EP0833656A2

EP0833656A2 - Pharmaceutical compositions comprising restrictin p/activin a and use thereof as antagonist of il-6 and/or il-11

Info

Publication number: EP0833656A2
Application number: EP96917638A
Authority: EP
Inventors: Dov Zipori; Yigal Burstein
Original assignee: Yeda Research and Development Co Ltd
Current assignee: Yeda Research and Development Co Ltd
Priority date: 1995-06-09
Filing date: 1996-06-09
Publication date: 1998-04-08
Also published as: JPH11507663A; WO1996041607A3; EP0833656A4; WO1996041607A2

Abstract

The pleiotropic cytokine restrictin P/activin A modulates the in vivo activity of IL-6 and/or IL-11 and can be used for the preparation of pharmaceutical compositions useful, for example, for blocking or reducing the deleterious effects of IL-6, e.g. in patients being treated with IL-6 while undergoing tumor therapy or bone marrow transplantation.

Description

PHARMACEUTICAL COMPOSITIONS COMPRISING RESTRICTIN P/ACTIVIN A AND USE THEREOF AS ANTAGONIST OF IL-6 AND/OR IL-11

Field of the Invention

The present invention relates to the plasmacytoma and hybridoma growth inhibitor cytokine restrictin-P, herein identified as being activin A of stromal origin and an antagonist of the cytokines interleukin(IL)-6 and/or IL-1 1, and in particular to pharmaceutical compositions and methods using said restrictin-P/activin A for modulation of the in vivo activity of IL-6 and/or IL-l 1. Background of the Invention

Regulation of hemopoiesis is mediated by cytokines that act through distinct mechanisms. Some, like colony-stimulating factors (CSFs) promote accumulation of hemo- poietic cells by inducing proliferation coupled with differentiation. Others, like tumor necrosis factor (TNF), may cause cell cycle arrest and thus limits cell accumulation. The outcome of the interaction between the growth factor and the cell often depends on the nature of the target cell; the same cytokine may be stimulatory to one cell type and inhibitory to the other. Whereas some inhibitors operate by slowing down cell growth or by induction of terminal differentiation, others cause cell death by inducing apoptosis.

Restrictin-P has formerly been described by one of the present inventors as an inhibitor of plasmacytoma cell growth (Zipori et al., 1986; Honigwachs-Sha'anani et al,

1991 ). The biological activity of this factor was first noticed through the selective ability of primary stromal cells to slow down the proliferation of plasmacytoma cells (Zipori, 1 80 and

1981). A similar function was exhibited by trypsin-TPCK released proteins obtained by mild treatment of MBA-2.1, a bone marrow derived stromal cell line of mouse origin (Zipori et al., 1986.). The released crude protein mixture inhibited the growth of a series of plasmacytomas and hybridomas but did not have significant effects on the growth of a variety of other leukemia cell lines of lymphoid. erythroid and myeloid origin (Zipori et al., 1986).

Similarly, no effect was observed on normal cell populations such as bone marrow cells responding to colony-stimulating factors or spleen cells induced by mitogens (Zipori et al,

1986). Factor(s) mediating the growth inhibition were found to be produced by the stromal cell line in minuscule amounts and thus, in order to isolate the active component, it was necessary to establish conditions for large scale production of the factor. It was found that the producer cell line MBA-2.1 could be propagated on a three-dimensional carrier of non- woven fabric of polyester loaded in a bioreactor system under complete protein-free conditions (Kadouri et al., 1992). The study of such bioreactors showed that the cells could be maintained under protein-free conditions for up to 10 months while producing restrictin-P activity along with transforming growth factor (TGF)β, macrophage (M)-CSF and EL-6. Restrictin-P obtained from the bioreactor system induced in its target cells early Gi/Go arrest, morphological changes and signs of cell damage (Honigwachs-Sha'anani et al, 1991) accompanied by intracellular ionic changes (Malik et al., 1992).

IL-6 is a pleiotropic cytokine that affects cells in different tissues and organs. IL-6 has growth-promoting effect on plasma-like cells and is involved, within the hemopoietic system, in processes such as induction of growth and differentiation as well as growth inhibition of a variety of hemopoietic cell types (Ikebuchi et al., 1987; Hoang et al., 1988; Bot et al, 1989). It is therefore expected that the activity of IL-6 would be tightly regulated. This may occur on the level of expression of the IL-6 gene as a result of activity of other cytokines or a variety of mediators. In the treatment of human patients with IL-6, e.g. in tumor therapy and in bone marrow transplantation, IL-6 may adversely affect healthy cells and tissues, and thus it is impoπant to block or reduce the deleterious effects of EL-6. Activins are members of a TGFβ family and are involved in the regulation of many biological events. Activin A, also known as FSH-releasing protein (FRP), is identical to erythroid differentiation factor (EDF) that was shown to induce the differentiation of Friend erythroleukemia cells (Murata et al., 1988; Yu et al., 1987; Eto et al., 1987) and to affect normal hemopoietic progenitor cells (Broxmeyer et al., 1988; Nakao et al., 1991). Activin A was found to be expressed by stromal cells (Yamashita et al., 1992). Activin A was further shown to kill the B9 plasmacytoma cells and the U266B1 myeloma cell line (Nishihara et al., 1993). The U266B1 cells are E -6 independent; they grow in medium supplemented with serum and do not require IL-6 for growth. In this same publication, the autors showed in short term experiments that B9 cells grown with or without IL-6, died to the same extent when exposed to activin A. It is further stated in said publication that EL-6 did not prevent apoptosis of B9 cells induced by activin. The conclusion is that although activin A kills plasmacytoma cells, this effect is unrelated to IL-6. Compositions comprising hoπnonally active human and porcine activin A as a product of recombinant host cell expression are described in US Patent No. 4,798,885 granted to Mason et al. US Patent No. 4.973,577 granted to Vale, Jr. et al. discloses a method of treatment of a vertebrate animal so as to increase the secretion of FSH therein, which method comprises administering an effective amount of a protein which is a dimer of activin A. US Patent No. 4,997,815 granted to Perrine et al. describes a method for inhibiting the γ-globin to β-globin switching in subjects afflicted with β-globin disorders which comprises introducing to said subject periodically during his lifetime a compound selected from activin, inhibin or a chain thereof. US Patent No. 5,032,507 granted to Yu et al. describes a method of increasing the population of erythrocvtes in a human which comprises contacting progenitor cells of said human with FSH-releasing protein (FRP, identical to Activin A). US Patent No. 5,102,868 granted to Woodruff et al. discloses a method for inhibiting the maturation of follicles in the ovary of a female mammal which comprises administering to the ovary of the mammal an effective amount of activin A. According to the present invention, it is herein shown for the first time that the pleiotropic cytokine activin A is identical with restrictin-P and that this molecule is an antagonist of EL-6 and/or EL-11. Summary of the Invention

