Development of spirulina for the manufacture and oral delivery of protein therapeutics.

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina.

Design of functionalised circular tandem repeat proteins with longer repeat topologies and enhanced subunit contact surfaces.

Circular tandem repeat proteins ('cTRPs') are de novo designed protein scaffolds (in this and prior studies, based on antiparallel two-helix bundles) that contain repeated protein sequences and structural motifs and form closed circular structures. They can display significant stability and solubility, a wide range of sizes, and are useful as protein display particles for biotechnology applications. However, cTRPs also demonstrate inefficient self-assembly from smaller subunits.

Designed protein logic to target cells with precise combinations of surface antigens.

Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells using specific combinations of proteins present on their surfaces. In this study, we design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations.

A potent peptide-steroid conjugate accumulates in cartilage and reverses arthritis without evidence of systemic corticosteroid exposure.

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints.

Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes.

Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12).

Publisher Correction: Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets.

In the original version of this Article the colour key for the amino acid enrichment score was inadvertently omitted from the lower panel of Figure 5b during the production process. This has now been corrected in the PDF and HTML versions of the Article.

Screening, large-scale production and structure-based classification of cystine-dense peptides.

Peptides folded through interwoven disulfides display extreme biochemical properties and unique medicinal potential. However, their exploitation has been hampered by the limited amounts isolatable from natural sources and the expense of chemical synthesis. We developed reliable biological methods for high-throughput expression, screening and large-scale production of these peptides: 46 were successfully produced in multimilligram quantities, and >600 more were deemed expressible through stringent screening criteria.

Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets.

Protein:protein interactions are among the most difficult to treat molecular mechanisms of disease pathology. Cystine-dense peptides have the potential to disrupt such interactions, and are used in drug-like roles by every clade of life, but their study has been hampered by a reputation for being difficult to produce, owing to their complex disulfide connectivity. Here we describe a platform for identifying target-binding cystine-dense peptides using mammalian surface display, capable of interrogating high quality and diverse scaffold libraries with verifiable folding and stability.

Control of Tumor Initiation by NKG2D Naturally Expressed on Ovarian Cancer Cells.

Cancer cells may co-opt the NKG2D lymphocyte receptor to complement the presence of its ligands for autonomous stimulation of oncogenic signaling. Previous studies raise the possibility that cancer cell NKG2D may induce high malignancy traits, but its full oncogenic impact is unknown. Using epithelial ovarian cancer as model setting, we show here that ex vivo NKG2D cancer cells have stem-like capacities, and provide formal in vivo evidence linking NKG2D stimulation with the development and maintenance of these functional states.

Anti-CD28 Antibody-Initiated Cytokine Storm in Canines.

Background: CD28 signal blockade following T cell receptor activation is under intense investigation as a tolerance-inducing therapy for transplantation. Our goal is to produce a CD28-specific reagent as a therapy for the prevention of graft rejection and graft-versus-host disease in the canine model of allogeneic hematopoietic cell transplantation (HCT).

Methods: We infused a monoclonal mouse anti-canine CD28 antibody (1C6 mAb) into four dogs and a fragment of antigen-binding (1C6 Fab) into two dogs.

Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10.

The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors.