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 Phe229 and Phe279 of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe279. 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 Phe279. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe279, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe229.

Biosensing Cytokine IL-6: A Comparative Analysis of Natural and Synthetic Receptors

“…It has been postulated that only in the hexameric quaternary structure is IL-6 able to function properly [ 41 , 42 ]. Several artificial receptors have been tested against IL-6, IL-6R, and gp-130 [ 18 ], and some of those entered and passed clinical trials [ 7 , 8 , 9 , 10 , 18 , 28 , 43 , 44 ]. All of these receptors show high affinity to the ligand and have specific inhibitory abilities.…”

“…61H7 and 68F2 are neutralizing IL-6 antibody fragments. Antibodies were obtained upon Llama immunization with human IL-6 and exhibit ultra-high affinity to the cytokine, with a K D = 3.3–6.3 pM (61H7) and K D = 13–21 pM (68F2) [ 44 ]. On the other hand, the way 61H7 and 68F2 bind IL-6 is quite different: both were selected to block Site I of the cytokine, i.e., mimicking the action of IL-6R, but while 61H7 binds the top of the IL-6 helix bundle, 68F2 is set transversally toward the same side (see the Appendix A , Figure A1 ).…”

Structural Understanding of Interleukin 6 Family Cytokine Signaling and Targeted Therapies: Focus on Interleukin 11

“…Structures show that the olokizumab Fab blocks site-III of IL-6, preventing formation of the IL-6 hexameric complex (291). Structures have also been solved of two anti-IL-6 Fabs, which bind site-I, mimicking the IL-6/IL-6Rα interaction (292). No structures are available of the FDA-approved anti-IL-6 signaling antibodies in complex with their antigen.…”

An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening

“…Limited number of factors and levels are systematically engaged in the design. Footprint screening, reverse screening, and grid screening can be classified as systematic screening. Footprint screening produces a solubility curve and a precipitate curve (crystallization phase diagram) based on the concentration of biomacromolecules and precipitants to search for suitable crystallization conditions of biomacromolecules, which is important for many biomacromolecules with difficulties in forming crystals .…”

“…However, its advantages still outweigh its disadvantages. In prevailing screening kits, about 60% of the kits are designed by sparse matrix screening, including Index HT (Hampton Research), Crystal Screen HT (Hampton Research), Wizard I–IV (Rigaku and Emerald BioSystems), Structure Screen (Molecular Dimensions), JBScreen Basic HT (Jena Bioscience), Classics and Classics II (Qiagen), and Xtalquest Kit (Xtalquest) …”