Recognition of Human Tumor Necrosis Factor α (TNF-α) by Therapeutic Antibody Fragment

Marušič, Jaka; Podlipnik, Črtomir; Jevševar, Simona; Kuzman, Drago; Vesnaver, Gorazd; Lah, Jurij

doi:10.1074/jbc.m111.318451

Cited by 18 publications

(15 citation statements)

References 38 publications

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“…7A). A previous study demonstrated a stabilizing effect of adalimumab on TNF towards denaturation with urea3, which is in line with our present findings. For infliximab, a crystal structure of its complex with TNF reveals only direct contacts between the Fab and one TNF monomer (Fig.…”

Section: Discussionsupporting

confidence: 94%

Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

Schie

Heer

Dijk

et al. 2016

Sci Rep

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Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimumab.

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Section: Discussionsupporting

confidence: 94%

Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

Schie

Heer

Dijk

et al. 2016

Sci Rep

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“…In our experiments we used recombinant human IL‐6 (21 kDa; BioLegend) and recombinant TNF‐α (17,3 kDa; BioLegend) as cytokine markers and observed that IL‐6 was removed more efficiently than TNF‐α, even though it has a higher molecular size. Our data suggest that bioactive TNF‐α exists as a trimeric form of approximately 52 kDa, as others described naturally occurring TNF‐α .…”

Section: Discussionsupporting

confidence: 83%

Reduction of Elevated Cytokine Levels in Acute/Acute‐on‐Chronic Liver Failure Using Super‐Large Pore Albumin Dialysis Treatment: An In Vitro Study

et al. 2013

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The removal of small water soluble toxins and albumin-bound toxins in acute liver failure patients (ALF) or acute-on-chronic liver failure (AocLF) patients has been established using extracorporeal liver support devices (e.g. Molecular Adsorbents Recirculating System; MARS). However, reduction of elevated cytokines in ALF/AocLF using MARS is still not efficient enough to lower patients' serum cytokine levels. New membranes with larger pores or higher cut-offs should be considered in extracorporeal liver support devices based on albumin dialysis in order to address these problems, as the introduction of super-large pore membranes could counterbalance high production rates of cytokines and further improve detoxification in vivo. Using an established in vitro two compartment albumin dialysis model, three novel membranes of different pore sizes were compared with the MARS Flux membrane for cytokine removal and detoxification qualities in vitro. Comparing the membranes, no improvement in the removal of water soluble toxins was found. Albumin-bound toxins were removed more efficiently using novel large (Emic2) to super-large pore sized membranes (S20; HCO Gambro). Clearance of cytokines IL-6 and tumor necrosis factor-α was drastically improved using super-large pore membranes. The Emic2 membrane predominantly removed IL-6. In vitro data suggest that the usage of larger pore sized membranes in albumin dialysis can efficiently reduce elevated cytokine levels and liver failure toxins. Using large to super-large pore membranes might exert effects on patients' serum cytokine levels. Combined with increased detoxification this could lead to higher survival in ALF/AocLF. Promising membranes for clinical evaluation have been identified.

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“…This reactivity pattern was also observed on another widely used human IgG1, anti-hTNFα (Invivogen, San Diego, CA), which also has a glutamate-terminal heavy chain and an aspartate-terminal light chain (Figure S13). 32 …”

Section: Resultsmentioning

confidence: 99%

Site-Specific Protein Transamination Using N-Methylpyridinium-4-carboxaldehyde

et al. 2013

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The controlled attachment of synthetic groups to proteins is important for a number of fields, including therapeutics, where antibody-drug conjugates are an emerging area of biologic medicines. We have previously reported a site-specific protein modification method using a transamination reaction that chemoselectively oxidizes the N-terminal amine of a polypeptide chain to a ketone or an aldehyde group. The newly introduced carbonyl can be used for conjugation to a synthetic group in one location through the formation of an oxime or a hydrazone linkage. To expand the scope of this reaction, we have used a combinatorial peptide library screening platform as a method to explore new transamination reagents while simultaneously identifying their optimal N-terminal sequences. N-methylpyridinium-4-carboxaldehyde benzenesulfonate salt (Rapoport's salt, RS) was identified as a highly effective transamination reagent when paired with glutamate-terminal peptides and proteins. This finding establishes RS as a transamination reagent that is particularly well suited for antibody modification. Using a known therapeutic antibody, herceptin, it was demonstrated that RS can be used to modify the heavy chains of the wild type antibody, or both the heavy and the light chains after N-terminal sequence mutation to add glutamate residues.

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Recognition of Human Tumor Necrosis Factor α (TNF-α) by Therapeutic Antibody Fragment

Cited by 18 publications

References 38 publications

Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

Reduction of Elevated Cytokine Levels in Acute/Acute‐on‐Chronic Liver Failure Using Super‐Large Pore Albumin Dialysis Treatment: An In Vitro Study

Site-Specific Protein Transamination Using N-Methylpyridinium-4-carboxaldehyde

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