Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

Abstract

Background: Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen.

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

“…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.…”

“…The generation of an 800E6 fiber‐expressing LV vector plasmid involved several cloning steps, which, in effect, amount to the following. The coding sequence for scFv800E6 was PCR‐amplified from pIVEX 2.4d‐His(6x)‐ad‐N‐ScFv800E6 39 using primers 800E6‐NcoI‐MluI‐F and 800E6‐BsrGI‐R (Table 1). Subsequently, employing the introduced Nco I site, the obtained scFv800E6 sequence was ligated at its 5′ end to a chimeric gene fragment (made by PCR‐mediated SOE) encoding the tail and first shaft repeat of the HAdV‐5 fiber protein followed by the coiled coil segment 13 and foldon domain of the bacteriophage T4 fibritin protein.…”

Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus

“…To date, scFv antibodies have been expressed in various systems, including mammalian cells (Ho et al, 2006), yeast (Koti et al, 2010(Koti et al, , 2011, plants (Galeffi et al, 2006), insect cells (Choo et al, 2002), and E. coli (Cao et al, 2003). Current knowledge of the genetics and biochemistry of E. coli make it a useful expression host (Baneyx, 1999).…”