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. In contrast to the ß sheet dyes, single-domain antibodies, found in camelids and a few other species, are highly specific, and their small size allows better brain entry and distribution than whole antibodies. Here, we have developed such imaging ligands via phage display libraries derived from llamas immunized with α-synuclein and tau preparations, respectively. These probes allow noninvasive and specific in vivo imaging of α-synuclein versus tau pathology in mice, with the brain signal correlating strongly with lesion burden. These small antibody derivatives have great potential for in vivo diagnosis of these diseases.

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. Overall, the survival curves differed significantly (p < .0001). Control flies not expressing htau survived the longest, whereas R406W expressing flies had the shortest lifespan, which was greatly prolonged by co-expressing the anti-tau scFv (p < .0001). Likewise, htau WT expressing flies had a moderately short lifespan, which was prolonged by co-expressing the anti-tau scFv (p < .01). In addition, the htau expression impaired wing expansion after eclosion (p < .0001), and caused progressive abdomen expansion (p < .0001). These features were more severe in htau R406W flies than in htau WT flies. Importantly, both phenotypes were prevented by co-expression of the anti-tau scFv (p < .01-0.0001). Lastly, brain analyses revealed scFv-mediated tau clearance (p < .05-0.01), and its prevention of tau-mediated neurotoxicity (p < .05-0.001). In summary, these findings support the therapeutic potential of an anti-tau scFv, including as gene therapies, and the use of Drosophila models for such screening.

In Vivo Imaging of Tauopathy in Mice.

Alzheimer's disease is characterized by amyloid-ß plaques and neurofibrillary tangles composed of tau aggregates. Several ß-sheet dyes are already in clinical use to detect amyloid-ß plaques by in vivo positron emission tomography (PET), and related dye compounds are being developed for targeting pathological tau aggregates. In contrast to ß-sheet binders, antibody-derived ligands should provide greater specificity for detecting tau lesions, and can be tailored to detect various pathological tau epitopes.For preclinical in vivo evaluation of these ligands prior to PET development, we have established an in vivo imaging system (IVIS) protocol to detect tauopathy in live mice. Antibodies and their derivatives are conjugated with a near infrared fluorescent dye and injected intravenously into anesthetized mice, which subsequently are imaged at various intervals to assess their pathological tau burden, and clearance of the ligand from the brain. The in vivo signal obtained through the skull correlates well with the degree of tau pathology in the mice, and the injected ligand can be found intraneuronally within the brain bound to tau aggregates. Control IgG and injections of the tau antibodies/fragments into wild-type mice or mice with amyloid-ß plaques lead to minimal or no signal, confirming the specificity of the approach.

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. RESULTS: Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. CONCLUSIONS: Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

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 ß-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. Furthermore, IAPP7-19-TT-immunized Tg females cleared a glucose bolus more efficiently than controls, while IAPP-immunized Tg females showed an impaired ability to clear a glucose bolus compared to their adjuvant injected Tg controls. Interestingly, IAPP or IAPP7-19-TT treatments had no effect on glucose clearance in Tg males. Overall, these beneficial effects of IAPP targeted immunization depend on Tg status, sex, and immunogen. Hence, future studies in this field should carefully consider these variables that clearly affect the therapeutic outcome. In conclusion, IAPP targeting immunotherapy may have benefits in patients with type 2 diabetes.

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. Peripheral injection of one of these scFv's consistently resulted in a strong in vivo brain signal in transgenic tauopathy mice, but not in wild-type or amyloid-ß plaque mice. The parent tau antibody provided similar results, albeit with a weaker signal intensity. The imaging signal correlated very well with colocalization of the probe with intraneuronal tau aggregates. Both were associated with markers of endosomes, autophagosomes, and lysosomes, suggesting their interaction in these degradation pathways. Such specific antibody-derived imaging probes have great potential as diagnostic markers for Alzheimer's disease and related tauopathies.

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. Because the injected antigen, nmAb-KT, is a mouse IgG, using a commercial antibody to measure the antibody titer always gave a false positive signal against control mouse serum antibody in parallel with the antigen-treated immunized serum antibodies. To get a reliable and clearly differentiable signal by ELISA, idiotypic antigen was labeled with HRP and HRP-conjugated-nmAb-KT used to measure the antibody titers in the antigen-treated mice. Compared with control mice, signals were found in high anti-nmAb-KT IgG responses in test mice; however, untreated control mice had a significant amount of purified non-specific IgG. This method is amenable to long read lengths and will likely enable anti-idiotypic antibody titer measurement in a more specific and cost effective way without requiring commercial antibody.

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. We routinely detect neuronal, and to a lesser extent microglial, antibody uptake correlating with tau pathology. Antibodies bind tau aggregates in the endosomal/lysosomal system, enhancing clearance presumably by promoting their disassembly. Extracellular clearance has recently been shown by others, using antibodies that apparently are not internalized. As most pathological tau is neuronal, intracellular targeting may be more efficacious. However, extracellular tau may be more accessible to antibodies, with tau-antibody complexes a target for microglial phagocytosis. The extent of involvement of each pathway may depend on numerous factors including antibody properties, degree of pathology, and experimental model. On the imaging front, multiple tau ligands derived from ß-sheet dyes have been developed by several groups, some with promising results in clinical PET tests. Postmortem analysis should clarify their tau specificity, as in theory and based on histological staining, those are likely to have some affinity for various amyloids. We are developing antibody-derived tau probes that should be more specific, and have in mouse models shown in vivo detection and binding to pathological tau after peripheral injection. These are exciting times for research on tau therapies and diagnostic agents that hopefully can be applied to humans in the near future.

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide.

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a ß-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall ß-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.

