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 Detection of Cystic Fibrosis Serological Biomarkers Using a T7 Phage Display Library

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Harvinder Talwar ◽  
Samer Najeeb Hanoudi ◽  
Andreea Geamanu ◽  
Dana Kissner ◽  
Sorin Draghici ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Phage Display ◽  
Phage Display Library ◽  
T7 Phage Display ◽  
Serological Biomarkers ◽  
T7 Phage

scholarly journals Next-Generation Sequencing of a Single Domain Antibody Repertoire Reveals Quality of Phage Display Selected Candidates

PLoS ONE ◽  
2016 ◽  
Vol 11 (2) ◽  
pp. e0149393 ◽  
Author(s):  
Kendrick B. Turner ◽  
Jennifer Naciri ◽  
Jinny L. Liu ◽  
George P. Anderson ◽  
Ellen R. Goldman ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Phage Display ◽  
Single Domain ◽  
Antibody Repertoire ◽  
Next Generation ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Generation Sequencing

scholarly journals Diagnostic value of protein chips constructed by lung-cancer-associated markers selected by the T7 phage display library

2015 ◽  
Vol 6 (4) ◽  
pp. 469-474 ◽  
Author(s):  
Hong-Mei Li ◽  
Kang Guo ◽  
Zhuang Yu ◽  
Rui Feng ◽  
Ping Xu
Keyword(s):  
Lung Cancer ◽  
Phage Display ◽  
Diagnostic Value ◽  
Phage Display Library ◽  
Protein Chips ◽  
T7 Phage Display ◽  
T7 Phage

scholarly journals Development and Evaluation of CART Targeting Bcma with Humanized Alpaca-Derived Single-Domain Antibody As Antigen Recognition Domain

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1976-1976 ◽  
Author(s):  
Lu Han ◽  
Quanli Gao ◽  
Keshu Zhou ◽  
Qingsong Yin ◽  
Baijun Fang ◽  
...  
Keyword(s):  
T Cells ◽  
Heavy Chain ◽  
Solid Phase ◽  
Tumor Cell Line ◽  
Phage Display Library ◽  
Single Domain ◽  
Single Chain ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Heavy Chain Antibody

Abstract Chimeric antigen receptor T cells (CART) targeting CD19 have shown substantial activity against leukemia and lymphoma, which motivated developing CART cell therapy for Multiple myeloma (MM). B cell maturation antigen (BCMA) is the target molecule in MM. Several kinds of CART targeting BCMA have been created from 2016. Among these, the Bluebird Bio uses the humanized murine BCMA scFv to make CART, the Nanjing Legend company uses the single-domain antibody targeting BCMA to make CART. The single-domain antibody refers to the heavy chain antibody naturally present in camel species, which has no light chain. All VHh (Variable region of Heavy chain in heavy chain antibody) domains from the immunized alpaca were cloned into the phage-display library, then specific VHhs were screened out by solid-phase ELISA. Not like scFv using the linker region to connect the VH and VL to obtain the single chain region, the VHh itself is one single chain, which make it very suitable for making the recognition domain of CART. In order to increase the avidity, the Nanjing Legend company uses two tandem VHhs for binding BCMA. However, the single domain antibody has the capacity to bind the target molecule with very high affinity, one VHh should be able to bind BCMA tightly. To this aim, using our platform of single-domain antibody discovery and CART development, we successfully obtain the CART targeting BCMA (CART-BCMA) just using one VHh for recognizing the BCMA on tumor cells. Firstly, we immunized the alpaca using the extracellular domain of BCMA, then the B cell RNA were extracted, and the VHh phage-display library were constructed and screened. Finally we got the 6 single-domain antibodies through solid-phase ELISA screening. After gene sequencing and humanization of VHh, we constructed 6 lentiviral vectors with CAR gene including different VHh sequence connecting with CD8 hinge and transmembrane region, CD137 intracellular region, and CD3ζ intracellular region. We used 293T to package the lentivirus, and infected the healthy T cells to make 6 kinds of CART-BCMA cells. With in vitro killing assay, we found the #3 CART-BCMA is the best (83% killing rate at the E:T ratio of 4:1). Simultaneously, we used the Biocore to test the binding affinity of single-domain antibodies, the #3 antibody's affinity is 1.14nM, which indicated only one #3 VHh domain already have enough binding ability for CART to recognize the BCMA positive tumor cells. Next, we did further experiment to demonstrate the function of #3 CART-BCMA. We used the MM tumor cell line MM.1S (expressing high BCMA), myeloid tumor cell line K562 (no BCMA expression) as the target, the results show that CART-BCMA kill the MM.1S cells in 42% rate at the low E:T ratio of 0.5:1, but the CART-BCMA cells can't kill K562 cells even in high 4:1 E:T ratio. Accordingly, the cytokine level of IFN-γ or TNF-α is elevated dramatically in CART-BCMA wells. In animal experiment, the 1.5x106 MM.1S-Luc were injected into one NSG mouse. 40 mice were separated into 5 groups for PBS, 10x106 Mock T, 1.0x106 CART, 5.0x106 CART, 10x106 CART treatment. The results show that three CART groups of 1.0x106, 5.0x106, 10x106 CART are effective. Using the purified lentivirus, we found the lentivirus can infect the T cells at the MOI of 3, the CAR positive rate is over 50% by FACS, this implied that the CAR gene can be easily expressed on T cell membrane. For T cell infection, CAR-BCMA lentivirus need less amount compared to traditional CAR virus derived from scFv. We performed the clinical pilot study to investigate CART-BCMA's safety and effect on MM patients pre-conditioned with Fludarabine/cyclophosphamide. Until the time of abstract submission, 4 patients were enrolled, and got the dose of 5x106/kg (Pt1), dose of 10x106/kg (Pt2 and Pt3, split dose of 50% and 50% in two days), dose of 10x106/kg (Pt4) CART-BCMA cells. Pt1 has the CR response, and has the fever after 14 days. Pt2 has the PR response with no CRS. Pt3 has the PR response with grade 2 CRS. Pt4 has the PR response with grade 3 CRS and neurotoxicity (see table). In summary, we developed one CART-BCMA for MM treatment, which has the BCMA antigen recognition domain derived from the alpaca. Especially, the lentivirus carrying the CAR-BCMA gene can infect the T cells at the low MOI of 3, which will save the amount of lentivirus. The clinical pilot study showed that 4 patients received the CART-BCMA has the objective response (1 CR, 3 PR), further follow-up and enrollment is being performed. Table. Table. Disclosures Zhou: Health and Family Planning Commission of Henan Province: Patents & Royalties: Scientific and technological innovative talents "51282" Project leaders; Affiliated Cancer Hospital of Zhengzhou University: Employment; Natural Science Foundation of China: Research Funding; Henan Cancer Hospital: Consultancy, Employment. Zhang:The Shenzhen Pregene Biopharma Company, Ltd.: Employment. Du:The Shenzhen Pregene Biopharma Company, Ltd.: Employment. Li:The Shenzhen Pregene Biopharma Company, Ltd.: Employment.

Escherichia coli F4 fimbriae specific llama single-domain antibody fragments effectively inhibit bacterial adhesion in vitro but poorly protect against diarrhoea

2005 ◽  
Vol 111 (1-2) ◽  
pp. 89-98 ◽  
Author(s):  
M.M. Harmsen ◽  
C.B. van Solt ◽  
A. Hoogendoorn ◽  
F.G. van Zijderveld ◽  
T.A. Niewold ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Adhesion ◽  
Antibody Fragments ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Domain Antibody
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