title: Pegylated Trastuzumab Fragments Acquire an Increased in Vivo Stability but Show a Largely Reduced Affinity for the Target Antigen.
abstract: PEGylation of biomolecules is a major approach to increase blood stream half-life, stability and solubility of biotherapeutics and to reduce their immunogenicity, aggregation potential and unspecific interactions with other proteins and tissues. Antibodies have generally long half-lives due to high molecular mass and stability toward proteases, however their size lowers to some extent their potential because of a reduced ability to penetrate tissues, especially those of tumor origin. Fab or otherwise engineered smaller fragments are an alternative but are less stable and are much less well retained in circulation. We have here investigated the effects of various PEGylations on the binding properties and in vivo half-life of Fab fragments derived from the enzymatic splitting of Trastuzumab. We find that PEGylation increases the half-life of the molecules but also strongly affects the ability to recognize the target antigen in a way that is dependent on the extent and position of the chemical modification. Data thus support the concept that polyethylene glycol (PEG) conjugation on Trastuzumab Fabs increases half-life but reduces their affinity and this is a fine balance, which must be carefully considered for the design of strategies based on the use of antibody fragments.
authors.affiliation: Bioker srl-Multimedica Group, c/o Institute of Genetics and Biophysics, National Research Council (CNR-IGB) via P. Castellino 111, 80131 Napoli, Italy. fabio.selis@multimedica.it.
authors.forename: Fabio
authors.initials: F
authors.lastname: Selis
authors.affiliation: Institute of Biostructure and Bioimaging, National Research Council (IBB-CNR), via Mezzocannone 16, 80134 Napoli, Italy. giuseppina.foca@gmail.com.|Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), University of Naples Federico II, via Mezzocannone 16, 80134 Napoli, Italy. giuseppina.foca@gmail.com.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Focà
authors.affiliation: Institute of Biostructure and Bioimaging, National Research Council (IBB-CNR), via Mezzocannone 16, 80134 Napoli, Italy. annamaria.sandomenico@gmail.com.|Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), University of Naples Federico II, via Mezzocannone 16, 80134 Napoli, Italy. annamaria.sandomenico@gmail.com.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Sandomenico
authors.affiliation: Bioker srl-Multimedica Group, c/o Institute of Genetics and Biophysics, National Research Council (CNR-IGB) via P. Castellino 111, 80131 Napoli, Italy. carlamarra78@yahoo.it.
authors.forename: Carla
authors.initials: C
authors.lastname: Marra
authors.affiliation: Department of Clinical Medicine and Surgery, University of Naples Federico II, 80134 Napoli, Italy. dimauro.co@alice.it.
authors.forename: Concetta
authors.initials: C
authors.lastname: Di Mauro
authors.affiliation: Department of Biomedical, Biotechnological and Translational Science (S.Bi.Bi.T.), Università di Parma, 43126 Parma, Italy. gloria.saccani@unipr.it.
authors.forename: Gloria
authors.initials: G
authors.lastname: Saccani Jotti
authors.affiliation: Bioker srl-Multimedica Group, c/o Institute of Genetics and Biophysics, National Research Council (CNR-IGB) via P. Castellino 111, 80131 Napoli, Italy. silvia.scaramuzza@multimedica.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Scaramuzza
authors.affiliation: Bioker srl-Multimedica Group, c/o Institute of Genetics and Biophysics, National Research Council (CNR-IGB) via P. Castellino 111, 80131 Napoli, Italy. annalisa.politano@gmail.com.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Politano
authors.affiliation: Bioker srl-Multimedica Group, c/o Institute of Genetics and Biophysics, National Research Council (CNR-IGB) via P. Castellino 111, 80131 Napoli, Italy. riccardo.sanna@multimedica.it.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Sanna
authors.affiliation: Institute of Biostructure and Bioimaging, National Research Council (IBB-CNR), via Mezzocannone 16, 80134 Napoli, Italy. menotti.ruvo@unina.it.|Centro Interuniversitario di Ricerca sui Peptidi Bioattivi (CIRPeB), University of Naples Federico II, via Mezzocannone 16, 80134 Napoli, Italy. menotti.ruvo@unina.it.
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
authors.affiliation: Bioker srl-Multimedica Group, c/o Institute of Genetics and Biophysics, National Research Council (CNR-IGB) via P. Castellino 111, 80131 Napoli, Italy. giancarlo.tonon@multimedica.it.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Tonon
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D000970:Antineoplastic Agents / Q000097:blood / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000097:blood / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D008297:Male; D011092:Polyethylene Glycols / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D017207:Rats, Sprague-Dawley; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D000068878:Trastuzumab / Q000097:blood / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D007140:Immunoglobulin Fab Fragments; D011092:Polyethylene Glycols; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab
keywords: Fab; PEGylation; antibody fragment; papain digestion; pepsin digestion; pharmacokinetics