Michael Thiele

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.

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.

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 A181: Oxidized macrophage migration inhibitory factor (oxMIF) is a previously unrecognized, disease-related isoform of MIF and a potential new drug target in cancer.

Background: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with pleiotropic biologic effects and which is pathologically associated with several types of cancer. MIF is markedly different from other cytokines in that it is constitutively expressed, is stored in the cytoplasm and is present in the circulation of healthy subjects. Thus, the concept of targeting MIF for therapeutic intervention suffers from the drawback of targeting a ubiquitous protein. Here we report the discovery of a previously unrecognized conformational isoform of MIF that can be mimicked in vitro by mild oxidation of recombinant MIF and has therefore been designated as oxidized MIF (oxMIF). Ex vivo studies show that oxMIF is produced in patients with cancer. Highly selective, fully human monoclonal antibodies that specifically target oxMIF, but do not bind the ubiquitous reduced MIF isoform (redMIF), were shown to inhibit downstream signaling pathways associated with tumor proliferation and progression in vitro and in vivo. Methods: New ELISA methods have been established that allow the detection of oxMIF and redMIF in plasma samples acquired from commercial providers. The effect of highly selective oxMIF-specific antibodies on human cancer cells was explored in vitro and in mouse xenograft models using the cell lines PC-3 (prostate cancer), IGROV-1 (ovarian cancer) and COLO-357 (pancreatic cancer). Results: The baseline level of MIF detected in the plasma of healthy subjects (median 5123 pg/ml, n=69) was as expected according to the literature. However, our analysis revealed that this represents the ubiquitous reduced isoform of MIF, redMIF. OxMIF was, in general, not detected in the plasma from healthy controls (median 0 pg/mL, n=69). By contrast, significantly increased concentrations of oxMIF were detected in plasma from patients with breast cancer (644 pg/mL, p Conclusion: Our studies demonstrate that the oxMIF isoform is associated with cancer. OxMIF is a potential marker for disease activity or disease progression and is a promising new drug target for the treatment of cancer. 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 Information: Mol Cancer Ther 2013;12(11 Suppl):A181. Citation Format: Michael Thiele, Patrice Douillard, Dirk Voelkel, Thorsten Hagemann, Michael Freissmuth, Hartmut Ehrlich, Friedrich Scheiflinger, Randolf J. Kerschbaumer. Oxidized macrophage migration inhibitory factor (oxMIF) is a previously unrecognized, disease-related isoform of MIF and a potential new drug target in cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A181.

Abstract 4841: Oxidized macrophage migration inhibitory factor (oxMIF) expressed by tumor stroma and tumor cells, contributes to tumor growth

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Macrophage migration inhibitory factor (MIF) was found to be upregulated in many cancers and to act in para- and autocrine loops within the tumor microenvironment. MIF has pleiotropic effects and plays a role in tumor growth via several mechanisms. (i) MIF acts directly on tumor cells by activating signaling pathways that promote cell proliferation and cell survival. (ii) MIF facilitates invasion of the extracellular matrix and induces angiogenesis and tumor vascularization. (iii) As a proinflammatory cytokine, MIF is one of the mediators of tumor micro-inflammation. However, MIF occurs in two immunologically distinct, redox-dependent isoforms. In its reduced form (redMIF), MIF is abundantly expressed and is present even in healthy subjects. In contrast, oxidized MIF (oxMIF) is found in patients with cancer. Highly selective, fully human monoclonal antibodies that specifically target oxMIF can counter-regulate the biological functions of MIF, indicating that oxMIF is the disease-related isoform of MIF and a relevant drug target. Animal studies and in vitro studies revealed that novel human antibodies that selectively target oxMIF, inhibit proliferation by reducing phosphorylation of ERK1/2 and AKT, promote apoptosis by activating caspase 3, inhibit angiogenesis and metastasis by reducing VEGF expression and blood vessel density within the tumor, and decrease cancer-associated inflammation of the tumor by downregulating the production of proinflammatory cytokines. We used MIF wild type (wt) and MIF knockout (KO) murine pancreatic cancer cells and MIF wt and MIF KO mice with C57Bl/6 background to dissect the contribution of stromal versus tumor derived MIF to tumor progression. Transfer of a pancreatic cancer cell line expressing MIF into wt mice or MIF KO mice resulted in aggressive tumor growth, liver metastasis, and survival of approximately 30 days in both models. Similar results were obtained by transferring a MIF KO pancreatic cancer cell line into MIF wt mice. Only transfer of a MIF KO cell line into MIF KO mice led to improved overall survival of approximately 80 days and completely abrogated metastasis. In a tumor xenograft model, an intravenously applied oxMIF-specific fully human antibody was able to penetrate the cancerous tissue and was detected in the stroma and the tumor. We conclude that both tumor stroma and tumor cells are an efficient source of oxMIF to promote tumor growth and metastasis, and that anti-oxMIF antibodies are able to neutralize oxMIF in the stroma and the tumor. 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][1]). Citation Format: Alexander Schinagl, Thorsten Hagemann, Patrice Douillard, Michael Thiele, Dirk Voelkel, Michael Freissmuth, Friedrich Scheiflinger, Randolf J. Kerschbaumer. Oxidized macrophage migration inhibitory factor (oxMIF) expressed by tumor stroma and tumor cells, contributes to tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4841. doi:10.1158/1538-7445.AM2014-4841 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01765790&atom=%2Fcanres%2F74%2F19_Supplement%2F4841.atom

