Functional expression of a single-chain antibody specific for the HER2 human oncogene in a bacterial reducing environment

A lentiviral vector‐based adenovirus fiber‐pseudotyping approach for expedited functional assessment of candidate retargeted fibers

“…The intactness of these functionalities suggests that the respective responsible functional domains, fibritin foldon and scFv800E6, can both fold correctly in the context of this particular chimeric fiber configuration. The indication that scFv800E6 is able to acquire a functional fold as part of an Ad capsid fusion protein would further illustrate the stability and versatility previously reported for this scFv 39, 53, 54. Thus, these preliminary characterizations suggested the suitability of the 800E6 fiber for Ad capsid incorporation and vector targeting.…”

“…With the fiber‐pseudotyping system described above in place, we subsequently used it for the functional assessment of a new candidate retargeted fiber. In this regard, we sought to functionally test a chimeric fiber molecule harboring a scFv directed against tumor antigen HER2/ neu 39, 53, 54. Importantly, this particular single‐chain antibody, named scFv800E6, was specifically chosen for its intrinsic capability to fold efficiently and functionally in a reducing environment, a recognized prerequisite for bona fide assembly into the Ad capsid as part of a genetic capsid protein fusion 37, 55–57.…”

“…Therefore, to obtain more clues with respect to the ability of 800E6 fiber (and in particular its scFv constituent) to fold properly, we sought to investigate its redox state. On this topic, scFv800E6 had previously been found to be functionally folded when produced within a reducing environment 39, 53, 54, a property on which the candidacy for being part of a capsid incorporable fiber chimera was based. This preserved functionality suggests that the correct folding of scFv800E6 is independent of the immunoglobulin‐superfamily canonical intradomain disulfide bridges, of which this scFv has the potential to form two per polypeptide.…”

“…In view of the favorable results obtained with stabilized scFvs, we hypothesized that scFv800E6 39, 53, 54, an intrinsically stable scFv directed to the important tumor associated antigen Her2/ neu 69, would also be suited for the purpose of genetic Ad capsid incorporation. This scFv was previously found to possess, by nature, a structural framework of particular robustness, and it was observed to functionally fold in a reducing environment in a variety of expression platforms 39, 53, 54. Thus, for reasons of both therapeutic relevance and technical feasibility, scFv800E6 represents an interesting moiety to test for functionality as a targeting ligand genetically displayed on an Ad capsid protein.…”

In vivo expression and binding activity of scFv-RWAV, which recognizes the coat protein of tomato leaf curl New Delhi virus (family Geminiviridae)

“…Although many recombinant antibodies have been expressed successfully in transgenic plants [21,22], they have generally been directed to different subcellular compartments [23][24][25] because the cytoplasm does not provide a favorable environment for protein folding and the formation of disulfide bonds [26]. Functional scFvs have been expressed in the cytoplasm of Escherichia coli [27], but only a small number have proven to be stable and functional in the plant cytoplasm [26,28,29]. The stability and activity of antibodies usually relies on the formation of intradomain disulfide bonds within the variable regions [30,31], and disulfide bonds form well in the oxidizing environment of the secretory pathway but not in the reducing environment of the cytosol [26,28,32,33].…”