Affinity fine-tuning anti-CAIX CAR-T cells mitigate on-target off-tumor side effects.

One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

In Vivo Transmigrated Human Neutrophils Are Highly Primed for Intracellular Radical Production Induced by Monosodium Urate Crystals.

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production.

Immune-restoring CAR-T cells display antitumor activity and reverse immunosuppressive TME in a humanized ccRCC mouse model.

One of the major barriers that have restricted successful use of chimeric antigen receptor (CAR) T cells in the treatment of solid tumors is an unfavorable tumor microenvironment (TME). We engineered CAR-T cells targeting carbonic anhydrase IX (CAIX) to secrete anti-PD-L1 monoclonal antibody (mAb), termed immune-restoring (IR) CAR G36-PDL1. We tested CAR-T cells in a humanized clear cell renal cell carcinoma (ccRCC) orthotopic mouse model with reconstituted human leukocyte antigen (HLA) partially matched human leukocytes derived from fetal CD34+ hematopoietic stem cells (HSCs) and bearing human ccRCC skrc-59 cells under the kidney capsule. G36-PDL1 CAR-T cells, haploidentical to the tumor cells, had a potent antitumor effect compared to those without immune-restoring effect. Analysis of the TME revealed that G36-PDL1 CAR-T cells restored active antitumor immunity by promoting tumor-killing cytotoxicity, reducing immunosuppressive cell components such as M2 macrophages and exhausted CD8+ T cells, and enhancing T follicular helper (Tfh)-B cell crosstalk.

High-throughput identification of repurposable neuroactive drugs with potent anti-glioblastoma activity.

Glioblastoma, the most aggressive primary brain cancer, has a dismal prognosis, yet systemic treatment is limited to DNA-alkylating chemotherapies. New therapeutic strategies may emerge from exploring neurodevelopmental and neurophysiological vulnerabilities of glioblastoma. To this end, we systematically screened repurposable neuroactive drugs in glioblastoma patient surgery material using a clinically concordant and single-cell resolved platform. Profiling more than 2,500 ex vivo drug responses across 27 patients and 132 drugs identified class-diverse neuroactive drugs with potent anti-glioblastoma efficacy that were validated across model systems. Interpretable molecular machine learning of drug-target networks revealed neuroactive convergence on AP-1/BTG-driven glioblastoma suppression, enabling expanded in silico screening of more than 1 million compounds with high patient validation accuracy. Deep multimodal profiling confirmed Ca2+-driven AP-1/BTG-pathway induction as a neuro-oncological glioblastoma vulnerability, epitomized by the anti-depressant vortioxetine synergizing with current standard-of-care chemotherapies in vivo. These findings establish an actionable framework for glioblastoma treatment rooted in its neural etiology.

A prognostic neural epigenetic signature in high-grade glioma.

Neural-tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients' survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma, primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like, astrocyte-like and oligodendrocyte-progenitor-like, alongside oligodendrocytes and excitatory neurons. In line with these findings, high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients' plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes.

Targeting the Siglec-sialic acid axis promotes antitumor immune responses in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor, for which effective therapies are urgently needed. Cancer cells are capable of evading clearance by phagocytes such as microglia- and monocyte-derived cells through engaging tolerogenic programs. Here, we found that high expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) correlates with reduced survival in patients with GBM. Using microglia- and monocyte-derived cell-specific knockouts of Siglec-E, the murine functional homolog of Siglec-9, together with single-cell RNA sequencing, we demonstrated that Siglec-E inhibits phagocytosis by these cells, thereby promoting immune evasion. Loss of Siglec-E on monocyte-derived cells further enhanced antigen cross-presentation and production of pro-inflammatory cytokines, which resulted in more efficient T cell priming. This bridging of innate and adaptive responses delayed tumor growth and resulted in prolonged survival in murine models of GBM. Furthermore, we showed the combinatorial activity of Siglec-E blockade and other immunotherapies demonstrating the potential for targeting Siglec-9 as a treatment for patients with GBM.

Epigenetic neural glioblastoma enhances synaptic integration and predicts therapeutic vulnerability.

Neural-tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is nascent. We present an epigenetically defined neural signature of glioblastoma that independently affects patients' survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals high abundance of stem cell-like malignant cells classified as oligodendrocyte precursor and neural precursor cell-like in high-neural glioblastoma. High-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature associates with decreased survival as well as increased functional connectivity and can be detected via DNA analytes and brain-derived neurotrophic factor in plasma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant.

Medication-Facilitated Advanced Airway Management with First-Line Use of a Supraglottic Device - A One-Year Quality Assurance Review.

