CHEMOKINE-SELECTIVE CXCR4 ECTODOMAIN-DERIVED (POLY)PEPTIDE
20230242616 · 2023-08-03
Inventors
- JÜRGEN BERNHAGEN (MÜNCHEN, DE)
- Omar El Bounkari (Diessen, DE)
- APHRODITE KAPURNIOTU (MÜNCHEN, DE)
- Christos Kontos (Freising, DE)
Cpc classification
A61P29/00
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
C07K2319/32
CHEMISTRY; METALLURGY
International classification
C07K14/715
CHEMISTRY; METALLURGY
A61P9/10
HUMAN NECESSITIES
Abstract
The present invention relates to a chemokine-selective CXCR4 ectodomain-derived (poly)peptide comprising or consisting of a first peptide of (X1)(X2)(X3)(X4)(X5)WYFGNF(X6)(X7)(X8) (SEQ ID NO: 1) linked via a linker to a second peptide of (Y1)(Y2)(Y3)(Y4)(Y5)D(Y6)FY(Y7)N(Y8)LW(Y9) (SEQ ID NO: 2), wherein (X1) is present or absent and, if present, is an amino acid, preferably D or A (X2) is present or absent and, if present, is an amino acid, preferably A or G (X3) is present or absent and, if present, is an amino acid, preferably V or A (X4) is present or absent and, if present, is an amino acid, preferably A or G (X5) is present or absent and, if present, is an amino acid, preferably N (X6) is an amino acid, preferably L or A (X7) is an amino acid, preferably C, A or S, more preferably C or A (X8) is an amino acid, preferably K or A (Y1) is present or absent and, if present, is an amino acid, preferably D or A (Y2) is present or absent and, if present, is an amino acid, preferably R or A (Y3) is present or absent and, if present, is an amino acid, preferably Y (Y4) is present or absent and, if present, is an amino acid, preferably I or A (Y5) is present or absent and, if present, is an amino acid, preferably C. A or S, more preferably C or A (Y6) is present or absent and, if present, is an amino acid, preferably D, R or A (Y7) is an amino acid, preferably P or A (Y8) is an amino acid, preferably D or A (Y9) is present or absent and, if present, is an amino acid, preferably V;
and wherein said linker has a length of 0.2 to 5 nm, preferably 1 nm to 5 nm, more preferably 2 to 4 nm, and most preferably about 2.358 nm.
Claims
1. A chemokine-selective CXCR4 ectodomain-derived (poly)peptide comprising or consisting of a first peptide of (X1)(X2)(X3)(X4)(X5)WYFGNF(X6)(X7)(X8) (SEQ ID NO: 1) linked via a linker to a second peptide of (Y1)(Y2)(Y3)(Y4)(Y5)D(Y6)FY(Y7)N(Y8)LW(Y9) (SEQ ID NO: 2), wherein (X1) is present or absent and, if present, is an amino acid, preferably D or A (X2) is present or absent and, if present, is an amino acid, preferably A or G (X3) is present or absent and, if present, is an amino acid, preferably V or A (X4) is present or absent and, if present, is an amino acid, preferably A or G (X5) is present or absent and, if present, is an amino acid, preferably N (X6) is an amino acid, preferably L or A (X7) is an amino acid, preferably C, A or S, more preferably C or A (X8) is an amino acid, preferably K or A (Y1) is present or absent and, if present, is an amino acid, preferably D or A (Y2) is present or absent and, if present, is an amino acid, preferably R or A (Y3) is present or absent and, if present, is an amino acid, preferably Y (Y4) is present or absent and, if present, is an amino acid, preferably I or A (Y5) is present or absent and, if present, is an amino acid, preferably C, A or S, more preferably C or A (Y6) is present or absent and, if present, is an amino acid, preferably D, R or A (Y7) is an amino acid, preferably P or A (Y8) is an amino acid, preferably D or A (Y9) is present or absent and, if present, is an amino acid, preferably V and wherein said linker has a length of 0.2 to 5 nm, preferably 1 nm to 5 nm, more preferably 2 to 4 nm, and most preferably about 2.358 nm.
2. The (poly)peptide of claim 1, wherein the linker comprises or consists of 1 to 8, and preferably 2 or 3 amino acids.
3. The (poly)peptide of claim 1, wherein the linker comprises or consists of non-natural amino acids.
4. The (poly)peptide of claim 3, wherein the non-natural amino acids are selected from the group consisting of 6-aminohexanoic acid (6-Ahx), 12-amino-dodecanoic acid (12-Ado) and 3,6-dioxaoctanoic acid (O20c).
5. The (poly)peptide of claim 1, wherein the linker comprises or consists of 6-Ahx-12-Ado or O2Oc-12-Ado, and preferably consists of 6-Ahx-12-Ado.
6. The (poly)peptide of claim 1, wherein the linker comprises or consists of three naturally-occurring amino acids, preferably selected from G, D, R and K, wherein the linker is most preferably selected from DDD and RRR.
7. The (poly)peptide of claim 1, wherein the (poly)peptide is a cyclic (poly)peptide.
8. The (poly)peptide of claim 7, wherein the (poly)peptide comprises two cysteines or homocysteines being linked by an S—S bond.
9. The (poly)peptide of claim 8, wherein the two cysteines or homocysteines are preferably located at an N-terminus of the first peptide and a C-terminus of the second peptide.
10. The (poly)peptide of claim 1, wherein the (poly)peptide is fused to (i) a component modulating serum half-life, wherein the component modulating serum half-life is preferably Fc domain of an antibody, an albumin binding tag, albumin or polyethylene glycol, (ii) a component increasing solubility of the (poly)peptide, wherein the component increasing the solubility of the (poly)peptide is preferably selected from a peptide comprising acids with positively and negatively charged side chains, betaines, polyionic tags, cyclodextrins, glycosyl moieties, and conjugated nanoparticles, and/or (iii) a diagnostic label, preferably a chromogenic label, a fluorogenic label, or an isotope.
11. The (poly)peptide of claim 1, wherein the (poly)peptide comprises a first peptide differing by no more than three, preferably by no more than two, more preferably by one amino acid mutation and most preferably by zero amino acid mutations from the peptide of DAVANWYFGNFLCK (SEQ ID NO: 3) and/or comprises a second peptide differing by no more than three, preferably by no more than two, more preferably by one amino acid mutation and most preferably by zero amino acid mutations from the peptide of DRYICDRFYPNDLWV (SEQ ID NO: 4).
12. A composition, preferably a pharmaceutical composition comprising the (poly)peptide of claim 1.
13-15. (canceled)
16. A method of treating a disease, comprising using the (poly)peptide of claim 1.
17. The method of claim 16, wherein the disease is an atherosclerotic disease, an inflammatory disease, a tumor, a neuroinflammatory or neuro-degenerative disease, or an autoimmune disease.
18. The method of claim 17, wherein the atherosclerotic disease is an atherosclerotic disease in individuals with a high-MIF expression genotype as defined by the CATT6-8 or CATT-non-5/5 promoter polymorphism; and/or wherein the tumor is cancer, preferably metastatic cancer.
Description
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[0172] The Examples illustrate the invention.
EXAMPLE 1—DESIGNING SOLUBLE CHEMOKINE-SELECTIVE CXCR4 ECTODOMAIN MIMICS
[0173] Previous peptide array and SAR studies of the inventors showed that residues 97-110 of ECL1 and 182-196 of ECL2 of the CXCR4 ectodomain contribute to the interface between MIF and CXCR4.sup.25. It was therefore speculated whether this could be a basis to engineer soluble MIF-binding CXCR4 mimics. Peptides ECL1[97-110] and ECL2[182-196] were synthesized by solid-phase peptide synthesis (SPPS) using Fmoc chemistry.sup.28.
[0174] A synthetic linker was chosen based on the CXCR4 X-ray structures.sup.28, 29, 30. The conformationally constrained ectodomain mimic CXCR4-ECL1[97-110]-6-Ahx-12-Ado-ECL2[182-196] was designed and generated (‘msR4M-L1’;
TABLE-US-00001 TABLE 1 Summary list and molecular masses of all synthesized CXCR4 ectodomain peptides applied in this study. Peptide [M+H].sup.+ [M+H].sup.+ Peptide acronym sequence theoretical.sup.[a] experimental.sup.[a] ECL1 NH.sub.2-DAVANWYFGNFLCK-CONH.sub.2 (SEQ 1646.79 1670.02.sup.[b] ID NO: 3) ECL2 NH.sub.2-DRYICDRFYPNDLWW-CONH.sub.2 1973.94 1974.38 (SEQ ID NO: 4) mSR4M-L1 ECL1-6-Ahx-12-Ado-ECL2 3912.92 3913.67 msR4M-L2 ECL1-O2Oc-12 Ado-ECL2 3944.93 3945.49 msR4M-L1ox
[0175] 3,6-dioxaoctanoic acid (O2Oc)/12-Ado was chosen as an alternative, more hydrophilic, linker (‘msR4M-L2’;
[0176] To determine whether the CXCR4 ectodomain mimics bind to MIF, fluorescence titration spectroscopy.sup.32, 33 was applied measuring changes in fluorescence emission of Fluos-labeled ectodomain peptide upon titration against MIF or CXCL12. Conversely, Alexa-Fluor 488-labeled MIF (Alexa-MIF) was titrated against unlabeled ectodomain peptides. Importantly, msR4M-L1 exhibited high affinity binding to MIF with an apparent (app.) K.sub.D<40 nM (app. K.sub.D Fluos-msR4M-L1/MIF=36.8±2.3 nM; app. K.sub.D msR4M-L1/Alexa-MIF=31.1±16.6 nM), whereas no binding to CXCL12 was observed (app. K.sub.D>5 μM) (Table 1;
TABLE-US-00002 TABLE 2 Binding affinities between the CXCR4 ectodomain peptides and MIF versus CXCL12 as determined by fluorescence titration spectroscopy. MIF or CXCL12 MIF CXCR4 Fluos-ECD CXCL12 .sup.[a] ectodomain peptide/ Alexa-MIF/ Fluos-ECD peptide/ (ECD) MIF .sup.[b] ECD peptide .sup.[c] CXCL12 peptide app. K.sub.D (nM) .sup.[d] app. K.sub.D (nM) app. K.sub.D (nM) msR4M-L1 36.8 ± 2.3 31.1 ± 16.6 >5000 msR4M-L2 18.6 ± 2.9 40.5 ± 7.6 >5000 msR4M-L1ox 28.9 ± 2.5 30.0 ± 6.3 84.6 ± 42.1 msR4M-L2ox 105.3 ± 44.9 59.6 ± 15.3 54.8 ± 10.3 msR4M-LS 6.9 ± 2.0 n.d. 17.4 ± 4.7 ECL1 n.d. 345.2 ± 79.4 n.d. ECL2 >5000 2458 ± 1054 n.d. Table legend. .sup.[a] Alexa-CXCL12 measurements were not pursued, because of the notion that Alexa labeling could interfere with the crucial residue Lys-1 of CXCL12.sup.30 as well as other binding-relevant lysines. .sup.[b] Fluos-labeled ECD peptides used at a concentration of 5 nM; .sup.[c] Alexa-MIF used at 10 nM. .sup.[d] Reported apparent K.sub.D values are means ± SD from three independent binding curves and were calculated as described.sup.32. ECD, extracellular domain; app., apparent; n.d., not determined.
