Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

The potent oncogene NPM-ALK mediates malignant transformation of normal human CD4(+) T lymphocytes.

With this study we have demonstrated that in vitro transduction of normal human CD4(+) T lymphocytes with NPM-ALK results in their malignant transformation. The transformed cells become immortalized and display morphology and immunophenotype characteristic of patient-derived anaplastic large-cell lymphomas. These unique features, which are strictly dependent on NPM-ALK activity and expression, include perpetual cell growth, proliferation, and survival; activation of the key signal transduction pathways STAT3 and mTORC1; and expression of CD30 (the hallmark of anaplastic large-cell lymphoma) and of immunosuppressive cytokine IL-10 and cell-surface protein PD-L1/CD274. Implantation of NPM-ALK-transformed CD4(+) T lymphocytes into immunodeficient mice resulted in formation of tumors indistinguishable from patients' anaplastic large-cell lymphomas. Our findings demonstrate that the key aspects of human carcinogenesis closely recapitulating the features of the native tumors can be faithfully reproduced in vitro when an appropriate oncogene is used to transform its natural target cells; this in turn points to the fundamental role in malignant cell transformation of potent oncogenes expressed in the relevant target cells. Such transformed cells should permit study of the early stages of carcinogenesis, and in particular the initial oncogene-host cell interactions. This experimental design could also be useful for studies of the effects of early therapeutic intervention and likely also the mechanisms of malignant progression.

Relation of clinical culture method to T-cell memory status and efficacy in xenograft models of adoptive immunotherapy.

BACKGROUND AIMS: Cytotoxic T lymphocytes modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in pre-clinical models, and this efficacy has translated to success in several clinical trials. Many early trials were disappointing in large part because of the lack of proliferation and subsequent persistence of transferred cells. Recent investigations have pointed to the importance of delivering highly proliferative cells, whether of naive or early memory phenotypes. METHODS: We investigated the influence of two common cell culturing methods used in early trials and their relationship to T-cell phenotype and pre-clinical efficacy. RESULTS: We observed that stimulation with soluble anti-CD3 antibody OKT-3 and high-dose interleukin-2 produces more effector memory-type T cells with shorter average telomeres when compared with cells generated with the use of CD3/CD28 beads. When used in xenograft models of leukemia, bead-stimulated cells proliferated earlier and to a higher degree than those generated with the use of OKT-3/IL2 and resulted in better disease control despite no difference in distribution or migration throughout the mouse. Inclusion of the known successful clinical 4-1BB endodomain in the CAR could not rescue the function of OKT-3/IL-2-cultured cells. T cells isolated from animals that survived long-term (>120 days) retained a central memory-like phenotype and demonstrated a memory response to a large re-challenge of CD19-positive leukemia. CONCLUSIONS: In summary, we confirm that cells with a younger phenotype or higher proliferative capacity perform better in pre-clinical models and that cell culturing influences cell phenotype seemingly independent of the 4-1BB endodomain in the CAR structure.

Mining Polyethylene Glycol-Based Thermosensitive Hydrogel Materials: Preparation and Flame Retardant Properties.

A new type of efficient and anti-extinguishing materials to inhibit coal spontaneous combustion is required because of the current situation of the short activity cycle of existing anti-extinguishing technology. Now, polyethylene glycol (PEG) was used as a water-absorbing monomer to polymerize various substances to prepare an AB-type mining thermosensitive hydrogel that was obviously thermoresponsive. The thermosensitive hydrogel, which is low-cost and stable, can be stored for a long time, and it is prepared by compounding A and B components. The orthogonal experiments determined the optimal ratio of component A, while the controlling variable experiments determined the optimal ratio of component B. The thermal stability and flame-retardant properties of the AB-type thermosensitive hydrogel were analyzed during the process of natural oxidation of coal, and the temperature responsiveness of thermosensitive hydrogels was investigated at different temperatures. The results showed that the optimal ratio of polyethylene glycol:methyl cellulose:sodium carboxymethyl cellulose:guar gum of component A was 6:6:1.2:1.5; and the ratio of bentonite:kaolin:Mg(OH)2 of component B was 2:1:1. When the ratio of component A to component B was 1:2, the AB-type thermosensitive hydrogel shows the best flame retardant properties. When this ratio of gel was applied to coal samples, the weight loss was just 6%, and the reduction of CO was as high as 72.6%. The gel, which was convenient for transportation in mining pipelines, had strong fluidity at low temperatures and rapid temperature response. As the temperature rose, a phase transition occurred gradually, and after the phase transition, a high-viscosity solid substance was formed, whose viscosity was approximately 11 times that of the room temperature. It plugged the pores effectively, and in the high-temperature region, the occurred phase transition gathered to extinguish the fire. It is a new type of high-efficiency anti-extinguishing material with excellent properties.

