Browsing by Author "Bigner, Darell D"
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Item Open Access A novel recombinant immunotoxin-based therapy targeting wild-type and mutant EGFR improves survival in murine models of glioblastoma.(Oncoimmunology, 2013-12) Chandramohan, Vidyalakshmi; Bigner, Darell DBoth the amplification of the gene coding for wild-type (wt) epidermal growth factor receptor (EGFR) and the overexpression of the EGFR deletion mutant, commonly known as EGFRvIII, are hallmarks of glioblastoma. We have recently reported a novel, recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, that targets both wt EGFR and EGFRvIII, exhibiting potent antineoplastic effects against established murine gliomas.Item Open Access A pilot study of IL-2Rα blockade during lymphopenia depletes regulatory T-cells and correlates with enhanced immunity in patients with glioblastoma.(PLoS One, 2012) Sampson, John H; Schmittling, Robert J; Archer, Gary E; Congdon, Kendra L; Nair, Smita K; Reap, Elizabeth A; Desjardins, Annick; Friedman, Allan H; Friedman, Henry S; Herndon, James E; Coan, April; McLendon, Roger E; Reardon, David A; Vredenburgh, James J; Bigner, Darell D; Mitchell, Duane ABACKGROUND: Preclinical studies in mice have demonstrated that the prophylactic depletion of immunosuppressive regulatory T-cells (T(Regs)) through targeting the high affinity interleukin-2 (IL-2) receptor (IL-2Rα/CD25) can enhance anti-tumor immunotherapy. However, therapeutic approaches are complicated by the inadvertent inhibition of IL-2Rα expressing anti-tumor effector T-cells. OBJECTIVE: To determine if changes in the cytokine milieu during lymphopenia may engender differential signaling requirements that would enable unarmed anti-IL-2Rα monoclonal antibody (MAbs) to selectively deplete T(Regs) while permitting vaccine-stimulated immune responses. METHODOLOGY: A randomized placebo-controlled pilot study was undertaken to examine the ability of the anti-IL-2Rα MAb daclizumab, given at the time of epidermal growth factor receptor variant III (EGFRvIII) targeted peptide vaccination, to safely and selectively deplete T(Regs) in patients with glioblastoma (GBM) treated with lymphodepleting temozolomide (TMZ). RESULTS AND CONCLUSIONS: Daclizumab treatment (n = 3) was well-tolerated with no symptoms of autoimmune toxicity and resulted in a significant reduction in the frequency of circulating CD4+Foxp3+ TRegs in comparison to saline controls (n = 3)( p = 0.0464). A significant (p<0.0001) inverse correlation between the frequency of TRegs and the level of EGFRvIII specific humoral responses suggests the depletion of TRegs may be linked to increased vaccine-stimulated humoral immunity. These data suggest this approach deserves further study. TRIAL REGISTRATION: ClinicalTrials.gov NCT00626015.Item Open Access Detection of amino-terminal extracellular domain of somatostatin receptor 2 by specific monoclonal antibodies and quantification of receptor density in medulloblastoma.(Hybridoma (Larchmt), 2009-12) Kuan, Chien-Tsun; Wikstrand, Carol J; McLendon, Roger E; Zalutsky, Michael R; Kumar, Ujendra; Bigner, Darell DSomatostatin receptor 2 (SSTR2) is expressed by most medulloblastomas (MEDs). We isolated monoclonal antibodies (MAbs) to the 12-mer (33)QTEPYYDLTSNA(44), which resides in the extracellular domain of the SSTR2 amino terminus, screened the peptide-bound MAbs by fluorescence microassay on D341 and D283 MED cells, and demonstrated homogeneous cell-surface binding, indicating that all cells expressed cell surface-detectable epitopes. Five radiolabeled MAbs were tested for immunoreactive fraction (IRF), affinity (KA) (Scatchard analysis vs. D341 MED cells), and internalization by MED cells. One IgG(3) MAb exhibited a 50-100% IRF, but low KA. Four IgG(2a) MAbs had 46-94% IRFs and modest KAs versus intact cells (0.21-1.2 x 10(8) M(-1)). Following binding of radiolabeled MAbs to D341 MED at 4 degrees C, no significant internalization was observed, which is consistent with results obtained in the absence of ligand. However, all MAbs exhibited long-term association with the cells; binding at 37 degrees C after 2 h was 65-66%, and after 24 h, 52-64%. In tests with MAbs C10 and H5, the number of cell surface receptors per cell, estimated by Scatchard and quantitative FACS analyses, was 3.9 x 10(4) for the "glial" phenotype DAOY MED cell line and 0.6-8.8 x 10(5) for four neuronal phenotype MED cell lines. Our results indicate a potential immunotherapeutic application for these MAbs.Item Open Access Development of Novel Antibody-Based Immunotherapies Targeting Human Chondroitin Sulfate Proteoglycan 4(2018) Yu, XinChondroitin sulfate proteoglycan 4 (CSPG4) is a promising target for cancer immunotherapy due to its high level of expression in a number of malignant tumors, and its essential role in tumor growth and progression. Clinical application of CSPG4-targeting immunotherapies is hampered by the lack of fully human CSPG4 antibodies or antibody fragments. In addition, the efficacy of cytotoxic monotherapies, such as the CSPG4-targeting immunotoxins (ITs), is limited by hyperactive anti-apoptotic pathways prevalent in tumor cells. Therefore, there is a need to discover novel, fully human antibodies for CSPG4-targeting immunotherapies and to develop new strategies that sensitize resistant CSPG4-expressing tumor cells to IT therapies.
