©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Combining Two Mutations of Human Interleukin-6 That Affect gp130 Activation Results in a Potent Interleukin-6 Receptor Antagonist on Human Myeloma Cells (*)

(Received for publication, November 8, 1994; and in revised form, January 26, 1995)

Marc Ehlers Floris D. de Hon (1) Hanny Klaasse Bos (1) Ursula Horsten (2) Günther Kurapkat (2) Hildegard Schmitz van De Leur (2) Joachim Grötzinger (2) Axel Wollmer (2) Just P. J. Brakenhoff (1) Stefan Rose-John (§)

From the  (1)I. Medical Clinic-Section-Pathophysiology, Mainz University, D-55101 Mainz, Germany, the Department of Autoimmune Diseases, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam 1066 CX, The Netherlands, and the (2)Department of Biochemistry, Rheinisch-Westfälische Technische Hochschule Aachen, D-52057 Aachen, Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The pleiotropic cytokine interleukin-6 (IL-6) interacts with the specific ligand binding subunit (IL-6Ralpha) of the IL-6 receptor, and this complex associates with the signal-transducing subunit gp130 (IL-6Rbeta). Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells. The construction of a set of chimeric human/murine IL-6 proteins has recently allowed us to define a region (residues 43-55) within the human IL-6 protein, which is important for the interaction with gp130. Subdividing this region shows that mainly residues 50-55 of the human IL-6 are necessary for this interaction. Recently, another human IL-6 double mutant (Q159E and T162P) showed reduced affinity to gp130 but residual activity on the human myeloma cell line XG-1. Into this IL-6 mutant we introduced the murine residues 43-49 or 50-55 together with two point mutations, F170L and S176A, which had been reported to increase the affinity of IL-6 to the IL-6Ralpha. The resulting IL-6 molecule, which contained the murine residues 50-55, was inactive on human myeloma cells and in addition completely inhibited wild type IL-6 activity on these cells. Such an antagonist may be used as a specific inhibitor of IL-6 activity in vivo.


INTRODUCTION

The plasma level of the pleiotropic cytokine IL-6 (^1)is increased upon injury or infection. IL-6 is involved in a spectrum of activities like immune defense, hematopoiesis, and acute phase response(1) . An increased expression of IL-6 has been reported for several diseases like plasmacytoma/myeloma, mesangial proliferative glomerulonephritis, osteoporosis, autoimmune diseases, and AIDS(1) .

IL-6 activates target cells via a transmembrane receptor, which consists of two glycoproteins. First, IL-6 binds with low affinity (K = 500 pM) to the specific ligand binding subunit gp80 (IL-6Ralpha). This complex binds with high affinity (K = 30-50 pM) the signal transduction subunit gp130 (IL-6Rbeta). IL-6 by itself has no measurable affinity to gp130(2) . IL-6/IL-6Ralpha mediated homodimerization of two gp130 molecules via a disulfide bridge induces the IL-6 signal(3) .

Interleukin-6 is a protein of 184 amino acids(4) . Secondary structure predictions point out that IL-6 belongs to a hemopoietic cytokine family characterized by four antiparallel alpha-helices (A, B, C, and D) (5) . This family also includes leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, growth hormone, prolactine, and granulocyte colony stimulating factor(5, 6, 7) . Structure function studies of human IL-6 showed that the COOH terminus and the end of the A-B loop/beginning of the B-helix (region 2c, residues Gly-Glu) are involved in the interaction with the IL-6Ralpha(8, 9, 10, 11, 12, 13, 14, 15) . These results are in good agreement with a recently published human IL-6 model (15) where these two regions are in close proximity. Point mutations in the middle of the D-helix (F170L, S176R) resulted in a 2-3-fold increased affinity to the IL-6Ralpha(12, 16) .

Human IL-6 acts on human and murine cells, whereas murine IL-6 is only active on murine cells (the identity between human and murine IL-6 is 42%(14) ). Recently, we have exchanged the human residues 43-55 (beginning of the A-B loop, region 2a) against the corresponding murine amino acids(15) . This chimeric protein showed an only 5-fold reduced affinity to the human IL-6Ralpha but a highly reduced human IL-6Ralphadependent interaction with gp130 and no bioactivity on the human hepatoma cell line HepG2. But on the more sensitive human myeloma cell line XG-1, this IL-6 mutant reached full bioactivity at a 100-fold higher concentration than human IL-6.