Biochar Decreases Cr Toxicity and Accumulation in Sunflower Grown in Cr(VI)-Polluted Soil.

Biochar is preferentially recommended for the remediation of heavy metal-polluted soils. Sunflower is an important high-biomass oil crop with a promising potential for phytoremediation of Cr(VI)-polluted soil. However, how biochar affects sunflower growth and Cr accumulation in Cr(VI)-polluted soil needs to be elucidated. Here, a pot culture experiment was conducted to study whether soil amendment with biochar (0, 0.1%, 1%, and 5%, w/w) can mitigate Cr toxicity and accumulation in sunflower seedlings grown in soils artificially polluted with different levels of Cr(VI) (0, 50, and 250 mg Cr(VI)/kg soil). The addition of Cr(VI) exhibited significant phytotoxicity, as evidenced by inhibited plant growth and even the death of seedlings at 250 mg/kg Cr(VI). Overall, biochar amendment showed positive effects on plant growth and Cr immobilization, dependent on both the biochar dose and Cr addition level. When 50 mg/kg Cr(VI) was added, 1% biochar showed positive effects similar to 5% biochar on improving plant growth and mineral nutrition (particularly K), reducing Cr content in shoots and roots, and decreasing Cr availability and Cr(VI) content in the soil. In comparison with non-amendment, 1% and 5% biochar caused 85% and 100% increase in shoot dry weights, and 75% and 86% reduction in shoot Cr concentrations, respectively. When 250 mg/kg Cr(VI) was added, a 5% dose produced much better benefits than 1%, while a 0.1% dose did not help plants to survive. Overall, an appropriate dose of biochar enhanced Cr(VI) immobilization and subsequently decreased its toxicity and accumulation in sunflower seedlings. Our findings confirm that biochar can be used as an efficient amendment for the remediation of Cr(VI)-polluted soils and cleaner production of sunflower oil and biomass.

Individualism Versus Collectivism and the Early-Stage Transmission of COVID-19.

We propose a perspective based on the individualism versus collectivism (IC) cultural distinction to understand the diverging early-stage transmission outcomes of COVID-19 between countries. Since individualism values personal freedom, people in such cultures would be less likely to make the collective action of staying at home and less likely to support compulsory measures. As a reaction to the public will, governments of individualistic societies would be more hesitant to take compulsory measures, leading to the delay of necessary responses. With processed COVID-19 data that can provide a fair comparison, we find that COVID-19 spread much faster in more individualistic societies than in more collectivistic societies. We further use pronoun drop and absolute latitude as the instruments for IC to address reverse causality and omitted variable bias. The results are robust to different measures. We propose to consider the role of IC not only for understanding the current pandemic but also for thinking about future trends in the world.

Effect of Water Evaporation on the Inhibition of Spontaneous Coal Combustion.

Spontaneous coal combustion is the primary cause of coal mine fires. During the production process, spontaneous coal combustion in the goaf is often affected by air leakage, which weakens or annuls the effect of inhibitors and leads to secondary oxidation. However, the action mechanism of inhibitors on secondary oxidation spontaneous coal combustion remains unclear. Thus, this study analyzes the influence of moisture evaporation on the performance of a high-water-content physical inhibitor (HWPI) using the Carbolite temperature-programmed experiment, differential scanning calorimetry, scanning electron microscopy, and a MINI MR test. The results demonstrate that as the moisture content of the inhibitor decreased, after being treated with the HWPI and drying for 24 h, the concentrations of O2, CO, and CO2 were found to be lower than the gas concentration of raw coal, which showed that although the moisture content is reduced, the treated coal sample still has a lower spontaneous combustion tendency than the raw coal. The apparent activation energy was reduced, and the heat absorption per unit time decreased, which eventually weakened or annulled the effect of the HWPI. Future research should further improve existing inhibitor types to reduce the impact of secondary oxidation on spontaneous coal combustion caused by water evaporation.

3D Printed Intragastric Floating and Sustained-Release Tablets with Air Chambers.

