Delivery of single-domain antibodies into neurons using a chimeric toxin–based platform is therapeutic in mouse models of botulism

2021 ◽  
Vol 13 (575) ◽  
pp. eaaz4197
Author(s):  
Shin-Ichiro Miyashita ◽  
Jie Zhang ◽  
Sicai Zhang ◽  
Charles B. Shoemaker ◽  
Min Dong
Keyword(s):  
Motor Neurons ◽  
Therapeutic Protein ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Exposure Treatment ◽  
Post Exposure ◽  
In Vitro Characterization ◽  
Endosomal Membranes ◽  
Delivery Platform

Efficient penetration of cell membranes and specific targeting of a cell type represent major challenges for developing therapeutics toward intracellular targets. One example facing these hurdles is to develop post-exposure treatment for botulinum neurotoxins (BoNTs), a group of bacterial toxins (BoNT/A to BoNT/G) that are major potential bioterrorism agents. BoNTs enter motor neurons, block neurotransmitter release, and cause a paralytic disease botulism. Members of BoNTs such as BoNT/A exhibit extremely long half-life within neurons, resulting in persistent paralysis for months, yet there are no therapeutics that can inhibit BoNTs once they enter neurons. Here, we developed a chimeric toxin–based delivery platform by fusing the receptor-binding domain of a BoNT, which targets neurons, with the membrane translocation domain and inactivated protease domain of the recently discovered BoNT-like toxin BoNT/X, which can deliver cargoes across endosomal membranes into the cytosol. A therapeutic protein was then created by fusing a single-domain antibody (nanobody) against BoNT/A with the delivery platform. In vitro characterization demonstrated that nanobodies were delivered into cultured neurons and neutralized BoNT/A in neurons. Administration of this protein in mice shortened duration of local muscle paralysis, restoring muscle function within hours, and rescued mice from systemic toxicity of lethal doses of BoNT/A. Fusion of two nanobodies, one against BoNT/A and the other against BoNT/B, created a multivalent therapeutic protein able to neutralize both BoNT/A and BoNT/B in mice. These studies provide an effective post-exposure treatment for botulism and establish a platform for intracellular delivery of therapeutics targeting cytosolic proteins and processes.

scholarly journals A therapeutic vascular conduit to support in vivo cell-secreted therapy

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Edward X. Han ◽  
Hong Qian ◽  
Bo Jiang ◽  
Maria Figetakis ◽  
Natalia Kosyakova ◽  
...  
Keyword(s):  
Vascular Graft ◽  
Large Scale ◽  
Biological Effects ◽  
Therapeutic Protein ◽  
Close Proximity ◽  
Delivery Platform ◽  
Stage Renal Disease ◽  
End Stage

AbstractA significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library

2021 ◽  
Vol 492 ◽  
pp. 112990
Author(s):  
Jothivel Kumarasamy ◽  
Samar Kumar Ghorui ◽  
Chandrakala Gholve ◽  
Bharti Jain ◽  
Yogesh Dhekale ◽  
...  
Keyword(s):  
Phage Display ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
T7 Phage Display ◽  
T7 Phage

scholarly journals Selection and Characterization of CSFV-Specific Single-Domain Antibodies and Their Application along with Immunomagnetic Nanobeads and Quantum Dots

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Shunli Yang ◽  
Li Yuan ◽  
Youjun Shang ◽  
Jinyan Wu ◽  
Xiangtao Liu ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Magnetic Beads ◽  
Molecular Probes ◽  
Single Domain ◽  
Molecular Probe ◽  
Economic Losses ◽  
Single Domain Antibody ◽  
Swine Industry ◽  
E2 Protein

Outbreak of classical swine fever (CSF) results in high mortality and thus causes severe economic losses in the swine industry. Single-domain antibody (sdAb) is the smallest antigen-binding molecule derived from camelid heavy-chain antibodies and has the potential to be used as a molecular probe for detection of CSF virus (CSFV). In this study, two sdAb fragments against the E2 antigen of CSFV were obtained, expressed in vitro. The functional characteristics analysis indicated that the recombinant sdAbE2-1 and sdAbE2-2 have excellent binding activity, specificity, and high affinity with equilibrium constant value of 3.34 × 10−7 and 1.35 × 10−8 M to E2 protein. Then, sdAbE2s were conjugated with quantum dots (QD)/AF488 to synthesize two molecular probes for imaging CSFV distribution in cells. The sdAbE2-1 was also labeled with carboxyl-magnetic beads to construct immunomagnetic nanobeads (IMNBs) able to capture CSFV virions and recombinant E2 protein. QD/AF455-sdAbE2s probes colocalised with CSFV virions in swine testis cells, and IMNBs were used as a detection template and proved to bind specifically with CSFV virions and E2 protein. The selected sdAb fragments and sdAb-based molecular probes may be used for the rapid identification of CSFV during field outbreaks and for research on CSFV and host interactions.

