Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase

Fontana, Angelo; Spolaore, Barbara; Mero, Anna; Veronese, Francesco M.

doi:10.1016/j.addr.2007.06.015

Cited by 235 publications

(188 citation statements)

References 134 publications

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“…Although the remaining tumors resumed growth after discontinuation of treatment, they were still responsive to a second treatment cycle, indicating that engineering the fusion toxin for further enhanced pharmacologic performance is warranted. Because nontargeted PEG 20kDa -Off7DM-ETA 00 did not affect tumor growth, a role of passive tumor targeting by the EPR effect (39), as described for other PEGylated nanomedicines (38,40,48,49), can be excluded.…”

Section: Discussionmentioning

confidence: 99%

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Simon

Stefan

Borsig

et al. 2014

Molecular Cancer Therapeutics

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Fusion toxins used for cancer-related therapy have demonstrated short circulation half-lives, which impairs tumor localization and, hence, efficacy. Here, we demonstrate that the pharmacokinetics of a fusion toxin composed of a designed ankyrin repeat protein (DARPin) and domain I-truncated Pseudomonas Exotoxin A (PE40/ETA 00 ) can be significantly improved by facile bioorthogonal conjugation with a polyethylene glycol (PEG) polymer at a unique position. Fusion of the anti-EpCAM DARPin Ec1 to ETA 00 and expression in methionine-auxotrophic E. coli enabled introduction of the nonnatural amino acid azidohomoalanine (Aha) at position 1 for strain-promoted click PEGylation. PEGylated Ec1-ETA 00 was characterized by detailed biochemical analysis, and its potential for tumor targeting was assessed using carcinoma cell lines of various histotypes in vitro, and subcutaneous and orthotopic tumor xenografts in vivo. The mild click reaction resulted in a welldefined mono-PEGylated product, which could be readily purified to homogeneity. Despite an increased hydrodynamic radius resulting from the polymer, the fusion toxin demonstrated high EpCAM-binding activity and retained cytotoxicity in the femtomolar range. Pharmacologic analysis in mice unveiled an almost 6-fold increase in the elimination half-life (14 vs. 82 minutes) and a more than 7-fold increase in the area under the curve (AUC) compared with non-PEGylated Ec1-ETA 00 , which directly translated in increased and longer-lasting effects on established tumor xenografts. Our data underline the great potential of combining the inherent advantages of the DARPin format with bioorthogonal click chemistry to overcome the limitations of engineering fusion toxins with enhanced efficacy for cancer-related therapy. Mol Cancer Ther; 13(2); 375-85. Ó2013 AACR.

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Section: Discussionmentioning

confidence: 99%

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Simon

Stefan

Borsig

et al. 2014

Molecular Cancer Therapeutics

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“…Thus, transglutaminase is capable of catalyzing the incorporation of PEG-alkylamine reagents into the protein glutamine residues, which can be either natural or genetically introduced [53]. The reaction offers high degree of specificity since only those glutamine residues that are encompassed in a flexible or unfolded region are modified [54]. Even more promising appears a two-enzyme step A short overview of PEGylation technologies used today is summarized in Table 1.…”

Section: Site-specific Pegylationmentioning

confidence: 99%

PEGylation of therapeutic proteins

Jevševar¹,

Kunstelj²,

Porekar

2010

Biotechnology Journal

636

481

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International audiencePEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins since the first PEGylated product was approved by Food and Drug Administration in 1990. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation as the most established technology for extension of drug residence in the body will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates

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“…TGase mediated PEGylation using monodisperse BOC-PEG-NH 2 Polydisperse PEG-NH 2 , & biotin-PEG-NH 2 located in disordered regions of the proteins Modified Gln residues of the investigated pharmaceutical proteins 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 Rapid accurate identification of the sites of TGase-mediated PEGylation by mass spectrometry.…”

Section: In Agreement With This Assumptionmentioning

confidence: 99%

SNIPER SHOT PEGylation: TGase mediated site-specific conjugation of PEG to proteins

Satwekar

2011

Nat Prec

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The commercially avaliable recombinant protein drugs often cause immune reactions in the body which reduces its efficiency. Protein drugs can be PEGylated, (attachment of polyethylene glycol) to overcome this problem. PEGylation increases bioavaliability by reduced immune reactions and decreased renal clearnace [1]. So far the traditional approaches for PEGylation involve harsh reaction conditions which provides a heterogenous product (PEG attached randomly to different sites) along with the formation of several byproducts. Due to heterogenousity, the PEGylated protein drug faces challenge for the FDA approval. Therfore, there is an immense need to develop an approach which could generate a homogenous PEGylated protein drug. The transglutaminase (TGase) is an enzyme which catalyses specifically the formation of a covalent bond (-CONH-) between the glutamine residue and the amine group of the lysine. This TGase reaction can be engineered by substituting the lysine with primary amines, which results into the formation of similar covalent bond between the glutamine and the primary amine [2]. We utilized this specificity of TGase by using primary PEG-amines for conjugation with the glutamine present in the model protein (apomyoglobin). The reaction conditions were optimised in order to get a mono conjugated PEGylated derivative. The site of conjugation was determined by affinity purification of the modified peptides and characterized by the ESI Q-TOF mass spectrometer. Therefore, we were able to develop a site specifically PEGylated apomyoglobin along with no byproducts, which eventually reduced the derivative purification steps as compared with the traditional PEGylation approaches. This strategy was further implemented on commercial pharmaceutical proteins.

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Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase

Cited by 235 publications

References 134 publications

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

Increasing the Antitumor Effect of an EpCAM-Targeting Fusion Toxin by Facile Click PEGylation

PEGylation of therapeutic proteins

SNIPER SHOT PEGylation: TGase mediated site-specific conjugation of PEG to proteins

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