Accordingly, the present invention provides a pharmaceutical composition comprising restrictin-P/activin A as active ingredient together with a pharmaceutically acceptable carrier, for modulation of the in vivo activity of DL-6 and/or LL-11.

In a preferred embodiment the composition is intended for blocking or reducing the deleterious effects of LL-6, for example in patients receiving IL-6 during tumor therapy or bone marrow transplantation. In another aspect, the invention relates to the use of the cytokine restrictin-P/activin

A for the preparation of a pharmaceutical composition for modulation of the in vivo activity of EL-6 and/or EL-11, for example in cases of abnormal or excessive proliferation of IL-6- or H-11 -dependent cells.

In a further aspect, the invention relates to a method of modulating the in vivo activity of IL-6 and/or EL- 11 , which comprises administering to a patient in need thereof an effective amount of restrictin-P/activin A. In particular, the method is suitable for blocking or reducing the deleterious effects of IL-6 in patients undergoing tumor therapy or bone marrow transplantation.

Brief Description of the Drawings

Figs. 1A-D show a series of elution profiles constituting the final purification of restrictin-P from stromal cell conditioned medium as described in Example 2, wherein Fig.

1A is an elution profile on a Q-Sepharose anion exchange chromatography column; Fig. IB on a Superdex 75 gel filtration; Fig. 1 C on a C-8 Reversed Phase HPLC-I (RP-HPLC I), and

Fig. ID on a C-8 Reversed Phase HPLC-II (RP-HPLC II) column.

Figs. 2 A-B show analysis of highly purified restrictin-P. Fig. 2A depicts the sequence that was obtained for restrictin-P as compared to the known sequence of activin A.

The Xs at positions 4, 1 1 and 12 are the expected positions of cystein residues which are usually undetectable in the sequencing machine, and the X in position 25 corresponds to a

Trp residue that could not be detected. Fig. 2B shows SDS-PAGE analysis of highly purified restrictin-P obtained from the RP-HPLC II column, as described in Example 2. Fig. 3 shows inhibition of MPC-1 1 plasmacytoma cell growth by purified restrictin-P

(triangles) and recombinant bovine activin A (circles) or control growth medium (x), as described in Example 3.

Figs. 4A-B show that restrictin-P antagonizes IL-6 (4 A) and IL-1 1 (4B) induced proliferation of B9 cells, as described in Example 4. B9 cells were treated with the indicated dilutions of recombinant human IL-6 (4A, open symbols) or recombinant human IL-l 1 (4B, open symbols) or with purified restrictin-P and IL-6 (4 A, closed symbols) or IL-l 1 (4B, closed symbols.

Figs. 5A-B show that restrictin-P does not interfere with the binding of IL-6 to B9 cells, in an experiment as described in Example 5. Figs. 6 depicts a Western blot showing that restrictin-P interferes with the IL-6 induced secretion of the acute phase proteins α-acid glycoprotein (AGP) and haptoglobin

(HG) in HepG2 hepatoma cells, as described in Example 6.

Fig. 7 shows that in HepG2 hepatoma cells restrictin-P did not interfere with STAT activation involved in EL-6 signaling. The amounts of either Stat3 and Statlα homodimers or Stat3 /Statlα heterodimers were followed under different incubation conditions as described in Example 7 Detailed Description of the Invention

According to the present invention, the activity, designated as restrictin-P, found in media conditioned by stromal cells, which causes growth arrest and subsequent cell death of mouse plasmacytomas and hybridomas, was found to be mediated by a protein that was purified to homogeneity from medium conditioned by the stromal cell line MBA-2.1 and said protein was found to have an N-terminal amino acid sequence identical to that of activin A monomer. Activin A was found to be expressed by stromal cells (Yamashita et al., 1992). Since the molecular weight of monomeric restrictin-P, as deduced from PAGE, was 15 kDa, a size similar to that of monomeric activin A (βA inhibin), it was concluded that these two molecules are identical. Indeed, recombinant activin A is shown hereinafter to be inhibitory to the MPC-11 plasmacytoma to the same extent as restrictin-P.

Restrictin-P in its purified form killed the factor dependent hybridoma cell line B9 by competing with externally added IL-6 or IL-l 1. On the basis of the inability of a 270 fold excess of partially purified restrictin-P to compete with r-Mu-[^-^I)-IL-6 binding (Fig. 5 A) or a 340 fold excess of highly purified restrictin-P to compete with r-Hu-[^^I]-EL-6 Viuteine (Fig. 5B) binding, it is concluded that restrictin-P does not exert its effect by competing with H-6 for high affinity IL-6 ligand binding sites.

The antagonistic effect of restrictin-P is specific to IL-6 and IL- 11 since restrictin-P did not affect the growth of other cytokine-dependent cell lines such as 14M1.4 macrophages that depend on M-CSF for growth, MC/9 mastocvtoma which are IL-3 dependent or NFS-60, GM-CSF dependent cells (results not shown).

