Engineering Human Interleukin-6 to Obtain Variants with Strongly Enhanced Bioactivity

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 1996 Jun 3

PMID 8654370

Citations 7

Authors

C Toniatti

A Cabibbo

E Sporena

A L Salvati

M Cerretani

S Serafini

A Lahm

R Cortese

G Ciliberto

Affiliations

Soon will be listed here.

Abstract

Interleukin-6 (IL-6) triggers the formation of a high affinity receptor complex with the ligand binding subunit IL-6Ralpha and the signal transducing chain gp130. Since the intracytoplasmic region of the IL-6Ralpha does not contribute to signaling, soluble forms of the extracytoplasmic domain (sIL-6Ralpha), potentiate IL-6 bioactivity and induce a cytokine-responsive status in cells expressing gp130 only. This observation, together with the detection of high levels of circulating soluble human IL-6Ralpha (shIL-6Ralpha) in sera, suggests that the hIL-6-shIL-6Ralpha complex is an alternative form of the cytokine. Here we describe the generation of human IL-6 (hIL-6) variants with strongly enhanced shIL-6Ralpha binding activity and bioactivity. Homology modeling and site-directed mutagenesis of hIL-6 suggested that the binding interface for hIL-6Ralpha is constituted by the C-terminal portion of the D-helix and residues contained in the AB loop. Four libraries of hIL-6 mutants were generated by each time fully randomizing four different amino acids in the predicted AB loop. These libraries were displayed monovalently on filamentous phage surface and sorted separately for binding to immobilized shIL-6Ralpha. Mutants were selected which, when expressed as soluble proteins, showed a 10- to 40-fold improvement in shIL-6Ralpha binding; a further increase (up to 70-fold) was achieved by combining variants isolated from different libraries. Interestingly, high affinity hIL-6 variants show strongly enhanced bioactivity on cells expressing gp13O in the presence of shIL-6Ralpha at concentrations similar to those normally found in human sera.

Citing Articles

Next-Generation Protein Sequencing and individual ion mass spectrometry enable complementary analysis of interleukin-6.

Skinner K, Fisher T, Lee A, Su T, Forte E, Sanchez A bioRxiv. 2025; .

PMID: 39975277 PMC: 11839055. DOI: 10.1101/2025.02.07.637157.

Unveiling novel insights into human IL-6 - IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis.

Li K, Cai J, Jiang Z, Meng Q, Meng Z, Xiao H Sci Rep. 2024; 14(1):18293.

PMID: 39112658 PMC: 11306327. DOI: 10.1038/s41598-024-69429-w.

Phagekines: Directed Evolution and Characterization of Functional Cytokines Displayed on Phages.

Rojas G, Carmenate T, Garcia-Perez G, Perez-Martinez D Methods Mol Biol. 2023; 2702:149-189.

PMID: 37679619 DOI: 10.1007/978-1-0716-3381-6_8.

Functional role of skeletal muscle-derived interleukin-6 and its effects on lipid metabolism.

Lin W, Song H, Shen J, Wang J, Yang Y, Yang Y Front Physiol. 2023; 14:1110926.

PMID: 37555019 PMC: 10405179. DOI: 10.3389/fphys.2023.1110926.

Cytokimera GIL-11 rescued IL-6R deficient mice from partial hepatectomy-induced death by signaling via non-natural gp130:LIFR:IL-11R complexes.

Rafii P, Seibel C, Weitz H, Ettich J, Minafra A, Petzsch P Commun Biol. 2023; 6(1):418.

PMID: 37061565 PMC: 10105715. DOI: 10.1038/s42003-023-04768-4.

References

Horiuchi S, Koyanagi Y, Zhou Y, Miyamoto H, Tanaka Y, Waki M . Soluble interleukin-6 receptors released from T cell or granulocyte/macrophage cell lines and human peripheral blood mononuclear cells are generated through an alternative splicing mechanism. Eur J Immunol. 1994; 24(8):1945-8. DOI: 10.1002/eji.1830240837. View

van Gameren M, Willemse P, Mulder N, Limburg P, Groen H, Vellenga E . Effects of recombinant human interleukin-6 in cancer patients: a phase I-II study. Blood. 1994; 84(5):1434-41. View

May L, Patel K, Garcia D, Ndubuisi M, Ferrone S, Mittelman A . Sustained high levels of circulating chaperoned interleukin-6 after active specific cancer immunotherapy. Blood. 1994; 84(6):1887-95. View

