A Novel Fusion of IL-10 Engineered to Traffic across Intestinal Epithelium to Treat Colitis.

IL-10 is a potent anti-inflammatory cytokine capable of suppressing a number of proinflammatory signals associated with intestinal inflammatory diseases, such as ulcerative colitis and Crohn's disease. Clinical use of human IL-10 (hIL-10) has been limited by anemia and thrombocytopenia following systemic injection, side effects that might be eliminated by a gut-restricted distribution. We have identified a transcytosis pathway used by cholix, an exotoxin secreted by nonpandemic forms of the intestinal pathogen Vibrio cholerae A nontoxic fragment of the first 386 aa of cholix was genetically fused to hIL-10 to produce recombinant AMT-101. In vitro and in vivo characterization of AMT-101 showed it to efficiently cross healthy human intestinal epithelium (SMI-100) by a vesicular transcytosis process, activate hIL-10 receptors in an engineered U2OS osteosarcoma cell line, and increase cellular phospho-STAT3 levels in J774.2 mouse macrophage cells. AMT-101 was taken up by inflamed intestinal mucosa and activated pSTAT3 in the lamina propria with limited systemic distribution. AMT-101 administered to healthy mice by oral gavage or to cynomolgus monkeys (nonhuman primates) by colonic spray increased circulating levels of IL-1R antagonist (IL-1Ra). Oral gavage of AMT-101 in two mouse models of induced colitis prevented associated pathological events and plasma cytokine changes. Overall, these studies suggest that AMT-101 can efficiently overcome the epithelial barrier to focus biologically active IL-10 to the intestinal lamina propria.

Membrane Association Transforms an Inert Anti-TCRß Fab' Ligand into a Potent T Cell Receptor Agonist.

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRß H57 Fab' ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab'-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab':TCR interactions are necessary to stimulate early T cell activation.

Extension of in vivo half-life of biologically active molecules by XTEN protein polymers.

XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.

DNA-mediated assembly of protein heterodimers on membrane surfaces.

We present a method based on self-assembling oligonucleotides to anchor proteins to a supported membrane surface. This anchoring method allows control of the surface density of multiple proteins. By incorporating additional recognition sequences into the DNA linkers, defined heterodimers can be produced upon the addition of a heterospecific DNA cross-linking strand. Characterization by fluorescence cross-correlation spectroscopy (FCCS) confirmed lateral mobility and the formation of specific heterodimers. We further demonstrate that proteins linked in this manner as either monomers or dimers can form functional interfaces with living cells.

Controlled integration of gold nanoparticles and organic fluorophores using synthetically modified MS2 viral capsids.

The placement of fluorophores in close proximity to metal nanoparticle surfaces is proposed to enhance several photophysical properties of the dyes, potentially leading to improved quantum yields and decreased photobleaching. It is difficult in practice, however, to establish and maintain the nanoscale distances that are required to maximize these effects. The type of metal, size, and shape of the nanoparticle, the physical distance separating the metal nanoparticle from the organic dye, and the spectral properties of the fluorophore itself are all proposed to influence the quantum yield and lifetime. This results in a complex behavior that can lead to either enhanced or quenched fluorescence in different contexts. In this report, we describe a well-defined system that can be used to explore these effects, while physically preventing the fluorophores from contacting the nanoparticle surfaces. The basis of this system is the spherical protein capsid of bacteriophage MS2, which was used to house gold particles within its interior volume. The exterior surface of each capsid was then modified with Alexa Fluor 488 (AF 488) labeled DNA strands. By placing AF 488 dyes at distances of 3, 12, and 24 bp from the surface of capsids containing 10 nm gold nanoparticles, fluorescence intensity enhancements of 2.2, 1.2, and 1.0 were observed, respectively. A corresponding decrease in fluorescence lifetime was observed for each distance. Because of its well-defined and modular nature, this architecture allows the rapid exploration of the many variables involved in metal-controlled fluorescence, leading to a better understanding of this phenomenon.

Supported membranes embedded with fixed arrays of gold nanoparticles.

We present a supported membrane platform consisting of a fluid lipid bilayer membrane embedded with a fixed array of gold nanoparticles. The system is realized by preforming a hexagonal array of gold nanoparticles (∼5-7 nm) with controlled spacing (∼50-150 nm) fixed to a silica or glass substrate by block copolymer lithography. Subsequently, a supported membrane is assembled over the intervening bare substrate. Proteins or other ligands can be associated with the fluid lipid component, the fixed nanoparticle component, or both, providing a hybrid interface consisting of mobile and immobile components with controlled geometry. We test different biochemical coupling strategies to bind individual proteins to the particles surrounded by a fluid lipid membrane. The coupling efficiency to nanoparticles and the influence of nanoparticle arrays on the surrounding membrane integrity are characterized by fluorescence imaging, correlation spectroscopy, and super-resolution fluorescence microscopy. Finally, the functionality of this system for live cell experiments is tested using the ephrin-A1-EphA2 juxtacrine signaling interaction in human breast epithelial cells.

Grade III radial collateral ligament injuries of the thumb metacarpophalangeal joint: treatment by soft tissue advancement and bony reattachment.

PURPOSE: The purpose of this study is to report the results of intact radial collateral ligament (RCL) soft tissue sleeve advancement with bony reattachment without additional soft tissue reinforcement as the sole surgical repair technique for the treatment of grade III RCL injuries to the thumb metacarpophalangeal (MCP) joint in acute, chronic, and late cases. METHODS: During a 20-year period, 38 of 45 patients (84%) with grade III RCL injuries were found to have tears at or adjacent to the metacarpal head (25 patients) or the proximal phalanx (13 patients) and underwent surgical repair at a mean of 10.8 months after injury. Patients were evaluated at a mean follow-up time of 3.8 years (minimum, 1 year), comparing pre- and postoperative standard thumb x-rays, varus (adduction) MCP joint stress x-rays, joint range of motion, pinch and grip strengths, clinical deformity, and subjective symptoms. RESULTS: Thirty-three patients (87%) were symptom free. Thirty-five patients (92%) regained normal pinch and grip strength. Thirty patients (79%) had full range of motion at the MCP joint, and no patient lost any interphalangeal joint motion. No recurrent symptomatic MCP joint instability was noted. Three patients (8%) had mild residual asymptomatic MCP joint volar subluxation. One patient had progression of pre-existing degenerative joint changes. CONCLUSION: Acute, chronic, and late grade III RCL instability of the thumb MCP joint can be successfully treated in the majority of cases by RCL soft tissue sleeve advancement and bony reattachment alone without the need for other soft tissue reinforcement.

Staged flexor tendon reconstruction fingertip to palm.

Thirty-five fingertip to palm staged flexor tendon reconstructions were performed between 1971 and 1998. Tendon injuries involved 5 avulsions and 30 lacerations, 9 of which had failed primary tenorrhaphies. Follow-up evaluation averaged 30 months. Thirteen patients had total active motion (TAM) of > or =220 degrees (excellent), 11 patients had TAM of 200 degrees to 219 degrees (good), 7 patients had TAM of 180 degrees to 199 degrees (fair), and 4 patients had TAM of <180 degrees (poor). Less favorable results occurred in those with a >1-year interval between injury and stage I, in those with a >6-month interval between stage I and stage II, and in those with a higher injury severity classification. Fingertip to palm staged flexor tendon reconstruction produced 69% good to excellent results. This technique allows the use of the injured digit profundus as the motor, preserves lumbrical function, and requires less tendon graft length (palmaris longus usually suffices).