A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the ...biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.
High-grade (WHO grades III-IV) glioma remains one of the most lethal human cancers. Adoptive transfer of tumor-targeting chimeric antigen receptor (CAR)-redirected T cells for high-grade glioma has ...revealed promising indications of anti-tumor activity, but objective clinical responses remain elusive for most patients. A significant challenge to effective immunotherapy is the highly heterogeneous structure of these tumors, including large variations in the magnitudes and distributions of target antigen expression, observed both within individual tumors and between patients. To obtain a more detailed understanding of immunotherapy target antigens within patient tumors, we immunochemically mapped at single cell resolution three clinically-relevant targets, IL13Rα2, HER2 and EGFR, on tumor samples drawn from a 43-patient cohort. We observed that within individual tumor samples, expression of these antigens was neither random nor uniform, but rather that they mapped into local neighborhoods – phenotypically similar cells within regions of cellular tumor – reflecting not well understood properties of tumor cells and their milieu. Notably, tumor cell neighborhoods of high antigen expression were not arranged independently within regions. For example, in cellular tumor regions, neighborhoods of high IL13Rα2 and HER2 expression appeared to be reciprocal to those of EGFR, while in areas of pseudopalisading necrosis, expression of IL13Rα2 and HER2, but not EGFR, appeared to reflect the radial organization of tumor cells around hypoxic cores. Other structural features affecting expression of immunotherapy target antigens remain to be elucidated. This structured but heterogeneous organization of antigen expression in high grade glioma is highly permissive for antigen escape, and combinatorial antigen targeting is a commonly suggested potential mitigating strategy. Deeper understanding of antigen expression within and between patient tumors will enhance optimization of combination immunotherapies, the most immediate clinical application of the observations presented here being the importance of including (wild-type) EGFR as a target antigen.
Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (< or =10 micromol/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. (c)2010 AACR.
Despite aggressive standard-of-care therapy, including surgery, radiation, and chemotherapy, glioblastoma recurrence is almost inevitable and uniformly lethal. Activation of glioma-intrinsic ...Wnt/β-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)/β-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.
Using multiple glioma patient-derived xenografts cell lines and murine tumors (GL261, K-Luc), we demonstrated
cytostatic effects and a switch from proliferation to differentiation after treatment with ICG-001.
In these glioma cell lines, we further demonstrated that ICG-001 downregulated the CBP/β-catenin target gene
a hallmark of Wnt/CBP/β-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.
We conclude that specific Wnt/CBP/β-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.
Glioblastoma (GBM) is the most common primary brain tumor, accounting for approximately 40% of all central nervous system malignancies. Despite standard treatment consisting of surgical resection, ...radiotherapy and/or chemotherapy, the prognosis for GBM is poor; with a median survival of 14.6 months. The cancer stem cell or cancer-initiating cell model has provided a new paradigm for understanding development and recurrence of GBM following treatment. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plant, and along with its derivatives, has been shown to exhibit antitumor activity in several cancers. Here, we reported that a novel synthetic Berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of cancer stem-like cells (CSCs) in a time- and dose-dependent manner when the CSCs from four GBM patients (PBT003, PBT008, PBT022, and PBT030) were cultured. These CSCs grew in neurospheres and expressed CD133 and nestin as markers. Treatment with BBMD3 destroyed the neurosphere morphology, and led to the induction of apoptosis in the CSCs. Induction of apoptosis in these CSCs is dependent upon activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). MicroRNA-4284 (miR-4284) was shown to be over-expressed about 4-fold in the CSCs following BBMD3 treatment. Furthermore, transfection of synthetic anti-sense oligonucleotide against human miR-4284 partially blocked the anticancer effects of BBMD3 on the GBM derived CSCs. BBMD3 also increased phosphorylation of the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in an increase expression of phosphorylated c-Jun and total c-Fos; the major components of transcriptional factor AP-1. The JNK-c-Jun/AP-1 signaling pathway plays an important role in the induction of apoptosis in response to UV irradiation and some drug treatments. Targeting glioblastoma stem-like cells with BBMD3 is therefore novel, and may have promise as an effective therapeutic strategy for treating GBM patients.
