ErbB2/HER2-specific NK cells for adoptive cancer immunotherapy

Poster presentation: 28th Annual Scientific Meeting of the Society for Immunotherapy of Cancer (SITC) Significant progress has been made over the last decade towards realizing the potential of natural killer (NK) cells for cancer immunotherapy. NK cells can respond rapidly to transformed and stressed cells, and have the intrinsic potential to extravasate and reach their targets in almost all body tissues. In addition to donor-derived primary NK cells, also continuously expanding cytotoxic cell lines such as NK-92 are being considered for adoptive cancer immunotherapy. High cytotoxicity of NK-92 has previously been shown against malignant cells of hematologic origin in preclinical studies, and general safety of infusion of NK-92 cells has been established in phase I clinical trials. To enhance their therapeutic utility, we genetically modified NK-92 cells to express chimeric antigen receptors (CAR) specific for tumor-associated surface antigens. Such CAR were composed of a tumor-specific scFv antibody fragment fused via hinge and transmembrane domains to intracellular signaling moieties such as CD3 zeta chain, or composite fusion molecules also containing a costimulatory protein domain in addition to CD3 zeta. For development towards clinical applications, here a codon-optimized second generation CAR was constructed that consists of an ErbB2-specific scFv antibody domain fused via a linker to a composite CD28-CD3 zeta signaling domain. GMP-compliant protocols for vector production, lentiviral transduction and expansion of a genetically modified NK-92 single cell clone (NK-92/5.28.z) were established. Functional analysis of NK-92/5.28.z cells revealed high and stable CAR expression, selective cytotoxicity against ErbB2-expressing but otherwise NK-resistant tumor cells of different origins in vitro, as well as homing to ErbB2-expressing tumors in vivo. Furthermore, antigen specificity and selective cytotoxicity of these cells were retained in vivo, resulting in antitumoral activity against subcutaneous and intracranial glioblastoma xenografts in NSG mice. Ongoing work now focuses on the development of these cells for adoptive immunotherapy of ErbB2-positive glioblastoma

Clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations

An expansion of the GGGGCC hexanucleotide in the non-coding region of C9orf72 represents the most common cause of familial amyotrophic lateral sclerosis. The objective was to describe and analyse the clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations in a large population. Between November 2011 and December 2020, clinical and genetic characteristics of n = 248 patients with amyotrophic lateral sclerosis carrying C9orf72 mutations were collected from the clinical and scientific network of German motoneuron disease centres. Clinical parameters included age of onset, diagnostic delay, family history, neuropsychological examination, progression rate, phosphorylated neurofilament heavy chain levels in CSF and survival. The number of repeats was correlated with the clinical phenotype. The clinical phenotype was compared to n = 84 patients with SOD1 mutations and n = 2178 sporadic patients without any known disease-related mutations. Patients with C9orf72 featured an almost balanced sex ratio with 48.4% (n = 120) women and 51.6% (n = 128) men. The rate of 33.9% patients (n = 63) with bulbar onset was significantly higher compared to sporadic (23.4%, P = 0.002) and SOD1 patients (3.1%, P < 0.001). Of note, 56.3% (n = 138) of C9orf72, but only 16.1% of SOD1 patients reported a negative family history (P < 0.001). The GGGGCC hexanucleotide repeat length did not influence the clinical phenotypes. Age of onset (58.0, interquartile range 52.0-63.8) was later compared to SOD1 (50.0, interquartile range 41.0-58.0;P < 0.001), but earlier compared to sporadic patients (61.0, interquartile range 52.0-69.0;P = 0.01). Median survival was shorter (38.0 months) compared to SOD1 (198.0 months, hazard ratio 1.97, 95% confidence interval 1.34-2.88;P < 0.001) and sporadic patients (76.0 months, hazard ratio 2.34, 95% confidence interval 1.64-3.34;P < 0.001). Phosphorylated neurofilament heavy chain levels in CSF (2880, interquartile range 1632-4638 pg/ml) were higher compared to sporadic patients (1382, interquartile range 458-2839 pg/ml;P < 0.001). In neuropsychological screening, C9orf72 patients displayed abnormal results in memory, verbal fluency and executive functions, showing generally worse performances compared to SOD1 and sporadic patients and a higher share with suspected frontotemporal dementia. In summary, clinical features of patients with C9orf72 mutations differ significantly from SOD1 and sporadic patients. Specifically, they feature a more frequent bulbar onset, a higher share of female patients and shorter survival. Interestingly, we found a high proportion of patients with negative family history and no evidence of a relationship between repeat lengths and disease severity. Wiesenfarth et al. report that amyotrophic lateral sclerosis patients with C9orf72 mutations differ significantly from sporadic patients and SOD1 gene carriers, including a higher share of bulbar onset, female patients, more severe neuropsychological deficits and shorter survival. No evidence of a relationship between repeat lengths and disease severity was found

Human cytomegalovirus infection in tumor cells of the nervous system is not detectable with standardized pathologico-virological diagnostics.