[0177] msR4M-L2 bound to MIF with similar affinity and also lacked CXCL12 binding (app. K.sub.D Fluos-msR4M-L2/MIF=18.6±2.9 nM; app. K.sub.D msR4M-L2/Alexa-MIF=40.5*7.6 nM; app. K.sub.D Fluos-msR4M-L2/CXCL12 >5 μM; Table 1;
[0178] These experiments suggested that msR4M-L1 and -L2 could represent promising CXCR4 mimics with high selectivity for MIF versus CXCL12. However, msR4M-L2 exhibited a higher self-assembly propensity than msR4M-L1 (app. K.sub.D Fluos-msR4M-L2/msR4M-L2=69.6±61.9 nM versus Fluos-msR4M-L1/msR4M-L1=142.0*48.9 nM;
[0179] Thus, msR4M-L1 was selected as a lead and wished to further confirm its ligand binding selectivity. When immobilized MIF and CXCL12 were probed on a dot-blot membrane with 5(6)-carboxytetra-methylrhodamine (TAMRA)-msR4M-L1. MIF was readily detected in a concentration-dependent manner, whereas TAMRA-msR4M-L1 showed no binding to CXCL12 (
[0180] Together, the data demonstrate that msR4M-L1, an engineered soluble CXCR4 ectodomain mimic, binds with high affinity to MIF, exhibiting binding selectivity for MIF versus the cognate ligand CXCL12, while not interfering with MIF/CD74 binding. This led us to prioritize msR4M-L1 for further analysis.
EXAMPLE 2—ENGINEERED CXCR4 MIMICS BIND TO A CORE BINDING REGION IN MIF
[0181] Next, the binding region in MIF that is necessary for interaction with the CXCR4 mimics was mapped. As previous structure-activity studies on the MIF/CXCR4 interface had provided evidence for a role of the N-like loop.sup.24, 25, the mapping started with a MIF peptide fragment spanning this region (
TABLE-US-00003 TABLE 3 Binding affinities (K.sub.D) between the CXCR4 ectodomain peptide msR4M-L1 and partial MIF peptides as determined by fluorescence titration spectroscopy. Overall screen of MIF(2-115) Screen of binding region MIF(38-80) App. K.sub.D App. K.sub.D MIF sequence (nM) MIF sequence (nM) 2-16 >20000 38-80 57.1 ± 7.8 6-23 >20000 38-60 >20000 13-27 >20000 38-64 >20000 18-32 >20000 38-68 696.3 ± 26.3 23-38 >20000 38-72* 160.7 ± 89.6 28-43 >20000 38-76 42.2 ± 27.9 38-80 57.1 ± 7.8 50-60 >5000 69-90 >20000 50-80 55.2 ± 9.9 76-90 >20000 51-67* >20000 81-94 >20000 54-80 70.6 ± 14.2 81-95 481.1 ± 43.5 55-80 479.4 ± 154.7 81-102 480.2 ± 83.1 56-69* 1819 ± 491 82-95 >10000 57-80 283.1 ± 57.7 86-100 >20000 58-80 540.4 ± 206.3 91-105 >10000 60-74* >20000 101-115 >20000 60-80 1758 ± 272 62-80 >5000 Table legend. Fluorescence spectroscopic analyses were performed as described in FIG. 2 of the main manuscript. Fluos-msR4M-L1 was generally applied at a concentration of 5 nM; the asterisk (*) denotes those peptide titrations for which Fluos-msR4M-L1 was used at 10 nM. The numbering of the sequence of human MIF (2-115) refers to the cDNA sequence and accounts for the notion that the N-terminal Met-1 residue is processed. MIF, human macrophage migration-inhibitory factor; app., apparent.
[0182] As the peptide lacks the 3D conformation of folded full-length MIF, this suggested that a locally-defined sequence was sufficient for the interaction with the ectodomain mimic. Moreover, detailed mapping of the msR4M-L1/MIF binding region by analyzing various 14-30-meric MIF peptide fragments spanning regions within and outside of sequence 38-80, narrowed the core binding region in MIF to sequence 50-80 or 54-80 (app. K.sub.D Fluos-msR4M-L1/MIF[50-80]=55.2±9.9 nM; app. K.sub.D Fluos-msR4M-L1/MIF[54-80]=70.6±14.2 nM;
[0183] Molecular docking simulations between CXCR4-ECL1 [97-110]-Gly.sub.(7)-ECL2[182-196], an msR4M-L1-like CXCR4 ectodomain mimic with a heptaglycine linker instead of the 6-Ahx-12-Ado spacer, suggested that msR4M-L1 has a reasonable energetic probability of interacting with MIF and confirmed the experimentally determined binding interface within sequence 54-80, with amino acids of this region among the top residues predicted to be involved in msR4M-L1 binding (
EXAMPLE 3—CXCR4 ECTODOMAIN MIMICS SELECTIVELY INHIBIT MIF-TRIGGERED CXCR4 BINDING, SIGNALING AND CHEMOTAXIS, BUT DO NOT INTERFERE WITH CXCL12/CXCR4 AND MIF/CD74 SIGNALING
[0184] To scrutinize whether selective msR4M-L1/MIF binding correlates with inhibition of MIF-triggered inflammatory and atherogenic effects, it was first examined whether msR4M-L1 interfered with MIF/CXCR4-specific cell signaling. Advantage was taken of a yeast strain that expresses human CXCR4 and specifically measures agonist-mediated activation of CXCR4 via a reporter plasmid.sup.25. Confirming previous data.sup.25, MIF triggered CXCR4-mediated signaling, but co-incubation of MIF with msR4M-L1 blocked the β-galactosidase reporter signal in a concentration-dependent manner (
[0185] Receptor signaling analysis in the disclosed yeast system is limited to GPCRs and not amenable to the non-GPCR receptor CD74. To verify that msRM4-L1 does not interfere with the MIF/CD74 axis in a cell-based system, we transfected HEK293 cells with a construct driving CD74 surface expression.sup.14. Alexa-MIF cell surface binding as measured by flow cytometry was elevated in a CD74-dependent manner. Of note, co-incubation of Alexa-MIF with a 5-fold molar excess of msR4M-L1 did not reduce surface binding of Alexa-MIF (
[0186] It was next asked whether msR4M-L1 also selectively inhibits MIF responses in mammalian cell systems expressing endogenous CXCR4. B lymphocytes express substantial levels of CXCR4 and MIF has been shown to trigger murine B-cell chemotaxis in a CXCR4-dependent manner.sup.34. Human and murine MIF share 90% amino acid identity and there is a high degree of cross-species receptor activity.sup.5. There also is a high degree of sequence identity between human and murine MIF in the binding region for msR4M-L1 (MIF(38-80): 86%; MIF(54-80): 89%;
[0187] MIF is a pro-atherogenic cytokine, but also has context-dependent ‘local’ protective activity on cardiomyocytes.sup.14, 15, 16, 17. Before further evaluating the translational potential of our findings, it was wished to exclude that msR4M-L1 interferes with protective MIF/CD74-mediated signaling in cardiomyocytes. Primary human cardiomyocytes (HCM; expressing CD74,
EXAMPLE 4—CXCR4 ECTODOMAIN MIMIC INHIBITS PRO-ATHEROGENIC MIF ACTIVITIES IN VITRO AND IN THE VASCULATURE EX VIVO
[0188] MIF is a driver of atherogenic monocyte activity and inhibition of monocyte-dependent atherogenic inflammation is a preferred strategy to limit atherosclerotic lesion formation. Monocyte/macrophage-expressed CXCR4 promotes atherogenesis via low density lipoprotein (LDL) uptake and foam cell formation, an effect specifically mediated by the MIF/CXCR4 axis but not by CXCL12/CXCR.sup.46.
[0189] Confirming previous findings.sup.36, uptake of fluorescently labeled LDL (Dil-LDL) by human macrophages derived from peripheral blood mononuclear cells (PBMCs) was markedly enhanced by MIF and this activity was blocked inhibitor by the pharmacological inhibitor AMD3100, verifying CXCR4 dependency. Of note, msR4M-L1 dose-dependently inhibited MIF-mediated Dil-LDL uptake (
[0190] It was next tested the potency of msR4M-L1 towards MIF-elicited three-dimensional (3D) chemotaxis of PBMCs. We applied 3D-chemotaxis methodology and assessed single-cell migration tracks via time-lapse microscopy. msR4M-L1 dose-dependently attenuated MIF-triggered motility of human monocytes as quantified by forward migration index. The pro-migratory effect of MIF was already ablated by a 2-fold molar excess of msR4M-L1 (
[0191] A major atherogenic process promoted by MIF is its effect on leukocyte adhesion in the atherosclerotic vasculature, an activity involving engagement of CXCR4.sup.14. To determine whether this function of MIF can be attenuated by CXCR4 mimics. MIF-triggered adhesion of MonoMac-6 monocytes on human aortic endothelial (HAoEC) monolayers under static conditions was assessed in the presence or absence of msR4M-L1.