INTRODUCTION: Airway management is a controversial topic in modern Emergency Medical Services (EMS) systems. Among many concerns regarding endotracheal intubation (ETI), unrecognized esophageal intubation and observations of unfavorable neurologic outcomes in some studies raise the question of whether alternative airway techniques should be first-line in EMS airway management protocols. Supraglottic airway devices (SADs) are simpler to use, provide reliable oxygenation and ventilation, and may thus be an alternative first-line airway device for paramedics. In 2019, Alachua County Fire Rescue (ACFR; Alachua, Florida USA) introduced a novel protocol for advanced airway management emphasizing first-line use of a second-generation SAD (i-gel) for patients requiring medication-facilitated airway management (referred to as "rapid sequence airway" [RSA] protocol). STUDY OBJECTIVE: This was a one-year quality assurance review of care provided under the RSA protocol looking at compliance and first-pass success rate of first-line SAD use. METHODS: Records were obtained from the agency's electronic medical record (EMR), searching for the use of the RSA protocol, advanced airway devices, or either ketamine or rocuronium. If available, hospital follow-up data regarding patient condition and emergency department (ED) airway exchange were obtained. RESULTS: During the first year, 33 advanced airway attempts were made under the protocol by 23 paramedics. Overall, compliance with the airway device sequence as specified in the protocol was 72.7%. When ETI was non-compliantly used as first-line airway device, the first-pass success rate was 44.4% compared to 87.5% with adherence to first-line SAD use. All prehospital SADs were exchanged in the ED in a delayed fashion and almost exclusively per physician preference alone. In no case was the SAD exchanged for suspected dislodgement evidenced by lack of capnography. CONCLUSION: First-line use of a SAD was associated with a high first-pass attempt success rate in a real-life cohort of prehospital advanced airway encounters. No SAD required emergent exchange upon hospital arrival.

Anti-CAIX BBζ CAR4/8 T cells exhibit superior efficacy in a ccRCC mouse model.

Improving CAR-T cell therapy for solid tumors requires a better understanding of CAR design and cellular composition. Here, we compared second-generation (BBζ and 28ζ) with third-generation (28BBζ) carbonic anhydrase IX (CAIX)-targeted CAR constructs and investigated the antitumor effect of CAR-T cells with different CD4/CD8 proportions in vitro and in vivo. The results demonstrated that BBζ exhibited superior efficacy compared with 28ζ and 28BBζ CAR-T cells in a clear-cell renal cell carcinoma (ccRCC) skrc-59 cell bearing NSG-SGM3 mouse model. The mice treated with a single dose of BBζ CD4/CD8 mixture (CAR4/8) showed complete tumor remission and remained tumor-free 72 days after CAR-T cells infusion. In the other CAR-T and control groups, tumor-infiltrating T cells were recovered and profiled. We found that BBζ CAR8 cells upregulated expression of major histocompatibility complex (MHC) class II and cytotoxicity-associated genes, while downregulating inhibitory immune checkpoint receptor genes and diminishing differentiation of regulatory T cells (Treg cells), leading to excellent therapeutic efficacy in vivo. Increased memory phenotype, elevated tumor infiltration, and decreased exhaustion genes were observed in the CD4/8 untransduced T (UNT) cells compared with CD8 alone, indicating that CD4/8 would be the favored cellular composition for CAR-T cell therapy with long-term persistence. In summary, these findings support that BBζ CAR4/8 cells are a highly potent, clinically translatable cell therapy for ccRCC.

DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils.

Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation. Neutrophils harbor two distinct pools of the membrane-localized oxidase components, one expressed in the plasma membrane and one in the membranes of intracellular granules. Assembly of active oxidase at either type of membrane leads to release of extracellular ROS or to the production of ROS inside intracellular compartments, respectively. The cytoplasmic NADPH oxidase subunit p40phox seems selectively critical for the ability to generate intracellular ROS, and the recent characterization of patients with p40phox deficiency implies that selective loss of intracellular neutrophil ROS leads to disease with pronounced hyperinflammatory features, suggesting that these ROS are critical for regulation of inflammation. This study aimed at characterizing two pharmacological NADPH oxidase inhibitors, the newly described GSK2795039 and the widely used diphenyleneiodonium (DPI), focusing on their abilities to inhibit human neutrophil ROS production extra- and intracellularly. Whereas GSK2795039 blocked extra- and intracellular NADPH oxidase activity equally, DPI was found to selectively interfere with intracellular ROS production. Selectivity for the intracellular NADPH oxidase was evident as a lower IC50 value, faster onset, and irreversibility of inhibition. We found no evidence of direct interactions between DPI and p40phox, but the selectivity of DPI confirms that regulation of NADPH oxidase activity in neutrophils differs depending on the subcellular localization of the enzyme. This information may be used to pharmacologically mimic p40phox deficiency and to further our understanding of how intracellular ROS contribute to health and disease.

Sensitivity towards the GRP78 inhibitor KP1339/IT-139 is characterized by apoptosis induction via caspase 8 upon disruption of ER homeostasis.

The ruthenium drug and GRP78 inhibitor KP1339/IT-139 has already demonstrated promising anticancer activity in a phase I clinical trial. This study aimed to identify mechanisms underlying increased sensitivity to KP1339 treatment. Based on a screen utilizing 23 cell lines, a small panel was selected to compare KP1339-sensitive and low-responsive models. KP1339 sensitivity was neither based on differences in ruthenium accumulation, nor sensitivity to oxidative stress or constituents of KP1339 (ruthenium chloride and indazole). Subsequently, the biochemical response to KP1339 was analyzed using whole genome expression arrays indicating that, while sensitive cell lines were characterized by "response to chemical stimuli" and "regulation of cell death", low-responsive cells preferentially activated pathways controlling cell cycle, DNA repair, and metabolism. Cell culture experiments confirmed that, while low-responsive cells executed cell cycle arrest in G2 phase, pronounced apoptosis induction via activation of caspase 8 was found in sensitive cells. Cell death induction is based on a unique disruption of the ER homeostasis by depletion of key cellular chaperones including GRP78 in combination with enhanced KP1339-mediated protein damage.