On-target restoration of a split T cell-engaging antibody for precision immunotherapy

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (V-L) or variable heavy (V-H) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies

Two-step self-assembly of a spider silk molecular clamp

Molecular details that underlie mechanical properties of spider silk are of great interest to material scientists. Here, the authors report a previously unknown three-state mechanism of folding and an expanded structure of a spider silk protein that may contribute to elasticity of spider silk

Conservation of folding and association within a family of spidroin N-terminal domains

Web spiders synthesize silk fibres, nature’s toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce

Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk

Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function

Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk

Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider’s dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function