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

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University of California Davis Medical Center, Sacramento, California 95816
Cite this: Bioconjugate Chem. 2005, 16, 1, 113–121
Publication Date (Web):December 31, 2004
https://doi.org/10.1021/bc0498121
Copyright © 2005 American Chemical Society
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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.

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 Corresponding author:  Sally J. DeNardo, M.D., Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California, Davis Medical Center, 1508 Alhambra Blvd., Rm. 3100, Sacramento, CA 95816. Telephone:  916-734-3787, Fax:  916-451-2857, E-mail:  [email protected]

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  13. Sylvia K. E. Messerschmidt, Anke Kolbe, Dafne Müller, Michael Knoll, Jürgen Pleiss and Roland E. Kontermann. Novel Single-Chain Fv′ Formats for the Generation of Immunoliposomes by Site-Directed Coupling. Bioconjugate Chemistry 2008, 19 (1) , 362-369. https://doi.org/10.1021/bc700349k
  14. Zahra Ghassemi, Samuel Ruesing, Jennie B. Leach, Silviya P. Zustiak. Stability of proteins encapsulated in Michael‐type addition polyethylene glycol hydrogels. Biotechnology and Bioengineering 2021, 118 (12) , 4840-4853. https://doi.org/10.1002/bit.27949
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  16. A. Srivastava. Chemistry of targeted immobilization of biomediators. 2018,,, 231-250. https://doi.org/10.1016/B978-0-08-100496-8.00013-5
  17. Emily E. Reichard, Nisha Nanaware-Kharade, Guillermo A. Gonzalez, Shraddha Thakkar, S. Michael Owens, Eric C. Peterson. PEGylation of a High-Affinity Anti-(+)Methamphetamine Single Chain Antibody Fragment Extends Functional Half-Life by Reducing Clearance. Pharmaceutical Research 2016, 33 (12) , 2954-2966. https://doi.org/10.1007/s11095-016-2017-y
  18. Kannika Khantasup, Warangkana Chantima, Chak Sangma, Kanokwan Poomputsa, Tararaj Dharakul. Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy 2015, 34 (6) , 404-417. https://doi.org/10.1089/mab.2015.0036
  19. Nadine Hammer, Ferdinand P. Brandl, Susanne Kirchhof, Viktoria Messmann, Achim M. Goepferich. Protein Compatibility of Selected Cross-linking Reactions for Hydrogels. Macromolecular Bioscience 2015, 15 (3) , 405-413. https://doi.org/10.1002/mabi.201400379
  20. Nadine Hammer, Ferdinand P. Brandl, Susanne Kirchhof, Achim M. Goepferich. Cleavable carbamate linkers for controlled protein delivery from hydrogels. Journal of Controlled Release 2014, 183 , 67-76. https://doi.org/10.1016/j.jconrel.2014.03.031
  21. Akshay Srivastava, Iain B. O’Connor, Abhay Pandit, J. Gerard Wall. Polymer-antibody fragment conjugates for biomedical applications. Progress in Polymer Science 2014, 39 (2) , 308-329. https://doi.org/10.1016/j.progpolymsci.2013.09.003
  22. Annabelle Patricia Herrington-Symes, Monika Farys, Hanieh Khalili, Steve Brocchini. Antibody fragments: Prolonging circulation half-life special issue-antibody research. Advances in Bioscience and Biotechnology 2013, 04 (05) , 689-698. https://doi.org/10.4236/abb.2013.45090
  23. Joan G. Schellinger, Avinash Kudupudi, Arutselvan Natarajan, Wenjun Du, Sally J. DeNardo, Jacquelyn Gervay-Hague. A general chemical synthesis platform for crosslinking multivalent single chain variable fragments. Org. Biomol. Chem. 2012, 10 (8) , 1521-1526. https://doi.org/10.1039/C0OB01259A
  24. Satya Prakash, Meenakshi Malhotra, Wei Shao, Catherine Tomaro-Duchesneau, Sana Abbasi. Polymeric nanohybrids and functionalized carbon nanotubes as drug delivery carriers for cancer therapy. Advanced Drug Delivery Reviews 2011, 63 (14-15) , 1340-1351. https://doi.org/10.1016/j.addr.2011.06.013
