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Background Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn). Methods The extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM. Results The novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM. Conclusion In a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.

Background Chimeric antigen receptor (CAR) T-cells are a promising approach in cancer immunotherapy, particularly for treating hematologic malignancies. Yet, their effectiveness is limited when tackling solid tumors, where immune cell infiltration and immunosuppressive tumor microenvironments (TME) are major hurdles. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) and various tumor cells, playing an important role in tumor growth and immunosuppression. Aiming to modulate the TME with increased clinical safety and effectiveness, we developed novel small and size-extended immunotheranostic UniCAR target modules (TMs) targeting FAP. Methods The specific binding and functionality of the αFAP-scFv TM and the size-extended αFAP-IgG4 TM were assessed using 2D and 3D in vitro models as well as in vivo. Their specific tumor accumulation and diagnostic potential were evaluated using PET studies after functionalization with a chelator and suitable radionuclide. Results The αFAP-scFv and -IgG4 TMs effectively and specifically redirected UniCAR T-cells using 2D, 3D, and in vivo models. Moreover, a remarkably high and specific accumulation of radiolabeled FAP-targeting TMs at the tumor site of xenograft mouse models was observed. Conclusions These findings demonstrate that the novel αFAP TMs are promising immunotheranostic tools to foster cancer imaging and treatment, paving the way for a more convenient, individualized, and safer treatment of cancer patients. Graphical Abstract

A missense mutation, S85C, in the MATR3 gene is a genetic cause for amyotrophic lateral sclerosis (ALS). It is unclear how the S85C mutation affects MATR3 function and contributes to disease. Here, we develop a mouse model that harbors the S85C mutation in the endogenous Matr3 locus using the CRISPR/Cas9 system. MATR3 S85C knock-in mice recapitulate behavioral and neuropathological features of early-stage ALS including motor impairment, muscle atrophy, neuromuscular junction defects, Purkinje cell degeneration and neuroinflammation in the cerebellum and spinal cord. Our neuropathology data reveals a loss of MATR3 S85C protein in the cell bodies of Purkinje cells and motor neurons, suggesting that a decrease in functional MATR3 levels or loss of MATR3 function contributes to neuronal defects. Our findings demonstrate that the MATR3 S85C mouse model mimics aspects of early-stage ALS and would be a promising tool for future basic and preclinical research. MATR3 is an ALS-linked gene. Here the authors describe a mutant MATR3 knockin mouse, which mimics some aspects of early-stage ALS.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo .

We use 7 years (2010–2016) of methane column observations from the Greenhouse Gases Observing Satellite (GOSAT) to examine trends in atmospheric methane concentrations over North America and infer trends in emissions. Local methane enhancements above background are diagnosed in the GOSAT data on a 0.5∘×0.5∘ grid by estimating the local background as the low (10th–25th) percentiles of the deseasonalized frequency distributions of the data for individual years. Trends in methane enhancements on the 0.5∘×0.5∘ grid are then aggregated nationally and for individual source sectors, using information from state-of-science bottom-up inventories. We find that US methane emissions increased by 2.5±1.4  % a−1 (mean ± 1 standard deviation) over the 7-year period, with contributions from both oil–gas systems (possibly unconventional oil–gas production) and from livestock in the Midwest (possibly swine manure management). Mexican emissions show a decrease that can be attributed to a decreasing cattle population. Canadian emissions show year-to-year variability driven by wetland emissions and correlated with wetland areal extent. The US emission trends inferred from the GOSAT data account for about 20 % of the observed increase in global methane over the 2010–2016 period.

Background Adoptive transfer of regulatory T cells (Tregs) has demonstrated safety, feasibility and early signs of efficacy in promoting immunological tolerance in inflammatory conditions such as graft-versus-host disease (GvHD). Chimeric antigen receptor (CAR)-engineered Tregs offer localized activation and suppression compared to polyclonal Tregs, but their clinical translation is limited by high manufacturing costs, lengthy developing times and fixed single-antigen specificity. To address these limitations, we employed the universal adapter Reverse CAR (RevCAR) system, which harbors a peptide epitope lacking intrinsic antigen specificity but provides flexibility in targeting through the use of an antigen-specific RevCAR Target Module (RevTM). As a proof-of-concept, we used a RevTM targeting carcinoembryonic antigen (CEA), which is highly expressed in the gastrointestinal (GI) tract, as a potential strategy to achieve localized immunosuppression in GI acute GvHD. Methods To support clinical translation, we established an automated, GMP-compatible, clinical-scale manufacturing process. Tregs were magnetically enriched from leukapheresis using the CliniMACS ® Plus, followed by high-purity sorting on the MACSQuant ® Tyto ® . The sorted cells were virally transduced and the RevCAR Tregs were expanded on the CliniMACS Prodigy ® to obtain clinically relevant cell numbers. The harvested products were evaluated for phenotype, stability, antigen specificity and suppressive function. Results Across five manufacturing runs, Tregs (CD4 + CD25 high CD127 low FOXP3 + ) with a median initial purity of 94% were expanded to achieve a median therapeutic yield of 602 × 10 6 cells. The final product maintained a high purity (median: 91.9%) and exhibited high RevCAR expression (median: 60% RevCAR + ). Mass cytometry analysis revealed that expanded RevCAR Tregs predominantly exhibited a central memory phenotype with high expression of functional and homing markers. Under experimental pro-inflammatory conditions, the cells maintained stable FOXP3 and Helios expression with minimal pro-inflammatory cytokine production. Importantly, RevCAR Tregs showed antigen-specific activation upon target engagement via the CEA-specific RevTM and robust, dose-dependent suppression. Conclusion The study establishes a scalable, GMP-compatible process for manufacturing pure, stable and functional universal RevCAR Tregs for clinical applications. Furthermore, the RevCAR system offers a promising approach toward an allogenic, off-the-shelf Treg therapy capable of treating diverse immune-mediated diseases with spatial precision.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as “on-target, off-tumor” toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on hea9lthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA + and EpCAM + cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.