[0192] To determine the functional consequence of this finding, leukocyte recruitment was studied in ex-vivo-mounted atherogenic carotid arteries using MPM. This involved injection of mice with msR4M-L1 three days before vessel preparation and visualization of in-situ adhering msR4M-L1- versus vehicle-exposed fluorescently labeled bone marrow-derived leukocytes in the vasculature under physiological flow conditions (
[0193] Together, these findings suggested that msR4M-L1 localizes to atherosclerotic plaque tissue in a MIF-specific manner and inhibits MIF-mediated atherogenic leukocyte recruitment by interfering with chemotactic migration and arterial adhesion.
EXAMPLE 5—THE ENGINEERED CXCR4 MIMIC REDUCES ATHEROSCLEROSIS AND INFLAMMATION IN VIVO AND MARKS STABLE HUMAN CAROTID ATHEROSCLEROTIC PLAQUE TISSUE
[0194] Peptides are sensitive to proteolysis by plasma proteases and clearance. Thus, before testing the potential therapeutic utility of msR4M-L1 in vivo, its proteolytic stability was examined. Biotin-msR4M-L1 was incubated with human plasma isolated from the blood of healthy donors for various time intervals. SDS-PAGE/Western blot analysis revealed that appreciable amounts of intact, undigested biotin-msR4M-L1 could be recovered up to 16 h of plasma exposure, indicating that this peptide was reasonably stable in plasma (
[0195] To examine the therapeutic capacity of the CXCR4 mimic, an established in-vivo mouse model of early atherosclerosis was employed, in which lesions develop in aortic root and arch over a 4-5-week time course of HFD.sup.39. Apoe.sup.−/− mice received msR4M-L1 (50 μg per mouse i.p., three times per week) or vehicle treatment in parallel to HFD for 4.5 weeks (
TABLE-US-00004 TABLE 4 Therapeutic treatment of atherogenic Apoe.sup.−/− mice with msR4M-L1 does not affect blood leukocytes and lipid levels. Blood cell count, body weight and serum lipid levels from mice Apoe.sup.−/− mice on cholesterol-rich high-fat diet (HFD) for 4.5 weeks and treated with msR4M-L1 or vehicle control. Vehicle msR4M-L1 P value Serum lipid levels Cholesterol (mg/dL) 693.4 ± 25.2 647.4 ± 19.2 0.1686 Triglycerides (mg/dL) 161.3 ± 5.7 156.7 ± 3.8 0.5203 Blood cell counts Leukocytes (per μL) 4874 ± 673 4632 ± 286 0.7490 Monocytes (per μL) 702 ± 128 552 ± 51 0.3096 Lymphocytes (per μL) 3070 ± 560 2347 ± 50 0.2343 Neutrophils (per μL) 2115 ± 272 1897 ± 46 0.4538 Body weight Weight (g) 22.8 ± 0.4 22.3 ± 0.2 0.2848 Table legend. Shown are means ± SD. P-values calculated by Student’s t-test. N = 5 (blood cell counts), n = 7 (body weight), and n = 11 (lipids) per group.
[0196] Importantly, atherosclerotic lesion size in aortic arch (
[0197] To further test the translational relevance of these findings, stable and unstable human carotid atherosclerotic plaque sections obtained from patients undergoing carotid endarterectomy (CEA) (Table 5) were probed with Fluos-msR4M-L1.
TABLE-US-00005 TABLE 5 Characteristics of the atherosclerotic patients undergoing carotid endarterectomy (CEA). Staining with Fluos-msR4M-L1 Staining with anti-MIF Stable Unstable Stable Unstable ( n = 9) (n = 15) P value.sup.2 (n = 11) (n = 15) P value.sup.2 Age (y).sup.1 66.1 ± 2.0 71.2 ± 12.6 0.178 66.6 ± 1.8 68.9 ± 2.7 0.524 Sex (male, %) 33.3 60.0 0.223 54.6 40.0 0.482 Neurological 22.2 33.3 0.582 9.1 26.7 0.280 symptoms (%) Hypertension (%) 77.8 80.0 0.902 81.8 93.3 0.384 Diabetes mellitus (%) 44.4 20.0 0.219 45.5 20.0 0.178 Hyperlipidemia (%) 66.7 66.7 0.999 63.6 60.0 0.858 Smoking (%) 66.7 40.0 0.223 45.5 46.7 0.954 CKD.sup.1 (%) 0 6.7 0.451 0 6.7 0.403 Coronary heart 0 6.7 0.451 9.1 6.7 0.827 disease (%) PAD.sup.2 (%) 0 6.7 0.451 0 13.3 0.223 Aspirin/Clopidogrel 100.0 93.3 0.451 100.0 85.7.sup.3 0.207 (%) Beta-blocker (%) 22.2 33.3 0.582 27.3 28.6.sup.3 0.946 ACE-inhibitors.sup.3 (%) 22.2 13.3 0.591 18.2 28.6 0.565 Statins (%) 88.9 86.7 0.880 90.9 85.7.sup.3 0.706 Diuretics (%) 0 6.7 0.451 18.2 14.3.sup.3 0.802 Table legend. All atherosclerotic carotid tissue samples used for analysis showed an advanced stage of atherosclerosis (types V-VII according to the American Heart Association (AHA) guidelines). Healthy controls were age-matched (57.3 ± 5.5 years). P values refer to stable versus unstable samples (unpaired t-test). Information regarding this parameter missing for one patient. Abbreviations: CKD, chronic kidney disease; ACE, angiotensin-converting enzyme; PAD, peripheral artery disease.
[0198] Stainings were compared with sections from healthy vessels and counter-staining against MIF was performed using an anti-MIF antibody. Based on histological characterization of plaque morphology, a total of 11 stable and 17 unstable carotid plaques were examined; 6 healthy vessels served as controls. Fluos-msR4M-L1 led to a pronounced staining of stable carotid plaque tissue that was higher than Fluos-msR4M-L1 positivity detected in unstable plaques and healthy vessels (
EXAMPLE 6—DISCUSSION
[0199] Anti-cytokine/-chemokine strategies represent promising therapeutic approaches for a variety of diseases, including cancer, inflammation, and cardiovascular diseases. In addition to SMDs and antibodies, soluble receptors are an important targeting approach to block pathogenic cytokine effects.sup.7, 41. While soluble cytokine receptors have been developed for single-membrane spanning receptors and are successfully used in the clinic against immune-mediated diseases, anti-chemokine strategies based on a soluble receptor principle are not established.
[0200] Herein a small engineered peptide-based, soluble chemokine receptor mimic is provided that distinguishes between two chemokines and features ligand- and receptor-selective anti-atherosclerotic capacities in vitro and in vivo. We focused on CXCR4, one of the most studied chemokine receptors.sup.42, 43. CXCR4 has critical ligand- and context-dependent roles in various diseases. Together with its ligand CXCL12, it is a promising target in tumor metastasis.sup.42 and small molecule CXCR4 inhibitors such as Plerixafor/AMD3100 are used as stem cell mobilizers for transplantation therapy of patients with specific cancers.sup.44. However, in atherosclerotic diseases, the CXCR4/CXCL12 axis has proven to be a difficult target, with both disease-promoting and protective properties. Genome-wide association studies (GWAS) and CXCL12 plasma level analysis revealed CXCL12 as a candidate gene associated with CAD.sup.45, 46, 47, and disease-exacerbating activities such as cardiac inflammatory cell recruitment have been implied for the CXCR4/CXCL12 axis.sup.48, 49. In contrast, beneficial activities include cardioprotective effects based on the contribution of CXCR4/CXCL12 to neoangiogenesis and cardiomyocyte survival.sup.43, 50, 51. Moreover, disruption of this axis promotes atherosclerotic lesion formation through deranged neutrophil homeostasis.sup.52 and loss of atheroprotection.sup.26. In this context, we have shown that atherogenesis-induced endothelial damage is counter-acted by unleashing CXCR4 activity and autocrine CXCL12 expression in endothelial cells through miR-126-containing apoptotic bodies.sup.27 and that CXCR4 on vascular cells maintains arterial integrity and limits atherosclerosis by preserving barrier function and a normal contractile vascular smooth muscle cell (VSMC) phenotype.sup.26.
[0201] Capitalizing on our earlier findings that CXCR4 engages MIF as a non-cognate ligand to drive atherogenic leukocyte recruitment.sup.14, 16, 17 and that CXCR4-supported endothelial barrier integrity is mediated by CXCL12 but not MIF.sup.26, we surmised that MIF-specific CXCR4 targeting might be a promising avenue to circumvent the complexity of the CXCR4/CXCL12 system in cardiovascular conditions. In fact, we previously demonstrated that MIF-blocking strategies are superior to CXCL12 blockade in inducing plaque regression.sup.6, 14 and that the foam cell-promoting activity of CXCR4 is primarily elicited by MIF and not CXCL12.sup.36. However, currently available MIF blocking strategies may not be optimal, as anti-MIF (Imalumab) or anti-CD74 (Milatuzumab) antibodies would potentially interfere with the cardioprotective MIF/CD74 axis.sup.15, 16. The same holds true for MIF-directed SMDs, which are designed to bind in MIF's conserved tautomerase pocket and interfere with MIF binding to CD74. However, modification of this cavity invokes conformational changes in MIF that impair binding to CD74.sup.53. AMD3100 partially interferes with MIF/CXCR4 binding.sup.14, 25, but this CXCR4 inhibitor has been found to impair the cardio- and atheroprotective activity spectrum of the CXCR4/CXCL12 axis.sup.26, 27, 52.