To discover fully human antibodies that can be developed into potential CSPG4-targeting therapeutics, my first aim is to develop novel human single-chain variable fragments (scFvs) with high binding affinity and specificity to the CSPG4 antigen. Affinity maturation was performed on a novel, fully human anti-CSPG4 scFv using the random mutagenesis approach. A yeast display library was constructed for the mutant clones, and screened using a modified whole-cell panning method followed by fluorescence-activated cell sorting (FACS). After six rounds of panning and sorting, the top seven mutant scFvs were isolated and their binding affinities were characterized by flow cytometry and surface plasmon resonance. These mutant clones were highly specific to the CSPG4 antigen, and displayed nanomolar to picomolar binding affinities. While each of them harbored only two to six amino acid substitutions, they represented approximately 270-3000-fold improvement in affinity compared to the parental clone. These affinity-matured scFvs can be potentially developed into diagnostic or therapeutic agents for evaluation and treatment of CSPG4-expressing tumors.
To facilitate the screening of scFv libraries targeting CSPG4, my second aim is to develop a cell-based fluorescent assay for high-throughput analysis of antibody affinity (KD) in the nanomolar range. In this method, fluorescently labelled antibodies were added to antigen-positive and antigen-negative cell lines fixed on 96-well plates. The fluorescent signals from nonspecific binding to negative control cell lines is subtracted from the specific binding to the antigen-positive cell lines. The results confirmed that the KD values obtained using this method were comparable with values obtained by the conventional flow cytometry and radioactive (I125) scatchard assays. This demonstrates that the cell-based fluorescent method allows for accurate and efficient identification of therapeutically relevant leads.
Finally, to improve the efficacy of ITs targeting CSPG4, especially in the IT-resistant tumor cells, my third aim is to evaluate a multi-pathway therapy that combines anti-CSPG4 ITs and small molecule Bcl-2 inhibitors. To enhance sensitivity of cancer cells to ITs, we combined ITs (9.2.27-PE38KDEL or Mel-14-PE38KDEL) targeting CSPG4 with a Bcl-2 inhibitor (ABT-737, ABT-263, or ABT-199) against patient-derived glioblastoma xenografts, melanoma cell lines, and breast cancer cell lines. Results from the in vitro cytotoxicity assays demonstrated that the addition of the ABT compounds, specifically ABT-737, sensitized all three tumors to the IT treatment, and in some cases improved the IC50 values of 9.2.27-PE38KDEL by over 1000-fold. Mechanistic studies using 9.2.27-PE38KDEL and ABT-737 revealed that the rate of IT internalization and the efficiency of cleaved exotoxin accumulation in the cytosol correlated with the enhanced sensitivity of the tumor cells to the combination treatment. Furthermore, the synergistic effect of 9.2.27-PE38KDEL and ABT-737 combination therapy was confirmed in an orthotopic GBM xenograft model and a model of melanoma metastasized to the brain. For the first time, our study compares the efficacy of ABT-737 and 9.2.27-PE38KDEL combination therapy in GBM and a different brain metastases model, providing insights into overcoming IT resistance in brain tumors.
In conclusion, I discovered novel human scFvs with high binding affinities to CSPG4, developed a cell-based fluorescent method for accurate and efficient affinity analysis of antibodies, and investigated combination immunotherapies that utilized Bcl-2 inhibitors to sensitize tumor cells to treatment by CSPG4-targeting ITs. The results from these studies helped to facilitate the development of novel antibody-based immunotherapies and combination immunotherapies for CSPG4-expressing tumors.
