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Cited by 151Advanced biosensing and bioanalysis techniquesDNA and Nucleic Acid ChemistryRNA Interference and Gene Delivery

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Chemically Modified DNA Aptamers Bind Interleukin-6 with High Affinity and Inhibit Signaling by Blocking Its Interaction with Interleukin-6 Receptor

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Gupta, Shashi, Hirota, Masao, Waugh, Sheela M., Murakami, Ikuo, Suzuki, Tomoki, Muraguchi, Masahiro, Shibamori, Masafumi, Ishikawa, Yuichi, Jarvis, Thale C., Carter, Jeffrey D., Zhang, Chi, Gawande, Bharat, Vrkljan, Michael, Janjic, Nebojsa, Schneider, Daniel J.·2014·Journal of Biological Chemistry
DOI 10.1074/jbc.m113.532580PMC PMC3961692
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Abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.

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FIGURE 1. Properties of SOMAmers discovered by the SELEX process. A, structure of Bn-dU and Nap-dU. B, IL-6 inhibition activity of six high affinity SOMAmers (three Bn-dU and three Nap-dU) in the luciferase gene reporter assay. Percent luciferase activity was plotted relative to a no-SOMAmer control sample. Each bar represents the mean ± S.E. with two replicates in three independent experiments. C, dose-dependent inhibition of IL-6 by lead SOMAmers SL1022 and SL1029 in the luciferase gene reporter assay. Percent IL-6 activity (relative to a no-SOMAmer control sample) was plotted as a function of SOMAmer concentration. Each data point represents the mean ± S.E. with four replicates for each condition. The data were fit to a four-parameter sigmoid dose-response model, and IC50 values were determined. The reported IC50 value for SL1022 is an apparent IC50 because incomplete inhibition (<100%) was observed at the highest SOMAmer concentration tested.
FIGURE 1. Properties of SOMAmers discovered by the SELEX process. A, structure of Bn-dU and Nap-dU. B, IL-6 inhibition activity of six high affinity SOMAmers (three Bn-dU and three Nap-dU) in the luciferase gene reporter assay. Percent luciferase activity was plotted relative to a no-SOMAmer control sample. Each bar represents the mean ± S.E. with two replicates in three independent experiments. C, dose-dependent inhibition of IL-6 by lead SOMAmers SL1022 and SL1029 in the luciferase gene reporter assay. Percent IL-6 activity (relative to a no-SOMAmer control sample) was plotted as a function of SOMAmer concentration. Each data point represents the mean ± S.E. with four replicates for each condition. The data were fit to a four-parameter sigmoid dose-response model, and IC50 values were determined. The reported IC50 value for SL1022 is an apparent IC50 because incomplete inhibition (<100%) was observed at the highest SOMAmer concentration tested.CC BY · PubMed Central
FIGURE 2. Conserved sequence patterns in lead SOMAmers. A, alignment of SL1022 (indicated with *) with additional sequences from the Bn-dU SELEX pool containing the consensus motif GGZZZGG (Z represents Bn-dU). The number of copies of each sequence in a pool of 14,404 is shown. B, alignment of SL1029 (indicated with *) with additional sequences from the Nap-dU SELEX pool containing the consensus motifs CGPAAGGCGGP and PPAPGPAG (P represents Nap-dU). The number of copies of each sequence in a pool of 7,758 is shown.
FIGURE 2. Conserved sequence patterns in lead SOMAmers. A, alignment of SL1022 (indicated with *) with additional sequences from the Bn-dU SELEX pool containing the consensus motif GGZZZGG (Z represents Bn-dU). The number of copies of each sequence in a pool of 14,404 is shown. B, alignment of SL1029 (indicated with *) with additional sequences from the Nap-dU SELEX pool containing the consensus motifs CGPAAGGCGGP and PPAPGPAG (P represents Nap-dU). The number of copies of each sequence in a pool of 7,758 is shown.CC BY · PubMed Central
FIGURE 3. Post-SELEX optimization of truncated IL-6 SOMAmers SL1023 and SL1030. For all panels, affinity ratios (Kdvariant/Kdparent) are reported and shaded with a color gradient from blue (affinity enhancement) to red (affinity loss), where the color intensity represents the magnitude of the affinity ratio. No affinity data were acquired at positions marked with -. A, 2′-OMe and C3-spacer substitution scans at A, C, and G positions of SL1023. The sequence of lead optimized truncate SL1025 is shown with modifications indicated (Bn, Bn-dU; Pe, Pe-dU; Nap, Nap-dU; mC, 2′-OMe C; mG, 2′-OMe G; mA, 2′-OMe A). B, 5-position modification scans at Bn-dU positions of SL1023. Structures of hydrophobic modifications Bn-dU, Pe-dU, Pp-dU, and Nap-dU are illustrated. C, 2′-OMe and C3-spacer substitution scans at A, C, and G positions of SL1030. The sequence of lead optimized truncate SL1032 is shown with modifications indicated (Nap, Nap-dU; L, C3-spacer). D, 5-position modification scans at Nap-dU positions of SL1030.
