Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity

Yamamoto, Yoko; Tsutsumi, Yasuo; Yoshioka, Yasuo; Nishibata, Toshihide; Kobayashi, Kyoko; Okamoto, Takayuki; Mukai, Yohei; Shimizu, Tomoe; Nakagawa, Shinsaku; Nagata, Satoshi; Mayumi, Tadanori

doi:10.1038/nbt812

Cited by 205 publications

(141 citation statements)

References 29 publications

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“…Although principally reversible, the dissociation of TNF is likely to favor rapid systemic clearance and enhanced denaturation. One approach evaluated to circumvent this problem was conjugation of TNF to polyethylene glycol resulting in both an improved plasma half-life and therapeutic potency of TNF (11). Another strategy used to improve antitumor efficacy and decrease systemic toxicity of TNF is the development of tumortargeted TNF-fusion proteins.…”

mentioning

confidence: 99%

Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

Krippner‐Heidenreich¹,

Grunwald

Zimmermann

et al. 2008

The Journal of Immunology

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The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.

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mentioning

confidence: 99%

Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

Krippner‐Heidenreich¹,

Grunwald

Zimmermann

et al. 2008

The Journal of Immunology

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“…There are many conjugation strategies and many PEG-based reagents that have been developed to address the central issue of site-specific PEGylation [10][11][12][13] . Based on their selective chemical reactivity, thiol reactive PEG reagents offer the best opportunity for efficient and site-specific PEGylation.…”

Section: Existing Pegylation Technologiesmentioning

confidence: 99%

PEGylation of native disulfide bonds in proteins

et al. 2006

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PEGylation has turned proteins into important new biopharmaceuticals. The fundamental problems with the existing approaches to PEGylation are inefficient conjugation and the formation of heterogeneous mixtures. This is because poly(ethylene glycol) (PEG) is usually conjugated to nucleophilic amine residues. Our PEGylation protocol solves these problems by exploiting the chemical reactivity of both of the sulfur atoms in the disulfide bond of many biologically relevant proteins. An accessible disulfide bond is mildly reduced to liberate the two cysteine sulfur atoms without disturbing the protein's tertiary structure. Site-specific PEGylation is achieved with a bis-thiol alkylating PEG reagent that sequentially undergoes conjugation to form a three-carbon bridge. The two sulfur atoms are re-linked with PEG selectively conjugated to the bridge. PEGylation of a protein can be completed in 24 h and purification of the PEG-protein conjugate in another 3 h. We have successfully applied this approach to PEGylation of cytokines, enzymes, antibody fragments and peptides, without destroying their tertiary structure or abolishing their biological activity

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“…A scFv gene with a 15-amino acid linker was cloned into pYas-PSIF vectors. 37 ScFvs, dscFvs, and scFv-PSIFs were purified from inclusion bodies in E coli according to the previously described methods. 37 The binding affinity of each recombinant protein was assessed by surface plasmon resonance using BIAcore3000 (GE Healthcare UK Ltd., Chalfont, United Kingdom).…”

Section: Immune Phage Antibody Librariesmentioning

confidence: 99%

Robo4 is an effective tumor endothelial marker for antibody-drug conjugates based on the rapid isolation of the anti-Robo4 cell-internalizing antibody

Yoshikawa

Mukai

Okada

et al. 2013

Blood

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• First therapeutic application that targets Robo4 on the tumor blood vasculature • High-throughput screening system to isolate cellinternalizing monoclonal antibodies useful to develop effective antibody-drug conjugatesMonoclonal antibodies (mAbs) that are internalized into cells are a current focus in the development of antibody-drug conjugates (ADCs). We describe a phage display-based high-throughput screening system to rapidly isolate cellinternalizing mAbs. We simultaneously examined the cell-internalizing activities of several hundred independent mAbs and successfully isolated cell-internalizing mAbs against the tumor endothelial markers Roundabout homolog 4 (Robo4) and vascular endothelial growth factor receptor 2 (VEGFR2). Tumor accumulation of mAbs with high cell-internalizing activity was significantly higher than that of mAbs with low cell-internalizing activity. Furthermore, the antitumor effects of ADCs of mAbs with high cell-internalizing activity were significantly stronger than those of mAbs with low cell-internalizing activity. Although anti-VEGFR2 therapy caused a significant loss of body weight, anti-Robo4 therapy did not. These findings indicate that cell-internalizing activity plays an important role in the biodistribution and therapeutic effects of ADCs. Further, Robo4 can be an effective marker for tumor vascular targeting. (Blood. 2013;121 (14):2804-2813

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Site-specific PEGylation of a lysine-deficient TNF-α with full bioactivity

Cited by 205 publications

References 29 publications

Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

Single-Chain TNF, a TNF Derivative with Enhanced Stability and Antitumoral Activity

PEGylation of native disulfide bonds in proteins

Robo4 is an effective tumor endothelial marker for antibody-drug conjugates based on the rapid isolation of the anti-Robo4 cell-internalizing antibody

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