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. Here we report the selection of Cryptococcus-specific targets by using phage-display panning from a cDNA library, where bactericidal antibodies have been developed against conserved surface-exposed antigens. A single-chain variable fragment (scFv) phage library was constructed from splenocyte of an immunized mouse by idiotypic vaccination with HM-1 killer toxin (HM-1) neutralizing monoclonal antibody (nmAb-KT) that was used for selection against Cryptococcus neoformans membrane fraction (CnMF). Key elements were the selection against antigen (nmAb-KT and CnMF) and the release of bound phages using competitive panning elution with CnMF at neutral pH condition. Isolated scFvs react specifically with C. neoformans and some other pathogenic and non-pathogenic fungal strain's cell wall receptors by exerting strong antifungal activity in vitro. A high affinity clone, designated M1 was selected for detailed characterization and tested anti-cryptococcal activity with IC(50) values at 5.33 × 10(-7) to 5.56 × 10(-7) M against C. neoformans. The method described here is a new technique for the isolation of cell membrane specific immunoreactive phages in the form of scFv using CnMF that contained cell membrane associated proteins.

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. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K(d)=1.07×10(-10) M) and antifungal activity was three- to fivefold more efficient (IC(50)=0.46×10(-6) to 1.17×10(-6) M) than that for the conventional antibody (K(d)=2.91×10(-8) M and IC(50)=2.14×10(-6) to 3.78×10(-6) M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.

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. The key step was elution of bound phages with phosphate buffered saline (PBS) at pH 7.0 containing AMF. The specificity of soluble scFv AF1 antibody to antigens was verified by ELISA, which specifically binds to both AMF and nmAb-KT. After nucleotide sequencing, clone expression and purification by HisTrap HP affinity column, scFv AF1 showed in vitro anti-fungal activity against A. fumigatus. By SPR analysis it showed high binding affinity to nmAb-KT (K(d)=5.22×10(-11) M). The method used to isolate scFv AF1 was a new method and we believe that it will be applicable to isolate the specific scFv against any kind of membrane protein of yeast or fungus.

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. The short and compact His(6)-tag was placed on the previously constructed expression vector pCANTAB 5 E that contained the large affinity E-tag sequence (13 amino acids) by PCR-based mutagenesis and was expressed in Escherichia coli. The recombinant protein can then be purified by immobilized metal affinity chromatography (IMAC) and be used for biochemical and other functional characterization. This His(6)-tagged short scFv-K2 antibody (20 kDa) had strong cytocidal activity against Saccharomyces and Candida species with a IC(50) value of 0.44x10(-6)M and 1.10 x 10(-6)M, respectively. Tag replacement facilitates the purification of a Camelid-like single-domain scFv antibody and after that meets its different functional characteristics. The present study reflects that the V(H) domain of the scFv antibody is mainly responsible for its biological activity and single-domain scFv antibody may acts as a potent antimicrobial agent.

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. The competitive panning elution contained acidic, neutral and basic pH buffers with original antigen HM-1 or HM-1 peptide 6 used to release phages bound with HM-1-neutralizing monoclonal antibody (nmAb-KT). For neutral pH eluted conditions, 87.5% of clones showed high-binding affinity against nmAb-KT by using ELISA, but was 16% and 26% for acidic and basic eluted conditions, respectively. After nucleotide sequencing, we obtained seven different anti-idiotypic antibodies from the different selection procedures. The clone expression and purification by using a HisTrap HP column, showed that clones scFv S3, S4 and S7 had in vitro antifungal activity against Saccharomyces cerevisiae, Candida albicans. The purified scFvs showed strong binding affinity with nmAb-KT by using ELISA. These results showed that changing the buffer pH with competing elements plays important role in elution of bound phages to targeted antigen and also in identification of positive scFv phages.

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. RESULTS: To produce more selective and specific anti-idiotypic scFv-antibodies from a cDNA library, constructed from HM-1 killer toxin (HM-1)-neutralizing monoclonal antibodies (nmAb-KT), the method was modified by using an elution buffer supplemented with HM-1 that shares structural and functional similarities with the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT allowed easy and quick dissociation of scFv-displayed phages from immobilized nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After modified panning, 80% clones (40/50) showed several times higher binding affinity to nmAb-KT than regular panning. The major populations (48%) of these clones (scFv K1) were genotypically same and had strong cytocidal activity against Saccharomyces and Candida species. The scFv K1 (K(d) value = 4.62 x 10(-8) M) had strong reactivity toward nmAb-KT, like HM-1 (K(d) value = 6.74 x 10(-9) M) as judged by SPR analysis. CONCLUSION: The scFv antibodies generated after modified subtractive panning appear to have superior binding properties and cytocidal activity than regular panning. A simple modification of the elution condition in the phage-display panning protocol makes a large difference in determining success. Our method offers an attractive platform to discover potential therapeutic candidates.

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. The positive phages were screened by using enzyme-linked immunosorbent assay, and after nucleotide sequencing, clone expression, and purification, clone scFv-C1 was selected for detailed characterization. The scFv-C1 showed IC(50) values for cell growth against various Candida species and Saccharomyces cerevisiae as 2.40 to 6.40microM and 2.20microM, respectively. By using surface plasmon resonance analysis, the scFv-C1 had a K(d) value of 3.09x10(-11)M to nmAb-KT, indicating a 260-fold higher affinity than for HM-1. These results showed the generated scFv-C1 mimicking HM-1-binding affinity to nmAb-KT and in vitro antifungal activity. We believe that the effectiveness of the competitive panning elution method and antigen-specific recombinant scFv antibodies obtained in this study are excellent candidates for antimycotic drugs.