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

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.

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In Vivo Imaging of Brain Cancer Using Epidermal Growth Factor Single Domain Antibody Bioconjugated to Near-Infrared Quantum Dots

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2014.9076 ◽

2014 ◽

Vol 14 (7) ◽

pp. 5355-5362 ◽

Cited By ~ 21

Author(s):

D. Fatehi ◽

T. N. Baral ◽

A. Abulrob

Keyword(s):

Quantum Dots ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Brain Cancer ◽

Near Infrared ◽

Single Domain ◽

Single Domain Antibody ◽

Domain Antibody ◽

Epidermal Growth

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Prolonged in vivo residence times of llama single-domain antibody fragments in pigs by binding to porcine immunoglobulins

Vaccine ◽

10.1016/j.vaccine.2005.05.017 ◽

2005 ◽

Vol 23 (41) ◽

pp. 4926-4934 ◽

Cited By ~ 54

Author(s):

Michiel M. Harmsen ◽

Conny B. Van Solt ◽

Helmi P.D. Fijten ◽

Marga C. Van Setten

Keyword(s):

Antibody Fragments ◽

Single Domain ◽

Residence Times ◽

Single Domain Antibody ◽

Domain Antibody

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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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-002131 ◽

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.

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Single-domain antibody screening by isPLA-seq

Life Science Alliance ◽

10.26508/lsa.202101115 ◽

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.

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Advanced genetic engineering to achieve in vivo targeting of adenovirus utilizing camelid single domain antibody

Journal of Controlled Release ◽

10.1016/j.jconrel.2021.04.009 ◽

2021 ◽

Vol 334 ◽

pp. 106-113

Author(s):

Myungeun Lee ◽

Zhi Hong Lu ◽

Charles B. Shoemaker ◽

Jacqueline M. Tremblay ◽

Bradley St. Croix ◽

...

Keyword(s):

Genetic Engineering ◽

Single Domain ◽

Single Domain Antibody ◽

Domain Antibody

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Faculty Opinions recommendation of Crystal structure of a shark single-domain antibody V region in complex with lysozyme.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1024018.285841 ◽

2005 ◽

Author(s):

Christopher Garcia

Keyword(s):

Crystal Structure ◽

Single Domain ◽

Single Domain Antibody ◽

Domain Antibody ◽

V Region

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CAR T cells targeting CD13 controllably induce eradication of acute myeloid leukemia with a single domain antibody switch

Leukemia ◽

10.1038/s41375-021-01208-2 ◽

2021 ◽

Author(s):

Xin He ◽

Zijie Feng ◽

Jian Ma ◽

Xuyao Zhang ◽

Sunbin Ling ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Single Domain ◽

Single Domain Antibody ◽

Car T Cells ◽

Domain Antibody ◽

Car T ◽

Acute Myeloid

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

Journal of Immunological Methods ◽

10.1016/j.jim.2021.112990 ◽

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

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A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms22115501 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5501

Author(s):

Yutong Xing ◽

Keyuan Xu ◽

Shixiong Li ◽

Li Cao ◽

Yue Nan ◽

...

Keyword(s):

Prostate Cancer ◽

Animal Studies ◽

Single Domain ◽

Soluble Form ◽

Therapeutic Window ◽

Single Domain Antibody ◽

Pseudomonas Exotoxin A ◽

Simple Strategy ◽

Domain Antibody ◽

Recombinant Immunotoxin

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

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