Computational Design of Phosphotriesterase Improves V-Agent Degradation Efficiency.

Organophosphates (OPs) are a class of neurotoxic acetylcholinesterase inhibitors including widely used pesticides as well as nerve agents such as VX and VR. Current treatment of these toxins relies on reactivating acetylcholinesterase, which remains ineffective. Enzymatic scavengers are of interest for their ability to degrade OPs systemically before they reach their target. Here we describe a library of computationally designed variants of phosphotriesterase (PTE), an enzyme that is known to break down OPs. The mutations G208D, F104A, K77A, A80V, H254G, and I274N broadly improve catalytic efficiency of VX and VR hydrolysis without impacting the structure of the enzyme. The mutation I106 A improves catalysis of VR and L271E abolishes activity, likely due to disruptions of PTE's structure. This study elucidates the importance of these residues and contributes to the design of enzymatic OP scavengers with improved efficiency.

Targeting Macrophages with CAR T Cells Delays Solid Tumor Progression and Enhances Antitumor Immunity.

Tumor-associated macrophages (TAM) are one of the most abundant cell types in many solid tumors and typically exert protumor effects. This has led to an interest in macrophage-depleting agents for cancer therapy, but approaches developed to date have had limited success in clinical trials. Here, we report the development of a strategy for TAM depletion in mouse solid tumor models using chimeric antigen receptor (CAR) T cells targeting the macrophage marker F4/80 (F4.CAR-T). F4.CAR-T cells effectively killed macrophages in vitro and in vivo without toxicity. When injected into mice bearing orthotopic lung tumors, F4.CAR-T cells infiltrated tumor lesions and delayed tumor growth comparably with PD-1 blockade, and significantly extended mouse survival. Antitumor effects were mediated by F4.CAR-T-produced IFNγ, which promoted upregulation of MHC molecules on cancer cells and tumor-infiltrating myeloid cells. Notably, F4.CAR-T promoted expansion of endogenous CD8 T cells specific for tumor-associated antigen and led to immune editing of highly antigenic tumor cell clones. Antitumor impact was also observed in mouse models of ovarian and pancreatic cancer. These studies provide proof of principle to support CAR T-cell targeting of TAMs as a means to enhance antitumor immunity.

Mosquito-larvicidal binary toxin receptor protein (Cqm1): crystallization and X-ray crystallographic analysis.

Cqm1 from Culex quinquefasciatus has been identified as the receptor for Lysinibacillus sphaericus binary toxin (BinAB). It is an amylomaltase that is presented on the epithelial membrane in the larval midgut through a glycosyl-phosphatidylinositol anchor. The active core of this protein (residues 23-560) was overexpressed in Escherichia coli, purified and successfully crystallized by the sitting-drop vapor-diffusion method using D-arabinose and CaCl2 as additives, as identified using high-throughput differential scanning fluorimetry analysis. X-ray diffraction data were collected to a resolution of 2.8 Šusing a laboratory X-ray source. The crystals had the symmetry of space group P212121, with unit-cell parameters a = 191.3, b = 205.3, c = 59.0 Šand with four monomers in the asymmetric unit. Structure refinement is in progress. This is the first structure report for a binary toxin receptor and for a member of the GH13_17 subfamily in the CAZy database.