Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

Cheng, Man Wai; Chan, Shirley; Zhao, Qi; Chan, Elaine; Au, Shannon Wing-Ngor; Lee, Susanna S.T.; Cheung, Wing‐Tai

doi:10.1371/journal.pone.0027406

Cited by 5 publications

(8 citation statements)

References 53 publications

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“…Primers designed for the amplification of mouse immunoglobulins 29,30 have been successfully applied for the generation of scFv based phage libraries from immunized sources 31,32 . However, few primer sets that specifically amplify immunoglobulin variable regions from rats have been described.…”

Section: Discussionmentioning

confidence: 99%

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Nannini

Parekh

Wawrzyniecka

et al. 2020

Sci Rep

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Antibody phage display is a powerful platform for discovery of clinically applicable high affinity monoclonal antibodies against a broad range of targets. Libraries generated from immunized animals offer the advantage of in vivo affinity-maturation of V regions prior to library generation. Despite advantages, few studies have described isolation of antibodies from rats using immune phage display. In our study, we describe a novel primer set, covering the full rat heavy chain variable and kappa light chain variable regions repertoire for the generation of an unbiased immune libraries. Since the immune repertoire of rats is poorly understood, we first performed a deep sequencing analysis of the V(D)J regions of VH and VLK genes, demonstrating the high abundance of IGVH2 and IGVH5 families for VH and IGVLK12 and IGVLK22 for VLK. The comparison of gene’s family usage in naïve rats have been used to validate the frequency’s distribution of the primer set, confirming the absence of PCR-based biases. The primers were used to generate and assemble a phage display library from human CD160-vaccinated rats. CD160 represents a valid therapeutic target as it has been shown to be expressed on chronic lymphocytic leukaemia cells and on the surface of newly formed vessels. We utilised a novel phage display panning strategy to isolate a high affinity pool (KD range: 0.399–233 nM) of CD160 targeting monoclonal antibodies. Subsequently, identified binders were tested for function as third generation Chimeric Antigen Receptors (CAR) T cells demonstrating specific cytolytic activity. Our novel primer set coupled with a streamlined strategy for phage display panning enable the rapid isolation and identification of high affinity antibodies from immunised rats. The therapeutic utility of these antibodies was demonstrated in CAR format.

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Section: Discussionmentioning

confidence: 99%

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Nannini

Parekh

Wawrzyniecka

et al. 2020

Sci Rep

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“…Total RNA was isolated from mouse splenocytes using Trizol reagent (Invitrogen, Carlsbad, CA) and 10∼15 µg of total RNA was used to prepare first strand cDNA [17] . The cDNA fragments encoding immunoglobulin variable regions were amplified by PCR using specific degenerate primer pairs as described previously [18] with minor modifications. Briefly, PCR amplification of Ig V H and V Lκ was separately carried out in 50 µL reaction volume with 1X PCR buffer (100 mM Tris-HCl; pH 8.3, 500 mM KCl) containing 1.5 mM MgCl 2 , 0.2 mM dNTP, 0.05 U/µL Taq polymerase, 5 µL of cDNA.…”

Section: Methodsmentioning

confidence: 99%

“…Then the linker-added V H and V Lκ DNA fragments were joined together using over-lapping extension PCR. The nucleotide sequences of those linkers and primer pairs, and the PCR protocols were detailed in our previous publication [18] . Phage-displayed scFv library was constructed using a recombinant phage antibody system following the manufacturer's specifications (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…Phage propagation, either as filamentous phage or in the form of phage-displayed scFv library, was performed as described previously [18] . To select anti-idiotype antibody, the phage-displayed library was separately panned against chimera anti-CD22 SM03 or mouse anti-human CD22 RFB4 on 24-well microplates (IWAKI, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…The medium was quickly discarded and the bacteria in each well were resuspended in 1 mL of 2×YT medium containing 100 µg/mL ampicillin and 50 µg/mL of kanamycin. The bacterial culture was incubated overnight at 37°C with shaking at 250 rpm, and culture medium containing the phages was collected and directly tested for antigen binding by phage-ELISA as described previously [18] . Briefly, phage-ELISA was carried out in a 96-well ELISA plate (Nunc, Denmark).…”

Section: Methodsmentioning

confidence: 99%

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Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

et al. 2014

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Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

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Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi‐synthetic single domain antibody library and development of a VHH‐based lateral flow assay

Wang,

et al. 2024

Veterinary Medicine & Sci

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BackgroundAntibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases.ObjectivesTo expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi‐synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study.MethodsThe semi‐synthetic single domain antibody sequences consist of two parts: one is the FR1‐FR3 region amplified by RT‐PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3‐FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy‐chain antibodies (VHHs). Y. pestis low‐calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi‐synthetic library herein was screened using recombinant (LcrV) as a target antigen.ResultsAfter four cycles of panning the library, four VHH binders targeting 1–270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH‐hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection.ConclusionsThese data demonstrate the great potential of the semi‐synthetic library for use in isolation of antigen‐specific nanobodies and the isolated specific VHHs can be used in antigen‐capture immunoassays.

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Construction and Characterization of Single-Chain Variable Fragment Antibody Library Derived from Germline Rearranged Immunoglobulin Variable Genes

Cited by 5 publications

References 53 publications

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

A primer set for the rapid isolation of scFv fragments against cell surface antigens from immunised rats

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi‐synthetic single domain antibody library and development of a VHH‐based lateral flow assay

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