Christine E. Brown

Specific Recognition and Killing of Glioblastoma Multiforme by Interleukin 13-Zetakine Redirected Cytolytic T Cells

The interleukin (IL) 13 receptor α2 (IL13Rα2) is a glioma-restricted cell-surface epitope not otherwise detected within the central nervous system. Here, we describe a novel approach for targeting glioblastoma multiforme (GBM) with IL13Rα2-specific cytolytic T cells (CTLs) by their genetic modification to express a membrane-tethered IL13 cytokine chimeric T-cell antigen receptor, or zetakine. Our prototype zetakine incorporates an IL13 E13Y mutein for selective binding to IL13Rα2. Human IL13-zetakine+CD8+ CTL transfectants display IL13Rα2-specific antitumor effector function including tumor cell cytolysis, TC1 cytokine production, and zetakine-regulated autocrine proliferation. The E13Y amino acid substitution of the IL13 mutein of the zetakine endows CTL transfectants with the capacity to discriminate between IL13Rα2+ GBM targets from targets expressing IL13Rα1. In vivo, the adoptive transfer of IL13-zetakine+CD8+ CTL clones results in the regression of established human glioblastoma orthotopic xenografts. Pilot clinical trials have been initiated to evaluate the feasibility and safety of local-regional delivery of autologous IL13-zetakine redirected CTL clones in patients with recurrent GBM. Our IL13-zetakine is a prototype of a new class of chimeric immunoreceptors that signal through an engineered immune synapse composed of membrane-tethered cytokine muteins bound to cell-surface cytokine receptors on tumors.

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

Bioactivity and Safety of IL13Rα2-Redirected Chimeric Antigen Receptor CD8+ T Cells in Patients with Recurrent Glioblastoma

Purpose: A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)–engineered, autologous primary human CD8+ cytotoxic T lymphocytes (CTL) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Experimental Design: Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8+ CTL targeting IL13Rα2. Patients received up to 12 local infusions at a maximum dose of 108 CAR-engineered T cells via a catheter/reservoir system. Results: We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine+ CTL clones into the resection cavity of 3 patients with recurrent disease was well-tolerated, with manageable temporary brain inflammation. Following infusion of IL13-zetakine+ CTLs, evidence for transient anti-glioma responses was observed in 2 of the patients. Analysis of tumor tissue from 1 patient before and after T-cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine+ T-cell administration. Conclusions: These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T-cell therapies can be applied. Clin Cancer Res; 21(18); 4062–72. ©2015 AACR.

Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.

Purpose: To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem–like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8+ CTLs (IL13-zetakine+ CTL) to target this therapeutically resistant glioma subpopulation. Experimental Design: A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine+ CTL-mediated killing in vitro and in vivo. Results: We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2pos cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine+ CTL were capable of efficient recognition and killing of both IL13Rα2pos GSCs and IL13Rα2pos differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine+ CTL displayed robust antitumor activity against established IL13Rα2pos GSC TS-initiated orthotopic tumors in mice. Conclusions: Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine+ CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. Clin Cancer Res; 18(8); 2199–209. ©2012 AACR.

