Introduction Biotinylated antibodies are commonly used reagents in research and today they are under investigation for clinical applications (immunotargeting). Promising results are reported in radioimmunodetection and radioimmunotherapy using biotinylated-antibody constructs with streptavidin-radionuclide conjugates. Biotinylated antibodies are normally produced by chemical conjugation of active biotin molecules to amino groups on protein surface. An alternative method, which can be used for site-specific biotinylation of recombinant antibodies, takes advantage of the capability of the enzyma biotin ligase to catalyze the attachment of a biotin to a unique lysine residue in specific protein/peptide substrates that can be genetically linked to the antibody to generate a fusion protein. We describe the expression of functional scFv and concomitant enzymatic biotinylation of it in bacterial cytoplasm. We have used the anti-PSMA (prostate specific membrane antigen specific) scFv that recognizes the extracellular domain of human PSMA. PSMA is a transmembrane protein that is largely restricted to prostatic epithelial cells in humans and is strongly upregulated on prostatic carcinoma cells. It is also expressed on the endothelium of tumor vasculature. Materials and methods The single chain antibody fragment (scFv) derived from the anti-human PSMA D2B hybridoma is generated using recombinant DNA technology. The plasmid used to obtain enzimatically mono-biotinylated scFv was generated modifying the pET 20b vector backbone. Recombinant protein is expressed in two E. coli strains: AVB101 (Avidity Inc. USA) or BL21(DE3)pLysS. AVB101 allows us to obtain the biotinylated protein directly from the bacterial culture. Induction of expression of both recombinant proteins scFv and biotin ligase is induced with IPTG (AVB101), whereas the protein produced in BL21(DE3)pLysS was biotinylated by enzymatic reaction. The protein was finally purified by sequential passages on anion-exchange column (CM), affinity chromatography and then concentrated/purified on centricon centrifuge filters. The avidity and the specificity of the protein was analized by flow cytometry. Results Using recombinant DNA technology the expression vector has been constructed for mono-biotinylated scFv. Different domains were cloned at the 3’ site of scFv in the following order: a semi-rigid hinge region derived from human IgA, BAD (Biotin Acceptor Domain), a cleavable domain from proteases enterokinase, factor Xa and a histidine tail for purification. Biotinylated protein was expressed in AVB101 or in BL21(DE3)pLysS; expression of both the recombinant scFv and the biotin ligase were induced with IPTG and, also, in the presence of D-biotin for AVB101 strain. The protein was purified on CM sepharose and then on CNBr-α-mouse affinity column or by centricon 100 to a purity of about 50%. The protein was biotinylated with the enzyme and the excess contaminanting proteins and non biotinilated scFv were removed using a Soft link column (Promega,Madison, WI). The yield of the “bacterial” biotinylation reaction is about 30%. The degree of protein purity was estimated by gel SDS-PAGE and the correct identity was confirmed by western Blot, using Ab anti-scFv and HRP conjugate streptavidin to detect biotinylated protein. The specificity and activity of biotynilated scFv sintethized were analyzed by FACS on PSMA+cells (LNCaP) and PSMA-(PC3). MFI (mean fluorescence intensity) on LNCaP is 161, 728, 314 respectively for the negative control (avidin-FITC), chemical biotinylated Ab anti-PSMA and enzymatically biotinylated scFv anti-PSMA. The MFI on a negative cell line is 132, 151,180,respectively. Conclusions The new vector is useful for producing scFv mono-biotinylated without altering the capacity of binding and manteining a good specificity and affinity. It will be therefore an important tool for the development of procedures of imaging and in therapy. However, we will have to improve the purification steps, the yield of the biotinylated protein and take measures to avoid the scFv aggregation.

