Evaluation of the Immunomodulatory Effects of Radiation for Chimeric Antigen Receptor T Cell Therapy in Glioblastoma Multiforme.

Standard-of-care treatment for Glioblastoma Multiforme (GBM) is comprised of surgery and adjuvant chemoradiation. Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated disease-modifying activity in GBM and holds great promise. Radiation, a standard-of-care treatment for GBM, has well-known immunomodulatory properties and may overcome the immunosuppressive tumor microenvironment (TME); however, radiation dose optimization and integration with CAR T cell therapy is not well defined. Murine immunocompetent models of GBM were treated with titrated doses of stereotactic radiosurgery (SRS) of 5, 10, and 20 Gray (Gy), and the TME was analyzed using Nanostring. A conditioning dose of 10 Gy was determined based on tumor growth kinetics and gene expression changes in the TME. We demonstrate that a conditioning dose of 10 Gy activates innate and adaptive immune cells in the TME. Mice treated with 10 Gy in combination with mCAR T cells demonstrated enhanced antitumor activity and superior memory responses to rechallenge with IL13Rα2-positive tumors. Furthermore, 10 Gy plus mCAR T cells also protected against IL13Rα2-negative tumors through a mechanism that was, in part, c-GAS-STING pathway-dependent. Together, these findings support combination conditioning with low-dose 10 Gy radiation in combination with mCAR T cells as a therapeutic strategy for GBM.

Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .

Targeting Wnt signaling for improved glioma immunotherapy.

Introduction: Despite aggressive standard-of-care therapy, including surgery, radiation, and chemotherapy, glioblastoma recurrence is almost inevitable and uniformly lethal. Activation of glioma-intrinsic Wnt/ß-catenin signaling is associated with a poor prognosis and the proliferation of glioma stem-like cells, leading to malignant transformation and tumor progression. Impressive results in a subset of cancers have been obtained using immunotherapies including anti-CTLA4, anti-PD-1, and anti-PD-L1 or chimeric antigen receptor (CAR) T cell therapies. However, the heterogeneity of tumors, low mutational burden, single antigen targeting, and associated antigen escape contribute to non-responsiveness and potential tumor recurrence despite these therapeutic efforts. In the current study, we determined the effects of the small molecule, highly specific Wnt/CBP (CREB Binding Protein)/ß-catenin antagonist ICG-001, on glioma tumor cells and the tumor microenvironment (TME)-including its effect on immune cell infiltration, blood vessel decompression, and metabolic changes. Methods: Using multiple glioma patient-derived xenografts cell lines and murine tumors (GL261, K-Luc), we demonstrated in vitro cytostatic effects and a switch from proliferation to differentiation after treatment with ICG-001. Results: In these glioma cell lines, we further demonstrated that ICG-001 downregulated the CBP/ß-catenin target gene Survivin/BIRC5-a hallmark of Wnt/CBP/ß-catenin inhibition. We found that in a syngeneic mouse model of glioma (K-luc), ICG-001 treatment enhanced tumor infiltration by CD3+ and CD8+ cells with increased expression of the vascular endothelial marker CD31 (PECAM-1). We also observed differential gene expression and induced immune cell infiltration in tumors pretreated with ICG-001 and then treated with CAR T cells as compared with single treatment groups or when ICG-001 treatment was administered after CAR T cell therapy. Discussion: We conclude that specific Wnt/CBP/ß-catenin antagonism results in pleotropic changes in the glioma TME, including glioma stem cell differentiation, modulation of the stroma, and immune cell activation and recruitment, thereby suggesting a possible role for enhancing immunotherapy in glioma patients.