Item Open Access Disruption of wild-type IDH1 suppresses D-2-hydroxyglutarate production in IDH1-mutated gliomas.(Cancer research, 2013-01) Jin, Genglin; Reitman, Zachary J; Duncan, Christopher G; Spasojevic, Ivan; Gooden, David M; Rasheed, B Ahmed; Yang, Rui; Lopez, Giselle Y; He, Yiping; McLendon, Roger E; Bigner, Darell D; Yan, HaiPoint mutations at Arg132 of the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase 1 (IDH1) occur frequently in gliomas and result in a gain of function to produce the "oncometabolite" D-2-hydroxyglutarate (D-2HG). The mutated IDH1 allele is usually associated with a wild-type IDH1 allele (heterozygous) in cancer. Here, we identify 2 gliomas that underwent loss of the wild-type IDH1 allele but retained the mutant IDH1 allele following tumor progression from World Health Organization (WHO) grade III anaplastic astrocytomas to WHO grade IV glioblastomas. Intratumoral D-2HG was 14-fold lower in the glioblastomas lacking wild-type IDH1 than in glioblastomas with heterozygous IDH1 mutations. To characterize the contribution of wild-type IDH1 to cancer cell D-2HG production, we established an IDH1-mutated astrocytoma (IMA) cell line from a WHO grade III anaplastic astrocytoma. Disruption of the wild-type IDH1 allele in IMA cells by gene targeting resulted in an 87-fold decrease in cellular D-2HG levels, showing that both wild-type and mutant IDH1 alleles are required for D-2HG production in glioma cells. Expression of wild-type IDH1 was also critical for mutant IDH1-associated D-2HG production in the colorectal cancer cell line HCT116. These insights may aid in the development of therapeutic strategies to target IDH1-mutated cancers.Item Open Access EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis.(The Journal of clinical investigation, 2014-09) Feng, Haizhong; Lopez, Giselle Y; Kim, Chung Kwon; Alvarez, Angel; Duncan, Christopher G; Nishikawa, Ryo; Nagane, Motoo; Su, An-Jey A; Auron, Philip E; Hedberg, Matthew L; Wang, Lin; Raizer, Jeffery J; Kessler, John A; Parsa, Andrew T; Gao, Wei-Qiang; Kim, Sung-Hak; Minata, Mutsuko; Nakano, Ichiro; Grandis, Jennifer R; McLendon, Roger E; Bigner, Darell D; Lin, Hui-Kuan; Furnari, Frank B; Cavenee, Webster K; Hu, Bo; Yan, Hai; Cheng, Shi-YuanAberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.Item Open Access EGFRvIII-specific chimeric antigen receptor T cells migrate to and kill tumor deposits infiltrating the brain parenchyma in an invasive xenograft model of glioblastoma.(PLoS One, 2014) Miao, Hongsheng; Choi, Bryan D; Suryadevara, Carter M; Sanchez-Perez, Luis; Yang, Shicheng; De Leon, Gabriel; Sayour, Elias J; McLendon, Roger; Herndon, James E; Healy, Patrick; Archer, Gary E; Bigner, Darell D; Johnson, Laura A; Sampson, John HGlioblastoma (GBM) is the most common primary malignant brain tumor in adults and is uniformly lethal. T-cell-based immunotherapy offers a promising platform for treatment given its potential to specifically target tumor tissue while sparing the normal brain. However, the diffuse and infiltrative nature of these tumors in the brain parenchyma may pose an exceptional hurdle to successful immunotherapy in patients. Areas of invasive tumor are thought to reside behind an intact blood brain barrier, isolating them from effective immunosurveillance and thereby predisposing the development of "immunologically silent" tumor peninsulas. Therefore, it remains unclear if adoptively transferred T cells can migrate to and mediate regression in areas of invasive GBM. One barrier has been the lack of a preclinical mouse model that accurately recapitulates the growth patterns of human GBM in vivo. Here, we demonstrate that D-270 MG xenografts exhibit the classical features of GBM and produce the diffuse and invasive tumors seen in patients. Using this model, we designed experiments to assess whether T cells expressing third-generation chimeric antigen receptors (CARs) targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, would localize to and treat invasive intracerebral GBM. EGFRvIII-targeted CAR (EGFRvIII+ CAR) T cells demonstrated in vitro EGFRvIII antigen-specific recognition and reactivity to the D-270 MG cell line, which naturally expresses EGFRvIII. Moreover, when administered systemically, EGFRvIII+ CAR T cells localized to areas of invasive tumor, suppressed tumor growth, and enhanced survival of mice with established intracranial D-270 MG tumors. Together, these data demonstrate that systemically administered T cells are capable of migrating to the invasive edges of GBM to mediate antitumor efficacy and tumor regression.Item Open Access Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas.(Oncotarget, 2012-07) Jiao, Yuchen; Killela, Patrick J; Reitman, Zachary J; Rasheed, Ahmed B; Heaphy, Christopher M; de Wilde, Roeland F; Rodriguez, Fausto J; Rosemberg, Sergio; Oba-Shinjo, Sueli Mieko; Nagahashi Marie, Suely Kazue; Bettegowda, Chetan; Agrawal, Nishant; Lipp, Eric; Pirozzi, Christopher; Lopez, Giselle; He, Yiping; Friedman, Henry; Friedman, Allan H; Riggins, Gregory J; Holdhoff, Matthias; Burger, Peter; McLendon, Roger; Bigner, Darell D; Vogelstein, Bert; Meeker, Alan K; Kinzler, Kenneth W; Papadopoulos, Nickolas; Diaz, Luis A; Yan, HaiMutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1, which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutations in many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas ( more than 10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.Item Open Access IDH1(R132) mutation identified in one human melanoma metastasis, but not correlated with metastases to the brain.(Biochemical and biophysical research communications, 2010-07-13) Lopez, Giselle Y; Reitman, Zachary J; Solomon, David; Waldman, Todd; Bigner, Darell D; McLendon, Roger E; Rosenberg, Steven A; Samuels, Yardena; Yan, HaiIsocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to alpha-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP+to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1(R132) or the homologous IDH2(R172). Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1(R132) and IDH2(R172) mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1(R132) and IDH2(R172) mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1(R132) or IDH2(R172) may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors.Item Open Access Immunotoxin Monotherapy and Combinatorial Therapy With Immune Checkpoint Inhibitors for Malignant Brain Tumors(2016) Bao, XuhuiGlioblastoma is the most common and aggressive malignant brain tumor among all primary brain and central nervous system (CNS) tumors. The median survival time for glioblastoma patients given the current standard of care treatment (surgery, radiation, and chemotherapy) is less than 15 months. Medulloblastoma is another major malignant brain tumor that most frequently occurs in children. Although recent advances in surgery, radiotherapy, and chemotherapy have led to an increase in 5-year survival rates of medulloblastoma patients, treatment-related toxicity often has a major impact on long-term quality of survival.
