Significance of interleukin-13 receptor alpha 2-targeted glioblastoma therapy.
ABSTRACT: Glioblastoma multiforme (GBM) remains one of the most lethal primary brain tumors despite surgical and therapeutic advancements. Targeted therapies of neoplastic diseases, including GBM, have received a great deal of interest in recent years. A highly studied target of GBM is interleukin-13 receptor ? chain variant 2 (IL13R?2). Targeted therapies against IL13R?2 in GBM include fusion chimera proteins of IL-13 and bacterial toxins, nanoparticles, and oncolytic viruses. In addition, immunotherapies have been developed using monoclonal antibodies and cell-based strategies such as IL13R?2-pulsed dendritic cells and IL13R?2-targeted chimeric antigen receptor-modified T cells. Advanced therapeutic development has led to the completion of phase I clinical trials for chimeric antigen receptor-modified T cells and phase III clinical trials for IL-13-conjugated bacterial toxin, with promising outcomes. Selective expression of IL13R?2 on tumor cells, while absent in the surrounding normal brain tissue, has motivated continued study of IL13R?2 as an important candidate for targeted glioma therapy. Here, we review the preclinical and clinical studies targeting IL13R?2 in GBM and discuss new advances and promising applications.
Project description:Glioblastoma multiforme (GBM) remains highly incurable, with frequent recurrences after standard therapies of maximal surgical resection, radiation, and chemotherapy. To address the need for new treatments, we have undertaken a chimeric antigen receptor (CAR) "designer T cell" (dTc) immunotherapeutic strategy by exploiting interleukin (IL)13 receptor ?-2 (IL13R?2) as a GBM-selective target.We tested a second-generation IL13 "zetakine" CAR composed of a mutated IL13 extracellular domain linked to intracellular signaling elements of the CD28 costimulatory molecule and CD3?. The aim of the mutation (IL13.E13K.R109K) was to enhance selectivity of the CAR for recognition and killing of IL13R?2(+) GBMs while sparing normal cells bearing the composite IL13R?1/IL4R? receptor.Our aim was partially realized with improved recognition of tumor and reduced but persisting activity against normal tissue IL13R?1(+) cells by the IL13.E13K.R109K CAR. We show that these IL13 dTcs were efficient in killing IL13R?2(+) glioma cell targets with abundant secretion of cytokines IL2 and IFN?, and they displayed enhanced tumor-induced expansion versus control unmodified T cells in vitro. In an in vivo test with a human glioma xenograft model, single intracranial injections of IL13 dTc into tumor sites resulted in marked increases in animal survivals.These data raise the possibility of immune targeting of diffusely invasive GBM cells either via dTc infusion into resection cavities to prevent GBM recurrence or via direct stereotactic injection of dTcs to suppress inoperable or recurrent tumors. Systemic administration of these IL13 dTc could be complicated by reaction against normal tissues expressing IL13Ra1.
Project description: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 ?2 (IL13R?2) for the treatment of GBM. Here, we describe the optimization of IL13R?2-targeted CAR T cells, including the design of a 4-1BB (CD137) co-stimulatory CAR (IL13BB?) 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 IL13BB?-CAR T cells improved anti-tumor activity and T cell persistence as compared to first-generation IL13?-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.
Project description:Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13R?2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13R?2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13R?2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ?40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.
Project description:Immunotherapy with T cells expressing chimeric antigen receptors (CARs) is an attractive approach to improve outcomes for patients with glioblastoma (GBM). IL13R?2 is expressed at a high frequency in GBM but not in normal brain, making it a promising CAR T-cell therapy target. IL13R?2-specific CARs generated up to date contain mutated forms of IL13 as an antigen-binding domain. While these CARs target IL13R?2, they also recognize IL13R?1, which is broadly expressed. To overcome this limitation, we constructed a panel of IL13R?2-specific CARs that contain the IL13R?2-specific single-chain variable fragment (scFv) 47 as an antigen binding domain, short or long spacer regions, a transmembrane domain, and endodomains derived from costimulatory molecules and CD3.? (IL13R?2-CARs). IL13R?2-CAR T cells recognized IL13R?2-positive target cells in coculture and cytotoxicity assays with no cross-reactivity to IL13R?1. However, only IL13R?2-CAR T cells with a short spacer region produced IL2 in an antigen-dependent fashion. In vivo, T cells expressing IL13R?2-CARs with short spacer regions and CD28.?, 41BB.?, and CD28.OX40.? endodomains had potent anti-glioma activity conferring a significant survival advantage in comparison to mice that received control T cells. Thus, IL13R?2-CAR T cells hold the promise to improve current IL13R?2-targeted immunotherapy approaches for GBM and other IL13R?2-positive malignancies.
