Dirk Völkel

Immunochemical determination of cellular prion proteinin plasma from healthy subjects and patients withsporadic CJD or other neurologic diseases

BACKGROUND: Creutzfeldt‐Jakob disease is thought to be caused by conversion of cellular prion protein (PrP) from its soluble form (PrPsen) to a pathologic form (PrPres). The occurrence of a new variant of CJD has increased the demand for a rapid assay capable of detecting a theoretical risk of transmission of the disease by blood or plasma.

Native Versus Recombinant High-Mobility Group B1 Proteins: Functional Activity In Vitro

To compare the functional activity of native HMGB1 proteins from eukaryotic sources with HMGB1 from prokaryotic sources the cDNAs of human and murine HMGB1 were cloned and the proteins expressed in bacteria. Tissue-derived HMGB1 from calf thymus and HMGB1 secreted from Chinese hamster ovary (CHO) cells were purified. Human whole blood, THP-1 cells, and NIH/3T3 cells were exposed to HMGB1 proteins and the induction of tumor necrosis factor-α (TNF-α) release in whole blood and monocytic THP-1 cells and a proliferation assay in NIH/3T3 cells were used to study functional activity of HMGB1s in vitro. Native and recombinant HMGB1s induced TNF-α release in human blood and in THP-1 cells dose-dependently, but recombinant HMGB1s were more effective. Cell proliferation was induced by native and recombinant HMGB1s. The native HMGB1 proteins from eukaryotic sources exert the same (though less pronounced) biological activity in vitro as recombinant HMGB1 proteins from prokaryotic sources.

Neutralization of macrophage migration inhibitory factor (MIF) by fully human antibodies correlates with their specificity for the β-sheet structure of MIF

Background: A diverse panel of fully human antibodies specific for the macrophage migration inhibitory factor (MIF) has been generated. Results: In vitro and in vivo studies revealed that antibodies specific for a β-sheet structure are potent inhibitors of MIF. Conclusion: This β-sheet structure is a promising target for anti-MIF antibody therapy. Significance: Fully human antibodies with high therapeutic potential have been identified. The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50–68 or 86–102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a β-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this β-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.

Human Anti-Macrophage Migration Inhibitory Factor Antibodies Inhibit Growth of Human Prostate Cancer Cells In Vitro and In Vivo

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine, originally discovered for its eponymous effect and now known for pleiotropic biologic properties in immunology and oncology. Circulating MIF levels are elevated in several types of human cancer including prostate cancer. MIF is released presumably by both stromal and tumor cells and enhances malignant growth and metastasis by diverse mechanisms, such as stimulating tumor cell proliferation, suppressing apoptotic death, facilitating invasion of the extracellular matrix, and promoting angiogenesis. Recently described fully human anti-MIF antibodies were tested in vitro and in vivo for their ability to influence growth rate and invasion of the human PC3 prostate cancer cell line. In vitro, the selected candidate antibodies BaxG03, BaxB01, and BaxM159 reduced cell growth and viability by inhibiting MIF-induced phosphorylation of the central kinases p44/42 mitogen-activated protein kinase [extracellular signal–regulated kinase-1 and -2 (ERK1/2)] and protein kinase B (AKT). Incubation of cells in the presence of the antibodies also promoted activation of caspase-3/7. The antibodies furthermore inhibited MIF-promoted invasion and chemotaxis as transmigration through Matrigel along a MIF gradient was impaired. In vivo, pharmacokinetic parameters (half-life, volume of distribution, and bioavailability) of the antibodies were determined and a proof-of-concept was obtained in a PC3-xenograft mouse model. Treatment with human anti-MIF antibodies blunted xenograft tumor growth in a dose-dependent manner. We therefore conclude that the anti-MIF antibodies described neutralize some of the key tumor-promoting activities of MIF and thus limit tumor growth in vivo. Mol Cancer Ther; 12(7); 1223–34. ©2013 AACR.

C-reactive protein and IL-6: new marker proteins for the diagnosis of CJD in plasma?

BACKGROUND: CJD is usually diagnosed by clinical and neuropathological findings. A number of proteins regarded as markers for neuronal damage in plasma or serum have recently been described. Markers typical for tissue damage, although not usually associated with CJD, are another possibility. An evaluation of the relative usefulness of markers of neuronal and tissue damage in identifying CJD could be beneficial.

Selective Targeting of a Disease-Related Conformational Isoform of Macrophage Migration Inhibitory Factor Ameliorates Inflammatory Conditions

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine and counterregulator of glucocorticoids, is a potential therapeutic target. MIF is markedly different from other cytokines because it is constitutively expressed, stored in the cytoplasm, and present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention is challenging because of the need to neutralize a ubiquitous protein. In this article, we report that MIF occurs in two redox-dependent conformational isoforms. We show that one of the two isoforms of MIF, that is, oxidized MIF (oxMIF), is specifically recognized by three mAbs directed against MIF. Surprisingly, oxMIF is selectively expressed in the plasma and on the cell surface of immune cells of patients with different inflammatory diseases. In patients with acute infections or chronic inflammation, oxMIF expression correlated with inflammatory flare-ups. In addition, anti-oxMIF mAbs alleviated disease severity in mouse models of acute and chronic enterocolitis and improved, in synergy with glucocorticoids, renal function in a rat model of crescentic glomerulonephritis. We conclude that oxMIF represents the disease-related isoform of MIF; oxMIF is therefore a new diagnostic marker for inflammation and a relevant target for anti-inflammatory therapy.

