Single domain antibody multimers confer protection against rabies infection

Post-exposure prophylactic (PEP) neutralizing antibodies against Rabies are the most effective way to prevent infection-related fatality. The outer envelope glycoprotein of the Rabies virus (RABV) is the most significant surface antigen for generating virus-neutralizing antibodies. The small size and uncompromised functional specificity of single domain antibodies (sdAbs) can be exploited in the fields of experimental therapeutic applications for infectious diseases through formatting flexibilities to increase their avidity towards target antigens. In this study, we used phage display technique to select and identify sdAbs that were specific for the RABV glycoprotein from a naïve llama-derived antibody library. To increase their neutralizing potencies, the sdAbs were fused with a coiled-coil peptide derived from the human cartilage oligomeric matrix protein (COMP48) to form homogenous pentavalent multimers, known as combodies. Compared to monovalent sdAbs, the combodies, namely 26424 and 26434, exhibited high avidity and were able to neutralize 85-fold higher input of RABV (CVS-11 strain) pseudotypes in vitro, as a result of multimerization, while retaining their specificities for target antigen. 26424 and 26434 were capable of neutralizing CVS-11 pseudotypes in vitro by 90–95% as compared to human rabies immunoglobulin (HRIG), currently used for PEP in Rabies. The multimeric sdAbs were also demonstrated to be partially protective for mice that were infected with lethal doses of rabies virus in vivo. The results demonstrate that the combodies could be valuable tools in understanding viral mechanisms, diagnosis and possible anti-viral candidate for RABV infection

Retargeting T cells for HER2-positive tumor killing by a bispecific Fv-Fc antibody.

To exploit the biological and pharmacological properties of immunoglobulin constant domain Fc fragment and increase the killing efficacy of T cells, a single chain variable fragment specific to CD3 was fused with Fcab (Fc antigen binding), a mutant Fc fragment with specificity against Human epidermal growth factor receptor 2 (HER2) developed by F-star. The bispecific fusion named as FcabCD3 was expressed by transient transfection in HEK-293T cells and purified by affinity chromatography. Specific cytolytic activity of retargeted T cells to kill HER2 positive SKBR3 cell line was evaluated in vitro. FcabCD3 was able to retarget T cells to kill both Herceptin insensitive Colo205-luc cell line and HER2 low expression MDA-MB-231-luc cell line. Furthermore, FcabCD3 was effective in eliminating the Colo205 tumor established on BALB/c nu/nu mice

A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity <i>In Vivo</i>

<div><p>MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity <i>in vivo</i> by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.</p></div

Inhibition of miR-155-5p with antagomir-155 rapidly releases DTR expression.

<p>LN cells of CMV-Cre x R26-YFP × R26-DTR-155T mice founder 47 were stimulated with anti-CD3 for 48 hours. The activated cells were then transfected with 200 nM antagomir-155 or 200 nM antagomir-142 as negative control. FACS analysis was performed after 18 hours of transduction for detecting DTR expression. Left panel shows the dot plots of DTR expressing cells transduced with indicated antagomir. Middle panel shows the overlaid histograms of DTR expression between antagomir-155 and antagomir-142 transfected cells. Right panel shows DTR MFI of indicated cells. Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ****P<0.0001.</p

Validation of miR-155-5p-OFF system <i>in vitro</i>.

<p>(A) Validation of miR-155-5p target using luciferase assay, HEK293T cells were transfected with pmirGLO-4xmiR-155-5pT plus pEF-BOS-EX-miR-155, pEF-BOS-EX-miR-146a, and pEF-BOS-EX-empty vector (control), respectively, at a molar ratio of 1:1. (B) Validation of miR-155-5p target by flow cytometry, expression vector for miR-155 (pEF-BOS-EX-miR-155) or a control vector (pEF-BOS-EX) was co-transfected into HEK293T cells with an expression vector for miR-155-5p target reporter sequence (pDTR.BFP-155T) at various molar ratios. The expression of BFP reporter signal was analyzed through flow cytometry after 48 hours of transfection. (C and D) Sensitivity of miR-155-5p-OFF system. Fold change of mean fluorescent intensity of DTR expression in HEK293T cells transfected with pDTR.BFP-155T-N1 plasmid with increasing concentrations (0, 1.25, 2.5, 5, 10, 20, and 40 nM) of synthetic miR-155 or miR control. Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ns: not significant; *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001.</p

Generation and Characterization of miR-155-5p sensor transgenic mice.

<p>(A) Schematic illustration of the BAC transgenic constructs. Left panel-R26-DTR-BAC lacking miR-155-5p target sequence. Right panel-R26-DTR-155T-BAC containing 4×miR-155-5p target sequence in the 3’-UTR of DTR-BFP fusion gene. The fusion cassette gene was then placed downstream of a STOP element flanked by LoxP sites driven by Rosa26 promoter. (B) Schematic illustration of the determination of miR-155-5p activity in single living cell by flow cytometry. The relative activity of miR-155-5p will be inversely proportional to the expression level of DTR or BFP. (C) Phenotype of R26-DTR-155T mice. Figures in the upper panel show FACS profiling of DTR expression in conventional (conv) CD4-gated cells and Treg-gated cells of different mice as shown. Two founders, 47 and 84, of the R26-DTR-155T mice and a R26-DTR mouse were crossed with CMV-Cre mice; lymph node (LN) cells of the littermates were stained with anti-DTR-Biotin and then conjugated with Streptavidin-PE along with anti-CD4 and anti-Foxp3 (intracellular staining) and analyzed through FACS Calibur. Figures in the lower panel represent DTR MFI of indicated cells (shown in the figures of upper panel). Significance was calculated by Student’s <i>t</i>-test using GraphPad Prism 5 software. N = 3, ns: not significant; *P<0.05; **P<0.01.</p

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown. Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9–donor analog–acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases. [Figure not available: see fulltext.

FcabCD3 directed killing of tumor cells by T cells at different bsAb concentrations.

<p>Dose-response analysis of serial dilutions of FcabCD3, Fcab and Herceptin on (A) SKBR3, (B) Colo205-luc, (C) MDA-MB-231-luc , (D) K562-luc and B16F10-luc cells at E:T10. Values are mean ±SD of triplicates. Statistical analysis for difference between specific tumor cell lysis was analyzed by one-way ANOVA method using SPSS software (SPSS, p<0.05).</p

Anti-tumor effect of the FcabCD3 in a mouse tumor model.

<div><p>A, The tumor growth suppression by FcabCD3 on Colo205-luc cells was monitored by IVIS spectrum in nude mice. BALB/C strain nu/nu (n=10) mice were inoculated s.c. with Colo205-luc cells, and then randomly divided into two groups. On day1, 8 and 15, the mice were treated with FcabCD3 or human IgG (2µg per mouse) armed human T cells. On day 3, 10, 17, the tumor bioluminescence was measured;.</p> <p>B, The average value of relative light units (RLUs) was plotted against time (Days) in each group. Y-axis was in logarithmic scale.</p></div