As a result, there is an urgent need to develop more efficient and novel therapeutic approaches that specifically target tumor cells while preserving the surrounding normal CNS to improve the poor survival and quality of life of patients with malignant brain tumors. To address this need, we have developed two novel targeted immunotoxins (ITs), D2C7-(scdsFv)-PE38KDEL (D2C7-IT) and NZ-1-(scdsFv)-PE38KDEL (NZ-1-IT). D2C7-IT was developed by fusing the single-chain variable fragment (scFv) of the D2C7 monoclonal antibody (mAb) with domains II and III of Pseudomonas exotoxin A (PE38KDEL), and NZ-1-IT was developed by fusing the scFv of the NZ-1 mAb with PE38KDEL. D2C7-IT reacts with both the wild-type epidermal growth factor receptor (EGFRwt) and the EGFR variant III (EGFRvIII), two overexpressed proteins in glioblastomas. NZ-1-IT reacts with podoplanin (PDPN), a protein that has a high expression in glioblastomas and medulloblastomas.
In vitro cytotoxicity data shows that both ITs effectively inhibited protein synthesis in a variety of epitope-expressing glioblastoma and medulloblastoma xenograft cells and human tumor cell lines. Furthermore, the direct anti-tumor efficacy of D2C7-IT was examined in orthotopic glioma models in immunocompromised mice, while the direct anti-tumor efficacy of NZ-1-IT was observed in medulloblastoma xenograft-bearing immunocompromised mice. Both immunotoxins showed a robust anti-tumor efficacy in the preclinical brain tumor models. D2C7-IT was first investigated in the subsequent studies to accelerate its translation to the clinic. The preclinical toxicity of intracerebral D2C7-IT infusion was subsequently determined in normal Sprague-Dawley (SD) rats. The maximum tolerated dose (MTD) of D2C7-IT was determined to be between a total dose of 0.10 and 0.35 μg, and the no-observed-adverse-effect level (NOAEL) of D2C7-IT was a total dose of 0.05 μg in SD rats. Both the MTD and NOAEL were utilized as references for the D2C7-IT clinical trial design.
In addition to direct tumor cell killing, immunotoxin monotherapy has been shown to induce a secondary anti-tumor immune response through the engagement of T cells. Therefore, the D2C7-IT-induced secondary anti-tumor immune response was investigated using syngeneic mouse glioma models in immunocompetent mice. Moreover, previous studies have demonstrated that immune checkpoint inhibitors have a robust anti-tumor efficacy by augmenting the T cell response to the tumor cells. Thus, immune checkpoint inhibitors were combined with D2C7-IT in order to enhance the immunotoxin-induced anti-tumor immune response to eliminate residual tumor cells and prevent tumor recurrence in the long term. Meanwhile, studies with NZ-1-IT remain preliminary; thus, this IT will not be as robustly discussed as D2C7-IT throughout this text.
Item Open Access Improved efficacy against malignant brain tumors with EGFRwt/EGFRvIII targeting immunotoxin and checkpoint inhibitor combinations.(Journal for immunotherapy of cancer, 2019-05-29) Chandramohan, Vidyalakshmi; Bao, Xuhui; Yu, Xin; Parker, Scott; McDowall, Charlotte; Yu, Yen-Rei; Healy, Patrick; Desjardins, Annick; Gunn, Michael D; Gromeier, Matthias; Nair, Smita K; Pastan, Ira H; Bigner, Darell DBackground
D2C7-IT is a novel immunotoxin (IT) targeting wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins in glioblastoma. In addition to inherent tumoricidal activity, immunotoxins induce secondary immune responses through the activation of T cells. However, glioblastoma-induced immune suppression is a major obstacle to an effective and durable immunotoxin-mediated antitumor response. We hypothesized that D2C7-IT-induced immune response could be effectively augmented in combination with αCTLA-4/αPD-1/αPD-L1 therapies in murine models of glioma.Methods
To study this, we overexpressed the D2C7-IT antigen, murine EGFRvIII (dmEGFRvIII), in established glioma lines, CT-2A and SMA560. The reactivity and therapeutic efficacy of D2C7-IT against CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII cells was determined by flow cytometry and in vitro cytotoxicity assays, respectively. Antitumor efficacy of D2C7-IT was examined in immunocompetent, intracranial murine glioma models and the role of T cells was assessed by CD4+ and CD8+ T cell depletion. In vivo efficacy of D2C7-IT/αCTLA-4/αPD-1 monotherapy or D2C7-IT+αCTLA-4/αPD-1 combination therapy was evaluated in subcutaneous unilateral and bilateral CT-2A-dmEGFRvIII glioma-bearing immunocompetent mice. Further, antitumor efficacy of D2C7-IT+αCTLA-4/αPD-1/αPD-L1/αTim-3/αLag-3/αCD73 combination therapy was evaluated in intracranial CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII glioma-bearing mice. Pairwise differences in survival curves were assessed using the generalized Wilcoxon test.Results
