Stabilization of a Broadly Neutralizing Anti-Chikungunya Virus Single Domain Antibody

A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT50 = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values ~1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10°C over the original CC3 (Tm = 62°C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.

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Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus

Antibodies ◽

10.3390/antib7040044 ◽

2018 ◽

Vol 7 (4) ◽

pp. 44 ◽

Cited By ~ 2

Author(s):

Jinny Liu ◽

Lisa Shriver-Lake ◽

Dan Zabetakis ◽

Ellen Goldman ◽

George Anderson

Keyword(s):

Phage Display Library ◽

Peripheral Blood Lymphocytes ◽

Encephalitis Virus ◽

Single Domain ◽

Equine Encephalitis Virus ◽

Western Equine Encephalitis Virus ◽

Recognition Elements ◽

Single Domain Antibodies ◽

Western Equine Encephalitis

In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

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Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis

Scientific Reports ◽

10.1038/s41598-021-04434-x ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Jinny L. Liu ◽

Dan Zabetakis ◽

Christina L. Gardner ◽

Crystal W. Burke ◽

Pamela J. Glass ◽

...

Keyword(s):

Chikungunya Virus ◽

Therapeutic Potential ◽

Phage Display Library ◽

Single Domain ◽

Lateral Spread ◽

Health Concern ◽

Venezuelan Equine Encephalitis ◽

Single Domain Antibody ◽

Neutralization Capacity ◽

Equine Encephalitis Virus

AbstractVenezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

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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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Western equine encephalitis virus virus‐like particles from an insect cell‐baculovirus system elicit the strong immune responses in mice

Biotechnology Journal ◽

10.1002/biot.202100008 ◽

2021 ◽

pp. 2100008

Author(s):

JinZhu Ma ◽

HuaLei Wang ◽

XueXing Zheng ◽

HongXia Wu ◽

SongTao Yang ◽

...

Keyword(s):

Immune Responses ◽

Insect Cell ◽

Encephalitis Virus ◽

Virus Like Particles ◽

Equine Encephalitis Virus ◽

Western Equine Encephalitis Virus ◽

Baculovirus System ◽

Western Equine Encephalitis

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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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