Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer

Abstract

Abstract. Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDSpolyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein. IntroductionMonoclonal antibodies have become an accepted modality of cancer therapy. Recombinant antibodies and antibody-based fusion proteins hold the promise of further extending the therapeutic possibilities of this modality. Single chain variable fragments (scFv) consist of the variable regions of an antibody's heavy and light chains fused together via a flexible linker, whose length determines the quaternary structure. Thus, they carry the complete antigen binding site in a single polypeptide chain of only about 30 kDa. In tumor targeting, scFv have demonstrated excellent tumor penetration, high ratios of tumor to normal tissue concentration, and low background (1,2). This makes them attractive targeting components of bifunctional fusion proteins such as those needed for antibodydirected enzyme-prodrug therapy (ADEPT). In ADEPT, after binding of an antibody-enzyme construct to the cognate tumor antigen, the enzyme component converts a prodrug into a cytotoxic drug, thus generating drug activity specifically in tumor tissue (3,4).Several ADEPT systems have shown promising in vivo efficacy in a number of tumor models (5) and in several xenograft systems in nude mice (4,6-8), demonstrating in principle that ADEPT can target tumor tissue with high selectivity and deliver chemotherapeutic drugs with high intratumoral concentrations.Senter's group first used bacterial cytosine deaminase for ADEPT to catalyze the deamination of 5-fluorocytosine (5-FC), which is non-toxic in mammals, into 5-fluorouracil (5-FU) (9). Clinical studies on ADEPT have pro...

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

“…Ab-fragments are especially amenable to genetic manipulations (see below) or covalent modifications (e.g., [13,30]) aimed at improving their affinity, stability, and function. Antibody fragments have been linked with enzymes [31], toxins [32], and radionuclides for cancer treatment [33][34][35], as well as viruses for gene therapy, liposomes or nanoparticles for improved drug delivery, and dye or other sensing substances [11,[36][37][38][39]. Figure 2 depicts some Ab-fragments.…”

“…Such affinity resins have been used in the noncommercial purification of scFv's [31,41,80,89,105,106], Fab's [91,[106][107][108], and single-domain antibodies [52,91,98,103,108,109].…”

Production of bifunctional single-chain antibody-based fusion proteins in Pichia pastoris supernatants

“…The bifunctional activity of both fusion proteins from pilot expressions has recently been demonstrated in vitro [24,25], so in this paper we focus on optimization and upscaling of the fermentation method.…”

“…The fusion protein alone or 5-FC alone had no toxic effect on the cells [25]. The cytotoxic effect of A33scFv::CDy followed by 5-FC could be completely inhibited by preincubation with excess A33scFv::GFP, but not by preincubation with HP19scFv, which does not bind to gpA33 (Fig 3).…”

“…antibody binding and fluorescence) by direct flow cytometry and fluorescence microscopy as previously described [24]. For A33scFv::CDy, a previously described in-vitro application Bioprocess Biosyst Eng (2008) 31:559-568 561 of the complete ADEPT system in a fluorescein diacetate assay was employed [25].…”