D2C7-IT effectively killed CT-2A-dmEGFRvIII (IC50 = 0.47 ng/mL) and SMA560-dmEGFRvIII (IC50 = 1.05 ng/mL) cells in vitro. Treatment of intracranial CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII tumors with D2C7-IT prolonged survival (P = 0.0188 and P = 0.0057, respectively), which was significantly reduced by the depletion of CD4+ and CD8+ T cells. To augment antitumor immune responses, we combined D2C7-IT with αCTLA-4/αPD-1 in an in vivo subcutaneous CT-2A-dmEGFRvIII model. Tumor-bearing mice exhibited complete tumor regressions (4/10 in D2C7-IT+αCTLA-4 and 5/10 in D2C7-IT+αPD-1 treatment groups), and combination therapy-induced systemic antitumor response was effective against both dmEGFRvIII-positive and dmEGFRvIII-negative CT-2A tumors. In a subcutaneous bilateral CT-2A-dmEGFRvIII model, D2C7-IT+αCTLA-4/αPD-1 combination therapies showed dramatic regression of the treated tumors and measurable regression of untreated tumors. Notably, in CT-2A-dmEGFRvIII and SMA560-dmEGFRvIII intracranial glioma models, D2C7-IT+αPD-1/αPD-L1 combinations improved survival, and in selected cases generated cures and protection against tumor re-challenge.Conclusions
These data support the development of D2C7-IT and immune checkpoint blockade combinations for patients with malignant glioma.Item Open Access MiR-215 Is Induced Post-transcriptionally via HIF-Drosha Complex and Mediates Glioma-Initiating Cell Adaptation to Hypoxia by Targeting KDM1B.(Cancer Cell, 2016-01-11) Hu, Jing; Sun, Tao; Wang, Hui; Chen, Zhengxin; Wang, Shuai; Yuan, Lifeng; Liu, Tingyu; Li, Hai-Ri; Wang, Pingping; Feng, Yukuan; Wang, Qinhong; McLendon, Roger E; Friedman, Allan H; Keir, Stephen T; Bigner, Darell D; Rathmell, Jeff; Fu, Xiang-Dong; Li, Qi-Jing; Wang, Huibo; Wang, Xiao-FanThe hypoxic tumor microenvironment serves as a niche for maintaining the glioma-initiating cells (GICs) that are critical for glioblastoma (GBM) occurrence and recurrence. Here, we report that hypoxia-induced miR-215 is vital for reprograming GICs to fit the hypoxic microenvironment via suppressing the expression of an epigenetic regulator KDM1B and modulating activities of multiple pathways. Interestingly, biogenesis of miR-215 and several miRNAs is accelerated post-transcriptionally by hypoxia-inducible factors (HIFs) through HIF-Drosha interaction. Moreover, miR-215 expression correlates inversely with KDM1B while correlating positively with HIF1α and GBM progression in patients. These findings reveal a direct role of HIF in regulating miRNA biogenesis and consequently activating the miR-215-KDM1B-mediated signaling required for GIC adaptation to hypoxia.Item Restricted MRP3: a molecular target for human glioblastoma multiforme immunotherapy.(BMC Cancer, 2010-09-01) Kuan, Chien-Tsun; Wakiya, Kenji; Herndon, James E; Lipp, Eric S; Pegram, Charles N; Riggins, Gregory J; Rasheed, Ahmed; Szafranski, Scott E; McLendon, Roger E; Wikstrand, Carol J; Bigner, Darell DBACKGROUND: Glioblastoma multiforme (GBM) is refractory to conventional therapies. To overcome the problem of heterogeneity, more brain tumor markers are required for prognosis and targeted therapy. We have identified and validated a promising molecular therapeutic target that is expressed by GBM: human multidrug-resistance protein 3 (MRP3). METHODS: We investigated MRP3 by genetic and immunohistochemical (IHC) analysis of human gliomas to determine the incidence, distribution, and localization of MRP3 antigens in GBM and their potential correlation with survival. To determine MRP3 mRNA transcript and protein expression levels, we performed quantitative RT-PCR, raising MRP3-specific antibodies, and IHC analysis with biopsies of newly diagnosed GBM patients. We used univariate and multivariate analyses to assess the correlation of RNA expression and IHC of MRP3 with patient survival, with and without adjustment for age, extent of resection, and KPS. RESULTS: Real-time PCR results from 67 GBM biopsies indicated that 59/67 (88%) samples highly expressed MRP3 mRNA transcripts, in contrast with minimal expression in normal brain samples. Rabbit polyvalent and murine monoclonal antibodies generated against an extracellular span of MRP3 protein demonstrated reactivity with defined MRP3-expressing cell lines and GBM patient biopsies by Western blotting and FACS analyses, the latter establishing cell surface MRP3 protein expression. IHC evaluation of 46 GBM biopsy samples with anti-MRP3 IgG revealed MRP3 in a primarily membranous and cytoplasmic pattern in 42 (91%) of the 46 samples. Relative RNA expression was a strong predictor of survival for newly diagnosed GBM patients. Hazard of death for GBM patients with high levels of MRP3 RNA expression was 2.71 (95% CI: 1.54-4.80) times that of patients with low/moderate levels (p = 0.002). CONCLUSIONS: Human GBMs overexpress MRP3 at both mRNA and protein levels, and elevated MRP3 mRNA levels in GBM biopsy samples correlated with a higher risk of death. These data suggest that the tumor-associated antigen MRP3 has potential use for prognosis and as a target for malignant glioma immunotherapy.Item Open Access Multiple phenotypic changes in mice after knockout of the B3gnt5 gene, encoding Lc3 synthase--a key enzyme in lacto-neolacto ganglioside synthesis.