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

Abstract 2475: A novel class of fully human monoclonal anti-oxMIF antibodies penetrates metastases and accumulates in tumor tissue

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine known to exacerbate tumor growth. MIF expression correlates with tumor aggressiveness and metastatic potential. We discovered a novel, disease-related conformational isoform of MIF which we have designated “oxMIF” because it can be mimicked in vitro by mild oxidation of recombinant MIF. A new class of fully human monoclonal antibodies (mAbs) specifically targeting oxMIF demonstrated efficacy in vivo and inhibited signaling pathways associated with tumor proliferation and progression in vitro. By developing novel immunohistochemistry (IHC) methods we were able to differentially detect oxMIF and to determine biodistribution of human anti-oxMIF mAbs in human and animal-derived tissue. First, we observed by IHC that in human pancreatic ductal adenocarcinoma (PDAC) tissue, oxMIF is specifically expressed in tumor cells and in tumor stroma within pancreatic intraepithelial lesions. In contrast, we could not detect oxMIF in healthy control tissue, whereas MIF (sum of oxMIF and its reduced isoform) was abundantly expressed in both, tissue from PDAC patients and tissue from healthy subjects. Then, in a murine genetic model of PDAC, we assessed the biodistribution of anti-oxMIF mAbs: intravenously injected I 131 -labelled anti-oxMIF mAbs significantly accumulated in the pancreas (primary tumor site), as well as in liver and lung (sites of metastasis). Subsequently, we investigated the biodistribution of anti-oxMIF mAbs in more detail using a syngeneic mouse model of chronic lymphocytic leukemia (eμ-myc CLL model): IHC analysis of lymph nodes from mAb treated mice confirmed co-localization of oxMIF and anti-oxMIF mAbs in cancerous tissue. Finally, we applied our IHC methods to assess the tissue penetration of anti-oxMIF mAb in liver metastases from late stage colorectal cancer patients (Ph 1 clinical study; ClinicalTrials.gov identifier: NCT01765790): we demonstrated that the antibody was able to penetrate metastases and to accumulate in tumor cells and in stromal tissue during the course of treatment. Our previous and current studies clearly demonstrate that oxMIF is a disease-related isoform of MIF that can be detected in malignant tissue and that treatment with fully human anti-oxMIF mAbs enables neutralization of oxMIF in tumor tissue. We therefore suggest that oxMIF reflects a potent new therapeutic target in solid tumors with diagnostic and prognostic value. A phase 1 clinical study of a novel fully human anti-oxMIF mAb is currently ongoing in patients with solid malignancies (ClinicalTrials.gov identifier: NCT01765790). Citation Format: Alexander Schinagl, Michael Thiele, Patrice Douillard, Deyaa Adib, Xiaochun Liu, Salim Yazji, Friedrich Scheiflinger, Randolf Kerschbaumer. A novel class of fully human monoclonal anti-oxMIF antibodies penetrates metastases and accumulates in tumor tissue. [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 2475. doi:10.1158/1538-7445.AM2015-2475

