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Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. We hypothesized that BiTE-secreting T cells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent T cells compared with bivalent CAR T cells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

In the nuclear reactor, flowing state of surrounding fluid will change structural vibration characteristics of fuel rods, which may lead to severe resonance and damage of fuel rods. Therefore, it is necessary to study structural vibration characteristics of fuel rods in different fluid flow states. In this paper, based on experiment, structural vibration characteristics of a single rod in different water flow states and different restraint modes are obtained by hammering method. The results show that different restraint modes have influence on dry mode, wet mode and structural vibration characteristics of the rod. Under condition of no excitation, when flow rate is lower than 1m/s, the response frequency and flow velocity change trend is the same under different restraint modes, response frequency of test rod with increases of flow rate. When flow rate is more than 1m/s, the response frequency varies with flow rate in different restraint modes. Under the fixed support, the excitation ways and excitation positions have no effect on natural frequency of rod. For simply supported, the excitation positions have great influence on the natural frequency of rod. It has reference significance for the safety design of fuel rods.

When a rock mass shears along a structural plane, the shear resistance of the structural plane is affected by the structural plane undulations and by the friction between the contact regions. During an earthquake, the seismic load (composed of the cyclic and dynamic loads) produces a dynamic deterioration of the mechanical properties of the structural plane, which is mainly reflected as follows: (1) under cyclic shear of the seismic load, the undulant angle αk decreases. (2) Under the dynamic load, the frictional coefficient of the structural surface is reduced. The dilatancy angle is generally used instead of the undulant angle. When calculating rock slope stability, the frictional angle is equivalent to the sum of the basic frictional angle and the undulant angle. In this study, the equations for calculating the dilatancy angle of a structural plane under cyclic shear loading are determined based on cyclic shear tests of a split structural plane. The basic frictional angle for calculating the cyclic shear is also determined, based on previous research. Furthermore, according to the dynamic model of a rock slope, a method to calculate the permanent displacement of the rock slope is proposed, considering the effect of structural plane deterioration. We found that the effect of structural plane deterioration under a seismic load directly affects the stability and permanent displacement of the rock slope. The feasibility and engineering practicalities of this method are also verified by comparing the proposed method with previously developed methods.

Introduction: Hypotension is one of the most common complications during perioperative obstetric surgery and can cause serious adverse reactions in mothers and infants. Phenylephrine and ephedrine are the most widely used vasoconstrictors for preventing and controlling hypotension. However, which of them is the best choice remains unclear due to the lack of solid data on the effects of phenylephrine and ephedrine on uterine placental blood flow. This study aims to investigate the vasoconstriction effect of phenylephrine and ephedrine on the uterine arteries of pregnant rabbits via computed tomography (CT) imaging. Methods: Fifteen near-term pregnant rabbits at 27–29 days of pregnancy were randomly divided into Group P, Group E and Group C. Phenylephrine, ephedrine and normal saline were injected. CT imaging was performed, and the diameters of the uterine arteries were measured to evaluate the contractile response to different drugs. Results: Both phenylephrine and ephedrine induced a similar degree of vasoconstriction on peripheral resistance of the vessels (rostral arteries) (p > 0.05). At 5 min and 10 min, the uterine arteries contracted more strongly in response to phenylephrine than ephedrine (p p > 0.05). Conclusion: In summary, compared with ephedrine, phenylephrine had a faster effect, and resulted in stronger contraction of the uterine arteries, indicating a greater impact on placental perfusion. Therefore, in clinical practice, ephedrine has more favorable effects than phenylephrine on uterine and placental circulation.