Resiliencia y percepción de la crianza parental como predictoras de la salud mental positiva en adolescentes mexicanos / Resilience and parenting perception as predictors of positive mentalhealth in Mexican adolescents

Antecedentes: en la adolescencia la salud mental positiva es indispensable para el desarrollo óptimo. Objetivo: se predijo la salud mental positiva en adolescentes a partir de la percepción de la crianza parental y la resiliencia. Método: participaron 426 estudiantes de Toluca, Estado de México entre los 14 y 18 años (M = 15.6 años; DE = 1.14),el 44% fueron hombres y 56% mujeres. Se les evaluó con la escala de percepción de crianza parental, el cuestionario de resiliencia y la escala de salud mental positiva. Resultados: a través de la regresión lineal múltiple por pasos se encontró que la variable criterio es predicha en mayor medida por los factores protectores internos de la resiliencia y en menor proporción por el interés del padre y la madre en las actividades del hijo. Este modelo que combina ambos constructos tiene una varianza explicada de 51.6%, lo que se considera un poder explicativo satisfactorio. Conclusiones: se enfatiza la influencia de los factores de protección internos de la resiliencia sobre la capacidad de resolución de problemas y actualización, el autocontrol y la autonomía de los adolescentes. Palabras clave: salud mental positiva; resiliencia; percepción de la crianza parental; adolescentes.

Background: During adolescence positive mental health is indispensable for optimal development. Objective: Positive mental health in adolescents was predicted from the perception of parenting and resilience. Methods: 426 students from Toluca, State of Mexico between 14 and 18 years of age (M = 15.6 years; SD = 1.14), 44% male and 56% female, were evaluated with the Perception of Parenting Scale, the Resilience Questionnaire and the Positive Mental Health Scale. Results: Through stepwise multiple linear regression, it was found that the criterion variable is predicted to a greater extent by the internal protective factors of resilience and to a lesser extent by the father's and mother's interest in the child's activities.This model combining both constructs has an explained variance of 51.6%, which is considered satisfactory explanatory power. Conclusions: The influence of internal protective factors of resilience on adolescents' problem-solving and actualization skills, self-control and autonomyis emphasized. Keywords: positive mental health; resilience; perceptionof parenting; adolescents.

Management of Antracnosis with Electrochemically Activated Salt Solutions (EASSs) on Bean Culture.

Common bean (Phaseolus vulgaris L.) is an important crop for food security and for national economics for several countries worldwide. One of the most important factors of risk in common bean production is the fungal disease anthracnose caused by Colletotrichum lindemuthianum, which, in some cases, causes complete yield losses; this kind of plant disease is usually managed through the application of chemical products such as fungicides that are commonly not accepted by society. This rejection is based on the relationship of pesticides with health damage and environmental contamination. In order to help in solving these drawbacks, the present work proposes the use of electrochemically activated salt solutions (EASSs) as a safer pathogen control agent in crops, due to it having shown an elicitor and biostimulant effect on plants. With this background, this manuscript presents in vitro results of the evaluation of the inhibitory effect for multiple bean pathogens and in vivo results of EASS in the common bean-Colletotrichum pathosystem by evaluation of the infection severity and defense activation, such as secondary metabolite production and antioxidant activity. EASS presence in growth media had a strong inhibitory effect at the beginning of experiments for some of the evaluated fungi. EASSs showed an effect against the development of the disease when applied in specific doses to prevent distress in plants.

Inclusion of 4-1BB Costimulation Enhances Selectivity and Functionality of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells.

Chimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand-based CAR targeting the cancer antigen IL13Rα2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13Rα2 versus the more ubiquitous unintended target IL13Rα1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3ζ-containing CARs (IL13ζ), second-generation 4-1BB (CD137)-containing or CD28-containing CARs (IL13-BBζ or IL13-28ζ), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BBζ). In vitro coculture assays at high tumor burden establish that second-generation IL13-BBζ or IL13-28ζ outperform first-generation IL13ζ and third-generation IL13-28BBζ CAR designs, with IL13-BBζ providing superior CAR proliferation and in vivo antitumor potency in human xenograft mouse models. IL13-28ζ displayed a lower threshold for antigen recognition, resulting in higher off-target IL13Rα1 reactivity both in vitro and in vivo. Syngeneic mouse models of GBM also demonstrate safety and antitumor potency of murine IL13-BBζ CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BBζ CARs for greater selective recognition of IL13Rα2 over IL13Rα1, higher proliferative potential, and superior antitumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition. Significance: This study reveals how modulating CAR design outside the antigen targeting domain improves selective tumor recognition. Specifically, this work shows improved specificity, persistence, and efficacy of 4-1BB-based IL13-ligand CARs. Human clinical trials evaluating IL13-41BB-CAR T cells are ongoing, supporting the clinical significance of these findings.