[0202] The disclosed engineering design was guided by the CXCR4 structures.sup.29, 30, 31 and SAR studies.sup.24, 25, highlighting CXCR4 ectodomain regions that may be harnessed to engineer a soluble receptor mimic to selectively target MIF and spare CXCL12. Approaches to utilize the ectodomains of single membrane-spanning type I cytokine receptors such as the TNF or IL-6 receptor have been successfully developed as immunomodulatory drugs.sup.7, 41. However, mimicking the ectodomain of seven-helix membrane-spanning GPCRs is inherently complex due to the discontinuous nature of the receptor backbone topology. Ligand binding in (poly)peptide-ligating GPCRs such as chemokine receptors typically involves several extracellular portions of the receptor, often a combination of residues of several ECLs and the N-terminal.sup.30. Only a handful of reports are available: the N-terminal and ECL3 elements of CXCR1 and CXCR2 were assembled on a soluble GPCR B1 domain scaffold protein.sup.54; based on the crystal structure of rhodopsin, all three predicted ECLs of CXCR4 were connected to form an HIV gp120-binding mimic.sup.55; and a construct mimicking corticotropin-releasing factor receptor-1 (CRF-R1) combined native chemical ligation and recombinant technology and encompassed the entire 23 kDa ectodomain of CRF-R1.sup.56. Such studies have remained explorative, led to constructs with micromolar binding affinities, and neither chemokine selectivity, nor in-vivo or disease relevance were addressed.
[0203] The engineered MIF-selective CXCR4 mimics reported here are highly reduced GPCR mimics of only 29 residues plus two non-natural amino acids of the linker moiety (molecular weight <4 kDa), reducing the size of CXCR4 by >90%. MIF selectivity over CXCL12 was achieved by combining only selected residues within ECL1 and ECL2. As determined by independent biophysical methods, the lead candidate mimics bind MIF with low nanomolar affinity (K.sub.D˜30 nM), in line with the reported K.sub.D value of 19 nM for MIF binding to full-length membrane CXCR4.sup.14, while binding to CXCL12 is essentially absent. This affinity is reasonable compared with that of Imalumab or the pre-clinical anti-MIF antibody NIH/III.D9 (K.sub.D˜1-3 nM).sup.57 and MIF-directed SMDs (micromolar K.sub.D).sup.16. Of note, despite its high affinity, msR4M-L1 neither affected MIF binding to CD74, nor did it impair MIF/CD74-mediated stimulation of AMPK phosphorylation in human cardiomyocytes as a correlate of MIF's cardioprotective activity.sup.15. Hence, msR4M-L1 has more favorable selectivity characteristics than the available anti-MIF therapeutic strategies. A molecular explanation for this selectivity comes from experiments mapping the MIF binding site, msR4M-L1 targets MIF region 54-80, a part of the N-like loop known to mediate MIF/CXCR4 binding, but not involved in MIF/CD74 binding, in line with data showing that the tautomerase site of MIF and residues 80-87 determine the MIF/CD74 binding interface.sup.21, 22, 55. Importantly, binding selectivity of msR4M-L1 for MIF versus CXCL12 was functionally paralleled in a number of inflammation- and atherosclerosis-relevant cell systems, i.e. GPCR/CXCR4 signaling, 2D lymphocyte chemotaxis, foam cell formation, monocyte adhesion, and 3D monocyte migration, representing MIF/CXCR4-mediated cell systems with disease relevance.sup.36.
[0204] Intriguing structural information also comes from mimics, in which we introduced a disulfide bridge between residues Cys-109 of ECL1 and Cys-186 of ECL2. In contrast to msR4M-L1 and -12 that are fully selective for MIF, introduction of the disulfide bridge led to a gain-of-CXCL12-binding activity, irrespective of the presence (msR4M-L1ox, msR4M-L2ox) or absence (msR4M-LS) of the spacer-mediated conformational constraint. This is in line with the identification of a Cys-109-Cys-188 disulfide in the X-ray structure of CXCR4.sup.29, 30 and structural insights on the CXCR4/CXCL12 interface.sup.58, and supports the notion that the natural CXCR4 receptor is ‘equipped’ to interact with both CXCL12 and MIF.sup.14. On the other hand, the K.sub.D for MIF binding dropped >10-fold, when the respective ECL1 and 2 sequences were not covalently linked. Together, these data indicate that the MIF binding-determining sequence elements within the CXCR4 mimics need to be covalently linked, but that conformational restriction needs to allow for a certain flexibility to guarantee selectivity between different CXCR4 chemokines. Comparison of the various synthesized mimics further instructs for future optimization towards higher potency, stability, or selectivity.sup.28.
[0205] The biochemical and cell-based experiments encouraged us to examine whether the mimics would be efficacious in a pathogenic ex-vivo organ or in-vivo setting. Using fluorescently labeled msR4M-L1 to stain atherosclerotic tissue sections from atherogenic mice and in-vivo-administration of this peptide verified that msR4M-L1 localizes to and marks atherosclerotic plaque tissue in a MIF-specific manner. Indeed. MIF has been shown to be upregulated in atherosclerotic lesions, where secreted MIF is deposited similar to classical arrest chemokines and localizes to plaque macrophages, foam cells, and VSMCs.sup.14, 59, 60. While these experiments do not fully exclude the possibility that msR4M-L1 also—partially—localizes to CXCL12+ regions, our biochemical data proving binding selectivity, suggest that this is unlikely. Furthermore, while the MIF homolog MIF-2/D-DT.sup.13 has not been studied in atherosclerosis, it may be of future interest to design mimics directed at MIF-2 for applications in MIF-2-dominated inflammatory conditions.
[0206] An MPM-based ex-vivo atherosclerotic carotid artery system was used to monitor luminal leukocyte adhesion under pathophysiologically relevant conditions and demonstrated that treatment with msR4M-L1 markedly attenuated adhering leukocyte numbers. Such systems have been powerful in demonstrating the leukocyte recruitment potential of MIF or classical arrest chemokines such CXCL1/KC.sup.14, 39, 52, 61, 62. In conjunction with the Fluos-msR4M-L1 plaque staining data, the MPM data indicate that msR4M-L1 blocks MIF-mediated atherogenic leukocyte recruitment. Important proof for a translational utility of the GPCR mimics reported here comes from testing msR4M-L1 therapeutically in a mouse model of atherosclerosis in vivo.sup.39. The chosen treatment regimen of three 50 μg-injections per week maintained circulating doses of the mimic in line with the determined K.sub.D/IC.sub.50 values. The mimic potently blocked atherosclerosis at key predilection sites, reduced lesional macrophage accumulation and circulating inflammatory cytokines/chemokines, while no effects on lipids or leukocyte counts were observed, suggesting that it specifically targeted a MIF-mediated pathogenic inflammatory effect in atherogenic lesions. The experiment constitutes a ‘proof-of-concept’ for such compounds in an in-vivo disease setting and is a good predictor for their efficacy in advanced atherosclerosis models, but also other models involving MIF-related chronic inflammation.sup.12, 14, 16, 17, 52, 63. In fact, a pilot study indicates a beneficial role of msR4M-L1 in a 9-week regression type of atherosclerosis model, although the data currently only suggests a trend and did not reach statistical significance (
[0207] The CANTOS trial has provided clinical proof that an immunotherapy-based targeting approach against IL-1α, a key inflammatory mediator, improves cardiovascular outcome in an at-risk population.sup.4, 65. However, treatment with Canakinumab did not improve mortality in atherosclerotic patients and caused an increase in infections, highlighting the need to identify additional drug targets and to develop anti-inflammatory strategies with a high selectivity profile that block atherosclerotic pathways. Engineering of CXCR4 mimics towards MIF specificity could be one such approach and represent a novel class of anti-atherogenic molecules based on the soluble GPCR ectodomain concept. MsR4Ms are peptide-based molecules and, while there are over 60 peptide drugs approved worldwide, there are pros and cons compared to antibodies and SMDs. Advantages are a good surface coverage and hence high selectivity and potency, favorable safety, and low-cost production; disadvantages are the limited proteolytic stability and bioavailability.sup.66. However, these issues can be overcome by peptide chemistry tools and peptide design strategies.sup.28, 66. Thus, msR4M-L1 should be viewed as a proof-of-concept inhibitor of MIF/CXCR4-specific atherogenesis, whose properties may be improved by designed second-generation mimics. Accordingly, studies in patients with atherosclerotic disease could be a future perspective. In fact, staining of human carotid artery samples from patients who underwent CEA with Fluos-msR4M-L1 revealed interesting clinical correlations with stable versus unstable plaque phenotypes that mirrored the MIF expression profile in these lesions. In accord, CXCL12 expression was previously found to be more prominently expressed in unstable plaque tissue.sup.40.
[0208] In conclusion, the designed MIF-selective soluble CXCR4 mimics are a novel class of anti-atherosclerotic/-inflammatory agents that could complement currently available inhibition strategies by antibodies or SMDs. It is demonstrated that these molecules can be engineered to be chemokine-selective, to exhibit high binding affinities, and to be potent in blocking atherogenic chemokine activities in vitro and in vivo, while sparing potentially contraindicative protective pathways through alternative receptors or ligands.
EXAMPLE 7—NEXT GENERATION MIMETICS
[0209] There are over 60 peptide drugs approved worldwide, but there are pros and cons compared to antibodies and SMDs. Advantages of peptide drugs are a good surface coverage and hence high selectivity and potency, favorable safety, and low-cost production; disadvantages are the limited proteolytic stability and bioavailability.sup.66. The disadvantages can be overcome by peptide chemistry tools and peptide design strategies.sup.28, 66. To this end, one typical approach pursued in the field is to shorten the bioactive peptide sequence, to identify required and dispensable residues, and to translate this information into the design of shorter, more stable peptide analogs that retain activity, and to design peptidomimetics. The second-generation mimics described herein are representative of such an approach and represent shorter peptides themselves with retained full activity.
[0210] On the basis of “msR4M-L1”
TABLE-US-00006 97 110 182 196 NH2-DAVANWYFGNFLCK-6-Ahx-12-Ado-DRYICDRFYPNDLWV- CONH2
[0211] residues and sequence positions in msR4M-L1 that are dispensable and are available for substitutions to introduce D-amino acids, non-natural amino acids, N-methylated amino acids, and amino acids that may be used for covalent cyclization were identified. Identification relied on various analyses, e.g. a sequence comparison between the presumed binding sites of CXCR4 for MIF versus CXCL12, on data from peptide arrays and from an alanine-scanning approach, and on data from a shortening approach of the msR4M-L1 sequence (“fragment approach”).
