Beyond direct killing-novel cellular immunotherapeutic strategies to reshape the tumor microenvironment.

The clinical use of cellular immunotherapies is gaining momentum and the number of approved indications is steadily increasing. One class of cellular therapies-chimeric antigen receptor (CAR)-modified T cells-has achieved impressive results in distinct blood cancer indications. These existing cellular therapies treating blood cancers face significant relapse rates, and their application beyond hematology has been underwhelming, especially in solid oncology. Major reasons for resistance source largely in the tumor microenvironment (TME). The TME in fact functionally suppresses, restricts, and excludes adoptive immune cells, which limits the efficacy of cellular immunotherapies from the onset. Many promising efforts are ongoing to adapt cellular immunotherapies to address these obstacles, with the aim of reshaping the tumor microenvironment to ameliorate function and to achieve superior efficacy against both hematological and solid malignancies.

Utilizing chemokines in cancer immunotherapy.

Immune checkpoint inhibition and chimeric antigen receptor (CAR) T cell therapy have demonstrated stunning clinical efficacy in many cancer types. However, most patients do not respond to immunotherapies or relapse after an initial response, stressing the need for improved strategies. Chemokines, as mediators of immune cell trafficking, play an important role in the composition of the tumor microenvironment and exert both pro- and antitumorigenic functions. Here, chemokines may represent valuable prognostic biomarkers of response to immunotherapy and a strategy to improve immunotherapies. In this review, the pleiotropic functions of chemokines in the tumor microenvironment (TME) and strategies of utilizing chemokines or chemokine antagonism in immunotherapy are discussed. The review highlights preclinical and clinical studies that apply or target chemokines in monotherapy or in combination therapies.

Broad T Cell Targeting of Structural Proteins After SARS-CoV-2 Infection: High Throughput Assessment of T Cell Reactivity Using an Automated Interferon Gamma Release Assay.

Background: Adaptive immune responses to structural proteins of the virion play a crucial role in protection against coronavirus disease 2019 (COVID-19). We therefore studied T cell responses against multiple SARS-CoV-2 structural proteins in a large cohort using a simple, fast, and high-throughput approach. Methods: An automated interferon gamma release assay (IGRA) for the Nucleocapsid (NC)-, Membrane (M)-, Spike-C-terminus (SCT)-, and N-terminus-protein (SNT)-specific T cell responses was performed using fresh whole blood from study subjects with convalescent, confirmed COVID-19 (n = 177, more than 200 days post infection), exposed household members (n = 145), and unexposed controls (n = 85). SARS-CoV-2-specific antibodies were assessed using Elecsys® Anti-SARS-CoV-2 (Ro-N-Ig) and Anti-SARS-CoV-2-ELISA (IgG) (EI-S1-IgG). Results: 156 of 177 (88%) previously PCR confirmed cases were still positive by Ro-N-Ig more than 200 days after infection. In T cells, most frequently the M-protein was targeted by 88% seropositive, PCR confirmed cases, followed by SCT (85%), NC (82%), and SNT (73%), whereas each of these antigens was recognized by less than 14% of non-exposed control subjects. Broad targeting of these structural virion proteins was characteristic of convalescent SARS-CoV-2 infection; 68% of all seropositive individuals targeted all four tested antigens. Indeed, anti-NC antibody titer correlated loosely, but significantly with the magnitude and breadth of the SARS-CoV-2-specific T cell response. Age, sex, and body mass index were comparable between the different groups. Conclusion: SARS-CoV-2 seropositivity correlates with broad T cell reactivity of the structural virus proteins at 200 days after infection and beyond. The SARS-CoV-2-IGRA can facilitate large scale determination of SARS-CoV-2-specific T cell responses with high accuracy against multiple targets.

Evidence for nutrient-dependent regulation of the COPII coat by O-GlcNAcylation.

O-linked ß-N-acetylglucosamine (O-GlcNAc) is a dynamic form of intracellular glycosylation common in animals, plants and other organisms. O-GlcNAcylation is essential in mammalian cells and is dysregulated in myriad human diseases, such as cancer, neurodegeneration and metabolic syndrome. Despite this pathophysiological significance, key aspects of O-GlcNAc signaling remain incompletely understood, including its impact on fundamental cell biological processes. Here, we investigate the role of O-GlcNAcylation in the coat protein II complex (COPII), a system universally conserved in eukaryotes that mediates anterograde vesicle trafficking from the endoplasmic reticulum. We identify new O-GlcNAcylation sites on Sec24C, Sec24D and Sec31A, core components of the COPII system, and provide evidence for potential nutrient-sensitive pathway regulation through site-specific glycosylation. Our work suggests a new connection between metabolism and trafficking through the conduit of COPII protein O-GlcNAcylation.

