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. 2018 Jun;32(6):3411-3422.
doi: 10.1096/fj.201701189R. Epub 2018 Jan 25.

Novel Half-Life Extended anti-MIF Nanobodies Protect Against Endotoxic Shock

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Free PMC article

Novel Half-Life Extended anti-MIF Nanobodies Protect Against Endotoxic Shock

Amanda Sparkes et al. FASEB J. .
Free PMC article

Abstract

Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

Keywords: LPS; MIF antagonist; Nbs.

Conflict of interest statement

The authors thank Yvon Elkrim, Ella Omasta, Marie-Thérèse Detobel, Maria Slazak, Victor Orimoloye, Natalia Śmiejkowska, Shahid Hussain, and Nadia Abou for technical and administrative assistance, as well as Dr. Alain Beschin for constructive discussions [all from the Vrije Universiteit Brussel (VUB) and the Flanders Institute for Biotechnology (VIB) Center for Inflammation Research]. This work, performed in the frame of an Interuniversity Attraction Pole Program (PAI-IAP N. P7/41; http://www.belspo.be/belspo/iap/index_en.stm), was supported by a grant from the Research Foundation–Flanders (FWO; KaN 1515813N), and a starting budget from Ablynx. B.S. was supported by the Strategic Research Program (SRP3; Vrije Universiteit Brussels). R.B. was supported by the U.S. National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.
Sequences of anti-MIF Nbs and their predicted binding regions on the MIF protein. A) Sequence alignment of the 11 different anti-MIF Nbs, which are organized into different families according to Kabat classification (74). Complementarity determinant region 1 (CDR1), CDR2, and CDR3 are indicated in blue, green, and orange, respectively. B) Sequence alignment of rhMIF (Homo sapiens) and rmMIF (Mus musculus). Residues involved in MIF’s tautomerase activity, CD74 binding, and the interaction with CXCR2 and CXCR4 are colored in red, green (including those indicated by the asterisk), and light and dark blue, respectively. Results of the performed epitope-mapping studies are indicated below the sequence alignment and are color coded (NbA2, pink; Nbs D2, D4, D12 and B5, purple; NbH9, light brown; Nbs E5 and E10, orange; NbC10, yellow; NbH5, dark brown; for NbF10, no definite region could be appointed). C) Results of the epitope-mapping experiment displayed on the structure of human MIF (62). MIF trimer is shown in surface representation, and each constituting monomer is shown in white, gray, and dark gray.
Figure 2.
Figure 2.
Inhibition of the biologic activities of MIF by anti-MIF Nbs. AC) Anti-MIF Nbs were found to reduce TNF secretion from LPS (10 ng/well)-stimulated THP-1 monocytic cells (A), human PBMCs (B), and RAW2647.7 macrophages (C). In brief, cells were treated with 500 nM of each anti-MIF Nb, and an irrelevant Nb was used as negative control. After 18 h of incubation, cell culture supernatants were collected for the determination of TNF concentration. Data are shown as means ± sem. Statistical analysis was performed by comparing TNF levels of Nb-treated LPS-stimulated cells with levels of untreated LPS-stimulated cells. The dashed line represents TNF levels (cutoff level) of mice that were stimulated with LPS alone. D) Inhibition of the d-dopachrome tautomerase activity of MIF by anti-MIF Nbs. ISO-1 was used as positive control. Activity was determined by semicontinuous reduction in signal (tautomerization) measured at OD450nm in the presence and absence of anti-MIF Nbs. Percentage activity was expressed in relationship to the tautomerase activity of rmMIF alone. The dashed line represents 100% activity (i.e., when only MIF was used). Data are shown as means ± sem and representative of 3 independent experiments (n = 2). *P < 0.05, **P < 0.01.
Figure 3.
Figure 3.
Anti-MIF Nbs can override the anti-immunosuppressive effects of MIF of glucocorticoids. RAW264.7 cells were treated with 10 ng LPS, with or without 500 nM anti-MIF Nb and/or 10 nM dexamethasone. After 18 h of incubation, cell culture supernatants were collected for the determination of TNF concentration. Data are shown as means ± sem. Statistical analysis was performed by comparing TNF levels of anti-MIF Nb-treated cells [NbE5 (gray bars) or NbE10 (white bars)] with irrelevant Nb-treated cells (black bars) when LPS was added alone or with dexamethasone. The red line represents TNF levels (cutoff level) of mice that were stimulated with LPS in the absence of Nb. The blue line represents TNF levels (cutoff level) of cells that were stimulated with LPS in the presence of dexamethasone. Data are representative of 3 independent experiments ± sem (n = 2). *P < 0.05, **P < 0.01, ****P < 0.0001.
Figure 4.
Figure 4.
Anti-MIF NbE10 constructs can inhibit TNF secretion from LPS-stimulated macrophages. PEMs from naive mice were treated with 1.67 µM of all anti-MIF NbE10 constructs. After 18 h of incubation, cell culture supernatants were collected for the determination of the TNF concentration. Data are representative of 2 independent experiments (n = 2) and are shown as means ± sem. Statistical analysis was performed by comparing the induction of TNF of LPS-stimulated cells in the presence of anti-MIF Nb constructs with that of LPS-stimulated cells alone. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5.
Figure 5.
A bivalent half-life extended anti-MIF NbE10 construct is protective in a mouse model of endotoxemia. A) C57BL/6 mice were administered a single injection of 12.5 mg/kg LPS alone or in combination with 25 mg/kg of all available NbE10 constructs and monitored for survival. Data are representative of 3 independent experiments (n = 5–13). B) Dose kinetics of NbE10-NbAlb8-NbE10, whereby C57BL/6 mice were administered a single coinjection of 12.5 mg/kg LPS and 25, 12.5, 6.25, or 2.5 mg/kg of the NbE10-NbAlb8-NbE10 construct and monitored for survival. Percentage survival was expressed as the percentage of mice that survived 60 h post-LPS injection. Of note, mice that survived after this time period recovered completely. Data are representative of 2 independent experiments (n = 5) and are shown as means ± sem. *P < 0.05, **P < 0.01.
Figure 6.
Figure 6.
NbE10-NbAlb8-NbE10 reduced peak serum TNF levels in endotoxin-treated mice. C57BL/6 mice were administered a single i.p. injection of 12.5 mg/kg LPS alone or in combination with 25 mg/kg NbE10-NbAlb8-NbE10, and at 90 min, mice were sacrificed and serum tested in a TNF ELISA. Data are representative of 2 independent experiments and expressed as means ± sem (n = 3). *P < 0.05.
Figure 7.
Figure 7.
A bivalent half-life extended anti-MIF NbE10 construct administered therapeutically attenuates endotoxemia. C57BL/6 mice were administered a single lethal intraperitoneal injection of 12.5 mg/kg LPS alone (block box) or followed by 25 mg/kg NbE10-NbAlb8-NbE10 administered 6 h post-LPS injection (gray box). Data are representative of 2 independent experiments (n = 5) and are shown as means ± sem. **P < 0.01.

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