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy.

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)-based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells.

BACKGROUND: Chimeric antigen receptor (CAR) T cells engineered to recognize and target tumor associated antigens have made a profound impact on the quality of life for many patients with cancer. However, tumor heterogeneity and intratumoral immune suppression reduce the efficacy of this approach, allowing for tumor cells devoid of the target antigen to seed disease recurrence. Here, we address the complexity of tumor heterogeneity by developing a universal CAR. METHOD: We constructed a universal Fabrack-CAR with an extracellular domain composed of the non-tumor targeted, cyclic, twelve residue meditope peptide that binds specifically to an engineered binding pocket within the Fab arm of monoclonal antibodies (mAbs). As this site is readily grafted onto therapeutic mAbs, the antigen specificity of these universal Fabrack-CAR T cells is simply conferred by administering mAbs with specificity to the heterogeneous tumor. RESULTS: Using in vitro and in vivo studies with multiple meditope-engineered mAbs, we show the feasibility, specificity, and robustness of this approach. These studies demonstrate antigen- and antibody-specific T cell activation, proliferation, and IFNγ production, selective killing of target cells in a mixed population, and tumor regression in animal models. CONCLUSION: Collectively, these findings support the feasibility of this universal Fabrack-CAR T cell approach and provide the rationale for future clinical use in cancer immunotherapy.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR T cells.

Unlimited generation of chimeric antigen receptor (CAR) T cells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR T cell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αß T cell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory T cells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αß-positive CAR T cell characteristics. Expanded iPSC CD19-CAR T cells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR T cells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR T cells also mediated potent antitumor activity in vivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR T cells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

Off-the-shelf, steroid-resistant, IL13Rα2-specific CAR T cells for treatment of glioblastoma.

BACKGROUND: Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety, and therapeutic potential of this approach. METHODS: We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day). RESULTS: The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 108 cells over a two-week period with aldesleukin (9 infusions ranging from 2500-5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects. CONCLUSIONS: This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified, and/or glucocorticoid resistant CAR T cells.

IFNγ Is Critical for CAR T Cell-Mediated Myeloid Activation and Induction of Endogenous Immunity.

Chimeric antigen receptor (CAR) T cells mediate potent antigen-specific antitumor activity; however, their indirect effects on the endogenous immune system are not well characterized. Remarkably, we demonstrate that CAR T-cell treatment of mouse syngeneic glioblastoma (GBM) activates intratumoral myeloid cells and induces endogenous T-cell memory responses coupled with feed-forward propagation of CAR T-cell responses. IFNγ production by CAR T cells and IFNγ responsiveness of host immune cells are 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 monocytes/macrophages through IFNγ signaling and induce the generation of tumor-specific T-cell responses in a responding patient with GBM. These studies establish that CAR T-cell 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-cell therapy in GBM. We establish that CAR T cells can activate resident myeloid populations and promote endogenous T-cell immunity, emphasizing the importance of host innate and adaptive immunity for CAR T-cell therapy of solid tumors.This article is highlighted in the In This Issue feature, p. 2113.

The Cerebroventricular Environment Modifies CAR T Cells for Potent Activity against Both Central Nervous System and Systemic Lymphoma.