Chimeric antigen receptor (CAR)-T cells have had a significant effect in hematological diseases, with a recent study confirming that the cells can last for ten years in vivo. Unfortunately, to date, CAR-T cells have had little effect in solid tumors. One cause of the treatment difficulty is antigenic heterogeneity. Our experience with CAR-T cells in glioblastoma suggests that addressing antigenic heterogeneity may improve the clinical efficacy. Targeting the epidermal growth factor receptor (EGFR) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) individually could lead to tumor control in the short term, but it will be marked by tumor recurrence through antigen loss or downregulation in the long term. This resistance mechanism can be inhibited by combinatorial targeting. To this end, we have designed a bicistronic construct that targets both EGFR and IL13Rα2 simultaneously, to decrease the potential of antigen escape and tumor recurrence. The bicistronic CAR-T cells employ an “OR” logic gate to modify the T cells and kill the tumor cells in vitro and in vivo, in order to retain effectiveness when one target antigen is lost but the second is still present. Our experimental results demonstrate that and validate the safety and possibility of clinical translation. In addition, the efficiency of transfecting T cells with the single bicistronic vector was higher than two single vectors on monovalent CARs. In summary, the bicistronic CAR-T structure is a promising strategy to address the clinical problem of targeting antigenic heterogeneity in solid tumors and potentially level the barriers for targeted T-cell therapy. Educational Objectives: After completion, participants will be able to explain the importance of antigenic heterogeneity in solid tumors in the context of tumor escape. Participants will also be able to identify a strategy to reducing the problem of antigen escape and tumor recurrence through the implementation of bicistronic CARs.

Wearable electrocardiogram devices are being developed rapidly as they can be used to monitor the user’s health in the long term. Their development is dependent on advances in automatic processing algorithms. Given that its intended use requires the wearable device to collect a large amount of data, and in light of the context where its hardware is limited by cost and power consumption, this paper proposes an algorithmic framework that can automatically identify position premature ventricular contraction (PVC) and supraventricular premature beat (SPB) at low computational cost by using 24-hour dynamic single-lead ECG recordings. We first use convolutional neural networks that require a small number of calculations to find problematic segments in the signal, and then use bidirectional long short-term memory networks to accurately locate the PVC and SPB. In addition to using the second-level structure that reduces the total amount of computation required by the task to 1/3.78, the algorithm has adopted focal loss to ensure the accuracy of classification of the disease samples. The algorithm was used in the Third China Physiological Signal Challenge (CPSC) 2020 and came in 10th place, which verified its effectiveness. We trained the model once again by using the data provided at CPSC 2020, and obtained the F1 scores of PVC and SPB were 0.6698 and 0.6111.

Glioblastoma is the most common and lethal brain cancer globally. Clinically, this cancer has heterogenous molecular and clinical characteristics. Studies have shown that UBE2S is highly expressed in many cancers. But its expression profile in glioma, and the correlation with clinical outcomes is unknown. RNA sequencing data of glioma samples was downloaded from the Chinese Glioma Genome Atlas and The Cancer Genome Atlas. A total of 114 cases of glioma tissue samples (WHO grades II-IV) were used to conduct protein expression assays. The molecular and biological characteristics of UBE2S, and its prognostic value were analyzed. The results showed that high UBE2S expression was associated with a higher grade of glioma and PTEN mutations. In addition, UBE2S affected the degree of malignancy of glioma and the development of chemo-radiotherapy resistance. It was also found to be an independent predictor of worse survival of LGG patients. Furthermore, we identified five UBE2S ubiquitination sites and found that UBE2S was associated with Akt phosphorylation in malignant glioblastoma. The results also revealed that UBE2S expression was negatively correlated with 1p19q loss and IDH1 mutation; positively correlated with epidermal growth factor receptor amplification and PTEN mutation. This study demonstrates that UBE2S expression strongly correlates with glioma malignancy and resistance to chemo-radiotherapy. It is also a crucial biomarker of poor prognosis.