[0212] This led to the identification of the following dispensable residues: D182, R183, I185, C186, R188, V196.
[0213] These are available for the above described substitutions towards more stable and more active second-generation mimics and also represent candidate residues for a shortening approach.
[0214] Depending on their role in peptide conformation, the substitution of individual residues in a peptide sequence also can lead to an “increase” in inhibitory activity of the peptide. Such residues have an intrinsic inhibitory activity and dampen the effect. The substitution of such “inhibitory” residues can lead to peptide variants with higher, “improved” binding activity. In the present invention, potential “Inhibitory” residues were identified by substitution with Ala. Accordingly, Ala substitutions in the following positions led to an increase in binding affinity to MIF compared to the parent sequence in msR4M-L1 or a respective shorter sequence: D97, A98, V99, (A100), L108, C109, K110, P191, D193 This analysis also led to the identification of shorter fragments of msR4M-L1 with partial MIF binding and partial inhibitory activity that can serve as a basis or scaffold for short next generation mimics.
[0215] Their binding activity as determined by fluorescence spectroscopic binding assay is summarized in Table 5.
[0216] Table 6: MIF binding activity of shortened ECL1 and ECL2 fragments of msR4M-L1 [0217] fragment 100-110: Kd=215+/−72 nM [0218] fragment 101-110: Kd=51 nM [0219] fragment 102-110: Kd=80+/−8 nM [0220] fragment 187-195: Kd=286+/−35 nM [0221] fragment 185-195: Kd=324+/−30 nM [0222] (comp. msR4M-L1: Kd=30-35 nM)
[0223] Their MIF binding potential is further confirmed in the Dil-LDL uptake-based assay, a surrogate assay representing atherogenic foam cell formation. The fragments of Table 5 exhibit a ca. 80% inhibitory capacity compared to full-length msR4M-L1 in the Dil-LDL foam cell assay.
[0224] Next, the shortened ECL fragments with partial MIF-binding/inhibitory activity were reconnected in an attempt to generate shortened msRM4 variants comprising the minimally required residues from both the ECL1 and ECL2 loop sequences. Table 6 summarizes the reconnected short “active” fragments:
TABLE-US-00007 TABLE 7 Reconnected short mimics msR4M-L3, -L4, and -L5: Atherogenic Binding affinity inhibitory Sequence & linker (fluorescence activity (ECL1 - spectroscopic (Dil-LDL Name linker - ECL2) titration) uptake) msR4M-L3 102-110-6-Ahx-12- Kd = 14 +/− 4 nM 80% of Ado-187-195 full-length msR4M-L1 msR4M-L4 102-110-8-Aoc-187- Kd = 12 +/− 5 nM 80% of 195 full-length msR4M-L1 msR4M-L5 102-110-O1-Pen-O1- Kd = 14 +/− 6 nM 30% of Pen-187-195 full-length msR4M-L1
[0225] As a next step, reconnected short mimics of -L1 with improved solubility properties were created by introducing solubility-enhancing linkers and tags, which introduce positive or negative charges. This was achieved by introducing three lysine residues (K3), three arginine residues (R3), three aspartic acid residues (D3), or three glycine residues (G3) as N-terminal tag, C-terminal tag, or as linker between the shortened ECL1 and 2 sequences. As Table 7 shows, the introduction of these solubility-enhancing residues in msR4M-G3, -D3, -R3, and -K3 retained the high binding affinity to MIF as determined by fluorescence spectroscopic binding assay and by CD spectroscopy:
TABLE-US-00008 TABLE 8 Reconnected short mimics with enhanced solubility: Sequence & Binding affinity Estimated linker (fluorescence solubility (ECL1 - spectroscopic index by Name linker - ECL2) titration) CD spectroscopy msR4M-G3 102-110-G-G-G- Kd = 35 +/− 6 nM >2× compared to 187-195 msR4M-L1 msR4M-D3 102-110-D-D-D- Kd = 33 +/− 21 nM >2× compared to 187-195 msR4M-L1 msR4M-R3 102-110-R-R-R- 22 +/− 2 nM >2× compared to 187-195 msR4M-L1 msR4M-L5 102-110-K-K-K- ca. 30-40 nM >2× compared to 187-195 msR4M-L1
[0226] Furthermore, second-generation mimics will feature advantageous properties such as improved proteolytic stability by introducing conformational constraints via lactam-bridge- or disulfide-mediated cyclization, while accounting for the required conformational flexibility as determined from the comparison of the structure-activity relationships between msR4M-L1 and -L2 with -L1ox, -LS, and L2ox (see Table 1).
EXAMPLE 8—THERAPEUTIC APPLICABILITY OF MSR4M-L1 IN A REGRESSION MODEL
[0227] To further test the therapeutic applicability of msR4M-L1, an in vivo test in a “regression setting” was applied to mimic the patient situation, who is typically seen by a physician only when symptoms occur. The real-life situation is thus one in which preformed plaques already exist, when a patient starts treatment. A regression model therefore better mimics the situation in patient with pre-existing atherosclerotic disease.
[0228] In a pilot study, atherogenic ApoE−/− mice were put on Western diet for 4.5 wks. Next, treatment with msR4M-L1 was performed (50 μg/mouse, 3× per week; 4-5 mice per group) in parallel with another 4.5 wks of Western diet. msR4M-L1-treated mice show a decreased plaque load (trend).
[0229] The study was extended to 14 mice per group and the data indicate that msR4M-L1 leads to a regression of atherosclerotic plaques as measured by ORO staining in aortic root, HE staining in aortic root, and HE staining in aortic arch. Intralesional inflammation as measured by CD68+ macrophage area was also reduced in the msR4M-L1 group. The effect was not as pronounced as in the early atherogenesis co-treatment model, but significant (
EXAMPLE 9—INHIBITORY CAPACITY OF MSR4M-L2 IN LEUKOCYTE CHEMOTAXIS ASSAY
[0230] msR4M-L2 (Table 2) has a similar binding affinity to MIF as msR4M-L1 (see Table 1), although it contains a spacer with different hydrophobicity. Analysis of msR4M-L2 in the MIF-elicited leukocyte chemotaxis assay showed that it has a similar inhibitory capacity as msR4M-L1 in controlling leukocyte recruitment (
EXAMPLE 10—APPLICATION TO OTHER GPCRS
[0231] Chemokine receptor and many other GPCRs display an overall similar structural architecture with a discontinuous extracellular domain (ECD) consisting of an N-domain and three extracellular loops (
[0232] Accordingly, the msR4M principle can be applied to other CXC or CC chemokine receptors, or other GPCRs. Moreover, the principle can be applied to hybrid receptors combining ECD regions from different receptors to tailor, enhance, or restrict ligand binding and inhibitory specificities. For example, a msRxM hybrid between CXCR4 and CXCR2 can inhibit atherogenic functions of MIF that are mediated by both CXCR4 and CXCR2.
EXAMPLE 11—NEXT GENERATION MIMETICS II
[0233] msR4Ms are ectodomain mimics of CXCR4, with their size varying from 3.9 to 4.3 kDa. Full-length msR4Ms such msR4M-L1 and -L2 consist of a 14-meric ECL1 and a 15-meric ECL2 covalently bonded by a non-natural linker; in msR4M-L1ox and -L2ox an additional disulfide bridge is introduced. Size optimization studies of the individual ECL1 and ECL2 loops by alanine scanning suggested that the 9-mers ECL1(102-110) and ECL2(187-195) are the shortest individual binders of MIF with a reasonable binding affinity to MIF. These fragments were then linked to shorter “next generation mimics” (NGMs or ngms) as summarized in
[0234] The linkers were chosen as follows starting our considerations from msR4M-L1:
[0235] 6-Ahx and 12-Ado formed the linker in msR4M-L1. Even though the determined length of the 6-Ahx-12-Ado linker is longer than the measured distance of ECL1(102-110) and ECL2(187-195) (according to the crystal structure data), a 6-Ahx-12-Ado linker was chosen to generate the next generation mimic msR4M-L3 or shortly L3. In next trying to imitate it the length of 0.95 nm between K110 and D187, the mono-unit spacer 8-Aoc and the tandem spacer O1Pen-O1Pen were introduced resulting in NGMs L4 and LS, respectively. To generate the NGMs LD3, LK3, and LR3 with predicted improved solubility properties three aspartic acid, three lysine or three arginine residues, respectively, were introduced as linker. LG3 with three glycine residues form synthesized for further comparison.
[0236] Table 9 summarizes the names, sequences, mass spec analysis, and apparent affinities (app. Kds) of interaction between NGMs and MIF, as determined by fluorescence spectroscopic titrations
TABLE-US-00009 Fluos- Alexa-488- ngm/MIF MIF/ngm Peptide [M + H].sup.+ [M + H].sup.+ app. K.sub.d app. K.sub.d Peptide sequence.sup.[a] abbreviation expected.sup.[b] found.sup.[b] (±SD) (nM) .sup.[c] (±SD) (nM) .sup.[c] [ECL1(102-110)] -6 Ahx- ngm-L3 2693.36 2693.54 44.4 (±16.4) 11.7 (±7.3) 12 Ado- [ECL2(187-195)] [ECL1(102-110)] - 8 Aoc- ngm-L4 2524.24 2524.38 11.9 (±4.8) 43.2 (±20.2) [ECL2(187-195)] [ECL1(102-110)]- O1pen- ngm-L5 2585.23 2586.08 14.3 (±5.7) 41.8 (±16.4) O1pen - [ECL2(187-195)] [ECL1(102-110)] - D-D-D - ngm-LD3 2728.22 2728.16 36.0 (±22.2) 246.5 (±21.7) [ECL2(187-195)] [ECL1(102-110)] - G-G-G - ngm-LG3 2554.20 2555.23 35.0 (±19.6) >5000 [ECL2(187-195)] [ECL1(102-110)] - K-K-K - ngm-LK3 2767.42 2767.26 36.4 (±7.5) 44.8 (±10.3) [ECL2(187-195)] [ECL1(102-110)] - R-R-R - ngm-LR3 2851.44 2851.82 16.8 (±6.2) 110.1 (±28.1) [ECL2(187-195)] Peptides were dissolved and analyzed by MALDI-TOF-MS; .sup.[a]Peptides were synthesized with free amino-N-terminal and amidated C-terminal; .sup.[b]monoisotopic molar mass with an additional hydrogen [M + H].sup.+; .sup.[c] App. K.sub.ds, are means (±SD) from three independent titration experiments which were performed in aqueous 1 × b, pH 7.4, containing 1% HFIP.