Project description:Glioblastoma (GBM), the deadliest form of brain cancer, presents long-standing problems due to its localization. Chimeric antigen receptor (CAR) T cell immunotherapy has emerged as a powerful strategy to treat cancer. IL-13-receptor-?2 (IL13R?2), present in over 75% of GBMs, has been recognized as an attractive candidate for anti-glioblastoma therapy. Here, we propose a novel multidisciplinary approach to target brain tumors using a combination of fluorescent, therapeutic nanoparticles and CAR T cells modified with a targeted-quadruple-mutant of IL13 (TQM-13) shown to have high binding affinity to IL13R?2-expressing glioblastoma cells with low off-target toxicity. Azide-alkyne cycloaddition conjugation of nanoparticles to the surface of T cells allowed a facile, selective, and high-yielding clicking of the nanoparticles. Nanoparticles clicked onto T cells were retained for at least 8 days showing that the linkage is stable and promising a suitable time window for in vivo delivery. T cells clicked with doxorubicin-loaded nanoparticles showed a higher cytotoxic effect in vitro compared to bare T cells. In vitro and in vivo T cells expressing TQM-13 served as delivery shuttles for nanoparticles and significantly increased the number of nanoparticles reaching brain tumors compared to nanoparticles alone. This work represents a new platform to allow the delivery of therapeutic nanoparticles and T cells to solid tumors.
Project description:The interleukin-13 receptor alpha2 (IL13R?2) is a cell surface receptor that is over-expressed by a subset of high-grade gliomas, but not expressed at significant levels by normal brain tissue. For both malignant and non-malignant cells, IL13R?2 surface expression is reported to be induced by various cytokines such as IL-4 or IL-13 and tumor necrosis factor (TNF). Our group has developed a therapeutic platform to target IL13R?2-positive brain tumors by engineering human cytotoxic T lymphocytes (CTLs) to express the IL13-zetakine chimeric antigen receptor. We therefore sought to investigate the potential of cytokine stimulation to induce IL13R?2 cell surface expression, and thereby increase susceptibility to IL13R?2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13R?2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13R?2, because its expression does not consistently correlate with IL13R?2 mRNA levels, it does not bind IL-13, and it is not recognized by IL13-zetakine CTL. Instead we demonstrate by immunoprecipitation experiments and mass spectrometry that the antigen recognized by the B-D13 antibody following cytokine stimulation is VCAM-1, and that VCAM-1, but not IL13R?2, is induced on glioma cells by TNF alone or in combination with IL-13 or IL-4. Further evaluation of several commercial B-D13 antibodies revealed that B-D13 is bi-specific, recognizing both IL13R?2 and VCAM-1. This binding is non-overlapping based on soluble receptor competition experiments, and mass spectrometry identifies two distinct heavy and light chain species, providing evidence that the B-D13 reagent is di-clonal. PE-conjugation of the B-D13 antibody appears to disrupt IL13R?2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13R?2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.
Project description:A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR)-engineered, autologous primary human CD8(+) cytotoxic T lymphocytes (CTL) targeting IL13R?2 for the treatment of recurrent glioblastoma (GBM).Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8(+) CTL targeting IL13R?2. Patients received up to 12 local infusions at a maximum dose of 10(8) CAR-engineered T cells via a catheter/reservoir system.We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13R?2-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 IL13R?2 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.These findings provide promising first-in-human clinical experience for intracranial administration of IL13R?2-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.
Project description:Background:Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric antigen receptor (CAR) T cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of 2 glioma antigens offsets antigen escape and enhances T-cell effector functions, the interpatient variability in surface antigen expression between patients hinders the clinical impact of targeting 2 antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. Methods:We analyzed the surface expression of 3 targetable glioma antigens (human epidermal growth factor receptor 2 [HER2], interleukin-13 receptor subunit alpha-2 [IL13R?2], and ephrin-A2 [EphA2]) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with 3 CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T cells). Results:Our data showed that co-targeting HER2, IL13R?2, and EphA2 could overcome interpatient variability by a tendency to capture nearly 100% of tumor cells in most tumors tested in this cohort. UCAR T cells made from GBM patients' blood uniformly expressed all 3 CAR molecules with distinct antigen specificity. UCAR T cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T cells per patient tumor profile. Lastly, low doses of UCAR T cells controlled established autologous GBM patient derived xenografts (PDXs) and improved survival of treated animals. Conclusion:UCAR T cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.
Project description:The high affinity interleukin-13 receptor ?2 (IL13R?2) is selectively expressed at a high frequency by glioblastoma multiforme (GBM) as well as several other tumor types. One approach for targeting this tumor-specific receptor utilizes the cognate ligand, IL-13, conjugated to cytotoxic molecules. However, this approach lacks specificity because the lower affinity receptor for IL-13, IL13R?1, is widely expressed by normal tissues. Here, we aimed to develop and characterize a novel monoclonal antibody (mAb) specific to IL13R?2 for the therapeutic purpose of targeting IL13R?2-expressing tumors. Hybridoma cell lines were generated and compared for binding affinities to recombinant human IL13R?2 (rhIL13R?2). Clone 47 demonstrated binding to the native conformation of IL13R?2 and was therefore chosen for further studies. Clone 47 bound specifically and with high affinity (K(D) = 1.39 × 10(-9) M) to rhIL13R?2 but not to rhIL13R?1 or murine IL13R?2. Furthermore, clone 47 specifically recognized wild-type IL13R?2 expressed on the surface of CHO and HEK cells as well as several glioma cell lines. Competitive binding assays revealed that clone 47 also significantly inhibited the interaction between human soluble IL-13 and IL13R?2 receptor. Moreover, we found that N-linked glycosylation of IL13R?2 contributes in part to the interaction of the antibody to IL13R?2. In vivo, the IL13R?2 mAb improved the survival of nude mice intracranially implanted with a human U251 glioma xenograft. Collectively, these data warrant further investigation of this novel IL13R?2 mAb with an emphasis on translational implications for therapeutic use.