Tumor heterogeneity is one of the key reasons for therapeutic failure in Glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell clinical trial (NCT02209376) against Epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood brain barrier into GBM active tumor sites. However, CART cell infiltration was associated with a selective loss of EGFRvIII+ tumor cells and upregulation of immunosuppressive molecules. Post-CAR T treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations. To study this further We generated two structurally different CARs by fusing the scFv of mAb806 to 4-1BB and Killer immunoglobulin like receptor (KIR) co-stimulatory domains. Both 4-1BB and KIR based EGFR806 CAR T cells specifically lysed tumor cells and secreted cytokines when co-cultured with U87-MG (expressing low levels of EGFR) and U87-MG EGFR (U87-MG transduced with EGFR wild type for amplified background) cell lines and engineered to ectopically express targeted EGFR-ECD missense mutations and EGFRvIII. Unlike EGFR-specific cetuximab based CAR, EGFR-806CART cells did not kill EGFR wild type expressing fetal brain primary astrocytes and keratinocytes in vitro. We further evaluated the in vivo efficacy of 806-CARs and EGFRvIII CART currently in clinic in NSG mice. While EGFRvIII CART cells cleared EGFRvIII+ tumors alone 806 CART cells controlled EGFRvIII, amplified EGFR and EGFRR108K-mutant bearing tumors. KIR-CAR treated tumors are GVHD free and enhanced survival compared to 4-1BB based CARs. The broad specificity of EGFR806 CART cells to amplified EGFR and its mutant variants gives us the potential to clear various forms of EGFR. The enhanced anti-tumor efficacy by KIR based CAR in vivo setting provides us with additional therapeutic options.

Cells wear fast in NAND flash memory of high storage density (HSD), so it is very necessary to have a long-term frequent in-time monitoring on its raw bit error rate (RBER) changes through a fast RBER estimation method. As the flash of HSD already has relatively lower reading speed, the method should not further degrade its read performance. This paper proposes an improved estimation method utilizing known data comparison, includes interleaving to balance the uneven error distribution in the flash of HSD, a fast RBER estimation module to make the estimated RBER highly linearly correlated with the actual RBER, and enhancement strategies to accelerate the decoding convergence of low-density parity-check (LDPC) codes and thereby make up the rate penalty caused by the known data. Experimental results show that when RBER is close to the upper bound of LDPC code, the reading efficiency can be increased by 35.8% compared to the case of no rate penalty. The proposed method only occupies 0.039mm2 at 40nm process condition. Hence, the fast, read-performance-improving, and low-cost method is of great application potential on RBER monitoring in the flash of HSD.

Chimeric antigen receptor T cells (CAR T cells) therapy is one kind of immunotherapy that has revolutionally changed the landscape of immunotherapy and been approved by the FDA from 2017 for several blood malignancies. To bring new CAR T cells to clinic, every new CAR need to test in vitro for antigen recognition, tumor cell killing capacity, and off-target cytotoxicity effect. Detecting the secretion of cytokines upon engagement of CAR T cells with tumor antigens is routinely applied to assess these CAR functions. Here we describe coupling of intracellular cytokine staining and multicolor flow cytometry to measure CAR T cells activation upon antigen stimulation.

Chimeric antigen receptor T cells (CAR T cells) therapy is one kind of immunotherapy that has revolutionally changed the landscape of immunotherapy and been approved by the FDA from 2017 for several blood malignancies. To bring new CAR T cells to clinic, every new CAR need to test in vitro for antigen recognition, tumor cell killing capacity, and off-target cytotoxicity effect. Detecting the secretion of cytokines upon engagement of CAR T cells with tumor antigens is routinely applied to assess these CAR functions. Here we describe coupling of intracellular cytokine staining and multicolor flow cytometry to measure CAR T cells activation upon antigen stimulation.

High grade glioma is the most common malignant primary brain tumor. Despite the best available therapy, the median survival is limited. Chimeric antigen receptor (CAR)-T cells effectively attack target positive tumor cells, demonstrating a promising possible treatment. Based on our previous clinical and pre-clinical studies, we utilized epidermal growth factor receptor variant III (EGFRvIII) and interleukin 13 receptor subunit alpha 2 (IL13Rα2) targeting single-chain variable fragments (scFvs) to generate bi-specific tandem CAR T cells and bi-specific parallel CAR T cells to decrease the potential of antigen escape and tumor recurrence. 5, 10, 15 or 20 flexible amino acids were utilized to link scFvs in tandem CARs. Ribosomal “skipping” 2A peptides were utilized to generate parallel CAR T cells. Flow-based IL13Rα2 staining on glioma stem cells (GSCs) showed 7 out 11 cases positive (64%) heterogeneous intratumoral expression. Tandem CAR T cells and parallel CAR T cells upregulated CD69 expression (P< 0.0001), generated IFNγ, interleukin 2 and TNFα (P< 0.0001), as well as exhibiting cytotoxic activity (P< 0.0001) when compared against un-transduced T cells, in co-culture with either single- or double-target positive GSCs. There was no significant difference between different lengths of linker in tandem CAR T cells. Parallel CAR T cells generated a larger cytokine response (P< 0.0001) and more effectively killed target positive cells (P< 0.05) than tandem CAR T cells. In summary, glioma associated target expression is heterogeneous in GSCs. Multivalent tandem CAR T cells and parallel CAR T cells effectively respond to target expressing tumor cells. The function of tandem CAR T cells is not relative with the length of linker. Parallel CAR T cells are a more promising strategy in generating gene modified multivalent targeting T cells.