BackgroundA major obstacle to successful CAR T cell therapy for glioblastoma (GBM) is effective tumor trafficking and infiltration, which is limited by the blood-brain and blood-CSF barriers. ...Further, the GBM tumor microenvironment (TME) is characterized by solid stress, vessel leakiness, hypoxia, low pH, and high interstitial fluid pressure, all which impact CAR T cell trafficking. In this study, we set out to address two clinical challenges related to CAR T cell trafficking and efficacy: 1) the detection of CAR T cell tumor infiltration and bioactivity using clinical translatable imaging techniques, such as advanced MRI; and 2) the optimization of the route of administration of CAR T-cells for improved trafficking and therapeutic effect.MethodsWe are evaluating CAR T-cells as a novel cell-based immunotherapy for treating glioblastoma (GBM) in early phase clinical trials. CAR T-cell therapy has been shown to induce complete regression in at least one case (Brown et al. 2016). These results have led to the initiation of a first-in-human phase I CAR T-cell trial for recurrent high-grade glioma patients at City of Hope (NCT02208362). In this study, perfusion imaging was performed on a subset of patients who received MRI pre-treatment and post-resection, and follow-up MRI after 3 treatment cycles roughly one month after initial imaging (n = 41).ResultsA decrease in MR-observed tumor volume was significantly correlated to a decrease in contrast leakage into the surrounding tissue (r = 0.369, p = 0.0177*). These results suggest preliminary evidence of vascular normalization in patients who had strong initial response to CAR-T therapy. Immunohistochemistry analysis of patient tumor tissue indicates that endogenous human T cells were distributed around CD31 stained blood vessels (surgical sample analysis of CAR T patients). To better understand how perfusion imaging relates to CAR T cell therapy, we used two syngeneic models of glioma, K-luc and GL261, and characterized fluid flow dynamics during tumor response versus progression, comparing both invasive (K-luc) versus bulky (GL261) tumor growth phenotypes. We also characterized endogenous immune cell subset distribution at the tumor edge and tumor center, such as T cells (CD3, CD4 and CD8), macrophages (CD68 and CD163) and tumor biomarkers-VEGFA, VEGFC, CD31, HIF1a by immunohistochemistry, which were changing with perfusion-diffusion kinetics of the tumor.ConclusionsOngoing studies are focused on further investigating interstitial fluid flow as an imaging biomarker predictive of response both clinically and pre-clinically.
BackgroundChimeric 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.MethodWe 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.ResultsUsing 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.ConclusionCollectively, these findings support the feasibility of this universal Fabrack-CAR T cell approach and provide the rationale for future clinical use in cancer immunotherapy.
BackgroundA major barrier to achieving effective therapies for patients with glioblastoma multiforme (GBM) is the phenotypic heterogeneity seen both between patients and within individual tumors, the ...later creating multiple cell subpopulations contributing to disease recurrence. One strategy for creating more efficacious therapies is to design novel immunotherapies targeting higher proportions of tumors and tumor cells than current options. Chlorotoxin (CLTX) is a 36-amino acid peptide component of scorpion venom that selectively binds to glioma cells of all malignancy grades while sparing normal brain cells and non-malignant tissues.1 2 With this understanding, we developed chimeric antigen receptor (CAR) T cells incorporating the CLTX peptide as a tumor-recognizing ligand, thereby redirecting T cells to target GBM cells and tumors.3 Preclinical studies suggested broad yet specific CLTX binding to patient-derived GBM cells. Preclinical data also suggested a role for cell surface matrix metalloproteinase-2 (MMP-2) in CLTX-CAR T cell activation.MethodsWe designed a non-randomized and dose escalating phase 1 trial evaluating intracavity/intratumoral (ICT) delivery of CLTX-CAR T cells to patients with recurrent/progressing GBM (NCT04214392; approved by the City of Hope National Medical Center protocol review committee, written consent obtained for leukapheresis and treatment), with the primary objectives of feasibility and safety.ResultsHere we report clinical outcomes and correlative observations for four lead-in research participants, all of whom had a histopathological diagnosis of glioblastoma, idh wild type, grade 4, and who had received prior temozolomide as well as radiation and other treatments. The key enrollment criterion was MMP-2 expression in tissue biopsies at levels greater than 20% (moderate and/or high expression) as assessed by immunochemistry. Participants received three infusions of 4, 20, and 20 x 10^6 CLTX-CAR T cells at weekly intervals. There were no CRS events or DLTs observed. Three of the four participants (75%) achieved stable disease. Participants survived a median of 5.75 months (min = 2.4, max = 20.5) after CAR T cell infusion. The presence of CLTX-CAR T cells in tumor fluid collected before and one day after each infusion indicated persistence of CLTX-CAR T cells. IgG1 was absent in these samples, suggesting non-immunogenicity of CLTX-CAR T cells despite the presence of the exogenous CLTX peptide.ConclusionsPhase 1 clinical observations to date confirm the feasibility and safety of CLTX-CAR T cell immunotherapy for patients with GBM. These studies will lead to determination of a maximum tolerated dose/maximum feasible dose.ReferencesLyons SA, O’Neal J, Sontheimer H. Chlorotoxin, a scorpion-derived peptide, specifically binds to gliomas and tumors of neuroectodermal origin. Glia. 2022;39:162–173.Veiseh M, Gabikian P, Bahrami SB, Veiseh O, Zhang M, Hackman RC, Ravanpay AC, Stroud MR, Kusuma Y, Hansen SJ, Kwok D, Munoz NM, Sze RW, Grady WM, Greenberg NM, Ellenbogen RG, Olson JM. Tumor paint: a chlorotoxin:Cy5.5 bioconjugate for intraoperative visualization of cancer foci, Cancer Res. 2007;67:6882–6888.Wang D, Starr R, Chang WC, Aguilar B, Alizadeh D, Wright SL, Yang X, Brito A, Sarkissian A, Ostberg JR, Li L, Shi Y, Gutova M, Aboody K, Badie B, Forman SJ, Barish ME, Brown CE. Chlorotoxin-directed CAR T cells for specific and effective targeting of glioblastoma. Sci Transl Med. 2020;12.Ethics ApprovalWe designed a non-randomized and dose escalating phase 1 trial evaluating intracavity/intratumoral (ICT) delivery of CLTX-CAR T cells to patients with recurrent/progressing GBM (NCT04214392; approved by the City of Hope National Medical Center protocol review committee, written consent obtained for leukapheresis and treatment), with the primary objectives of feasibility and safety.
