Wide-spread application of chimeric antigen receptor (CAR) T cell therapy for cancer is limited by the current use of autologous CAR T cells necessitating the manufacture of individualized ...therapeutic products for each patient. To address this challenge, we have generated an off-the-shelf, allogeneic CAR T cell product for the treatment of glioblastoma (GBM), and present here the feasibility, safety, and therapeutic potential of this approach.
We generated for clinical use a healthy-donor derived IL13Rα2-targeted CAR+ (IL13-zetakine+) cytolytic T-lymphocyte (CTL) product genetically engineered using zinc finger nucleases (ZFNs) to permanently disrupt the glucocorticoid receptor (GR) (GRm13Z40-2) and endow resistance to glucocorticoid treatment. In a phase I safety and feasibility trial we evaluated these allogeneic GRm13Z40-2 T cells in combination with intracranial administration of recombinant human IL-2 (rhIL-2; aldesleukin) in six patients with unresectable recurrent GBM that were maintained on systemic dexamethasone (4-12 mg/day).
The GRm13Z40-2 product displayed dexamethasone-resistant effector activity without evidence for in vitro alloreactivity. Intracranial administration of GRm13Z40-2 in four doses of 108 cells over a two-week period with aldesleukin (9 infusions ranging from 2500-5000 IU) was well tolerated, with indications of transient tumor reduction and/or tumor necrosis at the site of T cell infusion in four of the six treated research subjects. Antibody reactivity against GRm13Z40-2 cells was detected in the serum of only one of the four tested subjects.
This first-in-human experience establishes a foundation for future adoptive therapy studies using off-the-shelf, zinc-finger modified, and/or glucocorticoid resistant CAR T cells.
Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we ...report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 10
CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .
Glioblastomas have been proposed to be maintained by highly tumorigenic glioblastoma stem cells (GSCs) that are resistant to current therapy. Therefore, targeting GSCs is critical for developing ...effective therapies for glioblastoma. In this study, we identify the regulatory cascade of the nuclear receptor TLX and the DNA hydroxylase Ten eleven translocation 3 (TET3) as a target for human GSCs. We show that knockdown of TLX expression inhibits human GSC tumorigenicity in mice. Treatment of human GSC-grafted mice with viral vector-delivered TLX shRNA or nanovector-delivered TLX siRNA inhibits tumour development and prolongs survival. Moreover, we identify TET3 as a potent tumour suppressor downstream of TLX to regulate the growth and self-renewal in GSCs. This study identifies the TLX-TET3 axis as a potential therapeutic target for glioblastoma.
Single agent PD-1 inhibitors have yielded durable responses in a minority of gastroesophageal cancers. Radiation therapy has been recognized to promote antitumor immune responses and may synergize ...with anti-PD-1 agents. We sought to evaluate if combining palliative radiation therapy with pembrolizumab can augment antitumor immune responses in gastroesophageal cancer.
Patients had metastatic gastroesophageal cancer with indication for palliative radiation therapy with ≥2 disease sites outside of the radiation field assessable for abscopal response and biopsies for laboratory correlative analyses. Palliative radiation was delivered to a dose of 30 Gy over 10 fractions. Pembrolizumab, 200 mg, was administered concurrently intravenously every 3 weeks until disease progression, unacceptable toxicity, or study withdrawal, for up to 2 years. Endpoints included PD-L1 expression in pre- and posttreatment biopsies and abscopal objective response rate per Response Evaluation Criteria in Solid Tumors.
Of 14 enrolled patients, the objective response rate was 28.6% (95% confidence interval, 8.4%-58.1%), and the median duration of response was not reached (95% confidence interval, 6.9-NR months). Overall, 2 patients had treatment-related grade 3 to 4 adverse events with no grade 5 events. One patient discontinued therapy due to grade 4 colitis. We did not observe an association between radiation and abscopal changes in PD-L1 expression via assessment of an analogous PD-L1 Combined Positive Score, Tumor Proportion Score, Mononuclear Immune Cell Density Score, or proportion of PD-L1-expressing immune cells between pre- and posttreatment tumor biopsies.
Combining palliative radiation therapy and pembrolizumab provided promising durable responses in this patient population but we were unable to definitively distinguish abscopal biologic changes. Biomarker analyses beyond PD-L1 expression are needed to better understand putative mechanisms and identify patients who will benefit from this approach.