Heart sounds play an important role in the initial screening of heart diseases. However, the accurate diagnosis with heart sound signals requires doctors to have many years of clinical experience and relevant professional knowledge. In this study, we proposed an end-to-end lightweight neural network model that does not require heart sound segmentation and has very few parameters. We segmented the original heart sound signal and performed a short-time Fourier transform (STFT) to obtain the frequency domain features. These features were sent to the improved two-dimensional convolutional neural network (CNN) model for features learning and classification. Considering the imbalance of positive and negative samples, we introduced FocalLoss as the loss function, verified our network model with multiple random verifications, and, hence, obtained a better classification result. Our main purpose is to design a lightweight network structure that is easy for hardware implementation. Compared with the results of the latest literature, our model only uses 4.29 K parameters, which is 1/10 of the size of the state-of-the-art work.

The mechanical behavior of rock masses with different combined structural planes is poorly understood because of the difficulties associated with the performance of rock observations and analyses from the surface, collection of samples during field investigations, specimen preparation, and laboratory tests. The combination of rock mass structural planes has a significant effect on the rock mass mechanical properties. The three-dimensional (3D) printing technology can conveniently create 3D entities with complex structures, thereby providing an unprecedented opportunity to break through this bottleneck. In this study, model samples of structural surface models with different combinations are printed using the 3D printing technology, and a cement slurry with 3:1 water–cement ratio is used as the structural body to produce rock masses with different combined structural planes. The method replicates well the morphological features of natural joints. Moreover, the shear properties and the failure characteristics are stable and consistent. Direct shear tests are also performed on the rock masses of different combined structural planes from the intersection line parallel and perpendicular to the structural planes to obtain the relevant mechanical parameters and rock mass failure modes. The results prove the feasibility of the 3D printing technology in the experimental study of rock mass mechanics and show that the strength and failure modes of rock masses with different combined structural planes are different. A calculation method for the shear strength of rock masses with different combined structural planes is proposed herein based on the experimental data and the Mohr–Coulomb theory, and the method’s feasibility is verified by several examples.

