Purification and biologic characterization of a specific tumor necrosis factor α inhibitor

Seckinger, P; Isaaz, S; Dayer, Jean‐Michel

doi:10.1016/s0021-9258(18)80161-2

Cited by 275 publications

(22 citation statements)

References 45 publications

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“…The affinity of the interaction between TNF and the combining site of soluble TNF receptors has been estimated by several laboratories, and assigned a K, value between 109 and 101~ (6,7,21,22). Since antibodies typically exhibit combining group affinities in the range of 10s-106 K~, it might be expected that the chimeric protein produced in this study would form far more stable complexes.…”

Section: Discussionmentioning

confidence: 99%

“…TNF was allowed to preincubate with the inhibitor for 1 h (Figs. 3,[6][7][8] at 37~ in a 96-well microtiter plate. 50/~1 of FM containing 10% FCS and 5 x 104 SK MEL 109 cells was then added to each well.…”

Section: Preparation Of the Chimeric Constructmentioning

confidence: 99%

“…T he recent cloning of the 55-kD (1-3) and 75-kD (3)(4)(5) TNF receptors has opened the way for further studies of TNF effects and signal transduction. Moreover, it appears that truncated receptor molecules, lacking the transmembrane or cytoplasmic domains, are capable of interacting with TNF, and therefore have been isolated from urine (6,7) and serum (2) as TNF inhibitors. We considered that derivatives of such molecules might prove useful as antagonists of TNF action in vivo, as high affinity ligands to be applied as the basis of a more sensitive assay for TNF, and as reagents to be used in defining the molecular interaction between TNF and its receptor.…”

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confidence: 99%

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A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.

Peppel

Crawford

Beutler

1991

The Journal of Experimental Medicine

306

174

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SummaryUsing a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extraceUular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monodonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.T he recent cloning of the 55-kD (1-3) and 75-kD (3-5) TNF receptors has opened the way for further studies of TNF effects and signal transduction. Moreover, it appears that truncated receptor molecules, lacking the transmembrane or cytoplasmic domains, are capable of interacting with TNF, and therefore have been isolated from urine (6, 7) and serum (2) as TNF inhibitors. We considered that derivatives of such molecules might prove useful as antagonists of TNF action in vivo, as high affinity ligands to be applied as the basis of a more sensitive assay for TNF, and as reagents to be used in defining the molecular interaction between TNF and its receptor.Unfortunately, truncated forms of the TNF receptor are highly unstable in vivo, and therefore are poor substitutes for antibodies as a means of antagonizing TNF action in living animals. The naturally occurring TNF receptor fragments are univalent and therefore have an avidity that is effectively far lower than that of a bivalent ligand. The production of large quantities of a truncated receptor by recombinant means has, in our hands, been problematic, since the protein is produced in an inactive and insoluble form in bacteria (Peppel, K., and B. Beutler, unpublished observations). This difficulty may arise from the highly cysteine-rich structure of the receptor-binding domain (1, 2). When produced in roammalian cells through recombinant techniques, the soluble receptor fragment is active, but produced at low levels, and therefore difficult to purify (Beutler, B., and T. Brown, unpublished observations).To circumvent these problems, we have engineered a chimeric protein in which the extracellular domain of the TNF receptor, which normally engages the TNF molecu...

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Section: Discussionmentioning

confidence: 99%

Section: Preparation Of the Chimeric Constructmentioning

confidence: 99%

mentioning

confidence: 99%

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A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.

Peppel

Crawford

Beutler

1991

The Journal of Experimental Medicine

306

174

View full text Add to dashboard Cite

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“…There are recent reports in the literature of factors from the urine of febrile patients that inhibit the toxic effects of TNF to tumor cell targets. Such substances have been purified and appear to act by binding to the TNF molecule and preventing the interaction between TNF and its receptor on the target cell surface (22)(23)(24). The…”

Section: Resultsmentioning

confidence: 99%

A serum factor that suppresses the cytotoxic function of cytokine-stimulated human eosinophils.

