Role of the non-hypervariable FR3 D-E loop in single-domain antibody recognition of haptens and carbohydrates.

Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between ß-strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.

Listeria monocytogenes internalin B activates junctional endocytosis to accelerate intestinal invasion.

Listeria monocytogenes (Lm) uses InlA to invade the tips of the intestinal villi, a location at which cell extrusion generates a transient defect in epithelial polarity that exposes the receptor for InlA, E-cadherin, on the cell surface. As the dying cell is removed from the epithelium, the surrounding cells reorganize to form a multicellular junction (MCJ) that Lm exploits to find its basolateral receptor and invade. By examining individual infected villi using 3D-confocal imaging, we uncovered a novel role for the second major invasin, InlB, during invasion of the intestine. We infected mice intragastrically with isogenic strains of Lm that express or lack InlB and that have a modified InlA capable of binding murine E-cadherin and found that Lm lacking InlB invade the same number of villi but have decreased numbers of bacteria within each infected villus tip. We studied the mechanism of InlB action at the MCJs of polarized MDCK monolayers and find that InlB does not act as an adhesin, but instead accelerates bacterial internalization after attachment. InlB locally activates its receptor, c-Met, and increases endocytosis of junctional components, including E-cadherin. We show that MCJs are naturally more endocytic than other sites of the apical membrane, that endocytosis and Lm invasion of MCJs depends on functional dynamin, and that c-Met activation by soluble InlB or hepatocyte growth factor (HGF) increases MCJ endocytosis. Also, in vivo, InlB applied through the intestinal lumen increases endocytosis at the villus tips. Our findings demonstrate a two-step mechanism of synergy between Lm's invasins: InlA provides the specificity of Lm adhesion to MCJs at the villus tips and InlB locally activates c-Met to accelerate junctional endocytosis and bacterial invasion of the intestine.

The N-terminal ß-sheet of the hyperthermophilic endoglucanase from Pyrococcus horikoshii is critical for thermostability.

The ß-1,4-endoglucanase (EC 3.2.1.4) from the hyperthermophilic archaeon Pyrococcus horikoshii (EGPh) has strong hydrolyzing activity toward crystalline cellulose. When EGPh is used in combination with ß-glucosidase (EC 3.2.1.21), cellulose is completely hydrolyzed to glucose at high temperature, suggesting great potential for EGPh in bioethanol industrial applications. The crystal structure of EGPh shows a triosephosphate isomerase (TIM) (ß/α)(8)-barrel fold with an N-terminal antiparallel ß-sheet at the opposite side of the active site and a very short C-terminal sequence outside of the barrel structure. We describe here the function of the peripheral sequences outside of the TIM barrel core structure. Sequential deletions were performed from both N and C termini. The activity, thermostability, and pH stability of the expressed mutants were assessed and compared to the wild-type EGPh enzyme. Our results demonstrate that the TIM barrel core is essential for enzyme activity and that the N-terminal ß-sheet is critical for enzyme thermostability. Bioinformatics analyses identified potential key residues which may contribute to enzyme hyperthermostability.

Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors.

The PD-1/PD-L1 pathway is a key immune checkpoint that regulates T cell activation. There is strong rationale to develop PD-1 agonists as therapeutics against autoimmunity, but progress in this area has been limited. Here, we generated T cell receptor (TCR) targeting, PD-1 agonist bispecifics called ImmTAAI molecules that mimic the ability of PD-L1 to facilitate the colocalization of PD-1 with the TCR complex at the target cell-T cell interface. PD-1 agonist ImmTAAI molecules specifically bound to target cells and were highly effective in activating the PD-1 receptor on interacting T cells to achieve immune suppression. Potent PD-1 antibody ImmTAAI molecules closely mimicked the mechanism of action of endogenously expressed PD-L1 in their localization to the target cell-T cell interface, inhibition of proximal TCR signaling events, and suppression of T cell function. At picomolar concentrations, these bispecifics suppressed cytokine production and inhibited CD8+ T cell-mediated cytotoxicity in vitro. Crucially, in soluble form, the PD-1 ImmTAAI molecules were inactive and, hence, could avoid systemic immunosuppression. This study outlines a promising new route to generate more effective, potent, tissue-targeted PD-1 agonists that can inhibit T cell function locally with the potential to treat autoimmune and chronic inflammatory diseases of high unmet need.

A structural basis for interferon-alpha-receptor interactions.

Interferon (IFN)-alpha subtypes exhibit differences in biological potencies based on their affinity interactions with the IFN receptor subunits, IFNAR1 and IFNAR2. Using available three-dimensional structural information and computational biology, homology models of human IFN-alpha1, human IFN-alpha8, IFN alfacon-1, and murine IFN-alpha4 were derived and docked with the extracellular region of human IFNAR2 to evaluate the behavior of potential interacting residue pairs and characterize the nature of the IFN-IFNAR2 binding interfaces. The data suggest that IFN afacon-1 has 9 optimal interactions with IFNAR2, comprising hydrophobic, electrostatic, and hydrogen bonding. Human IFN-alpha2 exhibits 8 optimal interactions, human IFN-alpha1, 7, and murine IFN-alpha4 exhibits the least number of optimal interactions, at 5. A model of IFNAR1 was generated, taking into consideration the IFNAR1 extracellular domain interaction with cell surface glycosphingolipids, putative ligand interaction residues, and residues stabilizing the structural integrity of IFNAR. IFNAR1 was then docked with the various IFN-IFNAR2 complexes to describe the complete extracellular receptor pocket with bound IFN. These data provide insights into the species specificity of IFN-alphas: residues in murine IFN-alpha4 that preclude strong affinity interactions with human IFNAR because of steric crowding and residues in human IFN-alpha8 that resemble a receptor interactive domain in murine IFN-alpha4, are described.

