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In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.

ABSTRACT: As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohn's disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohn's disease led to detection of intestinal mTNF(+) immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF(+) cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent anti-TNF therapy as compared to patients with low amounts of mTNF(+) cells (15%). This clinical response in the former patients was sustained over a follow-up period of 1 year and was associated with mucosal healing observed in follow-up endoscopy. These data indicate that molecular imaging with fluorescent antibodies has the potential to predict therapeutic responses to biological treatment and can be used for personalized medicine in Crohn's disease and autoimmune or inflammatory disorders.

SUBMITTER: Atreya R

PROVIDER: S-EPMC4479137 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.

Atreya Raja R Neumann Helmut H Neufert Clemens C Waldner Maximilian J MJ Billmeier Ulrike U Zopf Yurdagül Y Willma Marcus M App Christine C Münster Tino T Kessler Hermann H Maas Stefanie S Gebhardt Bernd B Heimke-Brinck Ralph R Reuter Eva E Dörje Frank F Rau Tilman T TT Uter Wolfgang W Wang Thomas D TD Kiesslich Ralf R Vieth Michael M Hannappel Ewald E Neurath Markus F MF

Nature medicine 20140223 3

As antibodies to tumor necrosis factor (TNF) suppress immune responses in Crohn's disease by binding to membrane-bound TNF (mTNF), we created a fluorescent antibody for molecular mTNF imaging in this disease. Topical antibody administration in 25 patients with Crohn's disease led to detection of intestinal mTNF(+) immune cells during confocal laser endomicroscopy. Patients with high numbers of mTNF(+) cells showed significantly higher short-term response rates (92%) at week 12 upon subsequent an ...[more]

PMID: 24562382

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Project description:To investigate genetic factors that might help define which Crohn's disease (CD) patients are likely to benefit from anti-tumor necrosis factor (TNF) therapy.This was a prospective cohort study. Patients were recruited from a university digestive disease practice database. We included CD patients who received anti-TNF therapy, had available medical records (with information on treatment duration and efficacy) and who consented to participation. Patients with allergic reactions were excluded. Patients were grouped as ever-responders or non-responders. Genomic DNA was extracted from peripheral blood, and 7 single nucleotide polymorphisms (SNPs) were assessed. The main outcome measure (following exposure to the drug) was response to therapy. The patient genotypes were assessed as the predictors of outcome. Possible confounders and effect modifiers included age, gender, race, and socioeconomic status disease, as well as disease characteristics (such as Montreal criteria).121 patients were included. Twenty-one were non-responders, and 100 were ever-responders. Fas ligand SNP (rs763110) genotype frequencies, TNF gene -308 SNP (rs1800629) genotype frequencies, and their combination, were significantly different between groups on multivariable analysis controlling for Montreal disease behavior and perianal disease. The odds of a patient with a Fas ligand CC genotype being a non-responder were four-fold higher as compared to a TC or TT genotype (P = 0.009, OR = 4.30, 95%CI: 1.45-12.80). The presence of the A (minor) TNF gene -308 allele correlated with three-fold higher odds of being a non-responder (P = 0.049, OR = 2.88, 95%CI: 1.01-8.22). Patients with the combination of the Fas ligand CC genotype and the TNF -308 A allele had nearly five-fold higher odds of being a non-responder (P = 0.015, OR = 4.76, 95%CI: 1.35-16.77). No difference was seen for the remaining SNPs.The Fas-ligand SNP and TNF gene -308 SNP are associated with anti-TNF treatment response in CD and may help select patients likely to benefit from therapy.

Project description:BackgroundOne-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-? (TNF-?).MethodsMice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-?, were tested for their effects on breast cancer cell proliferation.ResultsWe produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-?, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-?.ConclusionsNovel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-? provide evidence for a complex interplay between HER2 and TNF-? signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

Project description:The expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) is associated with production losses in dairy cows and is a hallmark of early inflammatory processes. Reliable tools for the detection and quantification of soluble as well as cytoplasmatic bovine TNF-α are needed to deepen our understanding of inflammatory dynamics in dairy cows. The objective of this study was to generate a monoclonal antibody (mAb) pair that could be used to quantify bovine TNF-α in cell culture supernatants and plasma and to detect cytoplasmatic TNF-α in bovine leukocyte populations. One mouse was immunized with a recombinant fusion protein of bovine TNF-α and equine IL-4 generated in Chinese hamster ovary cells. Murine monoclonal antibodies specific to bovine TNF-α were produced in hybridoma cell lines and selected based on their specificity to the recombinant IL-4/TNF-α protein. Clones 197-1 and 65-2, both murine IgG1 isotypes, detected the bovine TNF-α fusion protein as well as the native protein produced by peripheral blood mononuclear cells (PBMC) stimulated with a combination of phorbol myristate acetate and ionomycin. Both mAbs were tested for and lacked cross-reactivity to equine IL-4 and 3 other recombinant bovine cytokines (IFN-γ, IL-10, and CCL5) and were used to develop a fluorescent bead-based assay. The range of bovine TNF-α detection in the assay was 0.2 to 620 ng/mL, and the test was used to quantify native bovine TNF-α in cell culture supernatants of stimulated PBMC and in plasma from ex vivo whole-blood stimulations. Sample matrices were spiked with TNF-α, with subsequent recovery rates (mean ± SD) of 89% ± 9 (n = 3) in culture medium and 94% ± 12 (n = 3) in heat-inactivated fetal bovine serum. Serial dilutions of plasma and cell culture supernatants from stimulated whole blood or PBMC indicated excellent accuracy for quantification of native TNF-α in bovine samples. Both bovine TNF-α mAbs also detected intracellular TNF-α in bovine CD14+ monocytes and CD4+/CD8+ lymphocytes. In conclusion, we demonstrated that the mAbs generated provide valuable new tools to quantify native bovine TNF-α in a wide concentration range and to characterize intracellular TNF-α expression in bovine leukocytes.

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In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.

Publications

In vivo imaging using fluorescent antibodies to tumor necrosis factor predicts therapeutic response in Crohn's disease.

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