Introduction: Glioblastoma (GBM) is malignant and aggressive brain tumor with a median overall survival of 14.6 months, despite the development of new treatments. Chimeric Antigen Receptor (CAR) modified T cells are powerful new class of immune based therapies that have shown a high rate of long term durable response in CD19 expressing B-cell malignancies. However, this success has yet to be translated to solid tumors, in part due to a lack of surface antigens specifically expressed on tumor cells while not present on normal tissue and an immunosuppressive tumor microenvironment. Approximately 60% of GBMs contain a mutation, rearrangement, splicing alteration, and/or amplification of EGFR. Investigation of 411 GBM patient samples from University of Pennsylvania identified EGFR amplification in 38% of cases and EGFRvIII in 25% of cases and most common oncogenic missense mutations in extracellular domain (ECD) A289D/T/V, R108G/K, and G598V in 12-13% of cases. ECD missense mutations are associated with increased invasiveness aided by ligand independent activation of receptor and decreased patient survival compared to patients that are wild type EGFR at R108, A289 and G59V locus. Methods: We generated two structurally different CARs by fusing the scFv of mAb806 to 4-1BB and Killer immunoglobulin like receptor (KIR) co-stimulatory domains. mAb806 recognizes a conformationally exposed epitope of wild-type EGFR when it is overexpressed on tumor cells or in the presence of oncogenic mutations such as EGFRvIII. We tested in vitro cytolytic activity of 806-EGFR-CART cells against U87-MG (expressing low levels of EGFR) and U87-MG EGFR (U87-MG parental cell line transduced with EGFR wild type for amplified background) cell lines. These lines were engineered to ectopically express targeted EGFR-ECD missense mutations and EGFRvIII. Results: Both 4-1BB and KIR based EGFR806 CAR T cells specifically lysed tumor cells, proliferated, and secreted cytokines when co-cultured with target-expressing cell lines. To test exclusive specificity of EGFR806 CART to targeted mutations, K562 cells were transduced with EGFR-ECD mutations and co-cultured with 4-1BB and KIR CART cells. While the EGFR806 lysed K562 cells expressing wild type EGFR, EGFR-ECD Mutants, and EGFRvIII, it did not demonstrate any activity against K562-Parental cells. Unlike EGFR-specific cetuximab based CAR, EGFR-806CART cells did not kill EGFR wild type expressing fetal brain astrocytes and keratinocytes in vitro. We further evaluated the in vivo efficacy of CARs in NSG mice by injecting U87-MG, U87-MG EGFR and U87-MG EGFR expressing EGFRvIII subcutaneously and assessed tumor burden and mice survival. Though both 4-1BB and KIR based EGFR806 CART cells control tumor growth, KIR based CAR outperformed 4-1BB based CAR in terms of tumor burden and improved mice survival. Conclusion: Though our CART clinical trial for EGFRvIII positive recurrent GBM patients demonstrated CART therapy is safe and CART cells are able to successfully traffic to regions of active GBM, antigen loss and tumor heterogeneity resulted in limited therapeutic success. Broad specificity of EGFR806 CART cells to Amplified EGFR, EGFRvIII and EGFR-ECD mutants gives us the potential to clear various forms of EGFR. The enhanced anti-tumor efficacy by KIR based CAR in vivo setting provides us with additional therapeutic options for GBM. Citation Format: Radhika Thokala, Zev Binder, Yibo Yin, Michael Milone, Donald M. Orourke. High affinity chimeric antigen receptors with cross reactivity to clinically relevant EGFR mutated proteins [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6596.

The reorganization of brain structures after intracerebral hemorrhage (ICH) insult is crucial to functional outcome. Although the pattern of neuronal rewiring is well-documented after ischemic stroke, the study of brain plasticity after ICH has been focusing on the enhancement of dendritic complexity. Here we hypothesized that functional restoration after ICH involves brain reorganization which may be favorably modulated by stem cell transplantation. In this study, bone marrow stromal cells (BMSCs) were transplanted into the perilesional sites of collagenase-induced ICH in adult rats one day after ICH injury. Forelimb functional recovery was monitored with modified limb placing and vibrissae-elicited forelimb placement tests. Anterograde and retrograde tracing were used to assess the reorganization of bilateral forelimb areas of the sensorimotor cortex. We found that in rats transplanted with BMSCs after ICH injury, axonal sprouting occurred in the contralateral caudal forelimb area of the cortex, and was significantly higher than in ICH rat models that received only the vehicle (P < 0.01). The number of positive neurons in the ipsilateral rostral forelimb area of the cortex of the BMSC group was 1.5– to 4.5-fold greater than in the vehicle group (P < 0.05). No difference was found between the BMSC and vehicle groups in hemispheric atrophy or labeled neurons in the ipsilateral caudal forelimb area (P = 0.193). Scores for improved functional behavior in the BMSC group were in accord with the results from histology. Neuronal plasticity of the denervated corticospinal tract at bilateral forelimb areas of the cortex in the collagenase-induced ICH rat models was significantly enhanced by BMSC transplantation. BMSC transplantation may facilitate functional recovery after ICH injury.