(BMC Dev Biol, 2010-11-18) Kuan, Chien-Tsun; Chang, Jinli; Mansson, Jan-Eric; Li, Jianjun; Pegram, Charles; Fredman, Pam; McLendon, Roger E; Bigner, Darell DBACKGROUND: Ganglioside biosynthesis occurs through a multi-enzymatic pathway which at the lactosylceramide step is branched into several biosynthetic series. Lc3 synthase utilizes a variety of galactose-terminated glycolipids as acceptors by establishing a glycosidic bond in the beta-1,3-linkage to GlcNaAc to extend the lacto- and neolacto-series gangliosides. In order to examine the lacto-series ganglioside functions in mice, we used gene knockout technology to generate Lc3 synthase gene B3gnt5-deficient mice by two different strategies and compared the phenotypes of the two null mouse groups with each other and with their wild-type counterparts. RESULTS: B3gnt5 gene knockout mutant mice appeared normal in the embryonic stage and, if they survived delivery, remained normal during early life. However, about 9% developed early-stage growth retardation, 11% died postnatally in less than 2 months, and adults tended to die in 5-15 months, demonstrating splenomegaly and notably enlarged lymph nodes. Without lacto-neolacto series gangliosides, both homozygous and heterozygous mice gradually displayed fur loss or obesity, and breeding mice demonstrated reproductive defects. Furthermore, B3gnt5 gene knockout disrupted the functional integrity of B cells, as manifested by a decrease in B-cell numbers in the spleen, germinal center disappearance, and less efficiency to proliferate in hybridoma fusion. CONCLUSIONS: These novel results demonstrate unequivocally that lacto-neolacto series gangliosides are essential to multiple physiological functions, especially the control of reproductive output, and spleen B-cell abnormality. We also report the generation of anti-IgG response against the lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1.Item Open Access Mutant IDH1 is required for IDH1 mutated tumor cell growth.(Oncotarget, 2012-08) Jin, Genglin; Pirozzi, Christopher J; Chen, Lee H; Lopez, Giselle Y; Duncan, Christopher G; Feng, Jie; Spasojevic, Ivan; Bigner, Darell D; He, Yiping; Yan, HaiFrequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target.Item Open Access Mutations in IDH1, IDH2, and in the TERT promoter define clinically distinct subgroups of adult malignant gliomas.(Oncotarget, 2014-03-30) Killela, Patrick J; Pirozzi, Christopher J; Healy, Patrick; Reitman, Zachary J; Lipp, Eric; Rasheed, B Ahmed; Yang, Rui; Diplas, Bill H; Wang, Zhaohui; Greer, Paula K; Zhu, Huishan; Wang, Catherine Y; Carpenter, Austin B; Friedman, Henry; Friedman, Allan H; Keir, Stephen T; He, Jie; He, Yiping; McLendon, Roger E; Herndon, James E; Yan, Hai; Bigner, Darell DFrequent mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and the promoter of telomerase reverse transcriptase (TERT) represent two significant discoveries in glioma genomics. Understanding the degree to which these two mutations co-occur or occur exclusively of one another in glioma subtypes presents a unique opportunity to guide glioma classification and prognosis. We analyzed the relationship between overall survival (OS) and the presence of IDH1/2 and TERT promoter mutations in a panel of 473 adult gliomas. We hypothesized and show that genetic signatures capable of distinguishing among several types of gliomas could be established providing clinically relevant information that can serve as an adjunct to histopathological diagnosis. We found that mutations in the TERT promoter occurred in 74.2% of glioblastomas (GBM), but occurred in a minority of Grade II-III astrocytomas (18.2%). In contrast, IDH1/2 mutations were observed in 78.4% of Grade II-III astrocytomas, but were uncommon in primary GBM. In oligodendrogliomas, TERT promoter and IDH1/2 mutations co-occurred in 79% of cases. Patients whose Grade III-IV gliomas exhibit TERT promoter mutations alone predominately have primary GBMs associated with poor median OS (11.5 months). Patients whose Grade III-IV gliomas exhibit IDH1/2 mutations alone predominately have astrocytic morphologies and exhibit a median OS of 57 months while patients whose tumors exhibit both TERT promoter and IDH1/2 mutations predominately exhibit oligodendroglial morphologies and exhibit median OS of 125 months. Analyzing gliomas based on their genetic signatures allows for the stratification of these patients into distinct cohorts, with unique prognosis and survival.Item Open Access Myeloablative temozolomide enhances CD8⁺ T-cell responses to vaccine and is required for efficacy against brain tumors in mice.