FIGURE 3. Post-SELEX optimization of truncated IL-6 SOMAmers SL1023 and SL1030. For all panels, affinity ratios (Kdvariant/Kdparent) are reported and shaded with a color gradient from blue (affinity enhancement) to red (affinity loss), where the color intensity represents the magnitude of the affinity ratio. No affinity data were acquired at positions marked with -. A, 2′-OMe and C3-spacer substitution scans at A, C, and G positions of SL1023. The sequence of lead optimized truncate SL1025 is shown with modifications indicated (Bn, Bn-dU; Pe, Pe-dU; Nap, Nap-dU; mC, 2′-OMe C; mG, 2′-OMe G; mA, 2′-OMe A). B, 5-position modification scans at Bn-dU positions of SL1023. Structures of hydrophobic modifications Bn-dU, Pe-dU, Pp-dU, and Nap-dU are illustrated. C, 2′-OMe and C3-spacer substitution scans at A, C, and G positions of SL1030. The sequence of lead optimized truncate SL1032 is shown with modifications indicated (Nap, Nap-dU; L, C3-spacer). D, 5-position modification scans at Nap-dU positions of SL1030.CC BY · PubMed Central
FIGURE 4. Improvements in IL-6 binding and inhibition activity after SOMAmer optimization. Dose-dependent binding and inhibition of IL-6 by Bn-dU SOMAmers (A) and Nap-dU SOMAmers (B) and their optimized variants. Equilibrium binding constants of truncated SOMAmers SL1023 and SL1030 and optimized variants were measured using a solution affinity assay. The fraction of complexed SOMAmer was plotted as a function of IL-6 concentration, and equilibrium binding constants (Kd) were determined by fitting the data to a four-parameter sigmoid dose-response model. Functional inhibitory activity (IC50) values of truncated SOMAmers SL1023, SL1030, and optimized variants were measured in the luciferase gene reporter assay as described in Fig. 1C. Each data point represents the mean ± S.E. with four replicates for each condition.
FIGURE 4. Improvements in IL-6 binding and inhibition activity after SOMAmer optimization. Dose-dependent binding and inhibition of IL-6 by Bn-dU SOMAmers (A) and Nap-dU SOMAmers (B) and their optimized variants. Equilibrium binding constants of truncated SOMAmers SL1023 and SL1030 and optimized variants were measured using a solution affinity assay. The fraction of complexed SOMAmer was plotted as a function of IL-6 concentration, and equilibrium binding constants (Kd) were determined by fitting the data to a four-parameter sigmoid dose-response model. Functional inhibitory activity (IC50) values of truncated SOMAmers SL1023, SL1030, and optimized variants were measured in the luciferase gene reporter assay as described in Fig. 1C. Each data point represents the mean ± S.E. with four replicates for each condition.CC BY · PubMed Central
FIGURE 5. Kinetic evaluation of SL1025 and SL1032 binding to IL-6. Kinetic constants (kon and koff) for the interaction between IL-6 and SOMAmer were determined by SPR analysis with a BiOptix 404pi biosensor. Raw data are shown in gray with curve fits overlaid in black and IL-6 concentrations indicated to the right of each curve. A, SL1025 data were fit globally to a one-site binding model to determine kinetic constants. B, SL1032 data were fit globally to a two-site binding model to determine kinetic constants.
FIGURE 5. Kinetic evaluation of SL1025 and SL1032 binding to IL-6. Kinetic constants (kon and koff) for the interaction between IL-6 and SOMAmer were determined by SPR analysis with a BiOptix 404pi biosensor. Raw data are shown in gray with curve fits overlaid in black and IL-6 concentrations indicated to the right of each curve. A, SL1025 data were fit globally to a one-site binding model to determine kinetic constants. B, SL1032 data were fit globally to a two-site binding model to determine kinetic constants.CC BY · PubMed Central
FIGURE 6. Cross-reactivity of SOMAmers with IL-6 from different species. A, equilibrium binding constants (Kd) of SL1025 and SL1032 to human, cynomolgus monkey, rat, and mouse IL-6 protein. Kd values were determined as described in Fig. 4A. B, dose-dependent inhibition of human, glycosylated human, and glycosylated cynomolgus monkey IL-6 by SL1026 in the luciferase gene reporter assay. IC50 values were determined as described in Fig. 1C. C, amino acid sequence alignment of mature human, cynomolgus monkey, rat, and mouse IL-6 proteins. Sequences of IL-6 proteins with Swiss-Prot accession numbers P05231 (human), P79341 (cynomolgus monkey), P20607 (rat), and P08505 (mouse) were aligned with UniProt after removing the signaling peptide sequence at the N terminus. Amino acid sequences are presented in single letter code, and residues that are identical with the sequence of human IL-6 protein are indicated with a star.