The strict specificity of killing by restrictin-P of plasmacytomas and hybridomas suggested that the factor detects some molecular machinery characteristic to this cell type. The growth dependence on EL-6 is a characteristic of many plasmacytomas and hybridomas. Indeed we show here that restrictin-P inhibits the growth of B9 cells by competing with the growth factors obligatory for the survival of the hybridoma. However, some cells like the MPC-11 clone, are cytokine independent, but their growth is nonetheless inhibited by restrictin-P.

Plasmacytomas are but one target cell type that responds to IL-6 signalling. The HepG2 hepatoma cells release acute phase proteins under the influence of IL-6 (Gauldie et al., 1987). an activity that can be inhibited by restrictin-P. In addition, restrictin-P can abolish both the high mitochondrial and growth inhibition activity of Ml myeloblastic cells and their differentiation into adherent monocytes, effects that are induced by IL-6. Restrictin-P is thus an universal antagonist to IL-6, inhibiting IL-6 activity in all systems in which IL-6 is active.

Restrictin-P, herein identified as a stromal activin A, is an antagonist of IL-6 and/or IL-l 1, which are growth factors for plasma-like cells, but it is not restrictive to other cells that depend on alternative growth factors.

Restrictin-P/activin A can thus be used, for example, in the treatment of immune disorders. As an antagonist of the growth factors required for the survival and functioning of normal plasma cells, it can be used to reduce the number and suppress the activity of these cells in pathological immune conditions in which the antibody is contributing to the disease. Thus, restrictin-P/activin A is recommended in each case where it is required to kill plasma cells without harming other cell types, thus avoiding the use of cytotoxic drugs and immunosuppressive agents with a wide spectrum of target cells. The following categories of immune disorders can be treated with restrictin P/activin A: a. Hypersensitivity type I: IgE antibodies sensitized mast cells produce an inflammatory reaction with symptoms such as asthma or rhinitis. The cytokine restrictin-P/ activin A may be used to reduce the load of viable antibody producing plasma cell. b. Hypersensitivity type II: antibody-dependent cytotoxicity by phagocytosis, killer cell activity or complement-mediated lysis. For example, antibodies to the cell surface of erythrocytes cause hemolytic disease of the newborn (anti Rh) and autoimmune hemolytic anemias of adults. Hyperacute graft rejection is also mediated by antibodies. The disease myasthenia gravis is due to antibodies to acetylchoiine receptors. As in the hypersensitivity type I disorders, restrictin-P should reduce in these cases the burden of plasma cells. c. Hypersensitivity type III: immune complex diseases. Complexes between antibodies and antigens may occur in chronic hepatitis B, in systemic lupus erythematosus etc.. Restrictin-P may be used in these cases to reduce the number of complex-producing plasma cells without the need of using cytotoxic drugs that would affect other lineages. d. Some autoimmune diseases, like thyroiditis, are known to be mediated by autoantibodies and restrictin-P/activin A may be used in these cases as well.

In another aspect, restrictin-P/activin A may be used to treat abnormal immuno- globulin synthesis-plasma cell dyscrasias. A series of diseases are associated with accumulation of a monoclonal immunoglobulin. Some of these situations are controlled and the amount of the monoclonal protein is tolerated well. Other situations result in severe diseases and cytotoxic drugs are used to suppress plasma cells. Restrictin-P/activin A may be a much better approach due to its killing specificity to plasma-like cells. The conditions that may be treated include chronic cold agglutinin syndrome, heavy-chain diseases, papular mucinosis, pyoderma gangrenosum. Waldenstrom macroglobulinemia, and immunoglobulin- related amyloidosis. In one preferred embodiment, restrictin-P/activin A will be used for modulation of the in vivo activity of EL-6, a pleiotropic cytokine known to be a regulator of hemopoietic stem cell proliferation and differentiation, and affecting many other cell types. Treatment of human patients with IL-6 faces therefore the problem of side effects caused by EL-6 activity on cells in which the treatment is not directed to. The use of restrictin-P/activin A is meant to block or reduce the effect of IL-6 upon in vivo administration, in cases such as treatment with LL-6 in the course of bone marrow transplantation and tumor therapy.

In another aspect, as an antagonist of IL-6 and EL-11, restrictin-P/activin A may be useful in immunomodulation in the case of the immune disorders described in a-d above.

The present invention encompasses the use of any activin A molecule, native as well as recombinant activin A from different species, such as human, porcine, bovine and rat activins. There is 100% amino acid conservation among these activins. Native and recombinant activins of different species are described in US 4,798,885 (Mason et al., assigned to Genentech), US 4,973, 577 (Vale, Jr. et al.), US 4,997,815 (Perrine et al.), US 5,032.507 (Yu et al.), US 5,071,834 (Burton et al., assigned to Genentech) and US 5,102,868 (Woodruff et al., assigned to Genentech), all of which are herein incorporated by- reference in their entirety.

