Protein Engineering vol. 16 no. 10 pp. 761-770, 2003
© 2003 Oxford University Press
Tailoring structure–function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation
Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA
1 To whom correspondence should be addressed. e-mail: david.filpula{at}enzon.com
The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-
scFv at the C-terminus or within the linker segments of both scFv orientations, VL–linker–VH and VH–linker–VL. High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide–PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Indepen dent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.
Received June 25, 2003; revised July 16, 2003; accepted July 20, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. J. DeNardo, R. Liu, H. Albrecht, A. Natarajan, J. L. Sutcliffe, C. Anderson, L. Peng, R. Ferdani, S. R. Cherry, and K. S. Lam 111In-LLP2A-DOTA Polyethylene Glycol-Targeting {alpha}4{beta}1 Integrin: Comparative Pharmacokinetics for Imaging and Therapy of Lymphoid Malignancies J. Nucl. Med., April 1, 2009; 50(4): 625 - 634. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Riley, K. P. Engelhardt, J. W. Saldanha, W. Qi, L. S. Cooke, Y. Zhu, S. T. Narayan, K. Shakalya, K. D. Croce, I. G. Georgiev, et al. Design and Activity of a Murine and Humanized Anti-CEACAM6 Single-Chain Variable Fragment in the Treatment of Pancreatic Cancer Cancer Res., March 1, 2009; 69(5): 1933 - 1940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Taguchi, S. Planque, G. Sapparapu, S. Boivin, M. Hara, Y. Nishiyama, and S. Paul Exceptional Amyloid {beta} Peptide Hydrolyzing Activity of Nonphysiological Immunoglobulin Variable Domain Scaffolds J. Biol. Chem., December 26, 2008; 283(52): 36724 - 36733. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Muller, A. Karle, B. Meissburger, I. Hofig, R. Stork, and R. E. Kontermann Improved Pharmacokinetics of Recombinant Bispecific Antibody Molecules by Fusion to Human Serum Albumin J. Biol. Chem., April 27, 2007; 282(17): 12650 - 12660. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Kubetzko, E. Balic, R. Waibel, U. Zangemeister-Wittke, and A. Pluckthun PEGylation and Multimerization of the Anti-p185HER-2 Single Chain Fv Fragment 4D5: EFFECTS ON TUMOR TARGETING J. Biol. Chem., November 17, 2006; 281(46): 35186 - 35201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E.-M. Krinner, J. Hepp, P. Hoffmann, S. Bruckmaier, L. Petersen, S. Petsch, L. Parr, I. Schuster, S. Mangold, G. Lorenczewski, et al. A highly stable polyethylene glycol-conjugated human single-chain antibody neutralizing granulocyte-macrophage colony stimulating factor at low nanomolar concentration Protein Eng. Des. Sel., October 1, 2006; 19(10): 461 - 470. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. DeFrees, Z.-G. Wang, R. Xing, A. E. Scott, J. Wang, D. Zopf, D. L. Gouty, E. R. Sjoberg, K. Panneerselvam, E. C. M. Brinkman-Van der Linden, et al. GlycoPEGylation of recombinant therapeutic proteins produced in Escherichia coli Glycobiology, September 1, 2006; 16(9): 833 - 843. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-Y. Xiong, A. Natarajan, X.-B. Shi, G. L. Denardo, and S. J. Denardo Development of tumor targeting anti-MUC-1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding Protein Eng. Des. Sel., August 1, 2006; 19(8): 359 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Mabry, M. Rani, R. Geiger, G. B. Hubbard, R. Carrion Jr, K. Brasky, J. L. Patterson, G. Georgiou, and B. L. Iverson Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region Infect. Immun., December 1, 2005; 73(12): 8362 - 8368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kubetzko, C. A. Sarkar, and A. Pluckthun Protein PEGylation Decreases Observed Target Association Rates via a Dual Blocking Mechanism Mol. Pharmacol., November 1, 2005; 68(5): 1439 - 1454. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. DeNardo, C. M. Richman, H. Albrecht, P. A. Burke, A. Natarajan, A. Yuan, J. P. Gregg, R.T. O'Donnell, and G. L. DeNardo Enhancement of the Therapeutic Index: From Nonmyeloablative and Myeloablative toward Pretargeted Radioimmunotherapy for Metastatic Prostate Cancer Clin. Cancer Res., October 1, 2005; 11(19): 7187s - 7194s. [Abstract] [Full Text] [PDF]
|
|