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.

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.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory and tumor-promoting cytokine that occurs in two redox-dependent immunologically distinct conformational isoforms. The disease-related structural isoform of MIF (oxMIF) can be specifically and predominantly detected in the circulation of patients with inflammatory diseases and in tumor tissue, whereas the ubiquitously expressed isoform of MIF (redMIF) is abundantly expressed in healthy and diseased subjects. In this article, we report that cysteine 81 within MIF serves as a "switch cysteine" for the conversion of redMIF to oxMIF. Modulating cysteine 81 by thiol reactive agents leads to significant structural rearrangements of the protein, resulting in a decreased β-sheet content and an increased random coil content, but maintaining the trimeric quaternary structure. This conformational change in the MIF molecule enables binding of oxMIF-specific antibodies BaxB01 and BaxM159, which showed beneficial activity in animal models of inflammation and cancer. Crystal structure analysis of the MIF-derived EPCALCS peptide, bound in its oxMIF-like conformation by the Fab fragment of BaxB01, revealed that this peptide adopts a curved conformation, making the central thiol protein oxidoreductase motif competent to undergo disulfide shuffling. We conclude that redMIF might reflect a latent zymogenic form of MIF, and formation of oxMIF leads to a physiologically relevant, i.e., enzymatically active, state.

New therapeutic agents are needed to overcome the toxicity and suboptimal efficacy observed in current treatment of glomerulonephritis (GN). BaxB01 is a fully human monoclonal antibody targeting a disease-related immunologically distinct isoform of Macrophage migration Inhibitory Factor (MIF), designated oxidized MIF (oxMIF) and locally expressed in inflammatory conditions. We report the pharmacokinetic profile of BaxB01, and its dose and exposure-related disease-modifying activity in experimentally induced rat GN. BaxB01 bound to rat oxMIF with high affinity and reduced rat macrophage migration in vitro. After intravenous administration in rats, BaxB01 demonstrated favorable pharmacokinetics, with a half-life of up to nine days. Disease modification was dose-related (≥ 10mg/kg) as demonstrated by significantly reduced proteinuria and diminished histopathological glomerular crescent formation. Importantly, a single dose was sufficient to establish an exposure-related, anti-inflammatory milieu via amelioration of glomerular cellular inflammation. Pharmacodynamic modeling corroborated these findings, consistently predicting plasma exposures that were effective in attenuating both anti-inflammatory activity and reducing loss of kidney function. This pharmacologic benefit on glomerular function and structure was sustained during established disease, while correlation analyses confirmed a link between the antibody's anti-inflammatory activity and reduced crescent formation in individual rats. Finally, safety assessment in rats showed that the experimental therapeutic was well tolerated without signs of systemic toxicity or negative impact on kidney function. These data define therapeutically relevant exposures correlated with mechanism-based activity in GN, while toxicological evaluation suggests a large therapeutic index and provides evidence for achieving safe and effective exposure to a MIF isoform-directed therapeutic in nephritis-associated disease.

Background: Macrophage Migration Inhibitory Factor (MIF) has been described as a pleiotropic cytokine known to exacerbate tumor growth (Chesney and Mitchell, 2015). Due to its ubiquitous nature, MIF can be considered as an inappropriate target for therapeutic intervention. However, we discovered oxidized MIF (oxMIF) as the disease-related isoform of MIF, which is specifically present in tumor tissue (Schinagl A et al., 2016). A 1st generation anti-oxMIF monoclonal antibody (mAb) demonstrated an acceptable safety profile and efficacy signal in a phase 1 clinical trial (Mahalingam D et al., 2020). Two bioengineered 2nd generation anti-oxMIF mAbs with highly improved biophysicochemical and biological properties were generated and compared to the 1st generation anti-oxMIF mAb. Both bioengineered mAbs share identical variable domains, but distinct heavy-chain constant regions, to increase effector functions and efficacy of the therapeutic mAb ON203 and to reduce interactions with FcγRs for the radio diagnostic mAb ON102. Methods: Hydrophobicity and stability were determined by HIC and SEC. ADCC and ADCP activity were investigated by reporter and PBMC-mediated cell killing assays. Tumor penetration was assessed using IRDye 800CW or Zr89-labeled mAb in tumor-bearing Balb/c or Balb/c nude mice. PK and bioavailability were assessed in Balb/c nude mice. Efficacy was determined in PC3 xenograft models in NMRI nude mice. Results: Bioengineering significantly reduced hydrophobicity of ON203 and ON102, leading to improved stability and strongly reduced aggregation but retaining the low nM affinity for oxMIF. This further resulted in a two-fold improved bioavailability and tumor accumulation after 24-48h and a three-fold enhanced retention on day 7 in mice harboring solid tumors of the colon, when compared to the 1st generation anti-oxMIF mAb. In contrast to previously described anti-oxMIF reference, ON203 and ON102 did not mediate any unspecific release of MCP-1, IL-6, or TNF-α from PBMCs. Reporter and PBMC-mediated cell killing assays proved ADCC activity of ON203 with EC50 values of 0.1-0.8 nM and ADCP activity with EC50 values of 2.3 nM, whereas the 1st generation anti-oxMIF mAb was at least 10-fold less potent. Our findings for ON203 translated into abolishment of tumor growth in NMRI nude mice harboring human PC3 prostate cancer cells, showing superior efficacy compared to the 1st generation anti-oxMIF mAb. Conclusion: ON203 has a high potential to significantly improve efficacy compared to the 1st generation anti-oxMIF mAb, with Zr89-ON102 as companion diagnostic for patient stratification. We aim to develop these anti-oxMIF mAbs together for clinical use to create new treatment options for patients with solid tumors, with a clear rationale to combine ON203 with other immunotherapies or checkpoint inhibitors (Noe and Mitchell, 2020). Citation Format: Alexander Schinagl, Michael Thiele, Irina Mirkina, Gregor Rossmueller, Alejandro A. Puchol Tarazona, Randolf J. Kerschbaumer. Novel bioengineered monoclonal antibodies targeting oxidized macrophage migration inhibitory factor as anti-cancer therapeutics and companion diagnostics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 313.

