Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use..

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The worldwide epidemic of overweight and obesity has led to an increase in associated metabolic comorbidities. Obesity induces chronic low-grade inflammation in white adipose tissue (WAT). However, the function and regulation of both innate and adaptive immune cells in human WAT under conditions of obesity and calorie restriction (CR) is not fully understood yet. Using a randomized interventional design, we investigated postmenopausal overweight or obese female subjects who either underwent CR for 3 mo followed by a 4-wk phase of weight maintenance or had to maintain a stable weight over the whole study period. A comprehensive immune phenotyping protocol was conducted using validated multiparameter flow cytometry analysis in blood and s.c. WAT (SAT). The TCR repertoire was analyzed by next-generation sequencing and cytokine levels were determined in SAT. Metabolic parameters were determined by hyperinsulinemic-euglycemic clamp. We found that insulin resistance correlates significantly with a shift toward the memory T cell compartment in SAT. TCR analysis revealed a diverse repertoire in SAT of overweight or obese individuals. Additionally, whereas weight loss improved systemic insulin sensitivity in the intervention group, SAT displayed no significant improvement of inflammatory parameters (cytokine levels and leukocyte subpopulations) compared with the control group. Our data demonstrate the accumulation of effector memory T cells in obese SAT and an association between systemic glucose homeostasis and inflammatory parameters in obese females. The long-standing effect of obesity-induced changes in SAT was demonstrated by preserved immune cell composition after short-term CR-induced weight loss.

Obesity is associated with adipose tissue inflammation, insulin resistance, and the development of type 2 diabetes (T2D). However, our knowledge is mostly based on conventional murine models and promising preclinical studies rarely translated into successful therapies. There is a growing awareness of the limitations of studies in laboratory mice, housed in abnormally hygienic specific pathogen-free (SPF) conditions, as relevant aspects of the human immune system remain unappreciated. Here, we assessed the impact of housing conditions on adaptive immunity and metabolic disease processes during high-fat diet (HFD). We therefore compared diet-induced obesity in SPF mice with those housed in non-SPF, so-called “antigen exposed” (AE) conditions. Surprisingly, AE mice fed a HFD maintained increased insulin levels to compensate for insulin resistance, which was reflected in islet hyperplasia and improved glucose tolerance compared to SPF mice. By contrast, we observed higher proportions of effector/memory T cell subsets in blood and liver of HFD AE mice accompanied by the development of non-alcoholic steatohepatitis-like liver pathology. Thus, our data demonstrate the impact of housing conditions on metabolic alterations. Studies in AE mice, in which physiological microbial exposure was restored, could provide a tool for revealing therapeutic targets for immune-based interventions for T2D patients.

Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

IntroductionThe ubiquitous Epstein–Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen.MethodsWe used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus.ResultsApplying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow.DiscussionThe two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

Obesity is a growing global health problem due to its association with chronic low-grade inflammation contributing to metabolic complications. Multiple studies indicate that white adipose tissue (WAT) inflammation can drive the pathogenesis of type 2 diabetes, including altered levels of cells of the innate and adaptive immune system. However, the function and regulation of both innate and adaptive immune cells in human WAT under conditions of obesity and calorie restriction (CR) is not fully understood yet. Using a randomized interventional design, we investigated postmenopausal obese women who either underwent CR for three months followed by a 4 weeks phase of weight maintenance or had to maintain a stable weight over the whole study period. A comprehensive immune phenotyping protocol was conducted using validated multiparameter flow cytometry analysis in blood and subcutaneous WAT (SAT) (n=21). The T cell receptor repertoire was analyzed by next generation sequencing (n=20) and cytokine levels were determined in SAT (n=22). Metabolic parameters were determined by hyperinsulinemic-euglycemic clamp and then correlated to immune cell subsets. We found that insulin resistance (IR) correlates significantly with a shift towards the memory T cell compartment in SAT. Among various T cell subsets, predominantly CD8+ effector memory T cells were associated with obesity-related IR. Interestingly, T cell receptor analysis revealed a diverse repertoire in SAT arguing against an antigen-driven intra-SAT expansion of effector memory T cells. Surprisingly, neither inflammatory cytokine levels nor leucocyte subpopulations were significantly altered upon CR. Our findings demonstrate the accumulation of effector memory T cells in obese SAT contributing to chronic inflammation. The long-standing effect of obesity-induced changes in SAT was demonstrated by preserved immune cell composition after short-term CR induced weight loss.

Background Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Results Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro. Conclusions We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.