Cloning Murine Antibody V-genes with Non-degenerate Primers and Conversion to a Recombinant Antibody Format

Bialon, Magdalena; Schellenberg, Ludmila; Herzog, Nicolas; Kraus, Stefan; Jörißen, Hannah; Fischer, Rainer; Stein, Christoph; Nähring, Jörg; Barth, Stefan; Püttmann, Christiane

doi:10.1089/mab.2014.0044

Cited by 6 publications

(4 citation statements)

References 30 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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“…The V H and V L sequences of the TTC-reactive antibody from the murine hybridoma cell line 5E4 were identified by V-gene rescue polymerase chain reaction (PCR) with the primer set described in our earlier report [ 50 ]. The V L and V H sequences were amplified using Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific), 200 nM of each primer and 1 μl of cDNA in a 50-μl reaction volume.…”

Section: Methodsmentioning

confidence: 99%

Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

et al. 2017

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The antigen-specific targeting of autoreactive B cells via their unique B cell receptors (BCRs) is a novel and promising alternative to the systemic suppression of humoral immunity. We generated and characterized cytolytic fusion proteins based on an existing immunotoxin comprising tetanus toxoid fragment C (TTC) as the targeting component and the modified Pseudomonas aeruginosa exotoxin A (ETA') as the cytotoxic component. The immunotoxin was reconfigured to replace ETA' with either the granzyme B mutant R201K or MAPTau as human effector domains. The novel cytolytic fusion proteins were characterized with a recombinant human lymphocytic cell line developed using Transpo-mAb™ technology. Genes encoding a chimeric TTC-reactive immunoglobulin G were successfully integrated into the genome of the precursor B cell line REH so that the cells could present TTC-reactive BCRs on their surface. These cells were used to investigate the specific cytotoxicity of GrB(R201K)-TTC and TTC-MAPTau, revealing that the serpin proteinase inhibitor 9-resistant granzyme B R201K mutant induced apoptosis specifically in the lymphocytic cell line. Our data confirm that antigen-based fusion proteins containing granzyme B (R201K) are suitable candidates for the depletion of autoreactive B cells.

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“…That is because hybridoma cultures produce low concentrations of antibodies and are prone to microbial contamination and genetic instability, and therefore cannot be used for large-scale antibody production. 8 In addition, the use of murine Mabs for human disease is limited due to the strong immune response elicited by mouse antibodies when administered to humans. In order to minimize the risk to humans while maintaining the antibody specificity and affinity, human/mouse chimeric antibodies (~65% human) and humanized Mabs (~95% human) are produced using recombinant cloning techniques.…”

Section: Introductionmentioning

confidence: 99%

“…10 PCR-based strategies are the most widely used technique to identify the variable region sequences and to clone the fragments into the appropriate antibody expression vectors, although other techniques have been developed. 8 In the absence of IgG isotype information, degenerate PCR primers are used to amplify the variable regions for cloning or sequencing. However, this can often result in mismatches in the primer binding site, leading to degradation of PCR products by proofreading DNA polymerases.…”

Section: Introductionmentioning

confidence: 99%

Expediting Antibody Discovery with a Cell and Bead Multiplexed Competition Assay

Liu

O’Rourke

2018

SLAS Discovery

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With advances in molecular engineering and humanization, monoclonal antibodies are one of the fastest-growing classes of biopharmaceuticals. During antibody discovery, antibody from hybridoma or primary B-cell supernatants is screened for the desired binding characteristics, and secondary screens measure antibody function and concentration, identify immunoglobulin G (IgG) isotype, and assess cell health. In order to expedite the antibody discovery process, we developed a high-throughput, multiplexed cell and bead-based competition assay that identifies and quantitates mouse IgG isotypes and assesses cell health. No differences in assay performance were observed between single and multiplex formats. The linear range of the assay was from 0.5 to 50 µg/mL, and washing was not required, decreasing assay time and variability. Slight modifications to the protocol allowed quantification of dilute antibody supernatants (0.1-5 µg/mL). Using hybridoma cultures, we showed that cell viability measurements in the assay did not interfere with the bead-based IgG measurements. The assay described here is a simple mix-and-read, no-dilution screen that can reduce the time to antibody cloning and production. The high-content data can differentiate monoclonal and polyclonal wells, determine IgG quantity for downstream functional assays, provide isotype information, and monitor cell proliferation and viability.

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Cloning Murine Antibody V-genes with Non-degenerate Primers and Conversion to a Recombinant Antibody Format

Cited by 6 publications

References 30 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 an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

Expediting Antibody Discovery with a Cell and Bead Multiplexed Competition Assay

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