scholarly journals Single-domain antibody screening by isPLA-seq

2021 ◽  
Vol 5 (1) ◽  
pp. e202101115
Author(s):  
Yueyuan Yin ◽  
Fei Yan ◽  
Ruimin Zhou ◽  
Mingchen Li ◽  
Jinyi Ma ◽  
...  
Keyword(s):  
Single Domain ◽  
Proximity Ligation Assay ◽  
Single Domain Antibody ◽  
Cellular Processes ◽  
Proximity Ligation ◽  
Domain Antibody ◽  
Functional Measure ◽  
Potential Applications

Single-domain antibody (sdAb) holds the promising strategies for diverse research and translational applications. Here, we describe a method for the adaptation of the in situ proximity ligation assay (isPLA) followed by sequencing (isPLA-seq) to facilitate screening of a high-sensitive, high-throughput sdAb library for a given protein at subcellular and single-cell resolution. Based on the sequence of complementarity-determining region 3 (CDR3), the recombinant sdAb can be produced for in vitro and in vivo utilities. This method provides a general means to identify the functional measure of sdAb and its complementary epitopes and its potential applications to investigate cellular processes.

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.

scholarly journals Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer

2021 ◽  
Vol 9 (6) ◽  
pp. e002131
Author(s):  
Tianhang Zhai ◽  
Chao Wang ◽  
Yifeng Xu ◽  
Weifeng Huang ◽  
Zhijun Yuan ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Single Domain ◽  
Single Domain Antibody ◽  
X Ray ◽  
X Ray Crystallography ◽  
Domain Antibody ◽  
Cell Assays

BackgroundThe discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%–30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003).MethodsTo determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further.ResultsPM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo.ConclusionsA unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

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 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 A single-domain antibody inhibits SFTSV and mitigates virus-induced pathogenesis in vivo

JCI Insight ◽  
2020 ◽  
Vol 5 (13) ◽  
Author(s):  
Xilin Wu ◽  
Yanlei Li ◽  
Bilian Huang ◽  
Xiaohua Ma ◽  
Linjing Zhu ◽  
...  
Keyword(s):  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody

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 Cytoplasmic p21 Mediates 5-Fluorouracil Resistance by Inhibiting Pro-Apoptotic Chk2

Cancers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 373 ◽  
Author(s):  
Arnatchai Maiuthed ◽  
Chuanpit Ninsontia ◽  
Katharina Erlenbach-Wuensch ◽  
Benardina Ndreshkjana ◽  
Julienne Muenzner ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Mechanisms ◽  
Chorioallantoic Membrane ◽  
Proximity Ligation Assay ◽  
Proximity Ligation ◽  
Cancer Aggressiveness ◽  
Localization Signal ◽  
Colorectal Cancer Patients

The oncogenic cytoplasmic p21 contributes to cancer aggressiveness and chemotherapeutic failure. However, the molecular mechanisms remain obscure. Here, we show for the first time that cytoplasmic p21 mediates 5-Fluorouracil (5FU) resistance by shuttling p-Chk2 out of the nucleus to protect the tumor cells from its pro-apoptotic functions. We observed that cytoplasmic p21 levels were up-regulated in 5FU-resistant colorectal cancer cells in vitro and the in vivo Chorioallantoic membrane (CAM) model. Kinase array analysis revealed that p-Chk2 is a key target of cytoplasmic p21. Importantly, cytoplasmic form of p21 mediated by p21T145D transfection diminished p-Chk2-mediated activation of E2F1 and apoptosis induction. Co-immunoprecipitation, immunofluorescence, and proximity ligation assay showed that p21 forms a complex with p-Chk2 under 5FU exposure. Using in silico computer modeling, we suggest that the p21/p-Chk2 interaction hindered the nuclear localization signal of p-Chk2, and therefore, the complex is exported out of the nucleus. These findings unravel a novel mechanism regarding an oncogenic role of p21 in regulation of resistance to 5FU-based chemotherapy. We suggest a possible value of cytoplasmic p21 as a prognosis marker and a therapeutic target in colorectal cancer patients.

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