  25. Arutselvan Natarajan, Rajeswari Sundrarajan, Sally J. DeNardo. Magnetic Nanoparticles for Cancer Imaging and Therapy. 2011,,https://doi.org/10.1002/9783527610419.ntls0168
  26. Stefano Salmaso, Paolo Caliceti. Peptide and Protein Bioconjugation. 2011,,, 247-290. https://doi.org/10.1016/B978-0-12-384935-9.10011-2
  27. Jun Jim Wu, Henry T. Peng, Pang N. Shek. Terminal-specific PEGylation of polypeptides in a dilute solution. Journal of Applied Polymer Science 2010, 118 (6) , 3269-3273. https://doi.org/10.1002/app.32709
  28. Diane E. Milenic. Antibody Engineering: Optimizing the Delivery Vehicle. 2010,,, 1-38. https://doi.org/10.1002/9780470613214.ch1
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  30. A. Constantinou, C. Chen, M. P. Deonarain. Modulating the pharmacokinetics of therapeutic antibodies. Biotechnology Letters 2010, 32 (5) , 609-622. https://doi.org/10.1007/s10529-010-0214-z
  31. Edwardraja Selvakumar, Neelamegam Rameshkumar, Sun-Gu Lee, Soo-Jae Lee, Hyung-Soon Park. In vivo Production of Functional Single-Chain Fv Fragment with an N-Terminal-Specific Bio-orthogonal Reactive Group. ChemBioChem 2010, 11 (4) , 498-501. https://doi.org/10.1002/cbic.200900685
  32. Arutselvan Natarajan, Sally J. DeNardo. PEGylation of Antibody Fragments to Improve Pharmacodynamics and Pharmacokinetics. 2010,,, 191-205. https://doi.org/10.1007/978-3-642-01147-4_15
  33. Sam P. Heywood, David P. Humphreys. Polymer Fusions to Increase Antibody Half-Lives: PEGylation and Other Modifications. 2009,,, 275-292. https://doi.org/10.1017/CBO9780511596773.022
  34. Pascal Bailon, Chee-Youb Won. PEG-modified biopharmaceuticals. Expert Opinion on Drug Delivery 2009, 6 (1) , 1-16. https://doi.org/10.1517/17425240802650568
  35. Stefano Salmaso, Alessandra Semenzato, Sara Bersani, Francesca Mastrotto, Anna Scomparin, Paolo Caliceti. Site-selective protein glycation and PEGylation. European Polymer Journal 2008, 44 (5) , 1378-1389. https://doi.org/10.1016/j.eurpolymj.2008.02.021
  36. Elza Friedländer, Márk Barok, János Szöllősi, György Vereb. ErbB-directed immunotherapy: Antibodies in current practice and promising new agents. Immunology Letters 2008, 116 (2) , 126-140. https://doi.org/10.1016/j.imlet.2007.12.001
  37. Roland Stork, Kirstin A. Zettlitz, Dafne Müller, Miriam Rether, Franz-Georg Hanisch, Roland E. Kontermann. N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies. Journal of Biological Chemistry 2008, 283 (12) , 7804-7812. https://doi.org/10.1074/jbc.M709179200
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  39. Rob Aitken. Antibody Phage Display. 2008,,, 563-585. https://doi.org/10.1007/978-1-60327-375-6_34
  40. Angelo Fontana, Barbara Spolaore, Anna Mero, Francesco M. Veronese. Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Advanced Drug Delivery Reviews 2008, 60 (1) , 13-28. https://doi.org/10.1016/j.addr.2007.06.015
  41. Laura Bass, Sa Ho, Jianming Mo, John Buckley, Rory Finn. PEGylation of Biological Macromolecules. 2007,,, 383-402. https://doi.org/10.1201/9781420008357.ch24
  42. Ofer Cohen, Chanoch Kronman, Arie Lazar, Baruch Velan, Avigdor Shafferman. Controlled Concealment of Exposed Clearance and Immunogenic Domains by Site-specific Polyethylene Glycol Attachment to Acetylcholinesterase Hypolysine Mutants. Journal of Biological Chemistry 2007, 282 (49) , 35491-35501. https://doi.org/10.1074/jbc.M704785200