This work aimed to use hot-melt extrusion (HME) and dual fused deposition modeling (FDM) 3D printing technology to develop a novel intragastric floating and sustained-release drug delivery system. The intragastric floating and sustained-release tablet was engineered by employing hydroxypropyl methylcellulose (AffinisolTM HPMC HME 15LV) for a drug-loaded core and polylactic acid (PLA) for an insoluble shell with an air chamber. Filaments for the drug-loaded core were compounded using a single-screw hot melt extruder. 3DMAX software was utilized to design a core with a complementary shell which consisted of a hollow chamber at the top and a drug-release window with different sizes (radius in 1.5, 2.5, 3, 3.5, 4.5 mm) at the bottom. Pharmaceutical characterization, solid dispersion evaluation, and drug release behavior were studied. The model drug in all formulations kept stable, and part of the drug in the extruded filaments and 3D printed tablets became amorphous. The introduction of an air chamber reduced the tablet density to below 0.9 g/cm3 and the 3D printed tablets floated immediately and continuously during the drug release process. The presence of the insoluble shell greatly prolonged the drug release time, and the drug release rate was positively correlated with the area of the release window. In addition, compared with shellless tablets, the 3D printed tablets with air chambers (radius in 4.5 mm) showed closer zero-order drug release for 24 h and released drug by diffusion-erosion combined mechanism. The developed intragastric floating and sustained-release tablets with air chambers could be applied to various drugs and provided a new way for the development of personalized drug delivery systems.

Experimental study on coal dust wettability strengthened by surface active ionic liquids.

The water wettability of coal dust was very important for dust control when using water-based dust suppressant materials. The coal dust wettability strengthened by surface active ionic liquid was studied in this paper. The surface activity of ten ionic liquids with different anions Cl-, Br-, [BF4]-, [NTf2]- and cations [HOEtMIm]+, [Cnmim]+ (n = 4, 12, 14, 16) was studied by surface tension test. The water wettability of raw coal dust can be improved individually by adding ionic liquid to water or pre-treating coal dust by ionic liquids. The wettability of lignite was improved little, but that of bituminous coal and anthracite were improved much. The dual strengthened effects of ionic liquids on coal dust wettability were studied by the wetting results between ionic liquids solutions and ionic liquid-treated coal samples. The wettability of lignite can be strengthened under the combined action of [HOEtMIm][NTf2] and [C12MIm]Br, while other dual effects were not satisfactory. All ionic liquids combination had strengthened effects on the wettability of bituminous coal and anthracite, especially the [C12MIm]Br treatment and [C12MIm]Br solutions together had the best dual effects. The functional groups results indicated that the hydrophilic oxygen-containing functional groups in treated coal samples increased, the hydrophobic aliphatic hydrocarbon functional groups decreased and part of ionic liquids were adsorbed on the coal surface. These changes together enhanced the wettability of coal with high coalification degrees.

Quantitative immunopeptidomics reveals a tumor stroma-specific target for T cell therapy.

T cell receptor (TCR)-based immunotherapy has emerged as a promising therapeutic approach for the treatment of patients with solid cancers. Identifying peptide-human leukocyte antigen (pHLA) complexes highly presented on tumors and rarely expressed on healthy tissue in combination with high-affinity TCRs that when introduced into T cells can redirect T cells to eliminate tumor but not healthy tissue is a key requirement for safe and efficacious TCR-based therapies. To discover promising shared tumor antigens that could be targeted via TCR-based adoptive T cell therapy, we employed population-scale immunopeptidomics using quantitative mass spectrometry across ~1500 tumor and normal tissue samples. We identified an HLA-A*02:01-restricted pan-cancer epitope within the collagen type VI α-3 (COL6A3) gene that is highly presented on tumor stroma across multiple solid cancers due to a tumor-specific alternative splicing event that rarely occurs outside the tumor microenvironment. T cells expressing natural COL6A3-specific TCRs demonstrated only modest activity against cells presenting high copy numbers of COL6A3 pHLAs. One of these TCRs was affinity-enhanced, enabling transduced T cells to specifically eliminate tumors in vivo that expressed similar copy numbers of pHLAs as primary tumor specimens. The enhanced TCR variants exhibited a favorable safety profile with no detectable off-target reactivity, paving the way to initiate clinical trials using COL6A3-specific TCRs to target an array of solid tumors.

In vitro and in vivo evaluation of levofloxacin sustained-release capsules.