The compositions to be used in the present invention will be formulated and dosed according to standard procedures, for example as described in US 5,102, 868, herein incorporated by reference. The daily dose will depend from the disease to be treated and the condition of the patient and will be in the range of about lμg/kg body weight to about lmg/kg body weight. In a preferred embodiment, the restrictin-P/activin A is formulated with a parenteral pharmaceutically acceptable carrier, e.g a solution that is isotonic with the patient's blood, such as phosphate-buffered saline, and is administered by injection, The invention will now be illustrated by the following non-limiting examples. EXAMPLES Materials and Methods

(i) Cell cultures: The MBA-2.1 stromal cell line (Zipori et al., 1984) was grown in 100 mm plates (Falcon 303, Oxnard, CA) and maintained by weekly passage in growth medium composed of Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% heat inactivated fetal calf serum (FCS) (Biolabs, Jerusalem. Israel). 14M1.4 macrophages (Zipori et al., 1984) were grown in the above mentioned medium, with the addition of 20% L-cell conditioned medium as a source of M-CSF. MPC-11 (Laskov and Scharff, 1970), SP-2, NSO, X-24, X-63 and P3.1 (Kohler and Milstein, 1976) plasmacytoma cell lines were grown in Roswell Park Memorial Institute (RPMI) 1640 (Gibco) with 10% FCS. ABLS-8 (Sklar et al., 1975) and AVRij-1 (Zipori and Bol, 1978) pre-B lymphoma cells and the T lymphomas BW-5147 (Hyman and Stallings, 1974), the myeloid tumor cell line WEHI-265.1 (Warner et al., 1965), the F4N Friend erythroleukemia cell line and the Pu-5-lR macrophage cell line (Ralph et al., 1976) were all grown in RPMI 1640 with 10%) FCS and 50 mM β-mercaptoethanol. B9 B cell hybridoma cells (Helle et al., 1988) were grown in RPMI 1640 supplemented with 10 % FCS, 50 mM β- mercaptoethanol and 10 IU/ml human recombinant IL-6. M1-S6 (a subclone of a mouse myelomonocytic leukemia Ml cell line (Ichikawa, 1969) was grown in RPMI 1640 supplemented with 10% FCS. HepG2 hepatoma cells were grown in DMEM supplemented with 8% FCS. All cell lines were incubated at 37°C in a humidified atmosphere of 10% CO2 in air, except for B9 cells which were incubated in 5% CO in air- (ii) Cytokine Biological Assays: Restrictin-P was monitored using either MPC-11 plasmacytoma or B9 hybridoma cells. MPC-11 cells were seeded at 8x10^ cells/ml in 96- well microtiter plates (100 mi/well) (Costar, Cambridge, MA) in RPMI supplemented with 7.5% FCS in the presence of serial dilutions of the restrictin-P containing samples, or buffer (20 mM Tris-HCl pH 7.8 or 20 mM Hepes pH 7.8). Viable cell number was determined following 4 days of incubation using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, which measures cell viability via mitochondrial activity. MTT (Sigma) (10 μl 100 μl medium; stock solution of 5 mg/ml in PBS) was added to each well, and plates were incubated at 37°C for 3 h. After incubation, 150 μl acid-isopropanol was added to each well. Optical density (OD) was then determined in an enzyme-linked immunosorbent assay (ELISA) reader (Titertek Twinreader; FLAB, Helsinki, Finland), using a test wavelength of 570 nm and a reference wavelength of 630 nm. One unit of activity was designated as the amount of protein which, under the above conditions, caused 50% growth inhibition relative to the control. The assay was essentially the same using B9 cells except that the culture conditions were as indicated above for the B9 hybridoma. The latter was also used to titrate IL-6 and IL-l 1 levels. Briefly, B9 cells (5xl0³ cells/200 μl) were cultured in 96 well plates in the presence of test samples and recombinant human IL-6 and

EL-11 controls. At 64 h of incubation, the cells were pulsed with -'H-thymidine (Rotem

Industries, Israel, 1 μCi/well) for 16 h. IU IL-6 and IL-l 1 were determined by relating to standard curves of human recombinant IL-6 and IL- 11 , respectively. (iii) Cytokines and Corresponding Neutralizing Antibodies: The antibodies to TGF-β used were rabbit anti-native porcine platelet TGF-β 1. neutralizing for both TGF-β 1 and

TGF-β2. These antibodies were purchased from British Biotechnology Ltd. (Abingdon,

Oxon, England). Human TGF-β 1 was obtained from the same source. Hamster anti-murine

IFN-γ antibodies were from Genzyme Corporation (Boston, MA). IL-3 was purchased from Peprotec. Recombinant human, and monoclonal rat anti-mouse neutralizing antibodies to mouse IL-6 were purchased from Genzyme Corporation (Boston, MA). Recombinant, N- terminally truncated, human EL-6 (Muteine) and basic fibroblast growth factor (bFGF) were kindly provided by Pharmacia Biocenter (Nerviano, Italy). Crude concentrated murine IL-6 was kindly provided by Dr. J. Lotem, Weizmann Institute, and murine EL-6 was obtained from Dr. J. Van Snizk, Ludwig Institute for Cancer Research, Brussels. Goat anti-M-CSF neutralizing antiserum was kindly provided by Dr. R.E. Stanley from the Albert Einstein

University. Recombinant mouse IL-lα, IL-2, IL-4 and E -7, and recombinant human IL-10,

EL- 11 and PDGF were purchased from Genzyme Corporation (Boston, MA). Human G-

CSF was kindly provided by Dr. S. Gillis of Immunex Corporation, Seattle. Recombinant bovine activin A was purchased from Innogenetics (Belgium).

(iv) Labeling of IL-6: EL-6 was labeled as previously described (Chen et al., 1991). Recombinant (r)-murine EL-6 (10 μg) or r-human EL-6 Muteine (10 μg) in 100 μl buffer (100 mM K phosphate pH=7.0), 100 mM Na acetate, 300 mM NaCl) was added to 20 μl of Chloramine T (0.5 mg/^'ml in PBS, phosphate buffered saline, no Ca ^-1", no Mg^"^) that had been preincubated for two minutes at 4° C with 1 mCi ¹²⁵I. Na metabisulfate (20 μl, 1 mg/ml in PBS) was added 45 seconds later immediately followed by 100 μl of KI (10 mg^'ml in PBS). The [¹²⁵I]-IL-6 in PBS with 0.25% gelatin and 0.02% Na azide was separated form free iodine using medium course Sephadex G-25 (Pharmacia).