  43. Yariv Mazor, Roy Noy, Winfried S. Wels, Itai Benhar. chFRP5-ZZ-PE38, a large IgG-toxin immunoconjugate outperforms the corresponding smaller FRP5(Fv)-ETA immunotoxin in eradicating ErbB2-expressing tumor xenografts. Cancer Letters 2007, 257 (1) , 124-135. https://doi.org/10.1016/j.canlet.2007.07.009
  44. Daniel A. Vallera, Andy R. Sicheneder, Elizabeth P. Taras, Martin W. Brechbiel, Jesse A. Vallera, Angela Panoskaltsis-Mortari, Linda J. Burns. Radiotherapy of CD45-Expressing Daudi Tumors in Nude Mice with Yttrium-90-Labeled, PEGylated Anti-CD45 Antibody. Cancer Biotherapy and Radiopharmaceuticals 2007, 22 (4) , 488-500. https://doi.org/10.1089/cbr.2007.366
  45. Jan C. Zillies, Klaus Zwiorek, Gerhard Winter, Conrad Coester. Method for Quantifying the PEGylation of Gelatin Nanoparticle Drug Carrier Systems Using Asymmetrical Flow Field-Flow Fractionation and Refractive Index Detection. Analytical Chemistry 2007, 79 (12) , 4574-4580. https://doi.org/10.1021/ac062135e
  46. Yan Guo, Ye Sun, Jianren Gu, Yuhong Xu. Capillary electrophoresis analysis of poly(ethylene glycol) and ligand-modified polylysine gene delivery vectors. Analytical Biochemistry 2007, 363 (2) , 204-209. https://doi.org/10.1016/j.ab.2007.01.024
  47. Ashwath Jayagopal, Prasad Shastri. Nanoengineering of Drug Delivery Systems. 2007,,, 99-109. https://doi.org/10.1201/9781420008449.ch7
  48. Yu Seok Youn, Dong Hee Na, Kang Choon Lee. High-yield production of biologically active mono-PEGylated salmon calcitonin by site-specific PEGylation. Journal of Controlled Release 2007, 117 (3) , 371-379. https://doi.org/10.1016/j.jconrel.2006.11.013
  49. Arutselvan Natarajan, Wenjun Du, Cheng-Yi Xiong, Gerald L. DeNardo, Sally J. DeNardo, Jacquelyn Gervay-Hague. Construction of di-scFv through a trivalent alkyne–azide 1,3-dipolar cycloaddition. Chem. Commun. 2007, 252 (7) , 695-697. https://doi.org/10.1039/B611636A
  50. Mansoor Amiji, Dinesh Shenoy, Sushma Kommareddy. Long-Circulating Polymeric Nanoparticles for Drug and Gene Delivery to Tumors. 2006,,, 231-242. https://doi.org/10.1201/9781420006636.ch13
  51. James E.S. Hainsworth, Peter Harrison, Stephen J. Mather. Novel preparation and characterization of a trastuzumab???streptavidin conjugate for pre-targeted radionuclide therapy. Nuclear Medicine Communications 2006, 27 (5) , 461-471. https://doi.org/10.1097/00006231-200605000-00008
  52. Jayant Khandare, Tamara Minko. Polymer–drug conjugates: Progress in polymeric prodrugs. Progress in Polymer Science 2006, 31 (4) , 359-397. https://doi.org/10.1016/j.progpolymsci.2005.09.004
  53. Robert Mabry, Mridula Rani, Robert Geiger, Gene B. Hubbard, Ricardo Carrion, Kathleen Brasky, Jean L. Patterson, George Georgiou, B. L. Iverson. Passive Protection against Anthrax by Using a High-Affinity Antitoxin Antibody Fragment Lacking an Fc Region. Infection and Immunity 2005, 73 (12) , 8362-8368. https://doi.org/10.1128/IAI.73.12.8362-8368.2005
  54. Sally J. DeNardo, Carol M. Richman, Huguette Albrecht, Patricia A. Burke, Arut Natarajan, Aina Yuan, Jeff P. Gregg, R.T. O'Donnell, Gerald L. DeNardo. Enhancement of the Therapeutic Index: From Nonmyeloablative and Myeloablative toward Pretargeted Radioimmunotherapy for Metastatic Prostate Cancer. Clinical Cancer Research 2005, 11 (19) , 7187s-7194s. https://doi.org/10.1158/1078-0432.CCR-1004-0013
  55. Anna M Wu, Peter D Senter. Arming antibodies: prospects and challenges for immunoconjugates. Nature Biotechnology 2005, 23 (9) , 1137-1146. https://doi.org/10.1038/nbt1141
  56. Laura SANZ, Ángel M CUESTA, Marta COMPTE, Luis ÁLVAREZ-VALLINA. Antibody engineering: facing new challenges in cancer therapy. Acta Pharmacologica Sinica 2005, 26 (6) , 641-648. https://doi.org/10.1111/j.1745-7254.2005.00135.x

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