential of combining the IAP antagonist birinapant with CAR T cells as a novel and feasible approach to overcoming tumor antigen heterogeneity and enhancing CAR T cell therapy for GBM.

Immunologic Features in De Novo and Recurrent Glioblastoma Are Associated with Survival Outcomes.

Glioblastoma (GBM) is an immunologically "cold" tumor characterized by poor responsiveness to immunotherapy. Standard of care for GBM is surgical resection followed by chemoradiotherapy and maintenance chemotherapy. However, tumor recurrence is the norm, and recurring tumors are found frequently to have acquired molecular changes (e.g., mutations) that may influence their immunobiology. Here, we compared the immune contexture of de novo GBM and recurrent GBM (rGBM) using high-dimensional cytometry and multiplex IHC. Although myeloid and T cells were similarly abundant in de novo and rGBM, their spatial organization within tumors differed and was linked to outcomes. In rGBM, T cells were enriched and activated in perivascular regions and clustered with activated macrophages and fewer regulatory T cells. Moreover, a higher expression of phosphorylated STAT1 by T cells in these regions at recurrence was associated with a favorable prognosis. Together, our data identify differences in the immunobiology of de novo GBM and rGBM and identify perivascular T cells as potential therapeutic targets. See related Spotlight by Bayik et al., p. 787.

Locally secreted BiTEs complement CAR T cells by enhancing killing of antigen heterogeneous solid tumors.

Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. We hypothesized that BiTE-secreting T cells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent T cells compared with bivalent CAR T cells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms.

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood-brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

Transcriptome signatures associated with meningioma progression.

Meningiomas are the most common primary brain tumor of adults. The majority are benign (WHO grade I), with a mostly indolent course; 20% of them (WHO grade II and III) are, however, considered aggressive and require a more complex management. WHO grade II and III tumors are heterogeneous and, in some cases, can develop from a prior lower grade meningioma, although most arise de novo. Mechanisms leading to progression or implicated in de novo grade II and III tumorigenesis are poorly understood. RNA-seq was used to profile the transcriptome of grade I, II, and III meningiomas and to identify genes that may be involved in progression. Bioinformatic analyses showed that grade I meningiomas that progress to a higher grade are molecularly different from those that do not. As such, we identify GREM2, a regulator of the BMP pathway, and the snoRNAs SNORA46 and SNORA48, as being significantly reduced in meningioma progression. Additionally, our study has identified several novel fusion transcripts that are differentially present in meningiomas, with grade I tumors that did not progress presenting more fusion transcripts than all other tumors. Interestingly, our study also points to a difference in the tumor immune microenvironment that correlates with histopathological grade.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Epidermal Growth Factor Receptor Extracellular Domain Mutations in Glioblastoma Present Opportunities for Clinical Imaging and Therapeutic Development.

We explored the clinical and pathological impact of epidermal growth factor receptor (EGFR) extracellular domain missense mutations. Retrospective assessment of 260 de novo glioblastoma patients revealed a significant reduction in overall survival of patients having tumors with EGFR mutations at alanine 289 (EGFRA289D/T/V). Quantitative multi-parametric magnetic resonance imaging analyses indicated increased tumor invasion for EGFRA289D/T/V mutants, corroborated in mice bearing intracranial tumors expressing EGFRA289V and dependent on ERK-mediated expression of matrix metalloproteinase-1. EGFRA289V tumor growth was attenuated with an antibody against a cryptic epitope, based on in silico simulation. The findings of this study indicate a highly invasive phenotype associated with the EGFRA289V mutation in glioblastoma, postulating EGFRA289V as a molecular marker for responsiveness to therapy with EGFR-targeting antibodies.

SHP2 regulates proliferation and tumorigenicity of glioma stem cells.

SHP2 is a cytoplasmic protein tyrosine phosphatase (PTPase) involved in multiple signaling pathways and was the first identified proto-oncogene PTPase. Previous work in glioblastoma (GBM) has demonstrated the role of SHP2 PTPase activity in modulating the oncogenic phenotype of adherent GBM cell lines. Mutations in PTPN11, the gene encoding SHP2, have been identified with increasing frequency in GBM. Given the importance of SHP2 in developing neural stem cells, and the importance of glioma stem cells (GSCs) in GBM oncogenesis, we explored the functional role of SHP2 in GSCs. Using paired differentiated and stem cell primary cultures, we investigated the association of SHP2 expression with the tumor stem cell compartment. Proliferation and soft agar assays were used to demonstrate the functional contribution of SHP2 to cell growth and transformation. SHP2 expression correlated with SOX2 expression in GSC lines and was decreased in differentiated cells. Forced differentiation of GSCs by removal of growth factors, as confirmed by loss of SOX2 expression, also resulted in decreased SHP2 expression. Lentiviral-mediated knockdown of SHP2 inhibited proliferation. Finally, growth in soft-agar was similarly inhibited by loss of SHP2 expression. Our results show that SHP2 function is required for cell growth and transformation of the GSC compartment in GBM.