Introduction: Our clinical program in chimeric antigen receptor T (CART) cell therapy has focused on targeting EGFR oncoproteins for glioblastoma (GBM). We have completed a phase I trial using CART cells directed to the EGFRvIII antigen and showed in situ uptake, proliferation, functional activation and antigen editing by CART cells in GBM tissue. This first clinical trial demonstrated two barriers to clinical efficacy. First, GBM heterogeneity was striking spatially and temporally, including the number of variants in EGFR. Second, CART cell therapy was associated with an adaptive anti-GBM response illustrated by an initial wave of CART activation followed by dramatic immunosuppression. These two features indicate that we need strategies to overcome GBM heterogeneity and the immunosuppressive tumor microenvironment (TME). Methods: We have used multiple CART variants, targeting both IL13Rα2 and EGFRvIII, in combination with selected immune checkpoint blockade inhibitors, to explore possible additive effects in both in vitro and in vivo GBM model systems on limiting the immunosuppressive GBM TME. Independently, we have developed a portfolio of CARTs with different binding specificities that target other homo- and heterodimers of EGFR oncoproteins, to address GBM heterogeneity. Results: Combination studies of CART cells with immune checkpoint blockade inhibitors revealed a non-homogenous response. Different CART structures and targets showed increased tumor killing activity with specific immune checkpoint blockade inhibition. Specifically, IL13Rα2-targeting CART cells had the best effect when paired with anti-CTLA4 treatment, while EGFRvIII-targeting CART cells had the largest effect when paired with anti-PD-1 treatment. In parallel to our combination therapy work, we have expanded our repertoire of CART constructs for targeting EGFR oncogenic alleles with extracellular domain mutations by utilizing antibody phage display technology. EGFR-targeting antibody sequences were incorporated into viral constructs and transduced into T cells. We have shown variable killing with these novel CART constructs in GSC/PDX models of GBM heterogeneity. Conclusion: Our initial experience with CART cells in GBM suggested that although a single intravenous infusion results in CART cell bioactivity in the brain, overcoming the adaptive changes in the local TME and addressing antigen heterogeneity may improve the clinical efficacy of CART-directed strategies. Our combination work showed that different CART constructs cooperate with immune checkpoint blockade inhibition differentially. This selective pairing suggests that a more personalized immunophenotypic assessment may result in higher efficacy therapeutic combinations. In addition, development of a broad portfolio of both selective and promiscuous CART constructs targeting EGFR in its various forms gives us the potential to cover a larger percentage of both the GBM tumor volume and regionally-specific tumor cells within a single GBM. Citation Format: Zev A. Binder, Yibo Yin, Radhika Thokala, Donald M. O'Rourke. Combinatorial platform for CART cell therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-340.

Some potential danger, such as robbery, steal, and drug-dealing activity, can be avoided effectively by detecting loitering behavior in advance. In this paper, we propose a loitering detection algorithm by analyzing the trajectory of pedestrian. Firstly, we propose a trajectory extraction method combined with motion estimation and object detection to adapt to the tracking under a static camera scene. Then we judge the loitering behavior by analyzing the trajectory of object through the duration time and angles variation between the initial point and remaining points on the trajectory. The experiments were performed using several surveillance cameras. And experimental results show that the proposed method provides superior performance in terms of accuracy, robustness, and speed.

152 Background: Glioblastoma (GBM) is a deadly cancer, with a 5-year survival rate of less than 10%. T cells can be redirected to kill cancer cells using chimeric antigen receptors (CAR), a promising method to treat solid tumors. IL13Rα2 is expressed in many solid tumors but not normal tissues, and provides a target for CAR T cells as a powerful immunotherapy treatment. Methods: Human and canine tumors were screened for IL13Rα2 expression by flow cytometry and RT-PCR. Different IL13Rα2 single-chain variable fragments (scFv) were tested in CARs in vitro against human and canine recombinant IL13Rα2 protein to identify a cross-reactive clone. IL13Rα2 CARs expressed in human and canine T cells and showed antigen-specific stimulation by cytokine secretion and target cell lysis. Solid tumors were used to establish in vivo subcutaneous and orthotopic xenograft models in NSG mice. Mice received a single treatment of 2-5 million CAR T cells IV with or without anti PD-1, CTLA-4 or TIM3 mAb checkpoint blockade. Results: IL13Rα2 was detected on three human GBM (D270, U251 and U87), four canine osteosarcomas (BW-, CS-, MC- and SK-KOSA) and two canine lung cancer cell lines (Cacal3, Cacal5). Two different IL13Rα2 CAR were generated that recognized human IL13Rα2 and not IL13Rα1, one also recognized canine IL13Rα2. Five million IL13Rα2 CAR T-cells delivered IV were able to eliminate established GBM tumors. Use of fewer CAR T cells initially controlled tumor growth, followed by tumor outgrowth correlated with expression of T cell exhaustion markers. Combined treatment with CARs plus checkpoint blockade mAb delivered IP had synergistic effects, restoring anergic T-cell function and improving tumor treatment. Conclusions: IL13Rα2 is highly expressed on human and canine tumors, but not normal tissues. IL13Rα2 specific CAR T cells successfully respond to human and canine tumors and treat GBM in a xenograft mouse model, although this effect was transient at lower treatment doses. Addition of checkpoint blockade mAbs to CAR therapy was beneficial for the treatment of solid tumors. We plan to utilize this treatment in a canine spontaneous cancer preclinical model and translate into clinical trials for patients with GBM.