Brakenhoff et al.(17) have shown that substitution of two residues at the beginning of the D-helix (Q159E/T162P) also reduced the affinity of the IL-6bulletIL-6Ralpha complex to gp130, whereas the affinity of IL-6 to the IL-6Ralpha was decreased only 3-4-fold. This IL-6 mutant could be used as an IL-6 receptor antagonist on human HepG2 cells and the human Epstein-Barr virus-transformed B cell line CESS, but it had full activity on the more sensitive human XG-1 cells when applied at sufficiently high concentrations. (^2)

In the present study, we subdivided the region 2a into two parts (2a1, residues 43-49; 2a2, residues 50-55) to analyze which part is the most important for the interaction with gp130. We exchanged each part in the human IL-6 against the corresponding murine sequence. In the next step, we wanted to construct an IL-6 molecule that should bind to the IL-6Ralpha, but which should not activate gp130. In addition, this IL-6 protein should block the IL-6Ralpha and thereby inhibit human IL-6 activity on the highly sensitive human myeloma cell line XG-1. For this reason, we combined the murine residues 43-49 or 50-55 with the two point mutations (Q159E/T162P) in the beginning of the D-helix, which also decrease the interaction of the IL-6bulletIL-6Ralpha complex with gp130 (16) . In addition, we introduced point mutations in the end of the D-helix (F170L/S176R) that increase the affinity to the IL-6Ralpha(12, 16) . These changes resulted in an effective IL-6 receptor antagonist.


MATERIALS AND METHODS

Construction of Expression Vectors

For the construction of the human/murine chimeric IL-6 cDNAs 2a1 and 2a2, discrete regions of the human IL-6 cDNA were replaced by homologous murine IL-6-cDNA fragments. To construct the plasmid pRSET 5d-huIL-6-2a2, four oligonucleotides were fused and ligated into EcoNI-NheI-digested pRSET 5d-huIL-6. The oligonucleotides were 5`-GAAAGGAGACATGTAACAAGAGT-3` sense, 5`-ATGTTACTCTTGTTACATGTCTCCTTT-3` antisense, 5`-AACATGTGTATGAACAACGATGATGCG-3` sense, and 5`-CTAGCGCATCATCGTTGTTCATACAC-3` antisense.

Construction of pRSET 5d-huIL-6-2a1 was as follows. Since there was no common restriction site in the human or murine IL-6-cDNA, we introduced the unique restriction site SacI at analogous positions using synthetic oligonucleotides. These nucleotide exchanges did not result in changes of the amino acid sequence. Chimeric human and murine IL-6-cDNA fragments were then produced by PCR using pRSET 5d-huIL-6 or pRSET 5d-huIL-6-2a as template(14, 15) . PCR reaction conditions were described(15) . The oligonucleotides hybridizing to the pRSET 5d vector, the human IL-6 cDNA or the mutant 2a cDNA, were 5`-TAATACGACTCACTATAG-3` sense (vector), 5`-CTCAGCTTCCTTTCGG-3` antisense (vector), 5`-TGTGAGAGCTCCAAAGAGGCAC-3` SacI sense (human IL-6), and 5`-CATCGGAGCTCTCACAATCAGA-3` SacI antisense (mutant 2a).

To introduce the two point mutations, F170L and S176R, into the huIL-6-Q159E/T162P cDNA (short designation, huIL-6-EP), a XbaI-Bsu36I fragment encoding the F170L and S176R substitutions was created with PCR technology. pRSET 6d-huIL-6-EP was used as a template, and the resulting fragment was subcloned into XbaI-Bsu36I-digested pRSET 6d-huIL-6-EP. PCR was carried out with Pyrococcus furiosus polymerase (Stratagene) under the following conditions: denaturation, 90 s at 95 °C; annealing, 60 s at 55 °C; extension, 60 s at 72 °C, 30 cycles. The following primers were used: a sense primer carrying the XbaI site (5`-GAATCTAGATGCAATAACCACC-3`) and an antisense primer encoding the two substitutions and the Bsu36I site (5`-GAAGAGCCCTCAGGCTGCGCTGCAGGAACTCCTTAAGGCTGCGCAGAATG-3`). The pRSET 6d-huIL-6-EP-F170L/S176R was called pRSET 6d-huIL-6-EP-LR.