T cells armed with C-X-C chemokine receptor type 6 enhance adoptive cell therapy for pancreatic tumours.

The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Combined tumor-directed recruitment and protection from immune suppression enable CAR T cell efficacy in solid tumors.

CAR T cell therapy remains ineffective in solid tumors, due largely to poor infiltration and T cell suppression at the tumor site. T regulatory (Treg) cells suppress the immune response via inhibitory factors such as transforming growth factor-ß (TGF-ß). Treg cells expressing the C-C chemokine receptor 8 (CCR8) have been associated with poor prognosis in solid tumors. We postulated that CCR8 could be exploited to redirect effector T cells to the tumor site while a dominant-negative TGF-ß receptor 2 (DNR) can simultaneously shield them from TGF-ß. We identified that CCL1 from activated T cells potentiates a feedback loop for CCR8+ T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-ß shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.

Gigaxonin glycosylation regulates intermediate filament turnover and may impact giant axonal neuropathy etiology or treatment

Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor­dependent manner. MS analyses of human gigaxonin revealed 9 candidate sites of O-GlcNAcylation, 2 of which ­ serine 272 and threonine 277 ­ are required for its ability to mediate IF turnover in gigaxonin-deficient human cell models that we created. Taken together, the results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the nongenetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.

A Label-Free, Quantitative Fecal Hemoglobin Detection Platform for Colorectal Cancer Screening.

The early detection of colorectal cancer is vital for disease management and patient survival. Fecal hemoglobin detection is a widely-adopted method for screening and early diagnosis. Fecal Immunochemical Test (FIT) is favored over the older generation chemical based Fecal Occult Blood Test (FOBT) as it does not require dietary or drug restrictions, and is specific to human blood from the lower digestive tract. To date, no quantitative FIT platforms are available for use in the point-of-care setting. Here, we report proof of principle data of a novel low cost quantitative fecal immunochemical-based biosensor platform that may be further developed into a point-of-care test in low-resource settings. The label-free prototype has a lower limit of detection (LOD) of 10 µg hemoglobin per gram (Hb/g) of feces, comparable to that of conventional laboratory based quantitative FIT diagnostic systems.

Inoperable hepatocellular carcinoma: transarterial 188Re HDD-labeled iodized oil for treatment--prospective multicenter clinical trial.

PURPOSE: To prospectively evaluate, in a multicenter clinical trial, dosimetry-guided transarterial radionuclide therapy (TART) with rhenium 188 ((188)Re) 4-hexadecyl 1,2,9,9-tetramethyl-4,7-diaza-1,10-decanethiol (HDD)-labeled iodized oil in inoperable hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Ninety-three patients were recruited from 2000 to 2005 for this ethics committee-approved study. Informed written consent was obtained. After complete clinical evaluation (including assessment of liver status, serum alpha-fetoprotein [AFP] level, tumor size, portal vein status, Child-Pugh classification, Okuda staging), radiation absorbed dose (RAD) to various organs, including tumor, was calculated after injecting 185 MBq of (188)Re HDD iodized oil via the hepatic artery. From this value, the maximum tolerable activity of (188)Re, defined as the amount of radioactivity delivering no more than 12 Gy of RAD to lungs, 30 Gy to normal liver, or 1.5 Gy to bone marrow, was calculated and injected. RESULTS: Mean patient age was 53 years (80 men and 13 women). Sixty-eight percent of patients had serologic evidence of hepatitis B and/or C; 40% had clinicoradiologic evidence of cirrhosis. Mean tumor diameter was 10.3 cm +/- 4.4, with 40% of patients having more than three lesions; in 50% of patients, tumor was either unilateral, occupying 50% or more of the liver, or bilateral. AFP was elevated in 68% of patients and was elevated to more than 300 ng/mL in 44% of patients. There was portal vein thrombosis in 38% of patients, Child-Pugh status B disease in 37% of patients, and Okuda stage II or III disease in 50% of patients. Mean first administered activity was 5.3 GBq +/- 1.6, which delivered 88 Gy of RAD to the tumor. Treatment was tolerated well. Of 66 patients in whom complete tumor response occurred, five (8%) had complete tumor mass ablation, 17 (26%) had a partial response (>50% tumor reduction), and 23 (35%) had stable disease. Only RAD to the tumors was found to be significantly (P = .001) associated with tumor and/or AFP response. Survival rates at 6, 9, 12, 24, and 36 months among patients with objective tumor response were 100%, 95%, 90%, 58%, and 30%, respectively, with a median survival of 980 days. CONCLUSION: TART appears to be a safe, effective, and promising therapeutic option in patients with inoperable HCC.