Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment.

In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. Here we present INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment.

Glycan masking in vaccine design: Targets, immunogens and applications.

Glycan masking is a novel technique in reverse vaccinology in which sugar chains (glycans) are added on the surface of immunogen candidates to hide regions of low interest and thus focus the immune system on highly therapeutic epitopes. This shielding strategy is inspired by viruses such as influenza and HIV, which are able to escape the immune system by incorporating additional glycosylation and preventing the binding of therapeutic antibodies. Interestingly, the glycan masking technique is mainly used in vaccine design to fight the same viruses that naturally use glycans to evade the immune system.

Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region.

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies.

Modeling Immunity with Rosetta: Methods for Antibody and Antigen Design.

Structure-based antibody and antigen design has advanced greatly in recent years, due not only to the increasing availability of experimentally determined structures but also to improved computational methods for both prediction and design. Constant improvements in performance within the Rosetta software suite for biomolecular modeling have given rise to a greater breadth of structure prediction, including docking and design application cases for antibody and antigen modeling. Here, we present an overview of current protocols for antibody and antigen modeling using Rosetta and exemplify those by detailed tutorials originally developed for a Rosetta workshop at Vanderbilt University.

HoLaMa: A Klenow sub-fragment lacking the 3'-5' exonuclease domain.

The design, construction, overexpression, and purification of a Klenow sub-fragment lacking the 3'-5' exonuclease domain is presented here. In particular, a synthetic gene coding for the residues 515-928 of Escherichia coli DNA polymerase I was constructed. To improve the solubility and stability of the corresponding protein, the synthetic gene was designed to contain 11 site-specific substitutions.