BACKGROUND Experimental findings have suggested that human cytomegalovirus (HCMV) infection of tumor cells may exert oncomodulatory effects that enhance tumor malignancy. However, controversial findings have been published on the presence of HCMV in malignant tumors. Here, we present the first study that systematically investigates HCMV infection in human nervous system tumors by highly sensitive immunohistochemistry in correlation with the HCMV serostatus of the patients. METHODS Immunohistochemical and quantitative PCR-based methods to detect different HCMV antigens and genomic HCMV DNA were optimized prior to the investigation of pathological samples. Moreover, the pathological results were matched with the HCMV serostatus of the patients. RESULTS HCMV immediate-early, late, and pp65 antigens could be detected in single cells from HCMV strain Hi91-infected UKF-NB-4 neuroblastoma cells after 1:1024 dilution with noninfected UKF-NB-4 cells. Genomic HCMV DNA could be detected in copy numbers as low as 430 copies/mL. However, we did not detect HCMV in tumors from a cohort of 123 glioblastoma, medulloblastoma, or neuroblastoma patients. Notably, we detected nonspecifically positive staining in tumor tissues of HCMV seropositive and seronegative glioblastoma patients. The HCMV serostatus of 67 glioblastoma patients matched the general epidemiological prevalence data for Western countries (72% of female and 57% of male glioblastoma patients were HCMV seropositive). Median survival was not significantly different in HCMV seropositive versus seronegative glioblastoma patients. CONCLUSIONS The prevalence of HCMV-infected tumor cells may be much lower than previously reported based on highly sensitive detection methods

Tp53-induced glycolysis and apoptosis regulator (TIGAR) protects glioma cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis

Altered metabolism in tumor cells is increasingly recognized as a core component of the neoplastic phenotype. Because p53 has emerged as a master metabolic regulator, we hypothesized that the presence of wild-type p53 in glioblastoma cells could confer a selective advantage to these cells under the adverse conditions of the glioma microenvironment. Here, we report on the effects of the p53-dependent effector Tp53-induced glycolysis and apoptosis regulator (TIGAR) on hypoxia-induced cell death. We demonstrate that TIGAR is overexpressed in glioblastomas and that ectopic expression of TIGAR reduces cell death induced by glucose and oxygen restriction. Metabolic analyses revealed that TIGAR inhibits glycolysis and promotes respiration. Further, generation of reactive oxygen species (ROS) levels was reduced whereas levels of reduced glutathione were elevated in TIGAR-expressing cells. Finally, inhibiting the transketolase isoenzyme transketolase-like 1 (TKTL1) by siRNA reversed theses effects of TIGAR. These findings suggest that glioma cells benefit from TIGAR expression by (i) improving energy yield from glucose via increased respiration and (ii) enhancing defense mechanisms against ROS. Targeting metabolic regulators such as TIGAR may therefore be a valuable strategy to enhance glioma cell sensitivity toward spontaneously occurring or therapy-induced starvation conditions or ROS-inducing therapeutic approaches

EGFR and mTOR as therapeutic targets in glioblastoma

The quest for new and improved therapies for glioblastoma (GB) has been mostly unsuccessful in more than a decade despite significant efforts. The few exceptions include the optimization of classical alkylating chemotherapy by including lomustine in the first line regimen for GB with a methylated MGMT promoter and tumor treating fields. The GB signaling network has been well-characterized and genetic alterations resulting in activation of receptor tyrosine kinases and especially epidermal growth factor receptor (EGFR) and downstream mammalian target of rapamycin complex 1 (mTORC1) signaling were found in the majority of GBs. ..

Knockdown of the TP53-induced glycolysis and apoptosis regulator (TIGAR) sensitizes glioma cells to hypoxia, irradiation and temozolomide

The TP53-induced glycolysis and apoptosis regulator (TIGAR) has been shown to decrease glycolysis, to activate the pentose phosphate pathway, and to provide protection against oxidative damage. Hypoxic regions are considered characteristic of glioblastoma and linked with resistance to current treatment strategies. Here, we established that LNT-229 glioma cell lines stably expressed shRNA constructs targeting TIGAR, and exposed them to hypoxia, irradiation and temozolomide. The disruption of TIGAR enhanced levels of reactive oxygen species and cell death under hypoxic conditions, as well as the effectiveness of irradiation and temozolomide. In addition, TIGAR was upregulated by HIF-1α. As a component of a complex network, TIGAR contributes to the metabolic adjustments that arise from either spontaneous or therapy-induced changes in tumor microenvironment