Single‐Domain Antibodies and Their Utility

Baral, Toya Nath; MacKenzie, Ross; Ghahroudi, Mehdi Arbabi

doi:10.1002/0471142735.im0217s103

Cited by 52 publications

(66 citation statements)

References 123 publications

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“…A phage-displayed sdAb library was constructed from the heavychain-only IgG repertoire of the TGF-β3-immunized llama as previously described (Arbabi-Ghahroudi et al, 2009a;Baral et al, 2013;Henry et al, 2015;Hussack et al, 2011). Briefly, total cellular RNA was extracted from approximately 8 × 10 7 PBMCs using a PureLink ® RNA Mini Kit (Life Technologies, Carlsbad, CA), reverse transcribed using qScript TM cDNA SuperMix (Quanta BioSciences, Gaithersburg, MD) as per the manufacturer's instructions and then pooled.…”

Section: Llama Immunization Library Construction and Panningmentioning

confidence: 99%

“…Rearranged and expressed V H H genes were amplified using semi-nested PCR and cloned into the pMED1 phagemid vector, then phage were rescued from library-bearing Escherichia coli TG1 cells by superinfection with M13KO7 helper phage (Life Technologies) and purified by polyethylene glycol precipitation. The phage-displayed sdAb library was panned in a single round against immobilized TGF-β3 as previously described (ArbabiGhahroudi et al, 2009a;Baral et al, 2013;Henry et al, 2015;Hussack et al, 2011) Creative Biolabs, Shirley, NY).…”

Section: Llama Immunization Library Construction and Panningmentioning

confidence: 99%

“…The next morning, periplasmic proteins were extracted by osmotic shock, dialyzed and purified on a HiTrap IMAC HP column (GE Healthcare, Piscataway, NJ) using an ÄKTA FPLC protein purification system (GE Healthcare) with yields ranging from 1.2 to 14 mg/250 mL. Prior to surface plasmon resonance (SPR), the sdAbs were purified by size exclusion chromatography as previously described (Arbabi-Ghahroudi et al, 2009a;Baral et al, 2013) using a Superdex TM 75 10/300 GL column (GE Healthcare) on an ÄKTA FPLC protein purification system (GE Healthcare). To determine the affinities of the sdAbs to TGF-β isoforms (all gifts from A. Hinck), a total of 369-530 resonance units (RUs) of each TGF-β protein were immobilized in 10 mM acetate buffer, pH 4.5, on CM5 sensor chips (GE Healthcare) using an amine coupling kit (GE Healthcare).…”

Section: Expression and Characterization Of Sdabsmentioning

confidence: 99%

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Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing

Henry

Hussack

Collins

et al. 2016

Protein Engineering, Design and Selection

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Vous avez des questions? Nous pouvons vous aider. Pour communiquer directement avec un auteur, consultez la première page de la revue dans laquelle son article a été publié afin de trouver ses coordonnées. Si vous n'arrivez pas à les repérer, communiquez avec nous à PublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. Questions? Contact the NRC Publications Archive team atPublicationsArchive-ArchivesPublications@nrc-cnrc.gc.ca. If you wish to email the authors directly, please see the first page of the publication for their contact information. NRC Publications Archive Archives des publications du CNRCThis publication could be one of several versions: author's original, accepted manuscript or the publisher's version. / La version de cette publication peut être l'une des suivantes : la version prépublication de l'auteur, la version acceptée du manuscrit ou la version de l'éditeur. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=646f2540-63b6-4b0e-a7ae-731f5c2a6082 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=646f2540-63b6-4b0e-a7ae-731f5c2a6082 Protein Engineering, Design & Selection, 2016, pp. 1-5 doi: 10.1093/protein/gzw043 AbstractThe epitope specificity of therapeutic antibodies is often critical to their efficacy and mode of action. Here, we report the isolation of single-domain antibodies (sdAbs) against a pre-specified epitope of TGF-β3: namely, the site of interaction between the cytokine and its cell-surface type II receptor. By panning a phage-displayed immune llama VHH library against TGF-β3 using competitive elution with soluble dimeric type II receptor ectodomain in tandem with next-generation DNA sequencing, we identified several sdAbs that competed with the receptor for TGF-β3 binding and neutralized TGF-β3i nin vitro cellular assays. In contrast, all other sdAbs identified using conventional panning approaches (i.e., without regard to epitope specificity) did not target the site of receptor:cytokine interaction. We expect this strategy to be generally applicable for identifying epitope-specific sdAbs when binding reagents directed against the epitope of interest are available. The sdAbs identified here are of potential interest as cancer immunotherapeutics.