[0237]
[0238] The binding affinity of the NGMs for MIF and that for CXCL12 were tested for comparison, to determine their affinity and selectivity for MIF. msR4M-L5 and msR4M-LD3 have high affinities for MIF but essentially no binding propensity for CXCL12 (see binding curves in
[0239] msR4M-L3 and -L4 also showed high affinity for MIF (Table 9) and had essentially no binding affinity for CXCL12, but had less favorable solubility properties. msR4M-LK3 and -LR3 have very good solubility properties and also bound to MIF with high affinity (Table 9), but were also found to have good binding affinity for CXCL12.
[0240] Overall, these binding and biophysical data therefore suggested that msR4M-L5 (containing a non-natural linker moiety) and msR4M-LD3 (containing a natural triple-Asp spacer conveying very good solubility properties to the mimic) were the most favorable mimics in terms of their binding affinity for MIF, their selectivity for MIF over CXCL12, and good biophysical properties. These were therefore tested for MIF-blocking activity in prototypical atherogenic assays.
[0241] The inhibitory potential of the mimics on the atherogenic activity of MIF was tested in an oxLDL-based foam cell assay, in which the MIF-triggered uptake of Dil-labelled oxLDL is measured by microscopic quantification. The inhibitory effect of msR4M-L5 and msR4M-LD3 were tested in comparison with msR4M-L1 (
[0242] Using a lower threshold for the effect size of the MIF/CXCR4 response in this assay of roughly 50% as related to the effect of AMD3100 (IC50 of 48.9 nM), IC50 values or 69.7 nM and 1.4 nM were determined for msR4M-L5 and msR4M-LD3, respectively, which compares well with that estimated for msR4M-L1 (although the dose curve was not fully titrated) of ca. 100 nM.
[0243] In a second type of atherogenic assay, the inhibitory capacity of msR4M-L5 and msR4M-LD3 was tested on MIF-triggered monocyte migration in a 3D migration setting (
EXAMPLE 12—METHODS
[0244] Cytokines/chemokines and reagents. Biologically active recombinant MIF was prepared as reported previously and exhibited a purity of ˜98%.sup.14, 35. For some of the biophysical methods, a 90-95% purified preparation was used. Fluorescently-labeled MIF.sup.18 and was generated using the Microscale Protein Labeling Kit from Invitrogen-Molecular Probes (Karlsruhe, Germany: Alexa-488-MIF) or Monolith Kit RED-NHS from NanoTemper (Munich, Germany: MST-Red-MIF). LPS content was tested by limulus amoebocyte assay (LAL, Lonza, Cologne, Germany) and verified to be <5 pg/μg. Cell culture-grade tumor necrosis factor (TNF)-α was purchased from Life Technologies (Carlsbad, United States). Recombinant CXCL12, prepared as described.sup.62, was a gift of Dr. von Hundelshausen (LMU Munich) or was purchased from Peprotech (Hamburg, Germany). Other reagents were obtained from Sigma, Merck, Roth, or Calbiochem, and were of the highest purity degree available.
[0245] Design, peptide synthesis, purification, and linker chemistry. Based on the crystal structures of human CXCR4 (codes 3ODU, 3OEU0, 3OE6, 3OE8, 3OE9, 4RWS) and previous SAR studies.sup.24, 25, CXCR4 ectodomain peptides were selected. The crystal structures were imported into PyMOL Molecular Graphics System (Version 1.8.2.2 Schrödinger, LLC) and Jmol (http://www.jmol.org) for determining the C-to-N distance between residues 97-110 and 182-196.sup.29, 30. Conjugates of 12-Ado with either 6-Ahx or O2Oc were visualized in three-dimensional space using Molview and Jmol as. The estimated distances between the N- and C-terminal in both conjugates were similar to the ECL1-ECL2 distance. All CXCR4-derived peptides were synthesized as C-terminal amides on Rink amide MBHA resin by SPPS using Fmoc chemistry as described.sup.28. Couplings of Fmoc-6-Ahx-OH, Fmoc-12-Ado-OH and Fmoc-O2Oc-OH (Iris Biotech GmbH, Marktredwitz, Germany) were carried out with 3-fold molar excess of 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and 4.5-fold molar excess of N,N-diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF). Fmoc-deprotection was carried out with 0.1 M hydroxybenzotiazole (HOBt) in 20% v/v piperidine in dimethylformamide (DMF) for 3 and 9 min to avoid aspartimide formation.sup.32, 5(6)-carboxy-fluorescein (Fluos)- and biotin-labeled ectodomain peptides were synthesized as described.sup.32, 5(6)-carboxytetramethylrhodamine (TAMRA, Novabiochem/Merck KGaA, Darmstadt, Germany) was coupled N-terminally to side chain-protected msR4M-L1 on solid phase, after Fmoc-deprotection. Disulfide bridges in msR4M-L1ox and msR4M-L2ox were formed in 1 mg/mL peptide solution in aqueous 3 M guanidinium hydrochloride (GdnHCl) in 0.1 M ammonium carbonate (NH.sub.4HCO.sub.3) solution, containing 40% dimethylsulfoxide (DMSO). msR4M-LS was produced similarly, using 0.3 mg/mL ECL1 and 0.5 mg/mL ECL2 and 20% DMSO. Reverse-phase high-performance liquid chromatography (RP-HPLC) was applied for the purification of crude and oxidized peptides by using Reprosil Gold 200 C18 (250×8 mm) or Reprospher 100 C18-DE (250×8 mm) columns with pre-column (30×8 mm) (Dr. Maisch-GmbH, Herrenberg, Germany). The mobile phase consisted of 0.058% (v/v) trifluoroacetic acid (TFA) in water (buffer A) and 0.05% (v/v) trifluoroacetic acid in 90% (v/v) acetonitrile and water (buffer B) (flow rate 2.0 mL/min). All peptides were purified with an elution program of 10% B for 1 min, followed by a gradient from 10% to 90% B over 30 min, except for msR4M-LS, which was eluted with 30% B for 7 min followed by an increase to 60% B over 30 min. Expected molecular weights were verified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).sup.28. Peptides were used as TFA salts. For in-vivo experiments, the TFA anion was exchanged to chloride by four cycles of dissolution/lyophilization of pure msR4M-L1 in aqueous 5 mM HCl and one cycle of bidistilled water.sup.28. MIF sequence-based peptides (Table 3) were synthesized on Wang resin or purchased from Peptide Specialities GmbH (PSL, Heidelberg, Germany). MIF-derived peptides were N-terminally acetylated and had a free carboxylate function.
[0246] Fluorescence spectroscopy. Fluorescence spectroscopic titrations were performed as described.sup.28, 32. Fluorescence spectra were recorded using a JASCO FP-6500 fluorescence spectrophotometer. MIF or CXCL12 were reconstituted in 20 mM sodium phosphate buffer, pH 7.2: peptide stocks were freshly made in HFIP at 4° C. as described.sup.28, 32. After mixing Fluos-labeled peptides or Alexa-MIF with their unlabeled titration partner in assay buffer, measurements were performed in 10 mM sodium phosphate buffer, pH 7.4, containing 1% HFIP. Fluos-labeled peptide was applied at 5 nM and Alexa-MIF at 10 nM unless indicated otherwise. For the titration with ISO-1, Alexa-MIF had a concentration of 50 nM and ISO-1 varied from 0.1 to 500 μM in 10 mM sodium phosphate buffer, pH 7.4, containing 0.5% DMSO. The excitation wavelength was 492 nm and emission spectra were obtained between 500 and 600 nm. Apparent K.sub.D values (app. K.sub.D) were calculated assuming a 1/1 binding model.sup.32.
[0247] Circular dichroism (CD) spectroscopy. CD spectra were obtained with a JASCO J-715 spectro-polarimeter (JASCO, Tokyo, Japan) applying an established protocol.sup.67. Far-UV CD measurements were carried out between 195 and 250 nm. The response time was set at 1 s, intervals at 0.1 nm, and bandwidth at 1 nm. All spectra were measured at RT and represent an average of three recorded spectra. Scans were recorded for the ectodomain mimic peptides at a concentration of 1-20 μM in 10 mM sodium phosphate buffer, pH 7.4, containing 1% HFIP, following dilution of freshly made peptide stock solution in HFIP (4° C.) into the buffer-containing cuvette. Singular ECL1 and ECL2 peptides were measured at 5 μM. The background spectrum of buffer/1% HFIP alone was subtracted from the spectra of the peptides. Dynode voltage was below 1000 and did not interfere with the measurements.
[0248] Dot blot. Different amounts (0-400 ng) of human MIF, mouse MIF, or human CXCL12 were spotted on a nitrocellulose membrane and membranes allowed to dry for 30 min. Non-specific binding was blocked with Tris-buffered saline (TBS), pH 7.4, containing 0.1% Tween-20 (TBS-T) and 1% BSA. TAMRA-msR4M-L1 was reconstituted at a concentration of 10 μM in PBS containing 2.5% HFIP, diluted to a 3 μM working solution in 1% BSA/TBS-T, and incubated with the membrane at 4° C. Fluorescence intensities were measured at 600 nm using an Odyssey® Fc imager (LICOR Biosciences, Bad Homburg, Germany). The total intensity of each spot was automatically corrected by the individual background signal. The signal intensity of 400 ng human MIF was set to 100%.
