Characterization of Site-Specific ScFv PEGylation for Tumor-Targeting Pharmaceuticals

Abstract

New radiopharmaceuticals are possible using site-specific conjugation of small tumor binding proteins and poly(ethylene glycol) (PEG) scaffolds to provide modular multivalent, homo- or heterofunctional cancer-targeting molecules having preferred molecular size, valence, and functionality. Residence time in plasma can be optimized by modification of the size, number, and charge of the protein units. However, random PEG conjugation (PEGylation) of these small molecules via amine groups has led to variations of structural conformation and binding affinity. To optimize PEGylation, scFvs have been recombinantly produced in a vector that adds an unpaired cysteine (c) near the scFv carboxy terminus (scFv-c), thus providing a specific site for thiol conjugation. To evaluate the general applicability of this unpaired cysteine for PEGylation of scFv-c, conjugation efficiency was determined for four different scFvs and several PEG molecules having thiol reactive groups. The effect of the PEG molecular format on scFv-c PEG malignant cell binding was also addressed. ScFvs produced as scFv-c and purified by anti E-TAG affinity chromatography were conjugated using PEG molecules with maleimide (Mal) or o-pyridyl disulfide (OPSS). Conjugations were performed at pH 7.0, with 2 molar excess TCEP/scFv and PEG-(Mal) or PEG-OPSS, using 5:1 (PEG/scFv). PEG-Mal conjugation efficiency was also evaluated with 1:5 (PEG/scFv). PEGylation efficiency was determined for each reaction by quantitation of the products on SDS-PAGE. ScFv-c conjugation with unifunctional maleimide PEGs resulted in PEG conjugates incorporating 30-80% of the scFv-c, but usually above 50%. Efficiency of scFv-c conjugation to both functional groups of the bifunctional PEG-(Mal)2 varied between the PEG and scFv-c molecules studied. A maximum of 45% of scFv-c protein was conjugated as PEG- (scFv-c)2 using the smallest PEG-(Mal)2 (2 kDa). No significant increase in scFv-c conjugation was observed by the use of greater than a 5 molar excess of PEG/scFv-c. Under the same conjugation conditions, PEG as OPSS yielded less than 10% PEG-scFv-c. PEG-(scFv)2 conjugates had increased binding in ELISA using malignant cell membranes, when compared with unmodified scFv-c. PEGylated-scFv binding was comparable with unmodified scFv-c. In summary, scFv-c can be PEGylated in a site-specific manner using uni- or bivalent PEG-Mal, either linear or branched. ScFv-c was most efficiently conjugated to smaller PEG-Mal molecules, with the smallest, 2 kDa PEG-Mal, usually PEGylating 60-90% of the scFv-c. ScFv-c conjugation to form PEG-(scFv-c)2 reached greatest efficiency at 45%, and its purified form demonstrated greater binding than the corresponding scFv-c.

Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region

“…The scAb form of M18 was fused to a His 6 tag followed by a terminal Cys residue for the conjugation of PEG-maleimide (1,37,54). We have found that the scAb form of antibodies, comprised of an scFv fragment fused to a human C domain (34), has superior expression, stability, and serum half-lives compared to scFv fragments (33).…”

“…The availability of maleimide-PEG has allowed conjugation to antibodies through reaction with free native or engineered cysteines (12). This chemistry has proven to be suitable not only for Fab but also for single-chain variable fragments (scFvs) (1,37,54), immunoliposomes (38), and other conjugates (19).…”

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

“…The unbound mPEG 2 -Mal was removed by IMAC purification. PEGylation was visualized by barium iodide staining of SDS-polyacrylamide gels as described (23).…”

Enhancement of the Therapeutic Index: From Nonmyeloablative and Myeloablative toward Pretargeted Radioimmunotherapy for Metastatic Prostate Cancer

“…Sitespecific conjugation studies of di-scFv-c to PEG-Mal indicate that the free thiol is accessible in these configurations with a scFv-c-scFv format providing >90% conjugation. PEGylation efficiency seems inversely proportional to the length of the PEG (65). The di-scFv-c module for site-specific and covalent attachment to a PEG scaffold provides flexibility in the use of various scFv units.…”