The vectors pRSET 6d-huIL-6-EP-2a1-LR and pRSET 6d-huIL-6-EP-2a2-LR were constructed by ligating an NcoI-XbaI cDNA fragment from pRSET 5d-huIL-6-2a1 or -2a2 into NcoI-XbaI-digested pRSET 6d-huIL-6-EP-LR.

The integrity of all constructs was verified by restriction fragment analysis and DNA sequencing(18) .

Binding and Biological Assays

The IL-6 variants were expressed, purified, and quantified as described(15) . Binding of IL-6 proteins to a soluble form of IL-6Ralpha, ternary complex formation of IL-6 proteins with soluble forms of IL-6Ralpha and gp130, and the assay with human hepatoma HepG2 cells were done as previously described (15) . The biological assays with the murine cell line B9 and the human myeloma cell line XG-1 were performed as in (17) .


RESULTS AND DISCUSSION

Region 2a2 in the Human IL-6 Protein Is More Important for the Activation of gp130 than Region 2a1

In previous studies with human/murine IL-6 chimeric proteins, we have defined a region mainly comprising the beginning of the A-B loop of human IL-6 (residues 43-55, region 2a) as being important for the interaction of the IL-6bulletIL-6Ralpha complex with the signal-transducing protein gp130(15) . To more precisely map this interaction site, we subdivided this region into two parts: 2a1 (residues 43-49) and 2a2 (residues 50-55) and introduced the corresponding murine residues into the human IL-6 protein.

First, we measured displacement of human I-IL-6 binding to a soluble form of the IL-6Ralpha protein(14, 15, 19, 20, 21, 22) by an excess of IL-6 mutants. As shown in Fig. 1A, human IL-6 displaced I-IL-6 binding to 50% when used in a 10-20-fold molar excess. In contrast, murine IL-6 failed to compete with human I-IL-6 for binding. Chimera 2a showed half-maximal displacement at an excess of approximately 60-fold whereas chimera 2a1 and 2a2 showed similar affinity to the IL-6Ralpha as human IL-6.


Figure 1: Binding and bioactivity of the mutants 2a1 and 2a2. A, binding of the IL-6 variants to a soluble human IL-6Ralpha. Recombinant human soluble IL-6Ralpha was incubated with human I-IL-6. Binding was competed with increasing amounts of IL-6 variants. Average values of three experiments are shown. B, proliferation of human XG-1 cells in response to IL-6 mutants. Cells were grown in the presence of increasing amounts of IL-6 variants, and proliferation was measured using [^3H]thymidine incorporation. One representative of three experiments is shown. C1, induction of haptoglobin mRNA expression in human hepatoma cells by IL-6 variants. C2, photograph of the ethidium bromide-stained RNA gel. D, complex formation between soluble forms of IL-6Ralpha and gp130 and the IL-6 variants was determined using metabolically labeled S-soluble IL-6Ralpha and a fusion protein of human sgp130 and human IgG1. Complexes were precipitated with protein A-Sepharose, separated by SDS-polyacrylamide gel electrophoresis, and visualized by fluorography.



Human and murine IL-6 and the mutants 2a, 2a1, and 2a2 stimulated the proliferation of murine B9 cells to a similar extent (data not shown), which demonstrated that their structures were intact. On human myeloma XG-1 cells, human IL-6 but not murine IL-6 induced proliferation (Fig. 1B). The specific activity of chimera 2a was reduced by a factor of approximately 300. Chimera 2a1 was 5-fold and chimera 2a2 30-fold less active than human IL-6. These results indicate that the properties of chimera 2a reside mainly in part 2a2.

A similar result was obtained when we examined the induction of the acute phase protein gene haptoglobin by 10 ng/ml of the IL-6 variants in human hepatoma cells (HepG2). Untreated cells and cells treated with murine IL-6 and chimera 2a showed no haptoglobin mRNA induction (Fig. 1C). Stimulation of cells with human IL-6 led to full stimulation of haptoglobin mRNA expression. Chimera 2a2 was less active than chimera 2a1.