(v) Competition Binding on B9 Cells: r-Mu-[^{1 5}I]-IL-6 (47,800 cpm 500 μl) was incubated with 2.5x10^ B9 cells for 1 hour on ice in binding buffer (RPMI, 20 mM Hepes, S% fetal calf serum, 0.02% Na azide). r-Hu-[¹²⁵I]-IL-6 (210,000 cpm 500 μl) was incubated with 3x10^ B9 cells for 30 minutes at 37° C in binding buffer with 0.1% Na azide. The B9 cells have been maintained in IL-6 free medium 24 hours before the assay and were washed 3 times with binding buffer before use. The amount of r-Mu-[^~^l]-IL-6 or r-

Hu-[l²5i]_iL-6 used was 30-40% of the amount that gave saturation binding. Under these conditions there were 2,070+530 high affinity binding sites and the Mu-IL-6 and Hu-IL-6 bound with apparent Kd's of 5.5x10^" 10 and 1.1x10^"^ M. respectively, as determined by the LIGAND program (Munson and Rodbard.1980). Specific high affinity binding was competed with varying concentrations of crude concentrated mouse IL-6, r-Hu-IL-6 Muteine, partially purified or highly purified restrictin-P preparations as indicated. The results of the competition binding studies were plotted as a function of fold excess where one unit of restrictin-P inhibits one unit of EL-6 by 50% on B9 cells. (vi) Protein Purification: MBA-2.1 conditioned medium (crude material: 1400 ml, 3200 mg protein, containing 2.5x10° units of restrictin-P) was prepared in a protein-free bioreactor system as previously reported (Kadouri et al., 1992). Aliquots of 90 ml were loaded on a preparative Q Sepharose column (100 ml, XK 50), using an FPLC system (with a UVM-II detector, Pharmacia LKB, Sweden). The loaded column was washed with 1200 ml of the initial buffer (20 mM Tris-Cl pH 7.8), at a flow rate of 8 ml/min. Restrictin-P was eluted with 400 ml of 0.05 M NaCl in the initial buffer, at the same flow rate. Elution of proteins was followed by their absorbency at 280 nm. The salt-eluted material was filter- sterilized and then desalted and concentrated using reversed phase high performance liquid chromatography (RP-HPLC, the system consisted of Model SP8700/SP8750 pump, Spectra- Physics, CA, and model 441 detector from Waters Associates, MA). Each sample (450-500 ml) was loaded onto an Aquapore RP-300 column (7 μ, 4.6 x 100 mm + 4.6 x 30 mm pre- column, Applied Biosystems, CA), at a flow rate of 1-3 ml/min. The loaded column was washed with 30 ml of an aqueous solution of acetonitrile (ACN, 5%) and trifluoroacetic acid (TFA. 0.1%1 Proteins were then eluted with a three step gradient of aqueous ACN in 0.1% TFA at a flow rate of 0.5 ml min: 5-35% ACN/10 min followed by an isocratic step of 35% ACN/20 min and finally 35-80% ACN/20min. Fractions of 1 ml were collected. Elution of proteins was followed by their absorbency at 214 nm. Protein content was determined according to Bradford reagent (Bio-Rad, Munich) and restrictin-P content was assayed as described above. Fractions were vacuum dried in a Savant SpeedVac centrifuge and kept at -20°C. Fractions with restrictin-P activity were dissolved in 20 mM Hepes (pH 7.8), pooled, centrifiiged, filtered and loaded onto a Superdex 75 gel filtration column (20/60, Pharmacia) in batches of about 10 mg protein in 5 ml each. The column was washed with 360 ml of 2 x PBS at a flow rate of 2 ml/minute/tube. Elution pattern was followed at 280 nm. Protein and restrictin-P content was measured as described above. Protein samples were further purified by RP-HPLC. The biologically active fractions from the gel filtration step (3.0-3.5 mg protein) were pooled and loaded onto an RP-300 column (7 μ, 4.6 x 100 mm) and the column was washed with an aqueous solution of 5% ACN in 0.1% TFA, at a flow rate of 0.5 ml/min. Proteins were then eluted with a multi-step linear gradient of aqueous ACN in 0.1% TFA. The gradient composition was 5-25%> ACN/5 minutes followed by 25-40%o ACN/30 min, and then 40-80% ACN/2 min and an isocratic step of 80% ACN. Fractions of 0.5 ml were collected every minute. Elution of proteins was followed by their absorbency in 214 nm. Biologically active fractions were pooled, vacuum dried and rechromatographed (RP-HPLC) under essentially identical conditions, except for the following minor modification: when the protein peak started to elute (usually at ca. 36% ACN) the gradient program was put on "hold". When the protein peak was completely eluted from the column (within ca. 5-8 min) the original gradient program was resumed.

Biologically active fractions were vacuum dried and kept frozen at -20 C. Polyacrylamide gel electrophoresis (PAGE) analyses of different protein fractions was performed using a Mini-Protean II gel apparatus (Bio-Rad Laboratories, CA). Silver staining was performed with a Quick-Silver kit (Amersham, UK) or a Silver Stain Plus Kit (Bio-Rad Laboratories, CA). Coomassie brilliant blue staining was performed using the Serva blue G stain. EXAMPLE 1. Target cell range of restrictin-P activity in conditioned medium of the stromal cell line MBA-2.1

Table 1: Target cell range of restrictin-P activity in conditioned media of the MBA-2.1 stromal cell line

Cell line % Growth with restrictin-P (μg/ml)

Cell type Name 0.6 5 40

Plasmacytoma MPC-11 1.7+0.004 0 0

-"- SP2 96.9+0.042 29.7+0.019 0.2±0

-"- X24 109.6 0.005 65.3±0.004 44.0±0.014

-"- X63 1 13.6±0.045 35.9±0.015 10.2±0.01 1

-"- NSO 34.9+0.008 0.4±0 0.5+0

-"- P3 47.0+0.03 0.2+0 0.2+0

Erythroleukemia F4N 96.6+0.07 88.5+0.01 62.9+0.015

Myeloid cell line Ml 83.7+0.012 80.2+0.007 94.4±0.016

WEHI-265.1 163.7+0.05 1 15.6+0.09 148.2+0.06

Pre-B lymphoma AVRij-1 108.9+0.03 116.9+0.08 97.2±0.03

T lymphoma BW-5147 95.6+0.02 96.3+0.01 63.1±0.015

Macrophage cell line Pu-5-lR 114.0±0.02 146.4+0.02 146.5±0.01

The various cell lines shown in Table 1 were grown as indicated in Materials and Methods, section (i). Cells were seeded in 96-well microtiter plates at 400 cells/100 μl/well.

