Brain tumors are the leading cause of cancer death in children and a significant cause of morbidity and mortality in adults. Conventional treatments are suboptimal, thus signifying the need for novel therapeutic strategies, such as immunotherapy. Chimeric antigen receptor (CAR) T cells represent a revolutionary class of immunotherapy, achieving considerable success in eliminating hematological cancers but generally failing to control solid tumors in part due to the lack of a suitably-expressed target antigen. Epidermal growth factor receptor (EGFR) is the most ubiquitous and homogeneous antigen on glial brain tumors, and EGFR-directed therapies have been hotly pursued. Moreover, the mutant EGFR variant, EGFRvIII, is present on a subset of pediatric and adult high grade gliomas, representing a targetable, tumor-specific antigen. Unfortunately, CAR T cells targeting EGFRvIII fail to treat tumors possessing as few as 5–10% EGFRvIII-negative cells due to antigen escape. Thus, a CAR T cell that can target both EGFR and EGFRvIII is expected to be superior to a CAR that targets EGFRvIII alone. In this study, we developed a novel third generation CAR T cell consisting of the D2C7scfv targeting moiety that binds a shared epitope between EGFR and EGFRvIII. This D2C7 CAR was able to specifically and potently kill tumor cells expressing wildtype EGFR or EGFRvIII. Importantly, D2C7 CAR significantly prolonged survival of mice bearing EGFR or EGFRvIII-expressing gliomas of both adult (U87) and pediatric (DAOY) origin. Toxicity experiments involving EGFR-expressing human skin grafts provided evidence that D2C7 CAR is safe and effective when administered intracranially to mice bearing intracranial tumors.
BACKGROUND Medulloblastoma is the most common malignant brain tumor in children 0-19 years of age and current treatment requires surgical resection, followed by high dose chemotherapy and radiotherapy. Therapy carries high morbidity, with late effects including neurocognitive decline, endocrine dysfunction, and subsequent malignancies. Chimeric antigen receptor (CAR) T cell therapy presents the potential for less toxic and more effective treatment for medulloblastoma. Epidermal growth factor (EGFR) is expressed in medulloblastoma derived cell lines and appears to serve an important role in the metastatic potential of this tumor type. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) with dual specificity, binding to wild type EGFR (EGFRwt) and its mutant EGFR variant III (EGFRvIII). We previously developed a novel, third-generation chimeric antigen receptor T-cell construct utilizing the D2C7 scFv and performed analysis on both glioblastoma and medulloblastoma tumor models. METHODS U87 and U87vIII glioblastoma and DAOY medulloblastoma cell lines were characterized by flow cytometry to evaluate for EGFRwt and EGFRvIII expression. Cytotoxicity assays were performed utilizing flow cytometry on serially diluted effector: target ratios of U87, U87vII, and DAOY mixed with D2C7 CAR. DAOY was orthotopically implanted into the frontal lobe of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. After tumor implantation, D2C7 CAR was introduced within the tumor bed. RESULTS EGFR was detected in U87, U87vIII, and DAOY on flow analysis. EGFRvIII was expressed only on U87vIII. D2C7 CAR demonstrated effective in vitro cytotoxicity against all three cell lines with >90% cytotoxicity achieved at an effector: target ratio of 2.5:1. In vivo, D2C7 CAR treatment significantly prolonged survival compared to treatment with mock CAR. CONCLUSION D2C7 CAR demonstrates efficacy in vitro and in mouse models of medulloblastoma and glioblastoma. Future steps in our work will set out to assess the D2C7 CAR in NSG mice with DAOY in the posterior fossa.
