Single-domain antibody-based noninvasive in vivo imaging of α-synuclein or tau pathology.

Intracellular deposition of α-synuclein and tau are hallmarks of synucleinopathies and tauopathies, respectively. Recently, several dye-based imaging probes with selectivity for tau aggregates have been developed, but suitable imaging biomarkers for synucleinopathies are still unavailable. Detection of both of these aggregates early in the disease process may allow for prophylactic therapies before functional impairments have manifested, highlighting the importance of developing specific imaging probes for these lesions.

Neuronally expressed anti-tau scFv prevents tauopathy-induced phenotypes in Drosophila models.

We have derived single-chain variable fragments (scFv) from tau antibody hybridomas and previously shown their promise as imaging diagnostic agents. Here, we examined the therapeutic potential of anti-tau scFv in transgenic Drosophila models that express in neurons wild-type (WT) human tau (htau) or the human tauopathy mutation R406W. scFv expressing flies were crossed with the tauopathy flies and analyzed.

In Vivo Imaging of Tauopathy in Mice.

Alzheimer's disease is characterized by amyloid-β plaques and neurofibrillary tangles composed of tau aggregates. Several β-sheet dyes are already in clinical use to detect amyloid-β plaques by in vivo positron emission tomography (PET), and related dye compounds are being developed for targeting pathological tau aggregates. In contrast to β-sheet binders, antibody-derived ligands should provide greater specificity for detecting tau lesions, and can be tailored to detect various pathological tau epitopes.

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

Background: A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures.

Sex and Immunogen-Specific Benefits of Immunotherapy Targeting Islet Amyloid Polypeptide in Transgenic and Wild-Type Mice.

Type 2 diabetes mellitus is characterized by the deposition of islet amyloid polypeptide (IAPP) as amyloid in islets, a process thought to be toxic to β-cells. To determine the feasibility of targeting these aggregates therapeutically, we vaccinated transgenic (Tg) mice that overexpress human IAPP and were fed a high-fat diet to promote their diabetic phenotype. Our findings indicate that prophylactic vaccination with IAPP and its derivative IAPP7-19-TT, protects wild-type female mice, but not males, from obesity-induced early mortality, and the derivative showed a strong trend for prolonging the lifespan of Tg females but not males.

Antibody-derived in vivo imaging of tau pathology.

Antibodies or their derivatives as imaging probes for pathological tau protein have great potential, but have not been well studied. In particular, smaller, single-chain-variable antibody fragments (scFv's) are attractive for detecting tau lesions in live subjects. Here, we generated libraries of scFv's and identified numerous phospho-tau-selective scFv's.

Antigen itself antibody: an alternative one-step immunoassay for measuring the anti-idiotypic antibody titer.

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti-idiotypic antibody. Our initial attempt to measure titers of mouse anti-idiotypic antibody after idiotypic vaccination with HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) failed.

Harnessing the immune system for treatment and detection of tau pathology.

The tau protein is an attractive target for therapy and diagnosis. We started a tau immunotherapy program about 13 years ago and have since demonstrated that active and passive immunotherapies diminish tau pathology and improve function, including cognition, in different mouse models. These findings have been confirmed and extended by several groups.

New avenues for phage-display library to produce a Cryptococcus-specific anti-idiotypic antibody of HM-1 killer toxin.

Existing antifungal drugs are notable for their inability to act rapidly, as well as their toxicity and limited spectrum. The identification of fungal-specific genes and virulence factors would provide targets for new and influential drugs. The display of repertories of antibody fragments on the surface of filamentous phage offers a new way to produce immunoreagents as defined specificities.

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent.

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species.

Isolation and characterization of recombinant single chain fragment variable anti-idiotypic antibody specific to Aspergillus fumigatus membrane protein.

Aspergillus fumigatus causes the highly lethal form of invasive aspergillosis (IA). In the present study to develop a novel anti-fungal drug for protection against invasive disease, we identified a single chain fragment variable (scFv) antibody (scFv AF1) by panning against A. fumigatus membrane fraction (AMF) or HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) as antigen.

Different buffer effects in selecting HM-1 killer toxin single-chain fragment variable anti-idiotypic antibodies.

We reported previously competitive panning elution with PBS (pH 7.0) that contains HM-1 killer toxin (HM-1) and Candida albicans membrane fraction (CaMF) to release phages bound with CaMF as an antigen. Here, as an alternative strategy, we isolated high-binding affinity recombinant single-chain fragment variables (scFvs) with in vitro anti-fungal activity from an scFv phage library.

Purification and functional characterization of a Camelid-like single-domain antimycotic antibody by engineering in affinity tag.

Single-domain single-chain variable fragment (scFv) antibody is sometimes critical for purification using affinity tagging strategy. We failed in our initial effort to purify a prematurely developed Camelid-like E-tagged short scFv-K2 antibody that contained a complete variable region of the heavy chain and partial region of the light chain by using an anti-E-tag affinity column. To expedite the purification of this altered but interesting antimycotic agent, we replaced a long and large E-tag by a short and hydrophilic 6x-Histidine (His(6)) affinity tag by polymerase chain reaction.

An improved phage-display panning method to produce an HM-1 killer toxin anti-idiotypic antibody.

Background: Phage-display panning is an integral part of biomedical research. Regular panning methods are sometimes complicated by inefficient detachment of the captured phages from the antigen-coated solid supports, which prompted us to modify. Here, we produce an efficient antigen-specific single chain fragment variable (scFv) antibody by using a target-related molecule that favored selection of recombinant antibodies.

Cloning antifungal single chain fragment variable antibodies by phage display and competitive panning elution.

Phage display and two competitive panning elution conditions were used to isolate Candida-specific single chain fragment variable (scFv) antibodies. An scFv phage library constructed from splenic lymphocytes of mice immunized by idiotypic vaccination with an HM-1 killer toxin (HM-1)-neutralizing monoclonal antibody (nmAb-KT) was used for panning against Candidaalbicans membrane fraction (CaMF). Key steps were specific elution conditions to separately release the bound phages with original antigen HM-1+HM-1 peptide 6 and CaMF.