OBJECTIVE: To reduce the frequency of administration and improve patient compliance, novel levofloxacin sustained-release capsules with suitable in vitro release profiles and good bioavailability were developed. MATERIALS AND METHODS: A fluidized bed was used to prepare levofloxacin pellets by spraying the drug solution onto blank pellets. Then the pellets were coated with either Surelease water dispersion or Eudragit® NE30D water dispersion to achieve sustained-release characteristics. The mixed pellets containing 15% of the uncoated pellets and 85% of the coated pellets were filled into the hard gelatin capsules. In vitro release test was performed with the capsules. A single-dose pharmacokinetic study of the capsules was carried out in beagle dogs. RESULTS: Although Eudragit® NE30D-coated pellets and Surelease-coated pellets showed similar sustained-release profiles in vitro, their in vivo pharmacokinetic characteristics exhibited significant difference. Unsuccessful in vivo-in vitro correlation was shown in Eudragit® NE30D-coated pellets with a relative bioavailability of only 41.5%, whereas Surelease-coated pellets achieved best sustained-release feature both in vitro and in vivo with a relative bioavailability of 103.0%. CONCLUSION: Statistical analysis indicated that the capsules containing Surelease-coated pellets had a satisfactory sustained-release behavior and a desired pharmacokinetic property.

Analysis of the spatial distribution of collectors in dust scrubber based on image processing.

The spatial distribution of the collectors in dust scrubber is key in determining the effectiveness of the dust removal process. In the present study, a high-speed camera was used to capture images of the distribution of the collectors. Some of the image information was extracted by image processing, such as the gray mean (GM), the angular second moment (ASM), and the entropy (ENT) from the gray-level co-occurrence matrix of the image. Subsequently, the spatial distribution rules of the collectors were studied by analyzing the spatial proportion, dispersion area, and uniformity and intensiveness of the collectors. It is an intuitive approach and a novel analysis method for the operating state of dust scrubber. The results show that the spatial distribution of the collectors could be better reflected by image processing methods. The dispersion area of the collectors expanded with an increase in the airflow velocity. When the initial liquid level (ILL) was higher, the collectors expanded in an approximate circular shape, and when the ILL was lower the collectors expanded in an approximate sector shape. In general, the variation trend in the spatial proportion enhanced with an increase in ILL and airflow velocity, which is consistent with the uniformity of collectors. When the liquid level was 0-20 mm and the airflow velocity was greater than 6.5 m/sec, the spatial proportion and uniformity of the collectors reached the highest degree. However, the growth rate of the spatial proportion and uniformity of the collectors slowed down and even led to negative growth when the ILL was lower and the airflow velocity was higher. The intensiveness of the collectors was great when the ILL was higher, which was free from the apparent influence of the airflow velocity and the ILL. However, when the ILL was lower, the intensiveness of the collectors was poor, intensifying as the airflow velocity and ILL increased. When the liquid level was -5-10 mm and the airflow velocity was greater than 8 m/sec, the intensiveness of the collectors reached the highest degree, indicating that a liquid level greater than 0 mm and a higher airflow velocity improved the spatial distribution of the collectors. Implications: This paper focuses on the spatial distribution of the collectors in dust scrubber. Some of the image information was extracted by image processing, such as the gray mean of the image, the angular second moment, and the entropy from the gray-level co-occurrence matrix of the image. The spatial distribution rules of the collectors were studied by analyzing the spatial proportion, the dispersion area, and the uniformity and intensiveness of the collectors.

Effect of Ionic Liquid Surfactants on Coal Oxidation and Structure.

The effects of six ionic liquids with surfactant property (1-hydroxyethyl-3-methyl imidazolium bis(trifluoromethylsulfonyl)imide ([HOEtMIm][NTf2]), 1-hydroxyethyl-3-methyl imidazolium tetrafluoroborate ([HOEtMIm][BF4]), 1-dodecyl-3- methyl imidazolium bromide ([C12MIm]Br), 1-tetradecyl-3- methyl imidazolium bromide ([C14MIm]Br), trioctyl methyl ammonium chloride ([N8,8,8,1])Cl, and tetraethyl ammonium chloride ([N2,2,2,2]Cl)) on the oxidation characteristics and functional groups of coal were studied by means of critical micelle concentration, surface tension, thermogravimetric analysis, temperature-programmed oxidation, and Fourier transform infrared spectroscopy (FTIR) measurements. The lower critical micelle concentration for the ionic liquids except the [N2,2,2,2]Cl suggests the favorable surface activity of these ionic liquids. The surface activities of [N8,8,8,1]Cl, [C14MIm]Br, [C12MIm]Br, and [HOEtMIm][NTf2] were high, while that of [N2,2,2,2]Cl was relatively lower. The thermal stabilities of [HOEtMIm][NTf2] and [HOEtMIm][BF4] were high, while those of [N8,8,8,1]Cl and [N2,2,2,2]Cl were lower. The oxidation activities of ionic liquid-mixed coals were weakened to different degrees except [N8,8,8,1]Cl-mixed coal, because of the poor thermal stability and decomposition of [N8,8,8,1]Cl accelerating the coal oxidation. The other five ionic liquids were suitable for inhibiting coal oxidation, particularly the [HOEtMIm][BF4] and [HOEtMIm][NTf2] with higher inhibition rate, longer inhibition time, and also better thermal stabilities. The activation energy results further confirmed such inhibition effect. The functional group results showed that treatment of ionic liquids on coal can change the contents of hydrogen bonds, aliphatic groups, and aromatic groups in coal. It was inferred that the [HOEtMIm][BF4], [HOEtMIm][NTf2], and [C14MIm]Br were more effectively to affect coal structure and decrease coal oxidation activity.