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

Neural stem cells home to gliomas in mice where they convert a prodrug to 5-fluorouracil, leading to tumor regression. Cellular Assassins Derived from the supporting cells of the brain, gliomas are deadly tumors that can be only temporarily held at bay, but not cured. New ways to treat these cancers are needed. To get regulatory approval to test a new stem cell–based therapy in patients, Aboody et al. performed a series of preclinical experiments in mice with artificially implanted gliomas in their brains. By mimicking closely the treatments that they hoped to perform in humans, these authors were able to show to the satisfaction of the regulatory agency that the treatment was safe and effective enough in the mice to warrant a first-in-human trial in patients. The authors used a neural stem cell line carrying a v-myc gene and a gene for cytosine deaminase. These cells exhibit tropism to human glioma cells. When injected into mice with gliomas, they migrate to the site of the tumor, even when the mice are treated with steroids or radiation, as might be the case for human patients. The cytosine deaminase in the cells provides another anticancer weapon. This enzyme converts the prodrug 5-fluorocytosine (5-FC) to the toxic 5-flurouracil (5-FU), delivering a high concentration of the therapeutic agent directly in and around the tumor and causing it to shrink significantly. Injection of excess numbers of cells or increasing the dose of 5-FU did not result in any abnormalities in the animals; in fact, by 12 weeks after injection, no cells were to be seen in the brain or elsewhere, even when a highly sensitive polymerase chain reaction method was used to look for the v-myc DNA. This targeted cell-based approach to cancer therapy that concentrates the therapeutic agent in the vicinity of the tumor is expected to reduce toxicity to other tissues. Thus, a higher local dose is possible, potentially improving efficacy against the tumor. The phase 1 trial derived from these preclinical results is ongoing; its end will allow evaluation of how well these preclinical in vivo studies set the stage for humans. High-grade gliomas are extremely difficult to treat because they are invasive and therefore not curable by surgical resection; the toxicity of current chemo- and radiation therapies limits the doses that can be used. Neural stem cells (NSCs) have inherent tumor-tropic properties that enable their use as delivery vehicles to target enzyme/prodrug therapy selectively to tumors. We used a cytosine deaminase (CD)–expressing clonal human NSC line, HB1.F3.CD, to home to gliomas in mice and locally convert the prodrug 5-fluorocytosine to the active chemotherapeutic 5-fluorouracil. In vitro studies confirmed that the NSCs have normal karyotype, tumor tropism, and CD expression, and are genetically and functionally stable. In vivo biodistribution studies demonstrated NSC retention of tumor tropism, even in mice pretreated with radiation or dexamethasone to mimic clinically relevant adjuvant therapies. We evaluated safety and toxicity after intracerebral administration of the NSCs in non–tumor-bearing and orthotopic glioma–bearing immunocompetent and immunodeficient mice. We detected no difference in toxicity associated with conversion of 5-fluorocytosine to 5-fluorouracil, no NSCs outside the brain, and no histological evidence of pathology or tumorigenesis attributable to the NSCs. The average tumor volume in mice that received HB1.F3.CD NSCs and 5-fluorocytosine was about one-third that of the average volume in control mice. On the basis of these results, we conclude that combination therapy with HB1.F3.CD NSCs and 5-fluorocytosine is safe, nontoxic, and effective in mice. These data have led to approval of a first-in-human study of an allogeneic NSC-mediated enzyme/prodrug-targeted cancer therapy in patients with recurrent high-grade glioma.

Sorafenib Induces Growth Arrest and Apoptosis of Human Glioblastoma Cells through the Dephosphorylation of Signal Transducers and Activators of Transcription 3

Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (≤10 μmol/L) inhibited cell proliferation and induced apoptosis in two established cell lines (U87 and U251) and two primary cultures (PBT015 and PBT022) from human glioblastomas. The effects of sorafenib on these tumor cells were associated with inhibiting phosphorylated signal transducers and activators of transcription 3 (STAT3; Tyr705). Expression of a constitutively activated STAT3 mutant partially blocked the effects of sorafenib, consistent with a role for STAT3 inhibition in the response to sorafenib. Phosphorylated Janus-activated kinase (JAK)1 was inhibited in U87 and U251 cells, whereas phosphorylated JAK2 was inhibited in primary cultures. Sodium vanadate, a general inhibitor of protein tyrosine phosphatases, blocked the inhibition of phosphorylation of STAT3 (Tyr705) induced by sorafenib. These data indicate that the inhibition of STAT3 activity by sorafenib involves both the inhibition of upstream kinases (JAK1 and JAK2) of STAT3 and increased phosphatase activity. Phosphorylation of AKT was also reduced by sorafenib. In contrast, mitogen-activated protein kinases were not consistently inhibited by sorafenib in these cells. Two key cyclins (D and E) and the antiapoptotic protein Mcl-1 were downregulated by sorafenib in both cell lines and primary cultures. Our data suggest that inhibition of STAT3 signaling by sorafenib contributes to growth arrest and induction of apoptosis in glioblastoma cells. These findings provide a rationale for potential treatment of malignant gliomas with sorafenib. Mol Cancer Ther; 9(4); 953–62. ©2010 AACR.