Background aims The molecular mechanisms by which stem cell transplantation improves functional recovery after intracerebral hemorrhage (ICH) are not well understood. Accumulating evidence suggests that microglia cells are activated shortly after ICH and that this activation contributes to secondary ICH-induced brain injury. We studied the effect of human amniotic epithelial stem cells (HAESCs) on microglia activation. Methods To study the effect of HAESCs in vitro, we used thrombin to activate the microglia cells. Twenty-four hours after thrombin treatment, the levels of tumor necrosis factor-α and interleukin-1β were measured by enzyme-linked immunosorbent assay. In vivo , the HAESCs were transplanted into the rat striatum 1 day after collagenase-induced ICH. The expression levels of matrix metalloproteinase (MMP)-12 and microglia infiltration in the peri-hematoma tissues were determined 7 days after ICH through the use of reverse transcriptase–polymerase chain reaction and immunohistochemical analysis, respectively. Results Thrombin-activated microglia expression of tumor necrosis factor-α, interleukin-1β and MMP-12 was significantly reduced through contact-dependent and paracrine mechanisms when the HAESCs were co-cultured with microglia cells. After transplantation of HAESCs in rat brains, the expression levels of MMP-12 and microglia infiltration in the peri-hematoma tissues were significantly reduced. Conclusions Our observations suggest that microglia activation could be inhibited by HAESCs both in vitro and in vivo , which may be an important mechanism by which the transplantation of HAESCs reduces brain edema and ameliorates the neurologic deficits after ICH. Therefore, we hypothesize that methods for suppressing the activation of microglia and reducing the inflammatory response can be used for designing effective treatment strategies for ICH.

Identifying factors that contribute to the aggressiveness of glioblastoma (GBM) is crucial for treating this deadly brain tumor. We demonstrate here that CD70, a member of the TNF family, is overexpressed by tumor cells in a subset of patients with low grade glioma and GBM. The elevated gene and protein expression is associated with increased tumor grade and recurrences. CD70 expression on primary GBM is correlated with T cells infiltration and tumor MHC class I expression which could potentially promote antitumor immunity based on our result that T cell infiltration is negatively associated with GBM proliferation. However, no correlation between T cell infiltration and prolonged survival is seen, suggesting that other factors may exist to suppress T cell function. Analysis from 155 primary GBM patients using RNA-seq data culled from TCGA indicates that the gene expression of CD70 is highly correlated with markers (CD4, CD25, CD163 and CD14) commonly expressed on immune suppressive cells such as Tregs and tumor associate macrophages. This result is in line with additional finding that CD70 positive tumors display a predominantly mesenchymal gene expression signature, a subtype of GBM that has greater infiltration by immune cells than other subtypes. In addition, tumors derived from patients with MGMT promoter un-methylation express high levels of CD70. Furthermore, our results suggest that CD70 is a key driver of chemokine mediated attraction for these immune suppressive cells infiltrating the tumor. Lastly, shorter overall survival was observed in these patients who have high CD70 expression compared to those with low CD70 expression on tumors. Taken together, the data suggest that CD70 plays a critical role in chemokine-mediated immune suppression and in GBM progression. Targeting GBM with a drug-conjugated CD70 antibody or CD70-CAR T-cells may improve the therapeutic efficacy for patients with GBM.