Adoptive immunotherapy: good habits instilled at youth have long-term benefits.

Many recent advances in basic cell biology and immunology are a harbinger of progress in adoptive cell therapy (ACT) including (1) the finding that host lymphodepletion enhances engraftment and efficacy, (2) the recognition that in vitro T cell functions may not correlate with in vivo efficacy, and (3) the development of advanced ex vivo culture methods to expand lymphocytes to therapeutically effective numbers. In this article, we focus on the development of artificial antigen presenting cells (aAPCs) in our laboratory and their applicability to augment ACT protocols. We also describe how aAPCs can be used to broaden ACT to treat patients with a wide variety of cancers, chronic infectious diseases, and autoimmune manifestations.

Mesoscale behavior study of collector aggregations in a wet dust scrubber.

In order to address the bottleneck problem of low fine-particle removal efficiency of self-excited dust scrubbers, this paper is focused on the influence of the intermittent gas-liquid two-phase flow on the mesoscale behavior of collector aggregations. The latter is investigated by the application of high-speed dynamic image technology to the self-excited dust scrubber experimental setup. The real-time-scale monitoring of the dust removal process is provided to clarify its operating mechanism at the mesoscale level. The results obtained show that particulate capturing in self-excited dust scrubber is provided by liquid droplets, liquid films/curtains, bubbles, and their aggregations. Complex spatial and temporal structures are intrinsic to each kind of collector morphology, and these are considered as the major factors controlling the dust removal mechanism of self-excited dust scrubbers. For the specific parameters of gas-liquid two-phase flow under study, the evolution patterns of particular collectors reflect the intrinsic, intermittent, and complex characteristics of the temporal structure. The intermittent initiation of the collector and the air hole formation-collapse cyclic processes provide time and space for the fine dust to escape from being trapped by the collectors. The above mesoscale experimental data provide more insight into the factors reducing the dust removal efficiency of self-excited dust scrubbers. IMPLICATIONS: This paper focuses on the reconsideration of the capturer aggregations of self-excited dust scrubbers from the mesoscale. Complex structures in time and space scales exist in each kind of capturer morphology. With changes of operating parameters, the morphology and spatial distributions of capturers diversely change. The change of the capturer over time presents remarkable, intermittent, and complex characteristics of the temporal structure.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas.

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells' efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition.

Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases.Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, ß-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Results: The CRISPR gene-edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene-edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells.Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255-66. ©2016 AACR.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB). The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2. Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity. Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells. In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells. Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy.

A versatile system for rapid multiplex genome-edited CAR T cell generation.

The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.

A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors.

Chimeric antigen receptor (CAR)-modified adoptive T-cell therapy has been successfully applied to the treatment of hematologic malignancies, but faces many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced in both naturally occurring and genetically modified tumor-infiltrating lymphocytes (TIL) by inhibitory receptors (IR), namely PD1. We hypothesized that interfering with PD1 signaling would augment CAR T-cell activity against solid tumors. To address this possibility, we introduced a genetically engineered switch receptor construct, comprising the truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28, into CAR T cells. We tested the effect of this supplement, "PD1CD28," on human CAR T cells targeting aggressive models of human solid tumors expressing relevant tumor antigens. Treatment of mice bearing large, established solid tumors with PD1CD28 CAR T cells led to significant regression in tumor volume due to enhanced CAR TIL infiltrate, decreased susceptibility to tumor-induced hypofunction, and attenuation of IR expression compared with treatments with CAR T cells alone or PD1 antibodies. Taken together, our findings suggest that the application of PD1CD28 to boost CAR T-cell activity is efficacious against solid tumors via a variety of mechanisms, prompting clinical investigation of this potentially promising treatment modality.