Lymphomas with central nervous system (CNS) involvement confer a worse prognosis than those without CNS involvement, and patients currently have limited treatment options. T cells genetically engineered with CD19-targeted chimeric antigen receptors (CAR) are effective against B-cell malignancies and show tremendous potential in the treatment of systemic lymphoma. We aimed to leverage this strategy toward a more effective therapy for patients with lymphoma with CNS disease. NOD-scid IL2Rgammanull (NSG) mice with CNS and/or systemic lymphoma were treated with CD19-CAR T cells via intracerebroventricular (ICV) or intravenous (IV) injection. CAR T cells isolated after treatment were rigorously examined for phenotype, gene expression, and function. We observed that CAR T cells infused ICV, but not IV, completely and durably eradicated both CNS and systemic lymphoma. CAR T cells delivered ICV migrated efficiently to the periphery, homed to systemic tumors, and expanded in vivo, leading to complete elimination of disease and resistance to tumor rechallenge. Mechanistic studies indicated that ICV-delivered CAR T cells are conditioned by exposure to cerebrospinal fluid in the ICV environment for superior antilymphoma activity and memory function compared with IV-delivered CAR T cells. Further analysis suggested that manipulating cellular metabolism or preactivating therapeutic CAR T cells with antigen ex vivo may improve the efficacy of CAR T cells in vivo Our demonstration that ICV-delivered CD19-CAR T cells had activity against CNS and systemic lymphoma could offer a valuable new strategy for treatment of B-cell malignancies with CNS involvement.

CRISPR Screening of CAR T Cells and Cancer Stem Cells Reveals Critical Dependencies for Cell-Based Therapies.

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.

Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma.

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.

IL15 Enhances CAR-T Cell Antitumor Activity by Reducing mTORC1 Activity and Preserving Their Stem Cell Memory Phenotype.

Improvements in the quality and fitness of chimeric antigen receptor (CAR)-engineered T cells, through CAR design or manufacturing optimizations, could enhance the therapeutic potential of CAR-T cells. One parameter influencing the effectiveness of CAR-T cell therapy is the differentiation status of the final product: CAR-T cells that are less-differentiated and less exhausted are more therapeutically effective. In the current study, we demonstrate that CAR-T cells expanded in IL15 (CAR-T/IL15) preserve a less-differentiated stem cell memory (Tscm) phenotype, defined by expression of CD62L+CD45RA+ CCR7+, as compared with cells cultured in IL2 (CAR-T/IL2). CAR-T/IL15 cells exhibited reduced expression of exhaustion markers, higher antiapoptotic properties, and increased proliferative capacity upon antigen challenge. Furthermore, CAR-T/IL15 cells exhibited decreased mTORC1 activity, reduced expression of glycolytic enzymes and improved mitochondrial fitness. CAR-T/IL2 cells cultured in rapamycin (mTORC1 inhibitor) shared phenotypic features with CAR-T/IL15 cells, suggesting that IL15-mediated reduction of mTORC1 activity is responsible for preserving the Tscm phenotype. CAR-T/IL15 cells promoted superior antitumor responses in vivo in comparison with CAR-T/IL2 cells. Inclusion of cytokines IL7 and/or IL21 in addition to IL15 reduced the beneficial effects of IL15 on CAR-T phenotype and antitumor potency. Our findings show that IL15 preserves the CAR-T cell Tscm phenotype and improves their metabolic fitness, which results in superior in vivo antitumor activity, thus opening an avenue that may improve future adoptive T-cell therapies.

PET of Adoptively Transferred Chimeric Antigen Receptor T Cells with 89Zr-Oxine.

