Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies

Abstract

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).

Keywords: antibody engineering; crystal structure; high affinity; immunology; interleukin 6 (IL-6); structure-function.

FIGURE 1.

Structure of the Fab complexes with IL-6. A, stereo view of 61H7 bound to IL-6. B, stereo view of 68F2 bound to IL-6. In each panel the cytokine is shown in red, the VH and CH1 domains of the Fabs are in blue, and the VL and CL domains are in green.

FIGURE 2.

Superposition of the IL-6 structure as found in the Fab cytokine complexes with published structures. IL-6 from the IL-6·IL-6R complex (Protein Data Bank code 1P9M) is shown in red, IL-6 as present in the complex with 61H7 is in yellow, and the cytokine from its complex with 68F2 is in cyan. The difference in position of the mini helix as found in 68F2·IL-6 complex is indicated with an arrow.

FIGURE 3.

Epitope mapping of the anti-IL-6 antibodies. A, the ability of increasing concentrations of antibodies 61H7 and 68F2 to compete with biotinylated IL-6 for binding to captured IL-6R was investigated using a competition-based ELISA. IL-6 binding was revealed using horseradish peroxidase-conjugated streptavidin and expressed as a percentage of IL-6 binding to IL6-R as compared with IL-6 alone (y axis). The x axis shows mAb concentrations (nm). B, antibodies 61H7 and 68F2 compete with each other for binding to immobilized IL-6 as demonstrated by SPR. 61H7 (50 μg/ml) was first allowed to bind to and nearly saturate IL-6 (injected 61H7). Then 61H7 (injected 61H7, lower curve) or 68F2 (injected 68F2, upper curve) was added, and no additional binding was observed, indicating recognition of similar binding sites on IL-6. Inj., injected; Resp. Diff., response difference.

FIGURE 4.

Fab epitopes mapped on the surface of IL-6. A, stereo view of a surface representation of IL-6 (free form; Protein Data Bank code 1ALU) with the surface covered by 61H7 colored yellow, and the surface covered by 68F2 is colored cyan. The overlap between both epitopes is colored green. B, identical representation of IL-6, but with the surface covered by IL-6R colored red and largely overlapping the one for 68F2. The epitope covered by olokizumab is shown in pink and located on the opposite side of IL-6.

FIGURE 5.

Mimicry of site 1 interactions. A, surface representation of IL-6 from the IL-6·IL-6R complex. The backbone trace of IL-6R residues 226–232 and 275–285 is shown in red. The side chains of Phe²²⁹ and Phe²⁷⁹ of IL-6R are shown in stick to illustrate their penetration into cavities on the site I surface of IL-6. B, equivalent surface representation, but for IL-6 in the IL-6·68F2 complex. Residues 31–35 of VL and 101–108 of VH of 68F2 are shown in cyan. Tyr³² of VL (indicated by Y32L) (Kabat numbering 30) and Val¹⁰⁴ of VH (indicated by V104H) (yellow; Kabat numbering 99) mimic the interactions of Phe²²⁹ and Phe²⁷⁹ from IL-6R, respectively. C, superposition of key residues of 68F2 (in cyan) and IL-6R (in red) when bound to IL-6. D, surface representation of IL-6 from the IL-6·61H7 complex. The backbone trace of 61H7 residues 100–106 of VH is shown in yellow. Here the side chain of Trp¹⁰² of VH (indicated by W102H) (Kabat numbering 98) mimics Phe²²⁹ from IL-6R. E, surface representation of human growth hormone shown in the same orientation as IL-6 in A, B, and D. The backbone of residues 163–171 of the human growth hormone receptor is shown in green. Trp¹⁶⁹ from the human growth hormone receptor penetrates into a cavity on the surface of human growth hormone that is located similarly as the site I cavity of IL-6, into which Phe²²⁹ of IL-6R penetrates. F, superposition of key residues of 68F2 (in yellow) and IL-6R (in red) when bound to IL-6.