Background: 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 disease related conformational isoform of MIF, which we designated “oxMIF” because it can be mimicked in vitro by mild oxidation of recombinant MIF (Thiele et al., 2015, J. Immunol.). We found oxMIF expressed in different types of cancer tissue but not in healthy control tissues. A new class of fully human monoclonal antibodies specifically targeting oxMIF (Kerschbaumer et al., 2012, J. Biol. Chem) demonstrated efficacy in vivo and inhibited tumor signaling pathways associated with tumor proliferation and progression in vitro (Hussain et al., 2013, Mol. Cancer Ther.). Imalumab is Baxalta's lead candidate anti-oxMIF antibody which is currently tested in a phase I clinical trial (ClinicalTrials.gov identifier: NCT01765790). Methods: Biodistribution of anti-oxMIF antibodies was assessed (i) in a murine genetic model of pancreatic cancer and (ii) in biopsies from phase 1 late stage patients with metastatic colorectal cancer (mCRC) who did receive imalumab intravenously (28-days cycles; 4 dose schedules) once weekly. Pre- and on-therapy tumor biopsies of CRC liver metastases from these treated patients were analyzed for antibody tissue penetration using newly developed immunohistochemistry (IHC) methods; in parallel biopsies were also analyzed for phosphoproteins and cytokines/chemokines content by multiplex. Results: In a murine genetic model of pancreatic cancer, intravenously injected I131-labelled anti-oxMIF antibody accumulated in the pancreas (primary tumor site), as well as in liver and lung (sites of metastasis). Analysis of biopsies from phase 1 patients with late stage mCRC revealed similar results as imalumab was able (i) to penetrate metastases, (ii) to co-localize with oxMIF in tumor cells and in stromal tissue and (iii) to accumulate during the course of treatment. While accumulating in tumor tissue, the antibody was cleared from the plasma with a half-life (half plasma steady-state concentration) of approximately 7 days. In parallel, analysis of intratumoral phosphoproteins and cytokines showed that imalumab down regulates tumorigenic and pro-inflammatory signals (e.g. Akt, MEK, MAPK, Btk) and pro-inflammatory cytokines (e.g. TNF-α and TNF-β) while up-regulating anti-inflammatory cytokines like IL-1ra and IL-10. Conclusion: Imalumab shows single agent antitumor activity in heavily pretreated patients with mCRC. These observations are in line with in vitro and animal studies using anti-oxMIF antibodies (Hussain et al., 2013, Mol. Cancer Ther.) and literature data on MIF tumorigenic functions. Based on these data, a 10 mg/kg dose has been recommended for a phase 2a study of imalumab in combination with 5-FU/Leucovorin or Panitumumab versus standard of care treatment in subjects with mCRC. Patient recruitment for this study is currently ongoing (ClinicalTrials.gov Identifier: NCT02448810) Citation Format: Patrice Douillard, Michael Thiele, Alexander Schinagl, Niels Halama, Dirk Jaeger, Salim Yazji, Friedrich Scheiflinger, Randolf Kerschbaumer. Imalumab, a first-in-class anti-oxidized macrophage migration inhibitory factor (oxMIF) antibody penetrates tumor tissues and shows antitumor activity in patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A153.

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). 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

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