(PLoS One, 2013) Sanchez-Perez, Luis A; Choi, Bryan D; Archer, Gary E; Cui, Xiuyu; Flores, Catherine; Johnson, Laura A; Schmittling, Robert J; Snyder, David; Herndon, James E; Bigner, Darell D; Mitchell, Duane A; Sampson, John HTemozolomide (TMZ) is an alkylating agent shown to prolong survival in patients with high grade glioma and is routinely used to treat melanoma brain metastases. A prominent side effect of TMZ is induction of profound lymphopenia, which some suggest may be incompatible with immunotherapy. Conversely, it has been proposed that recovery from chemotherapy-induced lymphopenia may actually be exploited to potentiate T-cell responses. Here, we report the first demonstration of TMZ as an immune host-conditioning regimen in an experimental model of brain tumor and examine its impact on antitumor efficacy of a well-characterized peptide vaccine. Our results show that high-dose, myeloablative (MA) TMZ resulted in markedly reduced CD4(+), CD8(+) T-cell and CD4(+)Foxp3(+) TReg counts. Adoptive transfer of naïve CD8(+) T cells and vaccination in this setting led to an approximately 70-fold expansion of antigen-specific CD8(+) T cells over controls. Ex vivo analysis of effector functions revealed significantly enhanced levels of pro-inflammatory cytokine secretion from mice receiving MA TMZ when compared to those treated with a lower lymphodepletive, non-myeloablative (NMA) dose. Importantly, MA TMZ, but not NMA TMZ was uniquely associated with an elevation of endogenous IL-2 serum levels, which we also show was required for optimal T-cell expansion. Accordingly, in a murine model of established intracerebral tumor, vaccination-induced immunity in the setting of MA TMZ-but not lymphodepletive, NMA TMZ-led to significantly prolonged survival. Overall, these results may be used to leverage the side-effects of a clinically-approved chemotherapy and should be considered in future study design of immune-based treatments for brain tumors.Item Open Access Polio Virotherapy of Malignant Glioma Engages the Tumor Myeloid Infiltrate and Triggers Global Microglia Activation(2022) Yang, YuanfanMalignant glioma formation involves an abundant inflammatory infiltrate dominated by glioma-associated macrophages and microglia (GAMM). GAMM constitutes a large portion of the glioma mass and tumor microenvironment. They are actively involved in tissue repair and immune surveillance, however in the tumor microenvironment (TME), they are subverted to promote tumor progression. The human poliovirus receptor, hCD155h, is constitutively expressed in members of the mononuclear phagocytic system and is upregulated ectopically in the neoplastic compartment of malignant gliomas (and solid cancers in general). Intratumor treatment with the highly attenuated rhino:poliovirus chimera, PVSRIPO, has a dual effect of releasing neoantigens by oncolysis and activating the GAMM component via sublethal infection, leading to a substantial but transient immune therapy effect. In a phase I clinical trial, PVSRIPO treatment resulted in 21% long-term survival with durable radiographic responses in patients with recurrent glioblastoma (Desjardins et al. New England Journal of Medicine, 2018). Therefore, studying the mechanisms of PVSRIPO immunotherapy in mouse brain tumor models to decipher contributions of viral infection to GAMM vs. malignant cells is critical to improving the therapeutic efficacy in ongoing clinical trials. We recapitulated the clinical trial scenario in an immunocompetent intracerebral mouse tumor model (CT2A-CD155) and obtained baseline and post treatment brain in a time series. Histopathology studies, combined with detailed multiplex IHC/IF and RNAseq were performed on tumor bearing brains. We found the PVSRIPO therapy induced intense engagement of the GAMM infiltrate accompanied by substantial, but transient tumor regression. There were extensive microglia activation and proliferation in adjacent brain parenchyma and even part of the contralateral cortex. This occurred against a backdrop of sustained innate antiviral inflammation and is associated with an induction of the PD-L1 immune checkpoint on GAMM. In contrast to transient antitumor effects observed after PVSRIPO monotherapy, combining PVSRIPO with PD1/PD-L1 blockade led to durable remission. Our work implicates GAMM as active drivers of inflammation and reveals broad neuroinflammatory activation of the CNS-resident myeloid compartment upon polio virotherapy of malignant glioma.