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 study reported here explored the design and realization of a low-cost, electromyographically controlled hand prosthesis for amputees living in developing countries. The developed prosthesis is ...composed of a light aluminum structure with opposing fingers connected to a DC motor that imparts only the movement of grasp. Problems associated with surface electromyographic signal acquisition and processing, motor control, and evaluation of grasp force were addressed, with the goal of minimizing cost and ensuring easy assembly. Simple analog front ends amplify and condition the electromyographic signals registered from two antagonist muscles by surface electrodes. Analog signals are sampled at 1 kHz and processed by a microcontroller that drives the motor with a supply voltage proportional to the muscular contraction, performing the opening and closing of the opposing fingers. Reliable measurements of the level of muscle contractions were obtained by specific digital processing: real-time operators implementing the root mean square value, mean absolute value, standard deviation, and mean absolute differential value were compared in terms of efficiency to estimate the EMG envelope, computational load, and time delay. The mean absolute value operator was adopted at a time window of 64 milliseconds. A suitable calibration procedure was proposed to overcome problems associated with the wide variation of electromyograph amplitude and background noise depending on the specific patient's muscles selected. A pulse-width modulated signal drives the DC motor, allowing closing and opening of the prosthesis. The relationship between the motor-driver signal and the actual hand-grasp force developed by the prosthesis was measured using a hand-held grip dynamometer. The resulting force was proportional to current for moderate values of current and then saturated. The motor torque, and, in turn, the force elicited, can be measured by sensing the current absorbed by the motor. Therefore, the grasp force can be opportunely limited or controlled. The cost of the only electronic and mechanical components of the electromyographically controlled hand was about US$50; other costs, such as the cost of labor to assemble the prosthesis and the production of adapters for patients, were not estimated.
Endometrial cancer is the most common gynecological cancer in Western industrialized countries. Cables, a cyclin-dependent kinase binding protein, plays a role in proliferation and/or ...differentiation. Cables mutant mice are viable, but develop endometrial hyperplasia and carcinoma in situ at a young age. Exposure to chronic low levels of estrogen results in development of endometrial cancer, similar to that observed in the postmenopausal female. In vitro and in vivo studies demonstrate that levels of Cables mRNA in benign human endometrial epithelium are up-regulated by progesterone and down-regulated by estrogen. Furthermore, nuclear immunostaining for Cables is lost in a high percentage of cases of human endometrial hyperplasia and adenocarcinoma, which are likely the product of unopposed estrogen. The loss of Cables immunostaining in the human endometrial cancer samples correlates with a marked decrease in Cables mRNA. Ectopic expression of Cables in human endometrial cells dramatically slows cell proliferation. Collectively, these data provide evidence that Cables is hormonally regulated and is involved in regulating endometrial cell proliferation. In addition, loss or suppression of Cables may be an early step in the development of endometrial cancer.
Early events involved in the pathogenesis of colorectal cancer include mutations in the Adenomatous Polyposis Coli tumor-suppressor gene and oncogenic KRAS mutations. Later events include deletions ...on chromosome 18q, which are observed in a high proportion of colorectal cancers. However, the important tumor suppressor genes targeted by these deletions have not been fully defined. A previous study found Cables is located on human chromosome 18q11-12. Loss of Cables expression as determined by immunohistochemical staining (IHC) occurred in 60-70% of sporadic colorectal cancers that were usually correlated to loss of heterozygosity at 18q. To determine if Cables is an important target for the chromosome 18q deletions, the susceptibility of Cables-/- mice to develop colon tumors was studied. A well characterized colonic carcinogen, 1,2-dimethylhydrazine (DMH) was used as a tumor initiator. Cables-/- mice (n = 25) and the Cables+/+ littermates (n = 25) were treated with subcutaneous DMH injections over 20 weeks to initiate tumorigenesis. The median survival after DMH injections was significantly shorter for the Cables-/- mice compared to Cables+/+ littermates. The total number of colorectal tumors that developed in the Cables-/- mice was 46 tumors versus 21 tumors. The increased numbers of colorectal tumors, as well as shorter survival of the Cables-/- mice provides compelling evidence that Cables could play an important role in the pathogenesis and progression of colon cancer in mice. These data coupled with previous observations support the hypothesis that Cables is a relevant target of the chromosome 18q deletions frequently seen in human colorectal cancer.
A methodology is presented for developing a digital signal-processing architecture capable of simultaneous and automated detection and classification of transient signals. The basic unit of the ...aforementioned architecture is the wavelet network, which combines the ability of the wavelet transform of analyzing nonstationary signals with the classification capability of artificial neural networks. By exploiting the modularity as well as original strategies concerning wavelet network implementation and training, the method succeeds in enhancing the classification performance with respect to other available solutions.
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