Chimeric antigen receptor (CAR) T cell therapy is a promising clinical approach for reducing tumor progression and prolonging patient survival. However, improvements in both the safety and the potency of CAR T cell therapy demand quantitative imaging techniques to determine the distribution of cells after adoptive transfer. The purpose of this study was to optimize 89Zr-oxine labeling of CAR T cells and evaluate PET as a platform for imaging adoptively transferred CAR T cells. Methods: CAR T cells were labeled with 0-1.4 MBq of 89Zr-oxine per 106 cells and assessed for radioactivity retention, viability, and functionality. In vivo trafficking of 89Zr-oxine-labeled CAR T cells was evaluated in 2 murine xenograft tumor models: glioblastoma brain tumors with intracranially delivered IL13Rα2-targeted CAR T cells, and subcutaneous prostate tumors with intravenously delivered prostate stem cell antigen (PSCA)-targeted CAR T cells. Results: CAR T cells were efficiently labeled (75%) and retained more than 60% of the 89Zr over 6 d. In vitro cytokine production, migration, and tumor cytotoxicity, as well as in vivo antitumor activity, were not significantly reduced when labeled with 70 kBq/106 cells. IL13Rα2-CAR T cells delivered intraventricularly were detectable by PET for at least 6 d throughout the central nervous system and within intracranial tumors. When intravenously administered, PSCA-CAR T cells also showed tumor tropism, with a 9-fold greater tumor-to-muscle ratio than for CAR-negative T cells. Conclusion:89Zr-oxine can be used for labeling and imaging CAR T cells while maintaining cell viability and function. On the basis of these studies, we conclude that 89Zr-oxine is a clinically translatable platform for real-time assessment of cell therapies.

Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity.

Chimeric antigen receptor-modified (CAR-modified) T cells have shown promising therapeutic effects for hematological malignancies, yet limited and inconsistent efficacy against solid tumors. The refinement of CAR therapy requires an understanding of the optimal characteristics of the cellular products, including the appropriate composition of CD4+ and CD8+ subsets. Here, we investigated the differential antitumor effect of CD4+ and CD8+ CAR T cells targeting glioblastoma-associated (GBM-associated) antigen IL-13 receptor α2 (IL13Rα2). Upon stimulation with IL13Rα2+ GBM cells, the CD8+ CAR T cells exhibited robust short-term effector function but became rapidly exhausted. By comparison, the CD4+ CAR T cells persisted after tumor challenge and sustained their effector potency. Mixing with CD4+ CAR T cells failed to ameliorate the effector dysfunction of CD8+ CAR T cells, while surprisingly, CD4+ CAR T cell effector potency was impaired when coapplied with CD8+ T cells. In orthotopic GBM models, CD4+ outperformed CD8+ CAR T cells, especially for long-term antitumor response. Further, maintenance of the CD4+ subset was positively correlated with the recursive killing ability of CAR T cell products derived from GBM patients. These findings identify CD4+ CAR T cells as a highly potent and clinically important T cell subset for effective CAR therapy.

Optimization of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells for Improved Anti-tumor Efficacy against Glioblastoma.

T cell immunotherapy is emerging as a powerful strategy to treat cancer and may improve outcomes for patients with glioblastoma (GBM). We have developed a chimeric antigen receptor (CAR) T cell immunotherapy targeting IL-13 receptor α2 (IL13Rα2) for the treatment of GBM. Here, we describe the optimization of IL13Rα2-targeted CAR T cells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BBζ) and a manufacturing platform using enriched central memory T cells. Utilizing orthotopic human GBM models with patient-derived tumor sphere lines in NSG mice, we found that IL13BBζ-CAR T cells improved anti-tumor activity and T cell persistence as compared to first-generation IL13ζ-CAR CD8+ T cells that had shown evidence for bioactivity in patients. Investigating the impact of corticosteroids, given their frequent use in the clinical management of GBM, we demonstrate that low-dose dexamethasone does not diminish CAR T cell anti-tumor activity in vivo. Furthermore, we found that local intracranial delivery of CAR T cells elicits superior anti-tumor efficacy as compared to intravenous administration, with intraventricular infusions exhibiting possible benefit over intracranial tumor infusions in a multifocal disease model. Overall, these findings help define parameters for the clinical translation of CAR T cell therapy for the treatment of brain tumors.