Rare,high-affinity mouse anti-PD-1 antibodies that function in checkpoint blockade,discovered using microfluidics and molecular genomics

Conventionally, mouse hybridomas or well-plate screening are used to identify therapeutic monoclonal antibody candidates. In this study, we present an alternative to hybridoma-based discovery that combines microfluidics, yeast single-chain variable fragment (scFv) display, and deep sequencing to rapidly interrogate and screen mouse antibody repertoires. We used our approach on six wild-type mice to identify 269 molecules that bind to programmed cell death protein 1 (PD-1), which were present at an average of 1 in 2,000 in the pre-sort scFv libraries. Two rounds of fluorescence-activated cell sorting (FACS) produced populations of PD-1-binding scFv with a mean enrichment of 800-fold, whereas most scFv present in the pre-sort mouse repertoires were de-enriched. Therefore, our work suggests that most of the antibodies present in the repertoires of immunized mice are not strong binders to PD-1. We observed clusters of related antibody sequences in each mouse following FACS, suggesting evolution of clonal lineages. In the pre-sort repertoires, these putative clonal lineages varied in both the complementary-determining region (CDR)3K and CDR3H, while the FACS-selected PD-1-binding subsets varied primarily in the CDR3H. PD-1 binders were generally not highly diverged from germline, showing 98% identity on average with germline V-genes. Some CDR3 sequences were discovered in more than one animal, even across different mouse strains, suggesting convergent evolution. We synthesized 17 of the anti-PD-1 binders as full-length monoclonal antibodies. All 17 full-length antibodies bound recombinant PD-1 with K_D < 500 nM (average = 62 nM). Fifteen of the 17 full-length antibodies specifically bound surface-expressed PD-1 in a FACS assay, and nine of the antibodies functioned as checkpoint inhibitors in a cellular assay. We conclude that our method is a viable alternative to hybridomas, with key advantages in comprehensiveness and turnaround time.     

A radioimmunoassay for arthropod moulting hormones, introducing a novel method of immunogen coupling

Inokosteron-26-oic acid was coupled to thyroglobulin in aqueous pyridine by a water-soluble carbodiimide. After exhaustive dialysis and gel filtration on Sephadex G-25 in the presence of sodium dodecylsulfate, a coupling ratio of 164 haptens per molecule of thyroglobulin was determined. In all three animals injected with the conjugate, ecdysone-binding antibodies were detected. After one booster injection the antiserum could be diluted 1 : 5000 (1 : 4000, or 1 : 2000) in order to get a 50% binding of [3H]ecdysone. The dissociation constant was calculated as 5.8 X 10(-10) MOL/L. The antiserum has a greater affinity for ecdysone and 22-isoecdysone than for all other ecdysteroids and steroids tested.     

Synthetic riboswitches that induce gene expression in diverse bacterial species

We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.     

Bacterial community shifts in taxa and diversity in response to localized organic loading in the deep sea

The deep sea is a unique and extreme environment characterized by low concentrations of highly recalcitrant carbon. As a consequence, large organic inputs have potential to cause significant perturbation. To assess the impact of organic enrichment on deep sea microbial communities, we investigated bacterial diversity in sediments underlying two whale falls at 1820 and 2893 m depth in Monterey Canyon, as compared with surrounding reference sediment 10–20 m away. Bacteroidetes, Epsilonproteobacteria and Firmicutes were recovered primarily from whale fall‐associated sediments, while Gammaproteobacteria and Planctomycetes were found primarily within reference sediments. Abundant Deltaproteobacteria were recovered from both sediment types, but the Desulfobacteraceae and Desulfobulbaceae families were observed primarily beneath the whale falls. UniFrac analysis revealed that bacterial communities from the two whale falls (～30 km apart) clustered to the exclusion of corresponding reference sediment communities, suggesting that deposition of whale fall biomass is more influential on deep sea microbial communities than specific seafloor location. The bacterial population at whale‐1820 at 7 months post deposition was less diverse than reference sediments, with Delta‐ and Epsilonproteobacteria and Bacteroidetes making up 89% of the community. At 70 months, bacterial diversity in reference sediments near whale‐2893 had decreased as well. Over this time, there was a convergence of each community's membership at the phyla level, although lower‐taxonomic‐level composition remained distinct. Long‐term impact of organic carbon loading from the whale falls was also evident by elevated total organic carbon and enhanced proteolytic activity for at least 17–70 months. The response of the sedimentary microbial community to large pulses of organic carbon is complex, likely affected by increased animal bioturbation, and may be sustained over time periods that span years to perhaps even decades.     

Differential Diffusional Properties in Loose and Tight Docking Prior to Membrane Fusion

Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps, including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study because of their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here, we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.     

An apparent core/shell architecture of polyQ aggregates in the aging Caenorhabditis elegans neuron

Huntington''s disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein which results in its abnormal aggregation in the nervous system. Huntingtin aggregates are linked to toxicity and neuronal dysfunction, but a comprehensive understanding of the aggregation mechanism in vivo remains elusive. Here, we examine the morphology of polyQ aggregates in Caenorhabditis elegans mechanosensory neurons as a function of age using confocal and fluorescence lifetime imaging microscopy. We find that aggregates in young worms are mostly spherical with homogenous intensity, but as the worm ages aggregates become substantially more heterogeneous. Most prominently, in older worms we observe an apparent core/shell morphology of polyQ assemblies with decreased intensity in the center. The fluorescence lifetime of polyQ is uniform across the aggregate indicating that the dimmed intensity in the assembly center is most likely not due to quenching or changes in local environment, but rather to displacement of fluorescent polyQ from the central region. This apparent core/shell architecture of polyQ aggregates in aging C. elegans neurons contributes to the diverse landscape of polyQ aggregation states implicated in Huntington''s disease.