One possible explanation for the discrepancy between binding of chimeras 2a1 and 2a2 to the IL-6Ralpha and their reduced bioactivity on human cells could be a reduced interaction of the IL-6bulletIL-6Ralpha complex with gp130. To measure ternary complex formation of the IL-6 mutants with soluble forms of IL-6Ralpha and gp130(19, 23, 24, 25, 26) , we used metabolically labeled S soluble human IL-6Ralpha protein and soluble human gp130 with the constant region of human IgG1 fused to its COOH terminus(15) . The complexes were precipitated with protein A-Sepharose.

No precipitation of sIL-6Ralpha occurred without IL-6 (Fig. 1D). In the presence of human but not of murine IL-6, the sIL-6Ralpha subunit was coprecipitated with soluble gp130. The 2a mutant did not induce measurable complex formation between IL-6Ralpha and gp130. Chimera 2a1 led to a slightly reduced and chimera 2a2 to a more reduced ternary complex formation. Therefore, region 2a2 is more important for the interaction of the IL-6bulletIL-6Ralpha complex with the gp130 molecule than region 2a1. Whether this region is part of a binding side for gp130 or it is necessary for stabilizing other epitopes responsible for gp130 interaction remains open.

Design of Human IL-6 Receptor Antagonists

Recently, we showed that the introduction of two point mutations (Q159E/T162P; short designation, EP) into the human IL-6 protein also resulted in highly reduced IL-6Ralpha-dependent complex formation with gp130(17) . The affinity of this mutant to the IL-6Ralpha was only 3-4-fold reduced. This IL-6 protein proved to be an inhibitor of human IL-6 activity on HepG2 and CESS cells but not on XG-1 cells, where it showed full activity when applied at sufficiently high concentrations. This activity could be inhibited by anti-gp130 mAbs.^2

So far, it is not clear why the sensitivity of IL-6-responsive cells varies by 2-3 orders of magnitude. Cells that proliferate in response to IL-6 show a half-maximal response at 1-20 pg/ml, whereas cells that differentiate or express acute phase proteins have a half-maximal response at 1-5 ng/ml(14, 15) . This, however, cannot be explained by different receptor numbers(1, 34) . It cannot be excluded that there are differences in signal transduction or that the presence of additional receptor subunits on cells that proliferate in response to IL-6 renders these cells more sensitive to the cytokine. This phenomenon of differential sensitivity of different cell types has of course important implications for the therapeutic application of IL-6 receptor antagonists.

Fig. 2shows a ribbon representation of the IL-6 model (15, 27) . In our model, the two point mutations (EP) and the regions 2a1 and 2a2 are in close proximity. Recently, the substitution of two amino acids in the A-helix (Y31D/G35F) (Fig. 2) also lead to a reduced affinity of the IL-6bulletIL-6Ralpha complex to gp130(28) . There is evidence that the ligand-induced receptor complex consists of two molecules of each, IL-6, IL-6Ralpha, and gp130(22, 29) . So far, it is unclear whether the mentioned regions are all involved in the interaction with one gp130 or whether one IL-6 protein touches two gp130 proteins or two IL-6 proteins touch each other.


Figure 2: Ribbon representation of the human IL-6 model. Representation of amino acids important for IL-6Ralpha binding and IL-6Ralpha-dependent gp130 interaction (15, 35) (helix A, 19-45; helix B, 80-101; helix C, 106-132; helix D, 157-184). The amino acids Phe, Ser, Gln, Thr, Phe, and Ser (see text) are shown. The regions 2a1 and 2a2 are marked by the amino acids Thr, Cys, and Glu (see text). C, COOH terminus; N, NH(2) terminus (corresponds to residue 17 of IL-6)(15) .



We introduced the murine region 2a2 and 2a1, respectively, into the mutant IL-6-EP together with the two point mutations (F170L/S176R; short designation, LR), which increase the affinity of human IL-6 to the IL-6Ralpha. The two resulting IL-6 mutant proteins were called IL-6-EP-2a1-LR and IL-6-EP-2a2-LR. Both mutants showed similar affinity to the IL-6Ralpha as human IL-6 (Fig. 3A). Mutant IL-6-EP-2a1-LR and IL-6-EP-2a2-LR fully induced proliferation of IL-6-dependent B9 cells albeit at 10-100-fold higher concentrations (data not shown). For mutant IL-6-EP-2a2-LR, no bioactivity on human XG-1 (Fig. 3B) and HepG2 (Fig. 3C) cells and no IL-6Ralpha-dependent ternary complex formation with gp130 (Fig. 3D) was detected. Mutant IL-6-EP-2a1-LR showed residual bioactivity on XG-1 cells. This was in line with the higher specific activity of 2a1 compared with 2a2.