[0249] Microscale thermophoresis. MST measurements were recorded on a Monolith NT.115 instrument with green/red filters (NanoTemper Technologies, Munich, Germany). MST power was set at 80% and LED power was at 95%: all measurements were performed at 37° C. MST traces were tracked for 40 s (laser-off: 5 s, laser-on: 30 s; laser-off: 5 s). A stock solution of 200 nM TAMRA-msR4M-L1 was prepared in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2% Tween-20. For titration of MIF, sub-stock solutions were prepared by serial 1:1 dilutions from a 20 μM stock solution in 20 mM sodium phosphate buffer, pH 7.2. TAMRA-msR4M-L1 and each MIF sub-stock were mixed at a 1:1 ratio, incubated for 10 min and loaded in the capillaries. Experimental measurement values by the temperature jump (T-Jump) setting.
[0250] The setup was similar for the titrations between MST-Red-MIF and soluble human CD74 (sCD74). Soluble CD74 has been described.sup.19 and is a fusion protein of an N-terminal HA-tag and CD74 residues 73-232 (R&D Systems, Minnesota, USA). The stock solution of sCD74 (4 μM) was prepared in PBS (1×, pH 7.2) and MST-Red-MIF dissolved at a concentration of 100 nM in PBS containing 0.01% BSA. Sub-stocks of sCD74 for titration were prepared by serial 1:1 dilution in 1×PBS, pH 7.2, containing 0.005% BSA. To test if MIF/sCD74 binding is affected by msR4M-L1, MST-Red-MIF (100 nM) was pre-mixed with msR4M-L1 (4 μM) and sCD74 titrations performed as above. App. K.sub.D values were calculated assuming a 1/1 binding model.
[0251] CXCR4-specific signaling in a yeast-based cell system. The yeast CXCR4-specific cell signaling system employing S. cerevisiae strain (CY12946), expressing functional CXCR4 that replaces the yeast STE2 receptor and is linked to a β-galactosidase (lacZ) signaling read-out, has been described.sup.24, 25. CXCL12 and MIF elicit a CXCR4-specific signaling response in this cell system.sup.25, 26. Briefly, yeast transformants stably expressing human CXCR4 were grown overnight at 30° C. in yeast nitrogen base selective medium (Formedium, UK). Cells were diluted to an OD.sub.600 of 0.2 and grown to an OD.sub.600 of 0.3-0.6. Transformants were incubated with 20 μM human MIF or 2 μM human CXCL12 in the presence or absence of different concentrations of msR4M-L1 for 1.5 h. OD.sub.600 was measured and activation of CXCR4 signaling quantified by β-galactosidase activity using a commercial BetaGlo Kit (Promega, Mannheim, Germany).
[0252] Cell culture and cell lines. Human aortic endothekal cells (HAoECs) were from PromoCel (Heidelberg, Germany). Cells were plated on collagen (Biochrom AG, Berlin, Germany) in endothelial cell growth medium (ECGM, PromoCell) and cultured as described.sup.68. The monocytic cell line MonoMac-6 was cultured in RPMI 1640 medium with 10% fetal calf serum (FCS) as established.sup.14. Primary human cardiac myocytes (HCM) isolated from the ventricles of the adult heart were from PromoCell and used at passage 2-8. They were cultured in myocyte basal medium (PromoCell), containing 5 μg/mL insulin, 5% FCS, 2 ng/mL fibroblast growth factor (FGF), and 0.5 ng/mL epidermal growth factor (EGF). Human embryonic kidney (HEK)-293 cells were cultured in DMEM-GlutaMAX (Life Technologies-Gibco) supplemented with 10% FCS and 1% penicillin/streptomycin. FCS was obtained from Invitrogen-Thermo Fisher Scientific. Miscellaneous cell culture reagents (media, supplements) were bought from Invitrogen and PAA (Pasching, Austria).
[0253] HEK293-CD74 surface binding assay. HEK293 cells were transiently transfected with 8 μg of the pcDNA3.1-CD74minRTS-FLAG plasmid using Polyfect (Qiagen. Hilden. Germany) and expressed surface CD74 after 24 h (efficiency 50-80%), as described.sup.69. HEK293-CD74 transfectants were washed and 3×10.sup.5 cells resuspended in ice-cold PBS containing 0.5% BSA, and incubated with 400 nM Alexa-488-labeled MIF in the presence or absence of msR4M-L1 (2 μM) on ice for 2 h. After washing in ice-cold PBS containing 0.1% BSA, the amount of Alexa-488-labeled MIF bound to the cell surface was quantified by flow cytometry using a FACS Verse instrument (BD Biosciences, Heidelberg, Germany). Binding of Alexa-488-MIF to non-transfected “wildtype” HEK293 cells, which do not express CD74, served as background control.
[0254] Mice. Mice were housed under standardized light-dark cycles in a temperature-controlled air-conditioned environment under specific pathogen-free conditions at the Center for Stroke and Dementia Research (CSD). Munich. Germany, with free access to food and water. AN mice used in this study were between 7-10 weeks or age and were on C57BL/6 background. Apoe.sup.−/− mice were initially obtained from Charles River Laboratories (Sulzfeld, Germany) and backcrossed within the CSD animal facility before use. The atherogenic Ldlr.sup.−/− and Ldlr.sup.−/− Mif.sup.−/− mice as well as Apoe.sup.−/− Mif.sup.−/− mice have been described previously.sup.14, 61. All mouse experiments were approved by the Animal Care and Use Committee of the local authorities and performed in accord with the animal protection representative at CSD.
[0255] Chemotaxis analysis of murine B cells. A Transwell-based assay was used as described previously.sup.34. Briefly, splenic B cells were isolated by negative depletion using a Pan B Cell Isolation Kit (Miltenyi Biotec. Bergisch Gladbach, Germany). Purity of the cells was between 95 and 99%. One-hundred μL of cell suspension containing 1×10.sup.6 cells in RPMI 1640/5% FCS was loaded into the upper chamber of a Transwell insert. Filters were transferred into the lower chambers containing MIF or CXCL12 in the presence or absence of ectodomain peptides. Chemotaxis was followed for 4 h at 37° C. in a humidified atmosphere of 5% CO.sub.2. Migrated cells were counted by flow cytometry using CountBright™ Absolute Counting Beads (Molecular Probes-Invitrogen).
[0256] CD74 signaling in human cardiomyocytes. Before stimulation, medium was replaced by fresh myocyte basal medium containing 0.05% FCS and HCMs rested for 16 h. Surface CD74 expression on HCMs was verified by flow cytometry (FITC-conjugated anti-human CD74, FITC-IgG2 (isotype control) (BD Pharmingen), 1 h/4° C. in the dark, BD FACSVerse™ flow cytometer, FlowJo software). AMPK signaling was elicited by addition of human MIF (16 nM, 60 min) following an established procedure.sup.15. To test for an influence of msR4M-L1, MIF was preincubated with 16 or 80 nM msR4M-L1 and mixtures added to HCMs. After treatment, cells were lysed and subjected to SDS-PAGE/Western blotting. AMPK activation was revealed with an antibody against phosphorylated AMPK (anti-pAMPKα, 1:1000, Cell Signaling Technologies, Heidelberg, Germany) and total AMPKα (anti-AMPKα, 1:1000), as well as actin detected for standardization. Anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (1:10000, GE Healthcare, Freiburg, Germany) was used for development and signals quantitated by chemiluminescence using an Odyssey® Fc imager.
[0257] Isolation of human peripheral blood-derived monocytes. Human peripheral blood-derived monocytes were isolated as described.sup.14. Briefly, blood was collected from healthy donors or buffy coat obtained from the blood bank of Munich University Hospital, mixed 1:1 with PBS, and PBMCs isolated by Ficoll-Paque Plus gradient (GE Healthcare). Monocytes were purified by negative depletion using the Monocyte Isolation Kit II (Miltenyi). Monocyte purity was verified by flow cytometry using an anti-CD14 antibody (Miltenyl) and was 95-98%. Purified cells were suspended in RPMI 1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin, 2 mM L-glutamine and 1% NEAA. The isolation of PBMCs from donor blood was approved by the local ethics committee of LMU Munich. 3D migration of human peripheral blood-derived monocytes by time-lapse microscopy. The 3D-migration behavior of human monocytes was assessed by time-lapse microscopy and individual cell tracking using the 3D chemotaxis μ-Slide system from Ibidi GmbH (Munich, Germany), adapting the established Ibidi dendritic cell protocol for human monocytes. Briefly, isolated monocytes (4×10.sup.6 cells) were seeded in rat tail collagen type-I gel in DMEM and subjected to a gradient or MIF or CXCL12 (64 nM) in the presence or absence of msR4M-L1. Cell motility was monitored performing time-lapse imaging every 1 min at 37° C. for 2 h using a Leica inverted DMi8-Life Cell Imaging System equipped with a DMC2900 Digital Microscope Camera with CMOS sensor and live cell-imaging software (Leica Microsystems, Wetzlar, Germany). Images were imported as stacks to ImageJ software and analyzed with the manual tracking and chemotaxis/migration tools (Ibidi GmbH).
[0258] Dil-LDL uptake/foam cell formation. MIF/CXCR4-dependent foam cell formation was assessed by measuring uptake of fluorescently labeled human low density lipoprotein particles (Dil-LDL) in primary human monocyte-derived macrophages following a described protocol.sup.36. Briefly, cells were incubated in culture medium (RPMI 1640-GlutaMAx medium containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.2% BSA) for 15 h at 37° C. and subsequently incubated in the same medium supplemented with 1% HPCD ((2-hydroxy)-β-cyclodextrin, Sigma-Aldrich) for 45 min. After washing with imaging solution (MEM without phenol red containing 30 mM HEPES, 0.5 g/L NaHCO.sub.3, pH 7.4, and 0.2% BSA), cells were exposed to 50 μg/mL 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocarbocyanine-labeled LDL (Dil-LDL) for 30 min at 4° C. followed by incubation at 37° C. for 20 min. Cells were washed with ice-cold imaging solution (pH 3.5), fixed, and counter-stained with Hoechst 33258.
