AAV-mediated expression of anti-tau scFvs decreases tau accumulation in a mouse model of tauopathy.

Tauopathies are characterized by the progressive accumulation of hyperphosphorylated, aggregated forms of tau. Our laboratory has previously demonstrated that passive immunization with an anti-tau antibody, HJ8.5, decreased accumulation of pathological tau in a human P301S tau-expressing transgenic (P301S-tg) mouse model of frontotemporal dementia/tauopathy. To investigate whether the Fc domain of HJ8.5 is required for the therapeutic effect, we engineered single-chain variable fragments (scFvs) derived from HJ8.5 with variable linker lengths, all specific to human tau. Based on different binding properties, we selected two anti-tau scFvs and tested their efficacy in vivo by adeno-associated virus-mediated gene transfer to the brain of P301S-tg mice. The scFvs significantly reduced levels of hyperphosphorylated, aggregated tau in brain tissue of P301S-tg mice, associated with a decrease in detergent-soluble tau species. Interestingly, these mice showed substantial levels of scFvs in the cerebrospinal fluid without significant effects on total extracellular tau levels. Therefore, our study provides a novel strategy for anti-tau immunotherapeutics that potentially limits a detrimental proinflammatory response.

Overexpression of cryoglobulin-like single-chain antibody induces morular cell phenotype via liquid-liquid phase separation in the secretory pathway organelles.

Cryoprecipitation of immunoglobulins is often reported in association with B-cell lymphoproliferative disorders and plasma cell dyscrasias. However, the biochemical basis of such cryoglobulin behaviors is not well understood because of a general lack of suitable experimental systems. Here, we report the identification and characterization of a single-chain antibody (scFv-Fc) that recapitulates cryoglobulin-like properties. When model scFv-Fc protein was engineered to multimerize, by appending the secretory tailpiece (stp) of human immunoglobulin µ-chain to the C terminus, the resulting oligomeric scFv-Fc-stp protein acquired two unexpected properties: the induction of a morular cell phenotype during protein biosynthesis and the cryoprecipitation of secreted proteins in harvested cell culture media. The turbidity of the culture media and the inclusion bodies that gave morular appearances were attributed to microscopic spherical protein droplet formation, a hallmark characteristic of liquid-liquid phase separation (LLPS) event. Mutagenesis approaches revealed that these two phenomena were independent of covalent protein oligomerization induced by stp. Disruption of the N-linked glycosylation motif in the stp region enhanced morular phenotype propensity but reduced protein secretion. Intermolecular disulfide bonds that stabilize Fc dimers and oligomers were necessary for efficient induction of LLPS, but their simultaneous elimination could not abrogate the LLPS propensity completely. Noncovalent protein-protein interactions between scFv-Fc-stp chains sufficiently established a basis for LLPS induction. Morular cell phenotypes and cryoprecipitation were clearly underpinned by intrinsic physicochemical properties embedded in the overexpressed cargo protein. Overproduction of condensation-prone secretory proteins that culminate in LLPS in the endoplasmic reticulum therefore serves as a path to produce morular Russell body phenotype.

Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter ß-barrel domain of IgAP gene from Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture.

Expression of sclerostin scFv and the effect of sclerostin scFv on healing of osteoporotic femur fracture in rats.

Osteoporosis is a systemic metabolic disease characterized by low bone mass with deterioration of the bony microstructure which leads to both bone brittleness and increased risk of fracture. Sclerostin is a protein encoded by the SOST gene which is specifically expressed in osteocyte. Monoclonal antibodies of sclerostin can promote bone formation by antagonizing its inhibitory action. However, the effectiveness of monoclonal antibodies to exert such effects are limited by the large molecular mass and high immunogenicity. Here, we report that we purified a high immune affinity, single-chain antibody of SOST: SOST-single-chain Fv (scFv). Real-time polymerase chain reaction amplification of the variable regions of the heavy- and light-chain gene from a secretory anti-SOST antibody was performed. Animal experiments showed that SOST-scFv promoted bone healing in a rat model of osteoporosis.

A mimotope peptide of Aß42 fibril-specific antibodies with Aß42 fibrillation inhibitory activity induces anti-Aß42 conformer antibody response by a displayed form on an M13 phage in mice.

In Alzheimer's disease (AD), amyloid-ß (Aß) peptides accumulate in the brain in different forms, including fibrils and oligomers. Recently, we established three distinct conformation-dependent human single-chain Fv (scFv) antibodies, including B6 scFv, which bound to Aß42 fibril but not to soluble-form Aß, inhibiting Aß42 fibril formation. In this study, we determined the mimotopes of these antibodies and found a common mimotope sequence, B6-C15, using the Ph.D.-C7C phage library. The B6-C15 showed weak homology to the C-terminus of Aß42 containing GXXXG dimerization motifs. We synthesized the peptide of B6-C15 fused with biotinylated TAT at the N-terminus (TAT-B6-C15) and characterized its biochemical features on an Aß42-fibrillation reaction in vitro. We demonstrated that, first, TAT-B6-C15 inhibited Aß42 fibril formation; secondly, TAT-B6-C15 bound to prefibril Aß42 oligomers but not to monomers, trimers, tetramers, fibrils, or ultrasonicated fragments; thirdly, TAT-B6-C15 inhibited Aß42-induced cytotoxicity against human SH-SY5Y neuroblastoma cells; and, fourthly, when mice were administered B6-C15-phages dissolved in phosphate-buffered saline, the anti-Aß42 conformer IgG antibody response was induced. These results suggested that the B6-C15 peptide might provide unique opportunities to analyze the Aß42 fibrillation pathway and develop a vaccine vehicle for Alzheimer's disease.