Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

Genetic engineering is an important tool for redirecting the function of various types of immune cells and their use for therapeutic purpose. Although NK cells have many beneficial therapeutic features, genetic engineering of immune cells for targeted therapy focuses mostly on T cells. One of the major obstacles for NK cell immunotherapy is the lack of an efficient method for gene transfer. Lentiviral vectors have been proven to be a safe tool for genetic engineering, however lentiviral transduction is inefficient for NK cells. We show in this study that lentiviral vector pseudotyped with a modified baboon envelope glycoprotein can transduce NK cells 20-fold or higher in comparison to VSV-G pseudotyped lentiviral vector. When we investigated the mechanism of transduction, we found that activated NK cells expressed baboon envelop receptor ASCT-2. Further analysis revealed that only a subset of NK cells could be expanded and transduced with an expression profile of NK56bright, CD16dim, TRAILhigh and CX3CR1neg. Using CD19-CAR, we could show that CD19 redirected NK cells efficiently and specifically kill cell lines expressing CD19. Taken together, result from this study will be important for future genetic modification and redirecting of NK cell function for therapeutic purpose.

Meeting abstracts Adoptive immunotherapy with chimeric antigen receptor (CAR) transduced T lymphocytes has shown promising results in both pediatric and adult B cell malignancies. Nevertheless, both CAR-based and antibody-based anti-CD19 therapies, e.g. blinatumomab, have seen treatment failures

Meeting abstracts Previous studies in adoptive T-cell transfer have suggested that persistence of the transduced T-cells is central to making this therapy a viable option. Understanding the behavior of tumor-reactive T-cells in cancer patients and measuring persistence are two objectives in a phase

Patients with Pompe disease, a rare progressive neuromuscular disorder, receive a considerable amount of informal care. In this study, we examined the impact of providing informal care to patients with Pompe disease.Caregivers were administered various instruments, which measured the (impact of) informal care in the context of Pompe disease. Patients' quality of life and use of a wheelchair and respiratory support were used to investigate the impact of disease severity on the burden and well-being of caregivers.Of all Dutch patients with Pompe disease, 88 indicated to receive informal care, of which 67 (76%; 67 caregivers) participated in this study. On average, caregivers provided 17.7 hours of informal care per week. Higher disease burden was associated with more hours of informal care. Caregivers experienced burden due to caregiving. Half of the informal caregivers reported mental health problems and problems with daily activities due to providing informal care. Physical health problems occurred in 40% of informal caregivers. Caregiver burden was higher for patients with a lower quality of life and for wheelchair dependent patients. Burden was not associated with respiratory support. Caregivers reported deriving personal fulfillment from caregiving and, on average, would become unhappier if someone else were to take over their care activities.The provision of informal care causes burden to caregivers. However, caregivers also value caring for their loved ones themselves. The study may help physicians and policy makers to design measures to support informal caregivers.

Lentiviral vectors (LVs) are widely recognized as the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Using erythropoietin (EPO), recombinant factor VIII (fVIII), and an anti-CD20 antibody as model proteins, we demonstrate advantages of LV-based gene delivery to achieve high production levels by transduced cells. Highly productive cell clones were able to incorporate up to 100 vector copies per cellular genome, without selection or gene amplification, and were isolated without extensive screening of a large number of clones. The LV transgenes were shown to be distributed throughout the genome, as visualized by fluorescent in situ hybridization. High-expressing clones producing 100-200 pg/cell/day of EPO were isolated and characterized. EPO production was demonstrated for at least 5½ months of continuous culture without selection, during which all the clones displayed high levels of glycosylation despite production levels at 10-20 g/liter. To demonstrate the utility of LV technology for multiple classes of proteins, cell lines producing fVIII and an anti-CD20 antibody were also developed. Cell clones demonstrating high levels of fVIII (100 clot units/ml and anti-CD20 antibody as high as 40-100 pg/cell/day) were isolated and characterized. LV-transduced cells and plasmid-transfected cells were compared for protein production per transgene copy. LV-transduced cells produced significantly higher levels of protein per copy of transgene than plasmid-transfected cells did. This study demonstrates the utility of LV technology for rapid generation of highly productive and stable cell lines over conventional plasmid transfection methods, significantly decreasing the time, cost, and risk of the manufacture of proteins and other complex biological molecules.