scholarly journals Inhibition of Tau seeding by targeting Tau nucleation core within neurons with a single domain antibody fragment

2021 ◽  
Author(s):  
Clément Danis ◽  
Elian Dupré ◽  
Orgeta Zejneli ◽  
Raphaëlle Caillierez ◽  
Alexis Arrial ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Optimal Strategies ◽  
Phage Display Library ◽  
Biochemical Properties ◽  
Single Domain ◽  
Tau Proteins ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Intracellular Expression

Tau proteins aggregate into filaments in brain cells in Alzheimer's disease and related disorders referred to as tauopathies. Here, we used fragments of camelid heavy-chain-only antibodies (VHHs or single domain antibody fragments) targeting Tau as immuno-modulators of its pathologic seeding. A VHH issued from the screen against Tau of a synthetic phage-display library of humanized VHHs was selected for its capacity to bind Tau microtubule-binding domain, composing the core of Tau fibrils. This lead VHH was optimized to improve its biochemical properties and to act in the intracellular compartment, resulting in VHH Z70. VHH Z70 was more efficient than the lead to inhibit in vitro Tau aggregation in heparin-induced assays. Expression of VHH Z70 in a cellular model of Tau seeding also decreased the fluorescence-reported aggregation. Finally, intracellular expression of VHH Z70 in the brain of an established tauopathy mouse seeding model demonstrated its capacity to mitigate accumulation of pathological Tau. VHH Z70, by targeting Tau inside brain neurons, where most of the pathological Tau resides, provides a new tool to explore the optimal strategies of immunotherapy in tauopathies.

scholarly journals NaLi-H1: A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Sandrine Moutel ◽  
Nicolas Bery ◽  
Virginie Bernard ◽  
Laura Keller ◽  
Emilie Lemesre ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Rho Gtpase ◽  
Fluorescent Proteins ◽  
Phage Display Library ◽  
Specific Protein ◽  
Single Domain ◽  
Direct Selection ◽  
Single Domain Antibody ◽  
Domain Antibody

In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.

scholarly journals Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Peng-Nien Huang ◽  
Hsiang-Ching Wang ◽  
Hui-Chen Hung ◽  
Sung-Nien Tseng ◽  
Teng-Yuan Chang ◽  
...  
Keyword(s):  
Emerging Infectious Diseases ◽  
Conformational Epitope ◽  
Single Domain ◽  
Therapeutic Interventions ◽  
Asia Pacific ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Enterovirus A71

ABSTRACT In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo. We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion’s icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.

Structure- and sequence-based design of synthetic single-domain antibody libraries

2020 ◽  
Vol 33 ◽  
Author(s):  
Alexander M Sevy ◽  
Ming-Tang Chen ◽  
Michelle Castor ◽  
Tyler Sylvia ◽  
Harini Krishnamurthy ◽  
...  
Keyword(s):  
In Vitro Selection ◽  
Amyloid Β ◽  
Single Domain ◽  
Amyloid Β Peptide ◽  
Sequencing Analysis ◽  
Single Domain Antibody ◽  
High Affinity ◽  
Domain Antibody ◽  
Combine Structure

Abstract Single-domain antibody fragments known as VHH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating VHH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VHH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated VHH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-β peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.

scholarly journals Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model

2021 ◽  
Vol 17 (10) ◽  
pp. e1009542
Author(s):  
Kei Haga ◽  
Reiko Takai-Todaka ◽  
Yuta Matsumura ◽  
Chihong Song ◽  
Tomomi Takano ◽  
...  
Keyword(s):  
Severe Pneumonia ◽  
The Elderly ◽  
Single Domain ◽  
Nasal Delivery ◽  
Single Domain Antibody ◽  
Viral Attachment ◽  
Drug Candidates ◽  
Domain Antibody ◽  
New Treatment

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

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