Silberstein

Minkoff

Creasey

et al. 1990

The Journal of Experimental Medicine

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A human subject (NR) was identified whose eosinophils and neutrophils failed to respond to TNF in vitro in 29 of 33 experiments, using several biological assays. There was a response rate to TNF of 100% among 37 control subjects whose leukocytes were tested in parallel. NR serum contained an activity that inhibited the cytotoxic function of TNF- and GM-CSF-stimulated normal human eosinophils. A similar activity was detected in 4 of 122 control sera and in sera of two subjects with hypereosinophilia. This activity (ECI) had an apparent molecular weight of 80,000-100,000 and was sensitive to heating at 80 degrees C or to trypsin treatment. HPLC sizing chromatography increased the titer of ECI by a factor of 50 to 2,000 in experiments using NR serum or other sera with detectable inhibitory activity. In seven experiments using sera with no inhibitory activity, HPLC generated ECI of the same apparent molecular weight. The effect of HPLC on ECI activity required the separation of serum components and did not result from exposure to HPLC system components or other sample processing methods. This suggests that ECI in serum can be stabilized in an inactive or partially active form and that HPLC removes the stabilizing component. ECI suppressed TNF-stimulated eosinophil cytotoxic function when added to cultures up to 4 h after exposure of eosinophils to cytokine. However, ECI did not protect L929 cells from the toxic effects of TNF. Thus, ECI did not act by preventing the initial interaction of TNF with eosinophils or by interfering with the binding of TNF to its receptor on L929 cells. The results suggest that ECI is a component of a feedback mechanism that suppresses functions of cytokine-activated eosinophils in inflammation.

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“…The cellular response to TNF-α is mediated by two receptors, TNF-receptor 1 and TNF receptor 2. [19][20][21][22] Hotamisligil et al reported an increase in mRNA levels of TNF-receptor 2 in fat tissue as well as an increase of soluble TNF-receptor in the blood of obese women. 23 Fernandez-Real et al showed a positive correlation between plasma soluble TNF-receptor 2 levels and body mass index (BMI).…”

Section: Discussionmentioning

confidence: 99%

High Body Mass Index is Associated with Shorter Retention of Tumor Necrosis Factor-Alpha Blocker Treatment in Rheumatoid Arthritis

Elalouf¹,

Lidar²,

Reitblat³

et al. 2021

BTT

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Purpose: To evaluate the association between body mass index (BMI) and tumor necrosis factor α (TNF-α) blockers retention in patients with rheumatoid arthritis (RA). Patients and Methods: This prospective cohort study analyzed data about patients with RA who initiated TNF blockers from the Israeli registry of inflammatory diseases from 2011 to 2019. Patients were grouped by BMI: normal (BMI <24.9 kg/m2), overweight (BMI 25-29.9 kg/m2), obese (BMI 30-34.9 kg/m2) and morbid obese (BMI ≥35 kg/m2). Treatment cessation due to inefficacy was defined as an "event" and therapy with a drug above 3 months was defined as a "course." Kaplan-Meier survival curve was used to describe drug survival. Event-free survival was calculated using Cox regression with a hazard ratio and confidence interval of 95%. Results: The final analysis included 521 RA patients (80% females) treated with etanercept, infliximab, adalimumab or golimumab. Eight hundred and eighteen treatment initiations were included in the final analysis, 334 (41%) in the normal weight group, 261 (32%) in the overweight, 144 (17%) in the obese and 79 (10%) in the morbid obesity group. Three hundred and twenty-six (40%) treatment initiations were with etanercept, 215 (26%) with adalimumab 197 (24%) with infliximab, and 80 (10%) with golimumab. BMI was inversely associated with drug survival. Morbid obese patients were more likely to discontinue treatment compared with normal weight patients HR 2.28 (95% CI 1.67-3.10, p<0.01). This association remained significant for each drug type (except for golimumab) in a subgroup analysis. Adalimumab switch rate was higher compared to etanercept with HR =1.51 (95% CI 1.20-1.91, p<0.01), no other significant differences were noted between the other drugs. Conclusion: Morbid obese RA patients have lower TNF-α blocker retention compared to normal weight patients.

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Purification and biologic characterization of a specific tumor necrosis factor α inhibitor

Cited by 275 publications

References 45 publications

A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.

A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.

A serum factor that suppresses the cytotoxic function of cytokine-stimulated human eosinophils.

High Body Mass Index is Associated with Shorter Retention of Tumor Necrosis Factor-Alpha Blocker Treatment in Rheumatoid Arthritis

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