Genomic DNA affinity chromatography: a technique to isolate interferon-inducible DNA binding factors.

Cytokines elicit responses in target cells by inducing changes in gene expression. For interferons (IFNs), this involves receptor-mediated activation of specific transcription factors, which then translocate into the nucleus to bind to cognate gene elements in the promoters of IFN-inducible genes. The prototypic IFN-inducible transcription factors are the signal transducer and activator of transcription (STAT) proteins. IFN-receptor interactions invoke Janus kinase activation via phosphorylation events, which in turn leads to the recruitment and phosphorylation of STAT proteins on tyrosine residues. Activated STATs then dimerize to form STAT complexes. IFNs-alpha/beta will activate STAT-1, STAT-2, STAT-3 ,and STAT-5, whereas IFN-gamma will predominantly activate STAT-1. In this chapter, we describe a procedure to identify IFN-inducible deoxyribonucleic acid (DNA) binding factors independently of any knowledge of their target DNA sequences. This procedure permits the identification of IFN-inducible STAT complexes as well as any other IFN-inducible DNA binding factors. This biochemical technique uses genomic DNA affinity chromatography to isolate DNA binding factors from IFN-inducible cytoplasmic or nuclear extracts.

High affinity anti-Internalin B VHH antibody fragments isolated from naturally and artificially immunized repertoires.

The need for rapid and easy technologies for the detection of food-borne and environmental pathogens is essential for safeguarding the health of populations. Furthermore, distribution of tainted food and water can have consequences which can affect whole economies. Antibodies and antibody fragments have been historically used in detection platforms due to their antigen specificity and robust physicochemical properties. In this study, we report the isolation and characterization of antibody fragments from the heavy chain antibody repertoire (VHH) of Camelidae which bind with specificity and high affinity to the Listeria monocytogenes invasin, Internalin B (InlB). To the best of our knowledge, this is the first report of anti-InlB VHHs from camelids. These anti-InlB VHHs were not cross-reactive to the structurally related Listeria invasin Internalin A (InlA) and are potential reagents to be used in the development of detection and medical technologies.

In situ proteolysis, crystallization and preliminary X-ray diffraction analysis of a VHH that binds listeria internalin B.

The variable region of camelid heavy-chain antibodies produces the smallest known antibody fragment with antigen-binding capability (a VHH). The VHH R303 binds internalin B (InlB), a virulence factor expressed by the pathogen Listeria monocytogenes. InlB is critical for initiation of Listeria infection, as it binds a receptor (c-Met) on epithelial cells, triggering the entry of bacteria into host cells. InlB is surface-exposed and is required for virulence, hence a VHH targeting InlB has potential applications for pathogen detection or therapeutic intervention. Here, the expression, purification, crystallization and X-ray diffraction of R303 are reported. Crystals of R303 were obtained following in situ proteolysis with trypsin. Gel filtration and SDS-PAGE revealed that trypsin removed the C-terminal tag region of R303, facilitating crystal formation. Crystals of R303 diffracted to 1.3 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a=46.4, b=31.2, c=74.8 Å, ß=93.8°. The crystals exhibited a Matthews coefficient of 1.95 Å3 Da(-1) with two molecules in the asymmetric unit.

Semiautomated panning of naive camelidae libraries and selection of single-domain antibodies against peptide antigens.

With the identification of vast numbers of novel proteins through genomic and proteomic initiatives, the need for efficient processes to characterize and target them has increased. Antibodies are naturally designed molecules that can fulfill this need, and in vitro methodologies for isolating them from either immune or naïve sources have been extensively developed. However, access to pure protein antigens for screening purposes is a major hurdle due to the limitations associated with recombinant production of eukaryotic proteins. Consequently, rational peptide design based on proteomic methodologies such as protein modeling, secondary sequence prediction, and hydrophobicity/hydrophilicity prediction, in combination with other bioinformatics data, is being explored as a viable solution to isolate specific antibodies against difficult antigens. Single-domain antibodies are becoming the ideal antibody format due to their structural advantages and ease of production compared to conventional antibodies and antibody fragments derived from conventional antibodies. For screening purposes, phage display technology is a well-established technique. With this technique, a repertoire of antibody fragments can be displayed on the surface of filamentous phages (f1, fd, M13) followed by screening against various antigenic targets. Furthermore, the technique can be expanded to a high-throughput scale using a magnetic-based, in-solution panning protocol which allows for the screening of multiple target antigens simultaneously. In this chapter, we describe a semiautomated panning method to screen a naïve Camelidae library against rationally designed peptide antigens, followed by preliminary characterization of isolated binders.

Investigation of the mechanism of binding between internalin B and heparin using surface plasmon resonance.

Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (>or=dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.