Partially purified conditioned medium from MBA-2.1 cells was added in the concentration indicated. Cultures were incubated for four days and viability was measured by the MTT colorimetric assay. Values represent the mean of triplicates + SD.

As shown in Table 1, the inhibitory activity of restrictin-P, as detected in conditioned media from MBA-2.1 cells, was specific to plasma-like tumor cell lines. A variety of other cell lines representing different hemopoietic lineages and stages of maturation were only slightly inhibited or were totally unaffected by this factor. To rule out the possibility that restrictin-P activity could be ascribed to one of the cytokines known to affect hemopoietic cells, we searched for factors that might have restrictin-P like activity. IL-l, IL-2, IL-3, EL- 4, IL-6. IL-7, H-10, IL-1 1, M-CSF, G-CSF, TGF β l, PDGF, bFGF, JTN-γ and LU were tested over a range of concentrations. These cytokines were found to be devoid of the ability to inhibit the growth of the MPC-1 1 plasmacytoma which is highly sensitive to restrictin-P (Zipori et al., 1986). In addition, neutralizing antibodies to TGF βl and β2, TNF, IL6, DrN-γ and M-CSF did not reduce restrictin-P -like activity in media conditioned by MBA-2.1 cells (results not shown).

The above conditioned media were used in an attempt to obtain some clue as to the mechanism by which the inhibition of plasma-like cells is mediated. The MBA-2.1 cells conditioned medium inhibited the growth of the MPC-1 1 plasmacytoma and, as detailed below, it also interfered with the growth promoting effect of IL-6 on B9 cells.

EXAMPLE 2. Purification of restrictin-P

To determine whether these two functions were mediated by the same molecule it was necessary to purify restrictin-P to homogeneity. We therefore constructed a bioreactor production system wherein restrictin-P activity could be observed in media conditioned by the cells in absolute protein-free conditions (Kadouri et al., 1992). A batch of 600 L of conditioned medium was concentrated by diafiltration and subjected to a further step of concentration by A icon ultrafiltration followed by fractionation using an automated FPLC column. The fractionation included anion exchange chromatography, gel filtration and finally two steps of purification to homogeneity by reverse phase HPLC.

The purification of restrictin-P from stromal cell conditioned medium is depicted graphically in Figs. 1A-D. Further details can be found in Material and Methods, section (vi). In the Q-Sepharose anion exchange chromatography (Fig. 1A), processed conditioned medium of MBA-2.1 cell line containing l .SxlO⁵ units of crude restrictin-P in

90 ml, was loaded on a preparative column of Q-Sepharose ( 100 ml, XK 50). The column was washed with 1200 ml of the initial buffer (20 mM Tris-Cl, pH 7.8), at a flow rate of 8 ml/min and then restrictin-P was eluted with 400 ml of 0.05 M NaCl in the initial buffer. Fractions with restrictin-P activity (eluted at 160-220 min) were pooled, desalted and concentrated by RP-HPLC.

In the Superdex 75 gel filtration (Fig. IB), fractions containing restrictin-P from the Q-Sepharose column, were dissolved in 20 mM Hepes (pH 7.8), and loaded (10 mg protein in 5 ml) onto a Superdex 75 gel filtration column (20/60, Pharmacia). The column was washed with 360 ml of 2 x phosphate buffered saline (PBS) at a flow rate of 2 ml/minute. Fractions of 8 ml were collected. Biologically active fractions (102-106 min) were pooled. In the C-8 Reversed-Phase HPLC-I (RP-HPLC I, Fig 1C), biologically active fractions from the Superdex 75 column were loaded onto an Aquapore RP-300 column (7 m, 4.6 x 100 mm + 4.6 x 30 mm pre-column), at a flow rate of 1-3 ml/min. The adsorbed proteins were eluted from the column with a multi-step gradient of aqueous ACN in 0.1% TFA at a flow rate of 0.5 ml/min: 5-30% ACN/5 min followed by 30-45% ACN/25 min, then 45-80% ACN/2 min and finally an isocratic step of 80% ACN/30. Fractions of 1 ml were collected. Restrictin-P bioactivity was eluted and collected at 31-35 min.

In the C-8 Reversed-Phase HPLC-II (RP-HPLC II, Fig. ID), partially purified restrictin-P was rechromatographed (RP-HPLC) under essentially identical conditions as in the previous step, except for the following minor modification: when the protein peak started to elute (usually at ca. 36% ACN) the gradient program was put on "hold". When the protein peak was completely eluted from the column (within ca. 5-8 min) the original gradient program was resumed. Biologically active fractions were collected at 33-34 min.

In Figs. 1A-D, broken lines indicate the absorption at 280 nm (Figs. 1A and IB) or 214 nm (Figs. 1C and ID); open circles and thick lines show the biological activity of restrictin-P as measured by the MPC-11 assay and thin lines indicate percentage of acetonitrile (Figs. 1C and D).

