Tumor-responsive, multifunctional CAR-NK cells cooperate with impaired autophagy to infiltrate and target glioblastoma

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking have rendered glioblastoma (GBM) highly resistant to therapy. As a result, GBM immunotherapies have failed to demonstrate sustained clinical improvements in patient overall survival (OS). To overcome these obstacles, here we describe a novel, sophisticated combinatorial platform for GBM: the first multifunctional immunotherapy based on genetically-engineered, human NK cells bearing multiple anti-tumor functions, including local tumor responsiveness, that addresses key drivers of GBM resistance to therapy: antigen escape, poor immune cell homing, and immunometabolic reprogramming of immune responses. We engineered dual-specific CAR-NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally-released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site specific activity in the tissue and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells, but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising new NK cell-based combinatorial strategy that can target multiple clinically-recognized mechanisms of GBM progression simultaneously

Where are we now with European forest multi-taxon biodiversity and where can we head to?

The European biodiversity and forest strategies rely on forest sustainable management (SFM) to conserve forest biodiversity. However, current sustainability assessments hardly account for direct biodiversity indicators. We focused on forest multi-taxon biodiversity to: i) gather and map the existing information; ii) identify knowledge and research gaps; iii) discuss its research potential. We established a research network to fit data on species, standing trees, lying deadwood and sampling unit description from 34 local datasets across 3591 sampling units. A total of 8724 species were represented, with the share of common and rare species varying across taxonomic classes: some included many species with several rare ones (e.g., Insecta); others (e.g., Bryopsida) were represented by few common species. Tree-related structural attributes were sampled in a subset of sampling units (2889; 2356; 2309 and 1388 respectively for diameter, height, deadwood and microhabitats). Overall, multi-taxon studies are biased towards mature forests and may underrepresent the species related to other developmental phases. European forest compositional categories were all represented, but beech forests were over-represented as compared to thermophilous and boreal forests. Most sampling units (94%) were referred to a habitat type of conservation concern. Existing information may support European conservation and SFM strategies in: (i) methodological harmonization and coordinated monitoring; (ii) definition and testing of SFM indicators and thresholds; (iii) data-driven assessment of the effects of environmental and management drivers on multi-taxon forest biological and functional diversity, (iv) multi-scale forest monitoring integrating in-situ and remotely sensed information

Association analyses identify 31 new risk loci for colorectal cancer susceptibility

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, and has a strong heritable basis. We report a genome-wide association analysis of 34,627 CRC cases and 71,379 controls of European ancestry that identifies SNPs at 31 new CRC risk loci. We also identify eight independent risk SNPs at the new and previously reported European CRC loci, and a further nine CRC SNPs at loci previously only identified in Asian populations. We use in situ promoter capture Hi-C (CHi-C), gene expression, and in silico annotation methods to identify likely target genes of CRC SNPs. Whilst these new SNP associations implicate target genes that are enriched for known CRC pathways such as Wnt and BMP, they also highlight novel pathways with no prior links to colorectal tumourigenesis. These findings provide further insight into CRC susceptibility and enhance the prospects of applying genetic risk scores to personalised screening and prevention

Design and characterisation of a prototype immobilised enzyme microreactor for the quantification of multi−step enzyme kinetics

The large number of novel biocatalyst candidates available due to advances in protein engineering and evolution has driven research on automated microwell techniques for rapid catalyst evaluation and quantification of enzyme kinetics and stability. Interest in the further reduction in volume to the microfluidic scale has complemented these microwell approaches for the development of bioprocess operations due to their potential as inexpensive analytical tools with minute volumes and high throughput as well as for their potential for mass replication. This project involves the design and characterisation of a prototype immobilised enzyme microreactor (IEMR) on the inner surface of a 200 μm ID fused silica capillary. Immobilisation is achieved through affinity-based interaction between His6- tags engineered on the transketolase (TK) and transaminase (TAm) enzyme variants and Ni-NTA groups on the derivatised capillary surface. The microreactor concept was validated with two reactions, namely the transketolase-catalysed conversion of hydroxypyruvate (HPA) and glycolaldehyde (GA) to produce L-erythrulose followed by the conversion of erythrulose to 2−amino−1,3,4−butanetriol (erythrulose−aminotriol) in the presence of methybenzylamine (MBA) by CV (Chromobacterium violaceum)-derived ω- transaminase. These keto- and aminodiol synthons are synthetically very useful in the production of a range of compounds with pharmaceutical application. The principles of stop-flow (batch) kinetics were initially investigated with respect to the catalytic performance of both enzymes, where the reaction was shown to depend on substrate concentration and residence time. TK kinetic parameters, evaluated based on a Michaelis−Menten model, in the IEMR (Vmax(app) = 0.1 ± 0.02 mmol.min-1, Km(app) = 26 ± 4 mM) were shown to be comparable to those measured in free solution. Furthermore, the kcat for the microreactor of 2.1 s−1 was similar to the value of 3.9 s−1 for the bioconversion in free solution. This was attributed to the controlled orientation and monolayer surface coverage of the His6−immobilised TK. Furthermore the quantitative elution of the immobilised TK and the regeneration and reuse of the derivatised capillary over 5 cycles were also demonstrated. Whilst slower than TK, the TAm reaction in the IEMR showed similar catalytic performance to a standard reaction in glass vials. Stopped−flow bioconversion results were complemented by continuous flow kinetics of the TK reaction with on-line UV detection (ActiPix, Paraytec), where the dependence of reaction kinetics on flow conditions was investigated. The Km(app), evaluated based on a continuous flow kinetic model, was shown to increase with flow rate, with the optimal being at the lowest flow rates used (0.2 μL.min-1). Furthermore, the value of Km(app) was shown to approach the value of the Michaelis constant of the free enzyme under zero flow (∼25 mM). The prototype microfluidic system was then implemented for the quantitative evaluation of multi-step TK-TAm bioconversion kinetics and the formation of chiral amino diol 2-amino-1,3,4- butanetriol (ABT) product from achiral substrates was demonstrated. The rate of accumulation of ABT (also referred to as EAT) by TAm was 0.02 mM.min-1.μgTAm -1, which was 4× slower than the rate of the TK−catalysed step. Demonstration of the synthesis of the product via the dual reaction and the monitoring of each component provided a full profile of the little known bioconversion and demonstrated the potential for creating novel multi−enzyme pathways in lab−on−a−chip systems, which further enables multi-substrate screening and screening of libraries of evolved enzymes of interest to be achieved rapidly and economically. This in vitro study of multi-step enzyme kinetics provides insight into the behaviour of these de novo engineered pathways to aid incorporation into suitable host cells

Quantification of kinetics for enzyme-catalysed reactions: implications for diffusional limitations at the 10 ml scale

The effects of different reaction scales [100 μl reactions in 96-standard round well (SRW) plates and 10 ml reactions in 24-square well (SW) plates] have been investigated using, as a model, transketolase (TK)-catalysed reaction producing l-erythrulose. Reactions were carried out under non-shaking, shaking and at 10 ml scale stirring conditions to assess the effect of diffusional limitations. Statistical analysis confirmed the significance of the observed difference in reaction rates under given conditions. Only when the laboratory scale system (10 ml) was well mixed did the reaction rate become comparable to that in the microwells, where there is negligible diffusional limitation. These findings have important implications for the scale-up (or scale-down) of enzyme-catalysed reactions. © 2008 Springer Science+Business Media B.V