Self-reactive IgE exacerbates interferon responses associated with autoimmunity.

Canonically, immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE), IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs), a type of cell of the immune system linked to viral defense, which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE, followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.

Experimentally guided computational antibody affinity maturation with de novo docking, modelling and rational design.

Antibodies are an important class of therapeutics that have significant clinical impact for the treatment of severe diseases. Computational tools to support antibody drug discovery have been developing at an increasing rate over the last decade and typically rely upon a predetermined co-crystal structure of the antibody bound to the antigen for structural predictions. Here, we show an example of successful in silico affinity maturation of a hybridoma derived antibody, AB1, using just a homology model of the antibody fragment variable region and a protein-protein docking model of the AB1 antibody bound to the antigen, murine CCL20 (muCCL20). In silico affinity maturation, together with alanine scanning, has allowed us to fine-tune the protein-protein docking model to subsequently enable the identification of two single-point mutations that increase the affinity of AB1 for muCCL20. To our knowledge, this is one of the first examples of the use of homology modelling and protein docking for affinity maturation and represents an approach that can be widely deployed.

The TGF-ß inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan.

Excessive transforming growth factor (TGF)-ß is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-ß activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-ß inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-ß activation utilized by an αvß8 targeting antibody, 37E1B5. This antibody blocks TGF-ß activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-ß activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibody's ability to inhibit latency-associated peptide (LAP) and αvß8 association, and TGF-ß activation in an αvß8-mediated TGF-ß signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvß8-mediated TGF-ß activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.

On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL.

COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.

The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators.

Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C(H)3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcgamma receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcgamma receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.

Structural characterization of a human Fc fragment engineered for lack of effector functions.

The first three-dimensional structure of a human Fc fragment genetically engineered for the elimination of its ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is reported. When introduced into the lower hinge and CH2 domain of human IgG1 molecules, the triple mutation L234F/L235E/P331S ('TM') causes a profound decrease in their binding to human CD64, CD32A, CD16 and C1q. Enzymatically produced Fc/TM fragment was crystallized and its structure was solved at a resolution of 2.3 A using molecular replacement. This study revealed that the three-dimensional structure of Fc/TM is very similar to those of other human Fc fragments in the experimentally visible region spanning residues 236-445. Thus, the dramatic broad-ranging effects of TM on IgG binding to several effector molecules cannot be explained in terms of major structural rearrangements in this portion of the Fc.

Capping transmembrane helices of MscL with aromatic residues changes channel response to membrane stretch.

Tyrosines and tryptophans that anchor both ends of the helices to membrane interfaces in many transmembrane proteins are not common in MscL and homologous mechanosensitive channels. This characteristic absence of two aromatic "belts" may be critical for MscL function as the opening transition is predicted to be associated with a strong helical reorientation. A single tyrosine (Y75) on the extracellular side of the M2 helix of pentameric EcoMscL is absent in TbMscL, which instead has a single tyrosine (Y87) on the cytoplasmic side of M2. Moving the tyrosine of EcoMscL to the intracellular side (Y75F/F93Y) or capping the TM2 helix on both sides (F93Y/W) slows the kinetics of gating and increases the threshold for activation, leading to a partial loss-of-function in osmotic shock survival assays. Increasing the distance between the caps (L98W, L102Y/W) partially restores channel function presumably by loosening restraints for tilting. Capping the TM2 helix with a charged residue (Y75E) causes a right shift of the activation curve ("stiff" phenotype) and abolishes function. Introducing a "cap" into the TM1 helix (I41W) decreases the activation threshold and shortens the mean open time but unexpectedly leads to a complete loss-of-function in vivo. The data are consistent with the view that restraining helical positions in MscL by introducing specific protein-lipid interactions at membrane interfaces compromises MscL function. Subtle differences in osmotic shock survival are more evident at low levels of mutant protein expression. We observed a correlation between the right shift of tension activation threshold and the loss-of-function channel phenotype, with a few exceptions that point to other parameters of gating that may define the osmotic rescuing ability in vivo.