No abstract

NATure BIoMedIcAl eNgINeerINgdeveloped an approach for in-cell site-specific protein phosphorylation to synthesize bioactive proteins fused with a phosphorylated alum-binding peptide (ABP) tag. We used this approach to produce a series of ABP-labelled cytokines, which rapidly adsorbed to alum after simple mixing, and upon i.t. injection were retained in tumours for more than a week. Applied to the cytokine IL-12, this approach dramatically increased i.t. retention of the cytokine and eliminated systemic toxicities seen upon i.t. injection of the free drug, while also increasing anti-tumour efficacy. Moreover, a single i.t. dose of alum-anchored IL-12 elicited strong IFN-γ-dependent collaboration between innate and adaptive immune cells, producing robust systemic anti-tumour responses in multiple poorly immunogenic preclinical models when combined with systemic checkpoint blockade therapy. ResultsTargeted phosphorylation via an in-cell approach is robust. A single kinase, Fam20C, is responsible for phosphorylation of

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

Network theory is now a method of choice to gain insights in understanding protein structure, folding and function. In combination with molecular dynamics (MD) simulations, it is an invaluable tool with widespread applications such as analyzing subtle conformational changes and flexibility regions in proteins, dynamic correlation analysis across distant regions for allosteric communications, in drug design to reveal alternative binding pockets for drugs, etc. Updated version of NAPS now facilitates network analysis of the complete repertoire of these biomolecules, i.e., proteins, protein–protein/nucleic acid complexes, MD trajectories, and RNA. Various options provided for analysis of MD trajectories include individual network construction and analysis of intermediate time-steps, comparative analysis of these networks, construction and analysis of average network of the ensemble of trajectories and dynamic cross-correlations. For protein–nucleic acid complexes, networks of the whole complex as well as that of the interface can be constructed and analyzed. For analysis of proteins, protein–protein complexes and MD trajectories, network construction based on inter-residue interaction energies with realistic edge-weights obtained from standard force fields is provided to capture the atomistic details. Updated version of NAPS also provides improved visualization features, interactive plots and bulk execution. URL: http://bioinf.iiit.ac.in/NAPS/

CD19-targeted CAR therapies have successfully treated B cell leukemias and lymphomas, but many responders later relapse or experience toxicities. CAR intracellular domains (ICDs) are key to converting antigen recognition into anti-tumor effector functions. Despite the many possible immune signaling domain combinations that could be included in CARs, almost all CARs currently rely upon CD3𝛇, CD28, and/or 4-1BB signaling. To explore the signaling potential of CAR ICDs, we generated a library of 700,000 CD19 CAR molecules with diverse signaling domains and developed a high throughput screening platform to enable optimization of CAR signaling for antitumor functions. Our strategy identifies CARs with novel signaling domain combinations that elicit distinct T cell behaviors from a clinically available CAR, including enhanced proliferation and persistence, lower exhaustion, potent cytotoxicity in an in vitro tumor rechallenge condition, and comparable tumor control in vivo. This approach is readily adaptable to numerous disease models, cell types, and selection conditions, making it a promising tool for rapidly improving adoptive cell therapies and expanding their utility to new disease indications.