Tumor-Derived Chemokine MCP-1/CCL2 Is Sufficient for Mediating Tumor Tropism of Adoptively Transferred T Cells

To exert a therapeutic effect, adoptively transferred tumor-specific CTLs must traffic to sites of tumor burden, exit the circulation, and infiltrate the tumor microenvironment. In this study, we examine the ability of adoptively transferred human CTL to traffic to tumors with disparate chemokine secretion profiles independent of tumor Ag recognition. Using a combination of in vivo tumor tropism studies and in vitro biophotonic chemotaxis assays, we observed that cell lines derived from glioma, medulloblastoma, and renal cell carcinoma efficiently chemoattracted ex vivo-expanded primary human T cells. We compared the chemokines secreted by tumor cell lines with high chemotactic activity with those that failed to elicit T cell chemotaxis (Daudi lymphoma, 10HTB neuroblastoma, and A2058 melanoma cells) and found a correlation between tumor-derived production of MCP-1/CCL2 (≥10 ng/ml) and T cell chemotaxis. Chemokine immunodepletion studies confirmed that tumor-derived MCP-1 elicits effector T cell chemotaxis. Moreover, MCP-1 is sufficient for in vivo T cell tumor tropism as evidenced by the selective accumulation of i.v. administered firefly luciferase-expressing T cells in intracerebral xenografts of tumor transfectants secreting MCP-1. These studies suggest that the capacity of adoptively transferred T cells to home to tumors may be, in part, dictated by the species and amounts of tumor-derived chemokines, in particular MCP-1.

Genome-Wide Profiling Identified a Set of miRNAs that Are Differentially Expressed in Glioblastoma Stem Cells and Normal Neural Stem Cells

A major challenge in cancer research field is to define molecular features that distinguish cancer stem cells from normal stem cells. In this study, we compared microRNA (miRNA) expression profiles in human glioblastoma stem cells and normal neural stem cells using combined microarray and deep sequencing analyses. These studies allowed us to identify a set of 10 miRNAs that are considerably up-regulated or down-regulated in glioblastoma stem cells. Among them, 5 miRNAs were further confirmed to have altered expression in three independent lines of glioblastoma stem cells by real-time RT-PCR analysis. Moreover, two of the miRNAs with increased expression in glioblastoma stem cells also exhibited elevated expression in glioblastoma patient tissues examined, while two miRNAs with decreased expression in glioblastoma stem cells displayed reduced expression in tumor tissues. Furthermore, we identified two oncogenes, NRAS and PIM3, as downstream targets of miR-124, one of the down-regulated miRNAs; and a tumor suppressor, CSMD1, as a downstream target of miR-10a and miR-10b, two of the up-regulated miRNAs. In summary, this study led to the identification of a set of miRNAs that are differentially expressed in glioblastoma stem cells and normal neural stem cells. Characterizing the role of these miRNAs in glioblastoma stem cells may lead to the development of miRNA-based therapies that specifically target tumor stem cells, but spare normal stem cells.

Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale.

A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8+ effector T cells derived from CD45RA− CD62L+ central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here, we describe a clinical scale, closed system, immunomagnetic selection method to isolate CD8+ TCM from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14+, CD45RA+, and CD4+ cells from PBMC, and then to positively select CD62L+ cells. The average purity and yield of CD8+CD45RA− CD62L+ TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8+ TCM are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3–6 weeks. The resulting CD8+ TCM-derived effectors are polyclonal, retain expression of CD62L and CD28, exhibit CAR-redirected antitumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified TCM cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19+ B-cell malignancies, and 3 clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.