BackgroundChimeric antigen receptor (CAR) T cell therapy is being explored in early-stage clinical trials as a strategy to improve treatment outcomes for high-grade gliomas (HGGs). We report here, a ...completed phase I trial (NCT02208362) evaluating locoregionally delivered IL13Rα2-targeted CAR T cells in 65 patients with recurrent HGG (rHGG), the majority being recurrent glioblastoma (rGBM).MethodsThis five-arm trial evolved to evaluate three routes of locoregional CAR T cell administration: (i) intratumoral (ICT) following tumor biopsy (Arm 1) or resection (Arm 2); (ii) intraventricular (ICV; Arm 3); and (iii) dual ICT/ICV (Arm 4). The final treatment arm (Arm 5) evaluated dual ICT/ICV delivery with a modified manufacturing process. Primary objectives were feasibility and safety. Secondary objectives evaluated therapy-related cytokine dynamics by cytometric bead array, CAR T cell persistence by flow cytometry and PCR, and clinical outcomes by radiographic imaging and survival. The pretreatment tumor immune landscape was evaluated by immunohistochemistry.ResultsFeasibility and safety were established for all three routes of locoregional CAR T cell delivery (ICT, ICV and dual ICT/ICV). IL13Rα2-CAR T cells were well-tolerated with clinically manageable adverse events at all dose levels, and no dose limiting toxicities observed. Stable disease or better was achieved in 50% of patients, with two partial responses, one complete response (CR), and a second CR after additional CAR T cycles off protocol therapy. Median overall survival (OS) for rGBM patients (68% treated at 2nd recurrence or later) was 7.7 mo. Post-hoc analysis revealed that Arm 5 rGBM patients exhibited the best median OS of 10.2 months, compared to 6.1 months for other treatment arms (Arms 1–4). Increase in inflammatory cytokines, including IFNγ, CXCL9, and CXCL10, was observed in the cerebrospinal (CSF) and tumor fluid after each infusion. Further, pre-treatment intratumoral CD3 T cell levels were positively associated with survival.ConclusionsWe report the largest CAR T cell clinical trial in brain tumors to date, assessing the feasibility, safety, and bioactivity of IL13Rα2-CAR T cells in rHGG. Key findings include: (1) repetitive locoregional administration of IL13Rα2-CAR T cells is feasible and safe, with clinical benefit observed in a subset of patients; (2) elevations in IFNγ-related chemokines in the CSF was associated with CAR T cell administration and bioactivity; and (3) tumor immune contexture was identified as a determinant of patient outcome to CAR T cell therapy. These findings advance our understanding of CAR T cell immunotherapy for malignant brain tumors.Ethics ApprovalThis study was conducted I accordance with the Institutional Review Board and Independent Ethics Committee at The City of Hope National Medical Center as well as the U.S. Food and Drug Administration (FDA). All subjects provided written informed consent.
The interleukin-13 receptor alpha2 (IL13Rα2) 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, IL13Rα2 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 IL13Rα2-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 IL13Rα2 cell surface expression, and thereby increase susceptibility to IL13Rα2-specific T cell killing. In the course of these experiments, we unexpectedly found that the commercially available putative IL13Rα2-specific monoclonal antibody B-D13 recognizes cytokine-induced VCAM-1 on glioblastoma. We provide evidence that the induced receptor is not IL13Rα2, because its expression does not consistently correlate with IL13Rα2 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 IL13Rα2, 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 IL13Rα2 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 IL13Rα2 recognition, while maintaining VCAM-1 specificity. While this work calls into question previous studies that have used the B-D13 antibody to assess IL13Rα2 expression, it also suggests that TNF may have significant effects on glioma biology by up-regulating VCAM-1.
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