FIGURE 6. Cross-reactivity of SOMAmers with IL-6 from different species. A, equilibrium binding constants (Kd) of SL1025 and SL1032 to human, cynomolgus monkey, rat, and mouse IL-6 protein. Kd values were determined as described in Fig. 4A. B, dose-dependent inhibition of human, glycosylated human, and glycosylated cynomolgus monkey IL-6 by SL1026 in the luciferase gene reporter assay. IC50 values were determined as described in Fig. 1C. C, amino acid sequence alignment of mature human, cynomolgus monkey, rat, and mouse IL-6 proteins. Sequences of IL-6 proteins with Swiss-Prot accession numbers P05231 (human), P79341 (cynomolgus monkey), P20607 (rat), and P08505 (mouse) were aligned with UniProt after removing the signaling peptide sequence at the N terminus. Amino acid sequences are presented in single letter code, and residues that are identical with the sequence of human IL-6 protein are indicated with a star.CC BY · PubMed Central
FIGURE 7. Stability of SOMAmers in human serum in vitro. A, gel images of truncated SOMAmers SL1023 and SL1031, dT controls SL1023dT and SL1031dT, and optimized SOMAmers SL1025 and SL1032 after serum exposure for the indicated amount of time. DNA markers (M) were run on a different part of the gel. Intact SOMAmers are indicated with arrows, and a stable metabolite of SL1023 is marked with an asterisk. B, graphical representation of PAGE results. The fraction of intact SOMAmer (relative to the zero time point control sample) was plotted as a function of time. The data were fit to a one-phase exponential decay model to determine half-life values.
FIGURE 7. Stability of SOMAmers in human serum in vitro. A, gel images of truncated SOMAmers SL1023 and SL1031, dT controls SL1023dT and SL1031dT, and optimized SOMAmers SL1025 and SL1032 after serum exposure for the indicated amount of time. DNA markers (M) were run on a different part of the gel. Intact SOMAmers are indicated with arrows, and a stable metabolite of SL1023 is marked with an asterisk. B, graphical representation of PAGE results. The fraction of intact SOMAmer (relative to the zero time point control sample) was plotted as a function of time. The data were fit to a one-phase exponential decay model to determine half-life values.CC BY · PubMed Central
FIGURE 8. Inhibition of tumor cell proliferation by SL1026, SL1033, and tocilizumab. A, inhibition of U266B1 (human myeloma) cell proliferation. Cell proliferation was measured with AlamarBlue, and percent IL-6 inhibition (relative to a no-inhibitor control sample) was plotted. Each bar represents the mean ± S.E. of three experiments. The data were analyzed by one-way analysis of variance (Dunnett's two-tailed), and each data point was compared with control data without IL-6 inhibitor. Statistically significant differences are denoted as *, p value < 0.05; **, p value < 0.01; ****, p value < 0.0001. Inhibition of U87MG (human glioma) and HepG2 (human hepatoma) cell proliferation are illustrated in B and C, respectively. Percent inhibition of cell growth (relative to a no-inhibitor control sample) was plotted.
FIGURE 8. Inhibition of tumor cell proliferation by SL1026, SL1033, and tocilizumab. A, inhibition of U266B1 (human myeloma) cell proliferation. Cell proliferation was measured with AlamarBlue, and percent IL-6 inhibition (relative to a no-inhibitor control sample) was plotted. Each bar represents the mean ± S.E. of three experiments. The data were analyzed by one-way analysis of variance (Dunnett's two-tailed), and each data point was compared with control data without IL-6 inhibitor. Statistically significant differences are denoted as *, p value < 0.05; **, p value < 0.01; ****, p value < 0.0001. Inhibition of U87MG (human glioma) and HepG2 (human hepatoma) cell proliferation are illustrated in B and C, respectively. Percent inhibition of cell growth (relative to a no-inhibitor control sample) was plotted.CC BY · PubMed Central
FIGURE 9. SL1026 and SL1033 compete with soluble IL-6 receptor for IL-6 binding. IL-6 binding to immobilized IL-6Rα was measured in the presence of SOMAmer or tocilizumab. Percent IL-6 binding (relative to a no-inhibitor control sample) was plotted as a function of inhibitor concentration. Each data point represents the mean ± S.E. with two replicates for each condition. The data were fit to a four-parameter sigmoid dose-response model.
FIGURE 9. SL1026 and SL1033 compete with soluble IL-6 receptor for IL-6 binding. IL-6 binding to immobilized IL-6Rα was measured in the presence of SOMAmer or tocilizumab. Percent IL-6 binding (relative to a no-inhibitor control sample) was plotted as a function of inhibitor concentration. Each data point represents the mean ± S.E. with two replicates for each condition. The data were fit to a four-parameter sigmoid dose-response model.CC BY · PubMed Central