As can be seen in Fig. 1, the last stage yielded a single peak of protein that coincided with the biological activity of restrictin-P, i.e. the capacity to inhibit the growth of the plasmacytoma cell line, MPC-11. SDS-PAGE analysis of this highly purified restrictin-P was carried out as follows:

An aliquot from the active fraction (RP-HPLC II , Figure ID) was loaded on 15% SDS- poly- acrvlamide gel under reducing conditions (Lane 1). Protein bands were viewed by silver staining (Lane 2). Molecular weight markers are: carbonic anhydrase 31 kDa, trypsin inhibitor 21.5 kDa, lysozyme 14.4 kDa, and aprotinin 6.5 kDa. As shown in Fig. 2B, the SDS-PAGE analysis of this product, under reducing conditions, revealed a single protein band at 15 kDa. This purified peptide was N-terminally sequenced and it was found, as shown in Fig. 2 A. that the first 35 residues were identical to those of the precursor of the β A subunit of inhibin which gives rise to a 15 kDa polypeptide (Mason et al., 1985; Robertson et al., 1992), the dimer of this subunit being activin A.

EXAMPLE 3. Inhibition of MPC-11 plasmacytoma cell growth bv purified restrictin- P and bv recombinant activin A.

Cells were seeded (4xl0-/well) in 96 well Falcon microtiter plates, in 100 μl of RPMI containing 10 % FCS with serial dilutions of restrictin-P (triangles), activin A (circles) or control growth medium (x). Cells were incubated for 4 days and their viability was estimated by the MTT assay. The bar lines represent the mean of duplicate determinations + SD. As can be seen in Fig. 3, it is clear that both factors have equal ability to suppress the growth of MPC-11 cells. This, considered together with results in Example 2, suggests that restrictin-P is identical to activin A.

EXAMPLE 4. Restrictin-P antagonizes IL-6 and IL-I 1 induced proliferation of B9 cells

To study the mechanism by which restrictin-P/ stromal activin A inhibits plasma-like cell growth, we examined whether its effect was mediated by reversible cytostasis or through a mechanism that involves cell destruction. Restrictin-P in its unpurified form appeared to induce ionic changes that are associated with apoptosis (Malik et al., 1992). This mode of cell death is known to occur in hemopoietic cells deprived of their specific growth factor. The B9 hybridoma is dependent for growth on IL-6 and was therefore used to examine the possibility that restrictin-P causes growth cessation by interfering with the action of the growth factor (IL-6).

B9 cells were washed 3 times with RPMI supplemented with 5% FCS (=growth medium), to remove traces of EL-6, and were incubated overnight in growth medium. They were then washed (x3) with growth medium and seeded in 96-microtiter well plates (5x10^J cells/200 μl/well) in growth medium supplemented with the indicated dilutions of recombinant human JX-6 (Fig. 4 A, open symbols) or with the same IL-6 dilutions and 1.5 units/well of purified restrictin-P (Fig. 4 A, closed symbols). A similar experiment was set with recombinant human EL-11. B9 cells were seeded with serial dilutions of this cytokine (Fig. 4B. open symbols) or with EL-1 1 and 0.018 units/well of restrictin-P (Fig. 4B, closed svmbols). This low concentration of restrictin-P is was used since IL-l 1 was a poor

15

SUBSTITUTE SHEET (RULE 2G stimulator of B9 cells and a^' relatively low concentration of restrictin-P was sufficient to cause complete growth inhibition. Cells were incubated for 48 hours and then pulsed with

^JH-thymidine (1 μCi/well) for 12 hours. Values represent the mean of triplicate determinations + SD. Thus, as shown in Fig. 4A, extensive proliferation of B9 cells was induced by 0.1

IU/well of IL-6 and only moderate growth stimulation is observed upon addition of increasing concentrations of the purified cytokine. Addition of restrictin-P to B9 cell cultures stimulated by 0.01-0.1 IU/well of JX-6 caused almost complete growth inhibition.

This inhibitory effect was gradually reduced with increasing concentrations of IL-6 and was almost abolished at 200 IU/ml of IL-6 (Fig. 4A).

IL-l 1 is an additional stimulator of plasma-like cells (Burger and Gramatzaki, 1993).

As shown in Fig. 4B, restrictin-P also inhibited the growth of IL-l 1 stimulated B9 cells and this inhibition was competed out by increasing the titer of IL-l 1.

EXAMPLE 5. Restrictin-P does not interfere with the binding of IL-6 to B9 cells

As shown above, restrictin-P counteracted the growth stimulating effect of IL-6 and IL-l 1 and these cytokines at high titers overcame the effect of restrictin-P. It therefore seems that the growth factors EL-6 and IL-l 1 and restrictin-P are competing on some target machinery used to generate a signaling pathway. A candidate target molecule for restrictin-P action was the IL-6 receptor complex. We studied the possibility that restrictin-P is a receptor antagonist by testing its ability to compete with radiolabled IL-6 for binding to its receptor on the surface of B9 cells.

The amount of r-Mu-[¹²⁵I]-IL-6 or r-Hu-[¹²⁵I]-IL-6 Muteine used (4.8xl0⁴ and l. lxlO⁵ cpm, respectively) was 30-40%) of the amount that gave saturation binding. B9 cells were weaned from IL-6 in the growth medium 24 hours before binding assay. Under these conditions, the cells have 2070+530 high affinity binding sites and both r-Mu-E -6 and r-Hu- L-6 bound with high affinity (apparent Kd's of 5.5x10"^ and l. lxl0^~l° M, respectively).