Ward L, Howlett G, Discolo G, Yasukawa K, Hammacher A, Moritz R . High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6, interleukin-6 receptor, and gp-130. J Biol Chem. 1994; 269(37):23286-9. View

Savino R, Ciapponi L, Lahm A, Demartis A, Cabibbo A, Toniatti C . Rational design of a receptor super-antagonist of human interleukin-6. EMBO J. 1994; 13(24):5863-70. PMC: 395561. DOI: 10.1002/j.1460-2075.1994.tb06931.x. View

Ward L, Howlett G, Hammacher A, Weinstock J, Yasukawa K, Simpson R . Use of a biosensor with surface plasmon resonance detection for the determination of binding constants: measurement of interleukin-6 binding to the soluble interleukin-6 receptor. Biochemistry. 1995; 34(9):2901-7. DOI: 10.1021/bi00009a021. View

Sui X, Tsuji K, Tanaka R, Tajima S, Muraoka K, Ebihara Y . gp130 and c-Kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells. Proc Natl Acad Sci U S A. 1995; 92(7):2859-63. PMC: 42318. DOI: 10.1073/pnas.92.7.2859. View

Ehlers M, de Hon F, Bos H, Horsten U, Kurapkat G, van De Leur H . Combining two mutations of human interleukin-6 that affect gp130 activation results in a potent interleukin-6 receptor antagonist on human myeloma cells. J Biol Chem. 1995; 270(14):8158-63. DOI: 10.1074/jbc.270.14.8158. View

DHondt V, Humblet Y, Guillaume T, Baatout S, Chatelain C, Berliere M . Thrombopoietic effects and toxicity of interleukin-6 in patients with ovarian cancer before and after chemotherapy: a multicentric placebo-controlled, randomized phase Ib study. Blood. 1995; 85(9):2347-53. View

10.

Paonessa G, Graziani R, DE SERIO A, Savino R, Ciapponi L, Lahm A . Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling. EMBO J. 1995; 14(9):1942-51. PMC: 398293. DOI: 10.1002/j.1460-2075.1995.tb07186.x. View

11.

Salvati A, Lahm A, Paonessa G, Ciliberto G, Toniatti C . Interleukin-6 (IL-6) antagonism by soluble IL-6 receptor alpha mutated in the predicted gp130-binding interface. J Biol Chem. 1995; 270(20):12242-9. DOI: 10.1074/jbc.270.20.12242. View

12.

Gordon M, Nemunaitis J, Hoffman R, Paquette R, Rosenfeld C, Manfreda S . A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia. Blood. 1995; 85(11):3066-76. View

13.

Hammacher A, Ward L, Weinstock J, Treutlein H, Yasukawa K, Simpson R . Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding. Protein Sci. 1994; 3(12):2280-93. PMC: 2142761. DOI: 10.1002/pro.5560031213. View

14.

Schobitz B, Pezeshki G, Pohl T, Hemmann U, Heinrich P, Holsboer F . Soluble interleukin-6 (IL-6) receptor augments central effects of IL-6 in vivo. FASEB J. 1995; 9(8):659-64. DOI: 10.1096/fasebj.9.8.7768358. View

15.

Mott H, Campbell I . Four-helix bundle growth factors and their receptors: protein-protein interactions. Curr Opin Struct Biol. 1995; 5(1):114-21. DOI: 10.1016/0959-440x(95)80016-t. View

16.

Schwabe M, Zhao J, Kung H . Differential expression and ligand-induced modulation of the human interleukin-6 receptor on interleukin-6-responsive cells. J Biol Chem. 1994; 269(10):7201-9. View

17.

Savino R, Lahm A, Salvati A, Ciapponi L, Sporeno E, Altamura S . Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation. EMBO J. 1994; 13(6):1357-67. PMC: 394953. DOI: 10.1002/j.1460-2075.1994.tb06389.x. View

18.

Sporeno E, Paonessa G, Salvati A, Graziani R, DELMASTRO P, Ciliberto G . Oncostatin M binds directly to gp130 and behaves as interleukin-6 antagonist on a cell line expressing gp130 but lacking functional oncostatin M receptors. J Biol Chem. 1994; 269(15):10991-5. View

19.

Ryffel B, Car B, Woerly G, Weber M, DiPadova F, KAMMULLER M . Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate--the marmoset. Blood. 1994; 83(8):2093-102. View

20.

Saggio I, Gloaguen I, Poiana G, Laufer R . CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction. EMBO J. 1995; 14(13):3045-54. PMC: 394365. DOI: 10.1002/j.1460-2075.1995.tb07307.x. View