[0259] Static monocyte adhesion. HAoECs were seeded at a density of 30.000 cells/well in 6 well μ-Ibidi Perfusion slides VI 0.4 (Ibidi GmbH). After overnight incubation, human TNF-α or MIF were added at a final concentration of 4 or 16 nM, respectively, in the presence versus absence of msR4M-L1 (320 nM), and cells incubated for 16 h. After perfusion of the chambers with fresh medium, MonoMac6 cells (1×10.sup.6 cells/mL) in PromoCell medium were added for 30 min. Non-adhering cells were flushed away by gentle perfusion using a 30 mL syringe. To quantify adherent monocytes, 10 individual images from each treatment were acquired using a Leica DMi8 inverted microscope with a 10× objective and cells quantified using Image J.
[0260] Staining of atherosclerotic plaque tissue with Fluos-msR4M-L1. Immunofluorescent staining of atherosclerotic tissue with Fluos-msR4M-L1 was performed with specimens from atherogenic Ldlr.sup.−/− and Apoe.sup.−/− mice. Ldlr.sup.−/− mice were on chow diet for 30 weeks and developed native atherosclerotic lesions as reported previously.sup.14. Mif-deficient mice (Ldlr.sup.−/− Mif.sup.−/−) were used for comparison. Aortic root sections were deparaffinized and rehydrated. For antigen retrieval, slides were boiled in sodium citrate buffer, pH 6.0, 0.05% Tween-20, and blocked with PBS, containing 5% donkey serum and 1% BSA. For staining, slides were incubated at 4° C. with Fluos-msR4M-L1 (5 μM) in blocking buffer. DAPI was used for nuclear counterstain and sections were imaged using a Leica DMI8 fluorescent microscope. The mean fluorescence intensity localized to the aortic vessel wall was quantified via Image J.
[0261] For Apoe.sup.−/− mice (and Apoe.sup.−/− Mif.sup.−/− as control.sup.61), cryo-conserved sections of advanced lesions from brachiocephalic artery (BC) were used from mice on Western-type high-rat diet (HFD, 1.25% cholesterol) for 24 weeks. Sides were fixed in ice-cold acetone, rehydrated in PBS, and blocked in PBS/1% BSA, incubated with 500 nM Fluos-msR4M-L1, and analyzed as above.
[0262] Fluos-msR4M-L1 staining and monocyte adhesion in atherosclerotic carotid arteries by multiphoton microscopy. Monocyte adhesion experiments in atherosclerotic carotid arteries under physiological flow conditions ex vivo have been established.sup.14, 70. Seven-week-old Apoe.sup.−/− mice were fed a Western-type HFD (0.2% cholesterol) for 12 weeks. The last three days before sacrifice, mice were injected with msR4M-L1 (100 μg, once daily) or sterile saline (control). On day 3, arteries were prepared and mounted into an arteriograph chamber as described.sup.70. Carotids were flushed with buffer containing msR4M-L1 (3 μM). Mouse leukocytes isolated from the bone marrow of msR4M-L1- or vehicle-treated atherogenic Apoe.sup.−/− mice were stained with fluorescent Green CMFDA or Red CMPTX (Thermo Fisher Scientific). After washing with Hank's Balanced Salt Solution (HBSS), stained leukocytes were incubated with 3 μM msR4M-L1 (red) or PBS (green, control) for 1 h at 37° C. The red- and green-stained cell pools were mixed at a 1:1 ratio and 3×10.sup.6 cells in 6 mL perfused into the artery of msR4M-L1- or vehicle-treated mice, respectively. Arteries were scanned by MPM using a multispectral TCS SP8 DIVE instrument with filter-free 4TUNE NDD detection module (Leica) and the number of adherent and transmigrated leukocytes determined by scanning multi-photon excitation. Vessel structure (and plaques) were visualized by second harmonic generation (SHG).
[0263] For plaque staining with Fluos-msR4M-L1 in carotid arteries ex vivo, Fluos-msR4M-L1 was i.p.-injected into aged atherogenic Apoe.sup.−/− mice (24-week HFD) three days before carotid preparation (50 μg per, once daily), arteries prepared and staining inspected by MPM as above.
[0264] Proteolytic stability assay. Human plasma was prepared from blood of healthy volunteers by standard procedure. Biotin-6-Ahx-msR4M-L1 was dissolved in PBS and mixed with PBS or human plasma (final concentration 70 μM) and solutions incubated for 0.5, 1, 4, or 16 h at 4° C. or 37′C. Samples were then diluted in 2× Novex Tricine SDS sample buffer (Life Technologies) at a ratio of 1:6. Samples were electrophoresed in a 10-20% Tricine gel, transferred to nitrocellulose, and biotin-6-Ahx-msR4M-L1 revealed by streptavidin-POD conjugate (Roche Diagnostics, Mannheim. Germany; 1:5000 dilution), using an Odyssey Fc imager.
[0265] Cytokine array. Cytokine/chemokine profiling was performed from plasma samples of msR4M-L1-versus vehicle-treated Apoe.sup.−/− mice using mouse cytokine array panel A (R&D Systems, ARY006) according to the manufacturer's instructions. Plasma samples were diluted (1:10) in array buffer; incubated with antibody detection cocktail for 1 h at RT, exposed to the blocked membranes (overnight, 4° C.), membranes washed and incubated with streptavidin-HRP conjugate working solution (30 min, RT). Membranes were developed with Chemi-Reagent Mix and analyzed by Odyssey® Fc imager. The average signal (pixel density) of duplicate spots was quantified by ImageJ.
[0266] In-Vivo Model of Atherosclerosis
[0267] Therapeutic injections of msR4M-L1 and aorta preparation. Seven-eight-week-old female Apoe.sup.−/− mice were randomly divided into two groups of 11-12 mice each and both groups put on a Western-type HFD (0.2% cholesterol) for 4.5 weeks. Mice develop early-to-intermediate atherosclerotic lesions in this model.sup.39. One group was i.p.-injected with 50 μg msR4M-L1 dissolved in saline every other day for 4.5 weeks; controls received saline. No toxicity or side effects were noted. At the end of the experiment, mice were sacrificed, blood collected by cardiac puncture and saved for blood cell and lipid measurements and mice transcardially perfused with saline. Hearts, proximal aortas and carotid arteries were prepared and fixed for plaque morphometry and lesion analysis.
[0268] Quantification of plaques and vessel morphometry (oil red O and H&E staining). Cut heart tissue containing aortic root were embedded in optimum cutting temperature (OCT) (Sakura Finetek, Osaka, Japan) and frozen at −80° C. Eight-μm sections were prepared for oil-red O (ORO) staining and plaque immune cell analysis. The accumulation of macrophages in aortic root lesions was determined by an anti-MAC-2 antibody followed by Cy5-conjugated secondary antibody. Nuclei were visualized with DAPI. The aortic arch was cut, fixed in 4% paraformaldehyde (PFA) and embedded in paraffin. Ten-μm sections containing the three branches (brachiocephalic, left common carotid, and left subclavian artery) were prepared and stained with hematoxylin/eosin (HE) for vessel morphometry. Images were captured with a Leica DMi8 microscope and quantified using Image J.
[0269] Blood cell counts, triglycerides and cholesterol levels. Blood was collected in EDTA tubes and leukocytes and plasma obtained by centrifugation at 630×g (10 min, 4° C.). For leukocyte counts, red blood cells (RBC) were depleted by RBC-lysis buffer (BioLegend) at RT, leukocytes washed and suspended in PBS containing 0.5% BSA. Cells were stained with an antibody cocktail comprising APC-Cy-7-conjugated anti-CD45, PE-conjugated anti-CD11b. APC-conjugated anti-CD19, FITC-conjugated anti-CD3. APC-conjugated anti-Ly6C. and PE-conjugated anti-Ly6G (BD Biosciences). Measurements were analyzed using a BD FACSVerse™ flow cytometer and data quantified using FlowJo software.
[0270] Total cholesterol and triglyceride concentrations were measured enzymatically using routine cholesterol fluorometric and triglyceride colorimetric assay kits, respectively (Cayman Chemical Company. Ann Arbor. USA).
[0271] Analysis of Human Carotid Atherosclerotic Plaques
[0272] Patient population, study groups and tissue samples. Carotid artery tissue samples (n=28) came from the Munich Vascular Biobank (MVB) and were from patients who underwent carotid endarterectomy (CEA) in the Department of Vascular and Endovascular Surgery at University Hospital of Technische Universität München. Preparation of samples for histological and IHC analysis has been reported.sup.40. Carotid specimens were fixed in formalin and embedded in paraffin (FFPE) and used to evaluate the expression of MIF by antibody or for staining with Fluos-msR4M-L1. Healthy FFPE carotid vessels were obtained from the Forensic Medicine Department (n=6). The type of atherosclerotic lesions in the CEA samples was determined according to the American Heart Association (AHA) guidelines using HE and Elastica-van-Gieson (EVG) staining procedures as described.sup.40. All carotid tissues used showed advanced atherosclerosis (stage V-VII). The study was approved by the local ethical committee of the University Hospital and followed the Guidelines of the World Medical Association Declaration of Helsinki. All patients provided informed consent. Immunohistochemistry/immunofluorescence staining. Immunofluorescence staining of human CEA tissues with Fluos-msR4M-L1 was performed using the same protocol as for paraffin-embedded specimens from Ldlr.sup.−/− mice (see above). Antibody-based detection of MIF was performed applying the DAB+ kit (Abcam, ab64238) following the standard protocol. MIF was detected with the polyclonal goat antibody N-20 (Santa Cruz, sc-16965; 1:100). HRP-conjugated polyclonal rabbit anti-goat immunoglobulin (DAKO, P0160, 1:1000) was used as secondary antibody. Slides were counterstained with Mayer hematoxylin and stainings analyzed with a Leica DMi8.
[0273] Statistical Analysis. Statistical analysis was performed using GraphPad Prism version 7 and 8 software. Data are represented as means±SD. After testing for normality, data were analyzed by two-tailed Student's t-test, Mann-Whitney U, or Kruskal-Wallis test as appropriate. Differences with p<0.05 were considered to be statistically significant.
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