Divalent Forms of CC49 Single-Chain Antibody Constructs in Pic) pastoris: Expression, Purification, and Characterization

Abstract

Single-chain variable fragments (scFvs) are tumor-recognition units that hold enormous potential in antibody-based therapeutics. Their clinical applications, however, require the large scale production and purification of biologically active recombinant scFvs. In the present study, we engineered and expressed divalent non-covalent [(scFv)(2)-His(6)] and covalent [sc(Fv)(2)-His(6)] scFvs of a tumor-associated monoclonal antibody (MAb) CC49 in Pichia pastoris. The purity and immunoreactivity of the scFvs were analyzed by SDS-PAGE, HPLC, and competitive ELISA. The binding affinity constant (K(A)), determined by surface plasmon resonance analysis using BIAcore, was 4.28 x 10(7), 2.75 x 10(7), and 1.14 x 10(8) M(-1) for (scFv)(2)-His(6), sc(Fv)(2)-His(6), and CC49 IgG, respectively. The expression of scFvs in P. pastoris was 30 to 40-fold higher than in Escherichia coli. Biodistribution studies in athymic mice bearing LS-174T human colon carcinoma xenografts showed equivalent tumor-targeting of CC49 dimers generated in yeast (scFv)(2)-His(6) and bacteria (scFv)(2) with 12.52% injected dose/gram (%ID/g) and 11. 42%ID/g, respectively, at 6 h post-injection. Interestingly, the pharmacokinetic pattern of dimeric scFvs in xenografted mice exhibited a slower clearance of His-tagged scFvs from the blood pool than scFvs lacking the His-tag (0.1 >/= p >/= 0.05). In conclusion, improved yields of divalent scFvs were achieved using the P. pastoris expression/secretion system. The in vitro and in vivo properties of these scFvs suggest possible therapeutic applications.

In Vivo Evaluation of Cysteine-Based Chelators for Attachment of 99mTc to Tumor-Targeting Affibody Molecules

“…Comparative biodistribution in normal mice (Figure 4) showed that the labeled conjugates had rather rapid blood clearance, with less than 1% IA/g at 4 h pi. This is in the same range (0.3-1.8% IA/g) as in mice for 99m Tc-labeled somatostatin analogues (37) and lower than the blood level reported at that time for 99m Tc-labeled scFv (38) or their multimeric constructs (39). The low blood radioactivity concentration suggested that, in agreement with the in Vitro serum stability test, no considerable transchelation to blood plasma proteins occurred.…”

Penetratin Improves Tumor Retention of Single-Chain Antibodies: A Novel Step toward Optimization of Radioimmunotherapy of Solid Tumors

“…Recently, patient trials have been done for an engineered anti-CEA minibody cT88.66, where 123 I-labeled minibody successfully targeted colorectal cancer and exhibited faster blood clearance than intact antibody (7). We have previously generated and evaluated monovalent, divalent, and tetravalent scFv constructs of anti-TAG-72 mAb CC49 (8)(9)(10). The divalent [sc (Fv) 2 ] and tetravalent ([sc(Fv) 2 ] 2 ) constructs are of particular interest due to the pharmacologic advantage of these molecules over larger IgG and smaller monovalent scFv fragments.…”

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

“…In a separate study by another group, using a tag-free anti-CD45 scFv, the uptake and binding by the liver was much lower at only < 5% of the injected dose [123]. Other studies, however, have not demonstrated an increase in liver uptake of scFvs that contain a poly-his tag [124,125]. Taken together, these results further suggest that the PK/BD of scFv fragments or scFv conjugates may be influenced by molecular tags within the constructs.…”