Cloning, expression and functional characterization of the full-length murine ADAMTS13

Summary.  Functional deficiency or absence of the human von Willebrand factor (VWF)‐cleaving protease (VWF‐cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non‐truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426‐amino‐acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose‐dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.

Immunochemical detection of prion protein on dipsticks prepared with crystalline bacterial cell-surface layers†

BACKGROUND: Transmissible spongiform encephalopathy (TSE) represents a spectrum of diseases affecting humans and animals. A definitive diagnosis of TSEs is only possible by postmortem identification of pathologic prion protein in brain tissue that has been treated with protease. The pathologic protein is detected by Western blot analysis or ELISA methods. The bovine spongiform encephalopathy crisis and occurrence of a new variant of CJD has increased demand for rapid and simple assays.

Oxidized macrophage migration inhibitory factor is a potential new tissue marker and drug target in cancer

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, which was shown to be upregulated in cancers and to exhibit tumor promoting properties. Unlike other cytokines, MIF is ubiquitously present in the circulation and tissue of healthy subjects. We recently described a previously unrecognized, disease-related isoform of MIF, designated oxMIF, which is present in the circulation of patients with different inflammatory diseases. In this article, we report that oxMIF is also linked to different solid tumors as it is specifically expressed in tumor tissue from patients with colorectal, pancreatic, ovarian and lung cancer. Furthermore, oxMIF can be specifically targeted by a subset of phage display-derived fully human, monoclonal anti-MIF antibodies (mAbs) that were shown to neutralize pro-tumorigenic activities of MIF in vivo. We further demonstrate that anti-oxMIF mAbs sensitize human cancer cell lines (LNCaP, PC3, A2780 and A2780ADR) to the action of cytotoxic drugs (mitoxantrone, cisplatin and doxorubicin) in vitro and in an A2780 xenograft mouse model of ovarian cancer. We conclude that oxMIF is the disease related isoform of MIF in solid tumors and a potential new diagnostic marker and drug target in cancer.

Abstract 3385: Cancerous tissue can be identified by the presence of oxMIF, the oxidized form of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and is pathologically associated with several types of cancer. We identified oxMIF, an immunological and conformationally distinct isoform of MIF that can be mimicked in vitro by mild oxidation of recombinant MIF. Fully human monoclonal antibodies that specifically target oxMIF were shown to inhibit signaling pathways associated with tumor proliferation and progression in vitro and in vivo in syngeneic or xenogeneic animal models. To evaluate the use of oxMIF as a biomarker of malignant tissue, we acquired plasma and tumor tissues from patients with colorectal cancer (CRC), ovarian, and non-small cell lung cancer (NSCLC), as well as control plasma and control tissue from healthy donors, i.e. in a normal state of health, with no apparent signs of disease. We developed a novel immunohistochemistry (IHC) method to detect oxMIF in malignant tissues and used a specific new ELISA to detect oxMIF in plasma. In patients with CRC, we detected oxMIF by IHC in primary tumors (31 of 39) and in liver, lung and lymph-node metastases (n = 10). Staining intensities showed variation between different tumor cells, as well as in the microenvironment. Similarly, we detected oxMIF by IHC in most types of ovarian cancer (n = 42), but not in controls (n = 4), and observed some variations in the localization and intensities of staining. We also detected oxMIF in liver metastases from patients with ovarian cancer (n = 2). In patients with NSCLC (n = 32), 22 samples showed a strong staining for oxMIF. In tissues from healthy donors (4 colon, 4 ovary and 3 lung), oxMIF was not or barely detectable, whereas total MIF (sum of reduced and oxidized MIF) was widely expressed in normal and tumor tissues and showed a more diffuse spatial distribution than oxMIF. In commercially acquired plasma samples from patients with CRC (n = 46) and NSCLC (n = 15), we did not observe a significant increase of oxMIF levels (median = 0 for both) compared with the plasma from healthy donors (median = 0; n = 69). In patients with ovarian cancer, significant increased levels of oxMIF were measured in plasma (median = 3.5ng/mL, n = 42), and more specifically from patients with papillary serous cystadenocarcinoma or serous cystadenocarcinoma compared with clear cell adenocarcinoma (medians = 3.5, 4.0, and 0 ng/mL respectively). Our studies demonstrated that oxMIF is a disease-related isoform of MIF that can be specifically detected in tissues from various types of cancer. We therefore suggest that oxMIF reflects a new biomarker in solid tumors and has potential diagnostic and prognostic value. A phase 1 clinical study of a novel human antibody that selectively targets oxMIF is currently ongoing in patients with solid malignancies (ClinicalTrials.gov identifier: NCT01765790). Citation Format: Nicolas Sabarth, Dirk Volkel, Michael Thiele, Alexander Schinagl, Patrice Douillard, Friedrich Scheiflinger, Randolf Kerschbaumer. Cancerous tissue can be identified by the presence of oxMIF, the oxidized form of macrophage migration inhibitory factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3385. doi:10.1158/1538-7445.AM2015-3385