The current study examined the effect of concentrate supplementation on beef cattle performance and methane (CH 4 ) emissions in traditional pastoral systems in Tanzania. The study used summarized data (least square means, SEM, n) from a previous experimental study conducted in Tanzania in 2007-08, as the raw data are not available. The experiment involved 60 Boran and 60 Tanzanian Shorthorn Zebu (TSHZ) cattle, assigned to five dietary treatments for 100 days: grazing alone (GrazC00), grazing with 50% concentrate (GrazC50), and ad libitum hay with 60%, 80%, and 100% concentrate supplementation (HayC60, HayC80, and HayC100, respectively). The concentrate levels (50%, 60%, 80%, and 100%) were determined relative to the ad-libitum concentrate intake established before the experiment. The experiment measured dry matter intake (DMI) and live weights throughout the experimental periods. The current study calculated average daily weight gain (ADG) and daily CH 4 production (DMP) following the 2019 Refinement to the 2006 Intergovernmental Panel on Climate Change (IPCC) guidelines. The estimated CH 4 emissions were then used to calculate emission intensity (EI) relative to weight gain (EI WG, g CH 4 /kg WG). Statistical analysis used the lme4 package in R and the lm function to fit ANOVA with breed and treatment main effects and their interaction, with significance set at p < 0.05. Results showed that concentrate supplementation increased ADG by 6%–33% and decreased DMP by 3%–35%, regardless of breed. Among treatments, HayC100 had the lowest DMP, indicating greater efficiency at higher concentrate levels. In both breeds, EI decreased as concentrate levels increased from 0% to 100%, with the lowest EI WG observed in the HayC100 treatment (from 396 at GrazC00 to 87 g CH 4 /kg WG at HayC100). The reduction in EI WG was consistent across both breeds, showing that the supplementation effect was similar regardless of breed differences. Overall, concentrate supplementation improved cattle performance and reduced DMP, due to decreased CH 4 conversion factor (Ym), and lower EIs. These findings suggest that concentrate supplementation could be an effective strategy for enhancing beef production efficiency and reducing environmental impact in Tanzania.

Applying CAR T-cell therapy to treat solid tumors is especially challenging due to the immunosuppressive tumor microenvironment (TME). While our modular RevCAR system enhances the safety and controllability of CAR T-cell therapy, effectively targeting solid tumors remains difficult. Since PD-L1 is an immune checkpoint frequently upregulated by cancer cells and their microenvironment, it is a relevant target for solid tumors. Here, we introduce a novel PD-L1 RevTM capable of redirecting RevCAR T-cells to specifically target and kill PD-L1-expressing tumor cells, becoming activated and secreting pro-inflammatory cytokines. This is shown in vitro with monolayer and 3D models, including patient-derived cultures, and in vivo. Furthermore, we demonstrate in vitro and in vivo an AND-gated targeting of cells simultaneously expressing PD-L1 and another tumor-associated antigen by the Dual RevCAR system. Our findings suggest that RevCAR-mediated targeting of PD-L1 could be a promising therapeutic approach for modulating the TME and improving solid tumor treatment.

In Africa, cattle are often fed low quality tropical roughages resulting in low-yielding animals with high methane (CH 4 ) emission intensity (EI, g CH 4 /per unit of product). Supplementation with protein is known to improve the nutritive value of the otherwise low-quality diets. However, animal nutrition studies in East Africa that are accompanied by CH 4 emission measurements are lacking. Thus, an animal experiment was conducted to quantify the effect of supplementing cattle fed mainly on low-quality Urochloa brizantha hay (control diet; CON; crude protein (CP) = 7.4%) or supplemented with either a tannin-rich leguminous fodder, Desmodium intortum hay (DES) or a commercial dairy concentrate (CUBES) on voluntary dry matter intake (DMI), nutrient apparent total tract digestibility, nitrogen (N) retention, enteric CH 4 production and animal performance (milk and average daily gain). Twelve mid-lactating crossbred (Friesian × Boran) cows (initial liveweight = 335 kg) were used in a 3×3 (Period × Diet) Latin square design with each period running for four weeks. Compared to CON, DES decreased nutrient (DM, OM, CP) intake, apparent total tract digestibility and daily milk yield. In contrast, CUBES increased nutrient intake and animal performance compared to CON, while nutrients’ apparent total tract digestibility was not different, except for CP digestibility that increased. Compared to CON, DES and CUBES improved overall N retention by the animals as a proportion of N intake. The DES diet compared with CON and CUBES, shifted the proportion of N excretion via urine to the fecal route, likely because of its tannin content. Both DES and CUBES, compared to CON, reduced methane yield (MY, g CH 4 /kg DMI) by 15% and 9%, respectively. The DES diet reduced absolute enteric CH 4 emissions by 26% while CUBES increased emissions by 11% compared to CON. Based on the present findings, high supplementation levels (>50%) of Desmodium intortum hay is not recommended especially when the basal diet is low in CP content. Supplementation with lower levels of better managed Desmodium intortum forage however, need to be investigated to establish optimal inclusion levels that will improve animal productivity and reduce environmental impact of livestock in smallholder tropical contexts.