Item Open Access Prognostic value of medulloblastoma extent of resection after accounting for molecular subgroup: a retrospective integrated clinical and molecular analysis.(The Lancet. Oncology, 2016-04) Thompson, Eric M; Hielscher, Thomas; Bouffet, Eric; Remke, Marc; Luu, Betty; Gururangan, Sridharan; McLendon, Roger E; Bigner, Darell D; Lipp, Eric S; Perreault, Sebastien; Cho, Yoon-Jae; Grant, Gerald; Kim, Seung-Ki; Lee, Ji Yeoun; Rao, Amulya A Nageswara; Giannini, Caterina; Li, Kay Ka Wai; Ng, Ho-Keung; Yao, Yu; Kumabe, Toshihiro; Tominaga, Teiji; Grajkowska, Wieslawa A; Perek-Polnik, Marta; Low, David CY; Seow, Wan Tew; Chang, Kenneth TE; Mora, Jaume; Pollack, Ian F; Hamilton, Ronald L; Leary, Sarah; Moore, Andrew S; Ingram, Wendy J; Hallahan, Andrew R; Jouvet, Anne; Fèvre-Montange, Michelle; Vasiljevic, Alexandre; Faure-Conter, Cecile; Shofuda, Tomoko; Kagawa, Naoki; Hashimoto, Naoya; Jabado, Nada; Weil, Alexander G; Gayden, Tenzin; Wataya, Takafumi; Shalaby, Tarek; Grotzer, Michael; Zitterbart, Karel; Sterba, Jaroslav; Kren, Leos; Hortobágyi, Tibor; Klekner, Almos; László, Bognár; Pócza, Tímea; Hauser, Peter; Schüller, Ulrich; Jung, Shin; Jang, Woo-Youl; French, Pim J; Kros, Johan M; van Veelen, Marie-Lise C; Massimi, Luca; Leonard, Jeffrey R; Rubin, Joshua B; Vibhakar, Rajeev; Chambless, Lola B; Cooper, Michael K; Thompson, Reid C; Faria, Claudia C; Carvalho, Alice; Nunes, Sofia; Pimentel, José; Fan, Xing; Muraszko, Karin M; López-Aguilar, Enrique; Lyden, David; Garzia, Livia; Shih, David JH; Kijima, Noriyuki; Schneider, Christian; Adamski, Jennifer; Northcott, Paul A; Kool, Marcel; Jones, David TW; Chan, Jennifer A; Nikolic, Ana; Garre, Maria Luisa; Van Meir, Erwin G; Osuka, Satoru; Olson, Jeffrey J; Jahangiri, Arman; Castro, Brandyn A; Gupta, Nalin; Weiss, William A; Moxon-Emre, Iska; Mabbott, Donald J; Lassaletta, Alvaro; Hawkins, Cynthia E; Tabori, Uri; Drake, James; Kulkarni, Abhaya; Dirks, Peter; Rutka, James T; Korshunov, Andrey; Pfister, Stefan M; Packer, Roger J; Ramaswamy, Vijay; Taylor, Michael DBackground
Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner.Methods
We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (<1·5 cm(2) tumour remaining), or sub-total resection (≥1·5 cm(2) tumour remaining). We did multivariable analyses of overall survival and progression-free survival using the variables molecular subgroup (WNT, SHH, group 4, and group 3), age (<3 vs ≥3 years old), metastatic status (metastases vs no metastases), geographical location of therapy (North America/Australia vs rest of the world), receipt of chemotherapy (yes vs no) and receipt of craniospinal irradiation (<30 Gy or >30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival.Findings
We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084).Interpretation
The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection.Funding
Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.Item Open Access Sym004-induced EGFR elimination is associated with profound anti-tumor activity in EGFRvIII patient-derived glioblastoma models.(Journal of neuro-oncology, 2018-07) Keir, Stephen T; Chandramohan, Vidyalakshmi; Hemphill, Carlee D; Grandal, Michael M; Melander, Maria Carlsen; Pedersen, Mikkel W; Horak, Ivan D; Kragh, Michael; Desjardins, Annick; Friedman, Henry S; Bigner, Darell DBackground
Sym004 is a mixture of two monoclonal antibodies (mAbs), futuximab and modotuximab, targeting non-overlapping epitopes on the epidermal growth factor receptor (EGFR). Previous studies have shown that Sym004 is more efficient at inducing internalization and degradation of EGFR than individual components, which translates into superior cancer cell inhibition. We investigated whether Sym004 induces removal of EGFRvIII and if this removal translates into tumor growth inhibition in hard-to-treat glioblastomas (GBMs) harboring the mutated, constitutively active EGFR variant III (EGFRvIII).Methods
To address this question, we tested the effect of Sym004 versus cetuximab in eight patient-derived GBM xenograft models expressing either wild-type EGFR (EGFRwt) and/or mutant EGFRvIII. All models were tested as both subcutaneous and orthotopic intracranial xenograft models.Results
In vitro studies demonstrated that Sym004 internalized and removed EGFRvIII more efficiently than mAbs, futuximab, modotuximab, and cetuximab. Removal of EGFRvIII by Sym004 translated into significant in vivo anti-tumor activity in all six EGFRvIII xenograft models. Furthermore, the anti-tumor activity of Sym004 in vivo was superior to that of its individual components, futuximab and modotuximab, suggesting a clear synergistic effect of the mAbs in the mixture.Conclusion
These results demonstrate the broad activity of Sym004 in patient-derived EGFRvIII-expressing GBM xenograft models and provide a clear rationale for clinical evaluation of Sym004 in EGFRvIII-positive adult GBM patients.