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Section: Llama Immunization Library Construction and Panningmentioning

confidence: 99%

Section: Llama Immunization Library Construction and Panningmentioning

confidence: 99%

Section: Expression and Characterization Of Sdabsmentioning

confidence: 99%

See 1 more Smart Citation

Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing

Henry

Hussack

Collins

et al. 2016

Protein Engineering, Design and Selection

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show abstract

“…No. HE2-H5225; ACROBiosystems, Beijing, China) as described previously [7,8]. All animal procedures were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O.…”

mentioning

confidence: 99%

“…Next, we panned the phage-displayed V H H libraries for four rounds against CD73 and HER2 directly adsorbed in wells of microtiter plates and eluted the bound sdAb-phage using triethylamine as previously described [7,8]. Library phage and phage particles eluted from each round of panning were used as PCR templates to amplify rearranged V H H genes using primers nano-MJ7 and nano-MJ8 (Table 1), as previously described [8,9].…”

mentioning

confidence: 99%

Oxford Nanopore Sequencing Enables Rapid Discovery of Single-Domain Antibodies from Phage Display Libraries

Lowden

Henry

2018

BioTechniques

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Antibody (Ab) repertoire sequencing using high-throughput massively parallel technologies has contributed substantially to the understanding of Ab responses following infection, vaccination and autoimmunity. Because individual B-cell receptors are recombined and diversified somatically, genomic comparisons are limited, and distinguishing rare variants from sequencing errors is a major challenge. Oxford Nanopore Technologies MinION is a highly portable and cost-effective third-generation sequencing instrument, but has not been used for Ab repertoire sequencing due to its high error rate (approximately 1/10 bases). Here, we applied nanopore sequencing to single-domain Ab (sdAb) repertoires and phage-displayed sdAb libraries. We show that despite low overall data fidelity, sdAb sequences could be reconstructed above a frequency threshold (100 copies); however, distinguishing clonal sdAb variants was not always possible. The data quality was sufficient to enable rapid identification of antigen-specific sdAb sequences enriched during panning of phage display libraries, obviating the need for screening single clones.

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Mouse Model for Pre‐Clinical Study of Human Cancer Immunotherapy

Hailemichael

Overwijk

et al. 2015

CP in Immunology

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This unit describes protocols for developing tumors in mice including subcutaneous growth, pulmonary metastases of B16 melanoma, and spontaneous melanoma in B-Raf V600E/PTEN deletion transgenic mouse models. Two immunization methods to prevent B16 tumor growth are described using B16.GM-CSF and recombinant vaccinia virus. A therapeutic approach is also included that uses adoptive transfer of tumor antigen-specific T cells. Methods including CTL induction, isolation, testing, and genetic modification of mouse T cells for adoptive transfer by using retrovirus expressing genes of interest are provided. Additional sections, including growing B16 melanoma, enumerating pulmonary metastases, tumor imaging technique and use of recombinant viruses for vaccination, are discussed together with safety concerns.

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Single‐Domain Antibodies and Their Utility

Cited by 52 publications

References 123 publications

Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing

Isolation of TGF-β-neutralizing single-domain antibodies of predetermined epitope specificity using next-generation DNA sequencing

Oxford Nanopore Sequencing Enables Rapid Discovery of Single-Domain Antibodies from Phage Display Libraries

Mouse Model for Pre‐Clinical Study of Human Cancer Immunotherapy

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