An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates.

The XpressCF+® cell-free protein synthesis system is a robust platform for the production of non-natural amino acids containing antibodies, which enable the site-specific conjugation of homogeneous antibody drug conjugates (ADCs) via click chemistry. Here, we present a robust and scalable means of achieving a 50-100% increase in IgG titers by combining the high productivity of cell-based protein synthesis with the unique ability of XpressCF+® reactions to produce correctly folded and assembled IgGs containing multiple non-natural amino acids at defined positions. This hybrid technology involves the pre-expression of an IgG light-chain (LC) protein in a conventional recombinant E. coli expression system, engineered to have an oxidizing cytoplasm. The prefabricated LC subunit is then added as a reagent to the cell-free protein synthesis reaction. Prefabricated LC increases IgG titers primarily by reducing the protein synthesis burden per IgG since the cell free translation machinery is only responsible for synthesizing the HC protein. Titer increases were demonstrated in four IgG products in scales ranging from 100-µL microplate reactions to 0.25-L stirred tank bioreactors. Similar titer increases with prefabricated LC were also demonstrated for a bispecific antibody in the scFvFc-FabFc format, demonstrating the generality of this approach. Prefabricated LC also increases robustness in cell-free reactions since it eliminates the need to fine-tune the HC-to-LC plasmid ratio, a critical parameter influencing IgG assembly and quality when the two IgG subunits are co-expressed in a single reaction. ADCs produced using prefabricated LC were shown to be identical to IgGs produced in cell-free alone by comparing product quality, in vitro cell killing, and FcRn receptor binding assays. This approach represents a significant step towards improving IgG titers and the robustness of cell-free protein synthesis reactions by integrating in vivo and in vitro protein production platforms.

Stochastic effects are important in intrahost HIV evolution even when viral loads are high.

Blood plasma viral loads and the time to progress to AIDS differ widely among untreated HIV-infected humans. Although people with certain HLA (HLA-I) alleles are more likely to control HIV infections without therapy, the majority of such untreated individuals exhibit high viral loads and progress to AIDS. Stochastic effects are considered unimportant for evolutionary dynamics in HIV-infected people when viral load is high or when selective forces strongly drive mutation. We describe a computational study of host-pathogen interaction demonstrating that stochastic effects can have a profound influence on disease dynamics, even in cases of high viral load and strong selective pressure. These stochastic effects are pronounced when the virus must traverse a fitness "barrier" in sequence space to escape the host's cytotoxic T-lymphocyte (CTL) response, as often occurs when a fitness defect imposed by a CTL-driven mutation must be compensated for by other mutations. These "barrier-crossing" events are infrequent and stochastic, resulting in divergent disease outcomes in genetically identical individuals infected by the same viral strain. Our results reveal how genetic determinants of the CTL response control the probability with which an individual is able to control HIV infection indefinitely, and thus provide clues for vaccine design.

Flow-Dependent Unfolding and Refolding of an RNA by Nonequilibrium Umbrella Sampling.

Nonequilibrium experiments of single biomolecules such as force-induced unfolding reveal details about a few degrees of freedom of a complex system. Molecular dynamics simulations can provide complementary information, but exploration of the space of possible configurations is often hindered by large barriers in phase space that separate metastable regions. To solve this problem, enhanced sampling methods have been developed that divide a phase space into regions and integrate trajectory segments in each region. These methods boost the probability of passage over barriers and facilitate parallelization since integration of the trajectory segments does not require communication, aside from their initialization and termination. Here, we present a parallel version of an enhanced sampling method suitable for systems driven far from equilibrium: nonequilibrium umbrella sampling (NEUS). We apply this method to a coarse-grained model of a 262-nucleotide RNA molecule that unfolds and refolds in an explicit flow field modeled with stochastic rotation dynamics. Using NEUS, we are able to observe extremely rare unfolding events that have mean first passage times as long as 45 s (1.1 × 10(15) dynamics steps). We examine the unfolding process for a range of flow rates of the medium, and we describe two competing pathways in which different intramolecular contacts are broken.