A Pentavalent Single-domain Antibody Approach to Tumor Antigen Discovery and the Development of Novel Proteomics Reagents

Zhang, Jianbing; Li, Qinggang; Nguyen, Thanh-Dung; Tremblay, Tammy-Lynn; Stone, Emily; To, Rebecca; Kelly, John F.; MacKenzie, Colin R.

doi:10.1016/j.jmb.2004.05.069

Cited by 44 publications

(41 citation statements)

References 36 publications

(36 reference statements)

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“…84,2010 RETARGETING ADENOVIRUS VECTORS THROUGH GENETIC FUSION 10081 A549 cells, CD66c is variably glycosylated and appears as a diffuse region of immunoreactivity by immunoblot analysis (Fig. 6B), as previously reported (69,86). AFAI is known to interact with a specific glycosylation variant of CD66c (86), and we were readily able to detect the interaction between the two proteins, suggesting that fusion of AFAI to pIX did not alter its ability to recognize CD66c.…”

Section: Mr1 Fused To Native Pix Does Not Recognize the Egfrviii Epitsupporting

confidence: 84%

“…pAFAI encodes a single-domain antibody (sdAb) identified through panning of a llama sdAb phage library on the non-small-cell lung carcinoma cell line A549 (86), and it was a kind gift of Jianbing Zhang (National Research Council, Ottawa, ON, Canada). This sdAb recognizes a variant of CD66c (carcinoembryonic antigen-related cell adhesion molecule 6 [CEACAM6 or CEA6]) (44).…”

Section: Methodsmentioning

confidence: 99%

“…The knob domains of Ad2 and AFAI sdAb were prepared in bacteria and purified as previously described (7,86). A549 cells were seeded into 35-mm dishes and, the next day, were preincubated with various quantities of knob or AFAI protein for 15 min at 37°C, at which time AdRP2218, AdRP2595, or AdRP2823 was added at an MOI of 10 PFU/cell.…”

Section: Methodsmentioning

confidence: 99%

“…To determine if pIX can be used to display sdAd on the capsid surface and lead to altered transduction characteristics, we generated a virus that contained a fusion protein composed of pIX and AFAI, an sdAb identified through phage panning of the A549 non-small-cell lung carcinoma cell line (86), and examined its ability to infect A549 cells. AFAI binds to CD66c (carcinoembryonic antigen-related cell adhesion molecule family member 6 [CEACAM6]), which is expressed on a subset of A549 cells in the culture (69,86). To determine if pIX-AFAI was efficiently incorporated into the Ad capsid, we examined the protein content of purified capsid by immunoblot analysis for the FLAG-tagged pIX-AFAI and fiber (loading control).…”

Section: Mr1 Fused To Native Pix Does Not Recognize the Egfrviii Epitmentioning

confidence: 99%

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Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Poulin

Lanthier

Smith

et al. 2010

J Virol

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Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

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Section: Mr1 Fused To Native Pix Does Not Recognize the Egfrviii Epitsupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mr1 Fused To Native Pix Does Not Recognize the Egfrviii Epitmentioning

confidence: 99%

See 2 more Smart Citations

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Poulin

Lanthier

Smith

et al. 2010

J Virol

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“…87 Due to their small size and very high stability, sdAbs are probably the only antibody fragments suitable to be fused to the B subunit of AB 5 toxins to form pentabodies. 88,89 Three pentabodies, with different affinities for bovine serum albumin (BSA), were constructed and evaluated in mice. The pentabody with the highest affinity to BSA induced mucosal response equivalent to that induced by CT as an adjuvant while the pentabodies with lower affinities did not enhance mucosal response.…”

Section: Exploitation Of Bacterial Ab 5 Toxins As Mucosal Adjuvantsmentioning

confidence: 99%

Recent advances in the development of novel mucosal adjuvants and antigen delivery systems

Chen¹,

Patel²,

Yan³

et al. 2010

Human Vaccines

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Single‐Domain Antibodies and Their Utility

2013

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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. NRC Publications Record / Notice d'Archives des publications de CNRC:http://nparc.cisti-icist.nrc-cnrc.gc.ca/eng/view/object/?id=e552ed0a-502f-47b3-a6f6-9daf5bd955d3 http://nparc.cisti-icist.nrc-cnrc.gc.ca/fra/voir/objet/?id=e552ed0a-502f-47b3-a6f6-9daf5bd955d3 ABSTRACTEngineered monoclonal antibody fragments have gained market attention due to their versatility and tailor-made potential and are now considered to be an important part of future immunobiotherapeutics. Single-domain antibodies (sdAbs), also known as nanobodies, are derived from VHHs [variable domains (V) of heavy-chain-only antibodies (HCAb)] of camelid heavy-chain antibodies. These nature-made sdAbs are well suited for various applications due to their favorable characteristics such as small size, ease of genetic manipulation, high affinity and solubility, overall stability, resistance to harsh conditions (e.g., low pH, high temperature), and low immunogenicity. Most importantly, sdAbs have the feature of penetrating into cavities and recognizing hidden epitopes normally inaccessible to conventional antibodies, mainly due to their protruding CDR3/H3 loops. In this unit, we will present and discuss comprehensive and step-by-step protocols routinely practiced in our laboratory for isolating sdAbs from immunized llamas (or other members of the Camelidae family) against target antigens using phage-display technology. Expression, purification, and characterization of the isolated sdAbs will then be described, followed by presentation of several examples of applications of sdAbs previously characterized in our laboratory and elsewhere.

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A Pentavalent Single-domain Antibody Approach to Tumor Antigen Discovery and the Development of Novel Proteomics Reagents

Cited by 44 publications

References 36 publications

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Retargeting of Adenovirus Vectors through Genetic Fusion of a Single-Chain or Single-Domain Antibody to Capsid Protein IX

Recent advances in the development of novel mucosal adjuvants and antigen delivery systems

Single‐Domain Antibodies and Their Utility

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