Fig. 5 A shows specific high affinity binding of a constant amount of r-Mu-[^²^I]-IL-

6 competed with varying amounts of crude concentrated mouse IL-6 (open symbols) or partially purified restrictin-P (closed symbols). Mean total r-Mu-EL-6 bound was 1120+48 cpm. Fig. 5B shows specific high affinity binding of a constant amount of r-Hu-[¹²^I]-IL-6

Muteine (open symbols) or purified restrictin-P (closed symbols). The mean total r-Hu-IL-6 binding was 2350+68 cpm which was competed to 123+30 with a 200 fold excess of crude concentrated murine IL-6. Results of the competition binding studies are plotted as a function of fold excess where one unit of restrictin-P inhibits one unit of JX-6 by 50% on B9 cells. Error bars indicate standard deviation of replicates. As can be seen in Fig. 5, "cold" IL-6 competed out the binding of radiolabeled IL-6 to its receptor as expected. On the other hand, restrictin-P, at concentrations that would completely abolish the growth stimulating effect of EL-6, failed to reduce the binding of radiolabeled IL-6 to its receptor. Thus, restrictin-P does not seem to interfere with ligand binding.

EXAMPLE 6. Restrictin-P interferes with the IL-6 induced secretion of the acute phase proteins α-acid glvcoprotein (AGP) and haptoglobin (HG).

HepG2 hepatoma releases acute phase proteins under the influence of IL-6. HepG2 cells were grown to confluence and stimulated with 100 units/ml of IL-6, or an equal amount of IL-6 with 312 units/ml restrictin-P. Conditioned media were collected at 24 hr, subjected to PAGE (10%) and 7% gels for AGP and HG. respectively) and were tested by Western blotting for the acute phase proteins using the corresponding antibodies (purchased from Sigma, Israel). As shown in Fig. 6, the secretion of both AGP and HG, induced in HepG2 cells by IL-6, was markedly reduced by addition of restrictin-P.

EXAMPLE 7. Effect of restrictin-P on STAT activation.

Janus Kinases (JAK) associate with IL-6 receptor. They couple ligand binding to tyrosine phosphorylation of transcription factors called signal transducers and activators of transcription (STAT). The possible interference of restrictin-P in the JAK/STAT pathways involved in IL-6 signaling was checked in HepG2 cells. HepG2 cells were incubated with human recombinant JL-6 ( 10 units/ml) and restrictin-P as indicated in Fig. 7. IL-6 and restrictin-P were either added simultaneously, or restrictin-P was added 30 min or 16 hr prior to E -6. Fifteen min after addition of IL-6 to the medium, the cells were harvested and nuclear extracts prepared therefrom by standard procedure. Ten μg protein were then analyzed in a gel retardation assay using a ^~2P-labeled oligonucleotide probe covering the c- fos promoter sis-induced element (SIE). The results are shown in Fig. 7, where the positions of Statlα DNA-protein complexes containing either Stat3 and Statlα homodimers. or Stat3/ Statlα heterodimers, are indicated, demonstrating that in HepG2 hepatoma cells restrictin-P did not interfere with the JAK/STAT pathway, and even moderately increased STAT protein activation.

EXAMPLE 8. Restrictin-P interferes with IL-6 augmented mitochondrial activity in Ml veloma cells.

Ml cells were seeded at 5x10⁴ /ml in microliter plates with the addition of restrictin- P and/or JX-6 as ndicated in Table 2. Restrictin-P was added at 130 units/well and EL-6 at 50 units/well. Under these conditions, no net change in cell number occurred at day 3 when cultures were tested by the MTT colorimetric assay (Materials and Methods, section (ii)). Data shown are from one out of 3 experiments performed, all showing similar results. The experiments indicate that restrictin-P interferes with the IL-6 mediated mitochondrial activity of Ml myeloblasts.

Table 2: Restrictin-P (RP) interferes with IL-6 augmented mitochondrial

activity in Ml cells.

Additions Activity (OD)±SE

none 0.185+ 0.05

RP 0.176± 0.024

JX-6 0.354± 0.03

RP+IL-6 0.217± 0.02

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Claims

CLAIMS:

1. A pharmaceutical composition for modulation of the in vivo activity of IL-6 and/or

IL-l 1 comprising restrictin-P/activin A and a pharmaceutically acceptable carrier 2. A pharmaceutical composition according to claim 1 wherein the activin A is selected from porcine, bovine and human activin A.

3. A pharmaceutical composition according to claim 2 wherein the activin A is recombinant human activin A.

4. A pharmaceutical composition according to claim 1 for blocking or reducing the deleterious effects of IL-6.

5. Use of restrictin-P/activin A for the manufacture of a pharmaceutical composition for modulation of the in vivo activity of IL-6 and/or IL-l 1.

6. Use according to claim 5 for blocking or reducing the deleterious effects of EL-6.

7. Use according to claim 5 or 6 wherein the activin A is selected from porcine, bovine and human activin A.

8. Use according to claim 7 of recombinant human activin A.

9. A method for modulating the in vivo activity of IL-6 and/or IL-l 1 which comprises administering to a patient in need thereof an effective amount of restrictin-P/activin A.

10. A method according to claim 9 wherein the activin is selected from porcine, bovine and human activin A.

11. A method according to claim 10 wherein the activin is recombinant human activin A.

12. A method for blocking or reducing the deleterious effects of 11-6 in a patient being treated by IL-6 while undergoing tumor therapy, which comprises administering to said patient an effective amount of restrictin-P/activin A.

13. A method according to claim 12 wherein the activin is selected from porcine, bovine and human activin A.

14. A method according to claim 13 wherein the activin is recombinant human activin A.

15. A method for blocking or reducing the deleterious effects of 11-6 in a patient being treated by EL-6 while undergoing bone marrow transplantation, which comprises administering to said patient an effective amount of restrictin-P/activin A.

16. A method according to claim 15 wherein the activin is selected from porcine, bovine and human activin A.

17. A method according to claim 16 wherein the activin is recombinant human activin A.

18. A method for immunomodulation which comprises administering to a patient afflicted with an immune disorder an effective amount of restrictin-P/activin a.

19. A method according to claim 18, wherein plasma cells are killed without affecting other cell types of the patient.

20. A method according to claim 18 or 19 wherein the activin is selected from porcine, bovine and human activin A.

21. A method according to claim 20 wherein the activin is recombinant human activin A.