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  1. Zhao, 2013, Anti-IL-6 antibody Clazakizumab is more potent than Tocilizumab in blocking in vitroex vivo IL-6-induced functions, Arthritis and Rheumatism, 65

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Metadata

Authors
Gupta, S., Hirota, M., Waugh, S. M., Murakami, I., Suzuki, T., Muraguchi, M., Shibamori, M., Ishikawa, Y., Jarvis, T. C., Carter, J. D., Zhang, C., Gawande, B., Vrkljan, M., Janjic, N., & Schneider, D. J.
Affiliations
SomaLogic (United States) (US)
Corresponding author
Schneider, Daniel J.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2014-03
Volume
289
Issue
12
Pages
8706-8719
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m113.532580
PMC
PMC3961692
OpenAlex
W2084203294
Cited by
151
References
52
Citations by year
2026: 3 · 2025: 13 · 2024: 7 · 2023: 10 · 2022: 16 · 2021: 9 · 2020: 13 · 2019: 17 · 2018: 14 · 2017: 13 · 2016: 14 · 2015: 16 · 2014: 6
FWCI
6.69
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
interleukin-6, SOMAmer, IL-6 receptor, cytokine signaling inhibition, SELEX, serum stability, tumor cell proliferation, inflammatory diseases
MeSH terms
Interleukin-6 Inhibitors, Amino Acid Sequence, Animals, Anti-Inflammatory Agents, Base Sequence, Cricetulus, Humans, Macaca fascicularis, Molecular Sequence Data, Interleukin-6, CHO Cells, Receptors, Interleukin-6, Serum, Mice, Rats, SELEX Aptamer Technique, Aptamers, Nucleotide, Drug Discovery

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