Glioblastoma (GBM) is the most aggressive primary brain cancer with a median survival of less than 16 months. Therefore, significant treatment advances are of critical need. Immunotherapies, such as chimeric antigen receptor (CAR) T cells, have achieved remarkable success in eliminating hematological cancers. Unfortunately, CARs have overall failed to control solid tumors. One major reason for this failure is tumor heterogeneity, a characteristic especially true of GBM. In GBM, EGFRvIII is a commonly targeted tumor antigen; however, only a subset of tumor cells will express EGFRvIII. Thus, EGFRvIII-negative cells easily escape EGFRvIII-targeting CAR therapies. A better approach would be to target multiple antigens or an antigen that is more homogeneously expressed throughout the tumor. More commonly than EGFRvIII expression, glioma cells overexpress the wildtype isoform of EGFR (EGFRwt). Simultaneously targeting EGFRvIII and EGFRwt, instead of targeting each independently, with a CAR will theoretically expand the amount of tumor that will be eliminated by the CAR. Here, we adapted the D2C7scFv that binds both EGFRwt and EGFRvIII into a third generation CAR. D2C7 CAR potently and specifically killed EGFRwt and EGFRvIII-expressing U87 glioma, but not U87 with EGFR knocked out. In xenograft models, intracranially administered D2C7 CAR significantly prolonged survival of mice bearing orthotopic U87vIII, U87 (EGFRwt), and heterogeneous U87/U87vIII tumors compared tocontrols. Additionally, D2C7 CAR was an effective treatment in an orthotopic EGFR-expressing medulloblastoma model. Furthermore, D2C7 CAR treatment led to a significant survival benefit in mice bearing intrancranial A431, an aggressive, EGFR-expressing carcinoma line, a model of metastasis to the brain. Importantly, D2C7 CAR did not show reactivity towards normal tissue with EGFR expression, suggesting a tumor-specific epitope of EGFR is being targeted. Together, these data provide evidence that D2C7 CAR can treat EGFR/EGFRvIII heterogeneous or homogeneous primary or metastatic brain tumors with potentially little-to-no off-tumor toxicity.
INTRODUCTION Chimeric antigen receptor (CAR) T-cells represent a revolutionary class of immunotherapy, achieving considerable success in hematological cancers but generally failing to control solid tumors, including gliomas, partly due to the lack of a ubiquitously-expressed target antigen. In this study, we engineered a novel CAR T-cell consisting of the D2C7scfv targeting moiety that binds a shared epitope between EGFR and EGFRvIII. EGFR is the most homogeneous antigen on glial brain tumors, and the mutant EGFR variant, EGFRvIII, is present on a considerable subset of high grade gliomas. CAR T-cells targeting EGFRvIII alone fail to treat tumors possessing as few as 5-10% EGFRvIII-negative cell. Thus, D2C7 CAR is expected to be superior to the EGFRvIII CAR. METHODS We retrovirally transduced T-cells with a vector encoding the D2C7scFv in tandem with intracellular signaling domains of CD28, 4-1BB, and CD3ζ to generate D2C7 CAR. We co-cultured D2C7 CAR or control CAR with fluorescently-tagged tumor cells expressing either EGFRwt or EGFRvIII to validate efficacy and specificity by flow cytometry. To determine in vivo efficacy, EGFRwt or EGFRvIII-expressing tumors were implanted intracranially in immunodeficient NSG mice. 48 hours later, D2C7 CAR, VIII CAR, or Mock CAR were administered intracranially and mice were monitored for survival. RESULTS D2C7 CAR specifically killed tumor cells that expressed either EGFRwt or EGFRvIII, but not cells that lacked EGFR. Intracranial D2C7 CAR administration resulted in significantly prolonged survival of mice bearing EGFRwt or EGFRvIII tumors compared to Mock CAR controls. Importantly, D2C7 CAR significantly benefitted mice bearing a heterogeneous mix of EGFRwt and EGFRvIII tumor cells, a model of tumor heterogeneity. CONCLUSIONS D2C7 CAR is efficacious against EGFRwt/EGFRvIII heterogeneous tumors. Intracranial administration of D2C7 CAR is predicted to safely and effectively treat a large cohort of patients due to the relatively high prevalence of EGFR and/or EGFRvIII-expressing brain tumors.
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