Production of a biotinylated anti-PSMA single-chain antibody fragment (scFv) for imaging and radioimmunotherapy

GIGLIO, BARBARA;FRACASSO, Giulio;CINGARLINI, Sara;ANSELMI, Cristina;CREMONESE, Giorgia;CETTO, Gianluigi;COLOMBATTI, Marco
2008

Abstract

Introduction Biotinylated antibodies are commonly used reagents in research and today they are under investigation for clinical applications (immunotargeting). Promising results are reported in radioimmunodetection and radioimmunotherapy using biotinylated-antibody constructs with streptavidin-radionuclide conjugates. Biotinylated antibodies are normally produced by chemical conjugation of active biotin molecules to amino groups on protein surface. An alternative method, which can be used for site-specific biotinylation of recombinant antibodies, takes advantage of the capability of the enzyma biotin ligase to catalyze the attachment of a biotin to a unique lysine residue in specific protein/peptide substrates that can be genetically linked to the antibody to generate a fusion protein. We describe the expression of functional scFv and concomitant enzymatic biotinylation of it in bacterial cytoplasm. We have used the anti-PSMA (prostate specific membrane antigen specific) scFv that recognizes the extracellular domain of human PSMA. PSMA is a transmembrane protein that is largely restricted to prostatic epithelial cells in humans and is strongly upregulated on prostatic carcinoma cells. It is also expressed on the endothelium of tumor vasculature. Materials and methods The single chain antibody fragment (scFv) derived from the anti-human PSMA D2B hybridoma is generated using recombinant DNA technology. The plasmid used to obtain enzimatically mono-biotinylated scFv was generated modifying the pET 20b vector backbone. Recombinant protein is expressed in two E. coli strains: AVB101 (Avidity Inc. USA) or BL21(DE3)pLysS. AVB101 allows us to obtain the biotinylated protein directly from the bacterial culture. Induction of expression of both recombinant proteins scFv and biotin ligase is induced with IPTG (AVB101), whereas the protein produced in BL21(DE3)pLysS was biotinylated by enzymatic reaction. The protein was finally purified by sequential passages on anion-exchange column (CM), affinity chromatography and then concentrated/purified on centricon centrifuge filters. The avidity and the specificity of the protein was analized by flow cytometry. Results Using recombinant DNA technology the expression vector has been constructed for mono-biotinylated scFv. Different domains were cloned at the 3’ site of scFv in the following order: a semi-rigid hinge region derived from human IgA, BAD (Biotin Acceptor Domain), a cleavable domain from proteases enterokinase, factor Xa and a histidine tail for purification. Biotinylated protein was expressed in AVB101 or in BL21(DE3)pLysS; expression of both the recombinant scFv and the biotin ligase were induced with IPTG and, also, in the presence of D-biotin for AVB101 strain. The protein was purified on CM sepharose and then on CNBr-α-mouse affinity column or by centricon 100 to a purity of about 50%. The protein was biotinylated with the enzyme and the excess contaminanting proteins and non biotinilated scFv were removed using a Soft link column (Promega,Madison, WI). The yield of the “bacterial” biotinylation reaction is about 30%. The degree of protein purity was estimated by gel SDS-PAGE and the correct identity was confirmed by western Blot, using Ab anti-scFv and HRP conjugate streptavidin to detect biotinylated protein. The specificity and activity of biotynilated scFv sintethized were analyzed by FACS on PSMA+cells (LNCaP) and PSMA-(PC3). MFI (mean fluorescence intensity) on LNCaP is 161, 728, 314 respectively for the negative control (avidin-FITC), chemical biotinylated Ab anti-PSMA and enzymatically biotinylated scFv anti-PSMA. The MFI on a negative cell line is 132, 151,180,respectively. Conclusions The new vector is useful for producing scFv mono-biotinylated without altering the capacity of binding and manteining a good specificity and affinity. It will be therefore an important tool for the development of procedures of imaging and in therapy. However, we will have to improve the purification steps, the yield of the biotinylated protein and take measures to avoid the scFv aggregation.
PSMA; imaging; biotinylation
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11562/340886
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