Abstract 2654: Human antibodies specific for oxidized macrophage migration inhibitory factor (oxMIF) synergize with chemotherapeutic agents in animal models of cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with pleiotropic biologic effects and is pathologically associated with several types of cancer. oxMIF is an immunological and conformational 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 downstream signaling pathways associated with tumor proliferation and progression in vitro and in vivo. We assessed the potential of anti-oxMIF antibodies combined with standard chemotherapeutic agents on the proliferation of cancer cell lines in vitro and in vivo in xenograft models. In vitro, anti-oxMIF sensitization of the prostate cancer cell lines PC3 or LnCAP significantly reduced the half maximal effective concentration (EC50) of mitoxantrone by 30% and 40%, respectively. In the ovarian cancer cell lines A2780, EC50 of cisplatin was reduced by 70% after sensitization with anti-oxMIF. In vivo, mice bearing pancreatic cancer xenografts (COLO357, BxPC3 or MiaPaCa2) treated with anti-oxMIF in combination with gemcitabine had a strong survival advantage compared with mice treated with either monotherapy. This effect was accompanied by a decrease of tumor volumes up to 70% and an increase in the expression of caspase 3 in mice bearing COLO357 xenografts. In addition, combining gemcitabine with anti-oxMIF treatment normalized blood vessel density inside the tumors, decreased their permeability by 25%, and decreased tumor VEGF levels by 40%, whereas gemcitabine alone had the opposite effect. In mice bearing the ovarian human cancer cell line A2780 xenografts, combining anti-oxMIF antibodies with cisplatin led to a 45% reduction of tumor weights. Anti-oxMIF specific antibodies decreased proliferation, angiogenesis and inflammation inside tumor xenografts, but were not cytotoxic (no antibody-dependent cell cytotoxicity or complement-dependent cytotoxicity). These experiments demonstrated synergistic effects between anti-oxMIF antibodies and cytotoxic chemotherapy. Combination treatments were superior to the corresponding monotherapies and led to a stronger inhibition of tumor growth and angiogenesis. 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][1]). Citation Format: Patrice Douillard, Thorsten Hagemann, Michael Freissmuth, Michael Thiele, Alexander Schinagl, Dirk Volkel, Friedrich Scheiflinger, Randolf Kerschbaumer. Human antibodies specific for oxidized macrophage migration inhibitory factor (oxMIF) synergize with chemotherapeutic agents in animal models of cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2654. doi:10.1158/1538-7445.AM2014-2654 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01765790&atom=%2Fcanres%2F74%2F19_Supplement%2F2654.atom

Abstract 1235: Human antibodies specific for a novel isoform of the macrophage migration inhibitory factor are potential therapeutics for prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Macrophage migration inhibitory factor (MIF) was found to be upregulated in many cancers and to act in para- and autocrine loops within the tumor microenvironment. Thereby, MIF contributes to the modulation of cytokine patterns and triggers anti-apoptotic and pro-proliferative cell signaling events. MIF was also described as a pro-angiogenic factor promoting neoangiogenesis and vascularization. In prostate cancer, MIF mRNA and protein levels were shown to be increased and to contribute to tumor development. We have discovered a novel, previously unrecognized conformational isoform of MIF (designated ‘active MIF’) that is produced in diseases and cannot be detected in healthy subjects. A set of phage display derived human monoclonal anti-MIF antibodies neutralizing MIF biological activity in vitro and in vivo specifically recognize active MIF. In vitro, these antibodies elicited both, a growth inhibitory effect and a pro-apoptotic response on PC3 and DU145 prostate cancer cells by interfering with ERK1/2 and Akt/PKB signaling pathways and by promoting activation of caspase-3. Furthermore, the antibodies inhibited tumor cell invasion of PC3 cells in a transwell assay in a dose-dependent manner. In vivo, treatment with human antibodies specific for active MIF dose-dependently inhibited the growth of the human prostate cancer cell line PC3 xenografted into MF1 nude mice. New ELISA methods have been established to allow the detection of active MIF as well as non-active MIF in plasma samples. In the plasma of PC3 xenografted nude mice, we observed a reduction of active MIF upon antibody treatment. In addition we were able to prove the occurrence of active MIF in the plasma of prostate cancer patients (median 2445 pg/mL, p < 0.002 versus controls), whereas active MIF was not detectable in plasma from healthy donors. These studies demonstrate that active MIF represents a new target in prostate cancer, as well as a promising marker for disease activity or disease progression. The data further suggest that our fully human anti-MIF antibodies have a good potential for clinical use in oncology. Citation Format: Michael Thiele, Michael Freissmuth, Filza Hussain, Patrice Douillard, Dirk Volkel, Hartmut Ehrlich, Friedrich Scheiflinger, Randolf Kerschbaumer. Human antibodies specific for a novel isoform of the macrophage migration inhibitory factor are potential therapeutics for prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2013-1235