Figure 3: Binding and bioactivity of the mutants IL-6-EP-2a1-LR and IL-6-EP-2a2-LR. A, binding of the IL-6 variants to the soluble human IL-6Ralpha. Average values of three experiments are shown. B, proliferation of human XG-1 cells in response to IL-6 variants. One representative of three experiments is shown. C1, induction of haptoglobin mRNA expression in human hepatoma cells by IL-6 variants. C2, photography of the ethidium bromide-stained RNA gel. D, induction of complex formation between soluble forms of IL-6Ralpha and gp130 and the IL-6 variants. See text and legend to Fig. 1for details.



To test for antagonistic activity, we added IL-6-EP-2a1-LR and IL-6-EP-2a2-LR at increasing concentrations, respectively, to human myeloma cells (XG-1) in the presence of 100 pg/ml human IL-6 (Fig. 4A). Mutant IL-6-EP-2a2-LR completely inhibited the bioactivity of IL-6 at an 100,000-fold excess, whereas mutant IL-6-EP-2a1-LR at this concentration only led to 70% reduction of cell proliferation. By increasing the amounts of human IL-6 to XG-1 cells in the presence of 10 µg/ml IL-6-EP-2a1-LR or IL-6-EP-2a2-LR, we observed that high concentrations of human IL-6 could completely overcome the antagonistic effect of both IL-6 variants (Fig. 4B).


Figure 4: Antagonistic effect of IL-6-EP-2a1-LR and IL-6-EP-2a2-LR on the human IL-6-induced proliferation of XG-1 cells. A, the indicated concentrations of IL-6-EP-2a1-LR and IL-6-EP-2a2-LR were added in the presence of 100 pg/ml human IL-6. One representative of two experiments is shown. B, the indicated concentrations of human IL-6 were added alone or in the presence of 10 µg/ml IL-6-EP-2a1-LR or IL-6-EP-2a2-LR.



IL-6-EP-2a2-LR behaved as a potent IL-6 receptor antagonist on XG-1 cells. Introduction of additional point mutations into IL-6 may further increase the affinity to IL-6Ralpha. This will reduce the amount of this antagonist necessary to inhibit IL-6 activity.

IL-6 plays a role in a variety of diseases, such as plasmacytoma/myeloma, mesangial proliferative glomerulonephritis, osteoporosis, autoimmune diseases, and AIDS(1) . The administration of IL-6-neutralizing monoclonal antibodies to patients with rheumathoid arthritis and multiple myeloma has highly improved the conditions of the patients for several weeks(30, 31) . But then the symptoms returned because the high stability of antibodies in plasma increased the level of IL-6, which normally is cleared from the circulation within several minutes(32) . No beneficial effect can be expected by using neutralizing monoclonal antibodies against IL-6Ralpha(33) . A natural soluble form of this receptor subunit together with IL-6 has been shown to behave as an agonist(19, 20, 21) . Neutralizing antibodies could lead to an accumulation of the soluble IL-6Ralpha in the plasma. Neutralizing monoclonal antibodies against the signal transduction protein gp130, which is also part of the receptor of leukemia inhibitory factor, ciliary neurotrophic factor, oncostatin M, and IL-11(34) , could provoke a lot of side effects in vivo. We therefore believe that the construction of IL-6 receptor antagonists is the most promising strategy to neutralize IL-6 activity in vivo. We have obtained such an IL-6 receptor antagonist, which can completely inhibit human IL-6 activity on human myeloma cells. The availability of such an antagonist may offer an approach to specifically inhibit IL-6 activity in vivo.


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany (to S. R.-J.) and the Netherlands Foundation for Fundamental Research (to J. P. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed. Tel.: 49-6131-173363; Fax: 49-6131-173364.

(^1)
The abbreviations used are: IL, interleukin; PCR, polymerase chain reaction.

(^2)
F. D. de Hon, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. P. Freyer for the synthesis of oligonucleotides, Christa Gerlach for help with the cell culture, Marcel Robertz for excellent assistance with the artwork, and Manfred Dewor for help with the amino acid analysis.


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