INTERLEUKIN

Abstract: A new interleukin called eosinophil differentiation factor has both eosinophil differentiation activity (EDA) and also B cell growth promoting activity of B cell growth factor II. Interleukins of both mouse and human origin have been sequenced and found to be substantially homologous and to have EDA in both mouse and human bone marrow cells. The interleukin is a glycoprotein with a mean MW of about 46000 containing a polypeptide of mean MW about 30000.

Description: DΠΈSLEOKIN This invention relates to an interleukin and in particular to novel compositions comprising the interleukin and to uses thereof. Interleukins are soluble factors which are secreted by leucocytes (white blood cells) and serve either as a means of intercellular communication within the immune system or as effector molecules of immunity. As such interleukins are involved in control of the proliferation, differentiation and functional activation of leucocytes such, as lymphocytes; for instance activation to produce antibody against an invading antigen or to otherwise neutralise adventitious foreign material such as infecting microorganisms. Interleukins, which include lymphokines such as interferon gamma (IFN γ) are currently the subject of intense study in view of their central role as mediators of the immune responses of higher organisms and their consequential potential application as therapeutic agents for influencing these responses. A crucial factor in the development of interleukin research in recent years has been the advances which have been made in recombinant DNA technology. Thus the genes which code for particular interleukins, such as IFN γ and interleukin 1, 2 and 3 (ILl, IL2 and IL3), have been cloned permitting not only the molecular characterisation of these interleukins but also their production by recombinant DNA techniques. This has provided material of defined interleukin composition for further study of biological function as well as a source of material for use in therapeutic applications. For instance, IFN γ has been produced by recombinant DNA techniques and has been proposed and tested as an anti-cancer agent. Early studies of interleukins involved testing of crude cell supernatants, often containing complex mixtures of factors, in imprecise biological assays. Numerous 'interleukins' have been indentified on the basis of their activities in these assays. With the development of preparations of defined interleukin composition and improved assays particular interleukins have been further characterised. In some cases i has been found that differing interleukin activities, originally believed to be caused by different factors, are in fact different properties of the same molecule. For example, IFN γ inhibits viral replication, activates macrophages, increases expression of class 2 MHC antigens and is involved in B cell activation. To date, however, relatively few interleukins e.g. IFN γ and IL1, IL2 and IL3, have been characterised to any extent and even so the biological functions of these factors is poorly understood. Many of the interleukins which have been identified still remain as ill-characterised molecules, often characterised only in terms of their activities in notoriously imprecise biological assays. Some time ago Swain and Dutton (Susan . Swain and Richard W. Dutton, J. Exp. Med., Volume 156, December 1982, pages 1821 - 1834) described a new B cell-growth-promoting activity contained in culture supernatants from a long-term alloreactive T cell line, the Dennert line C.C3. 11. 75 (DL) . They called this activity (DL) BCGF, though the factor responsible for this activity is now generally referred to as B Cell Growth Factor II (BCGF II) . This activity was defined in terms of activities in a series of biological assays and could be assayed on normal B cells or on the BCLi tumour line. Stimulation of the B cell tumour (BCLi) cell line is now an accepted assay used to define ECGF II. This factor has not been definitively characterised. Very recently, Sanderson e_t al. (Colin J. Sanderson, David J. Warren and Malcolm Strath, J. Exp. Med., Volume 162, July 1985, pages 60 - 74) have identified a novel interleukin, present in cell supernatants of certain marine lymphocytes, provisionally called eosinophil differentiation factor (EDF) of molecular weight (M r ) 46,000. This factor was shown to stimulate production of eosinophils in long-term bone marrow cultures probably acting on committed precursors in the bone marrow. The identification of EDF as a novel interleukin was facilitated by preparation of a T cell hybrid (NIMP - THl) which produces EDF but does not appear to produce other interleukins. Eosinophils..are bone marrow-derived cells which are present at low- levels in normal animals but are produced at high levels in association with helminthic infections and allergic responses. The specific role of eosinophils is not well understood, but it is believed that these cells may be important in the immune defenses of animals against helminthic infections. Subsequently EDF has been further characterised and purified and further studies have been conducted which have lead to the conclusion that the B cell growth promoting activity of BCGF II and the eosinophil differentiating activity of EDF are different activities of a single molecule which has been provisionally named EDF (hereinafter referred to as ©f for convenience). Furthermore it has now been shown that purified murine ∑b|F is also active on human cells in a lineage specific way, inducing proliferation, differentiation and activation of eosinophils with no detectable effect on neutrophils indicating the existance of this lymphokine in other species besides the mouse. Further studies of EDF have been published by Lopez et al, J. Exp. Med., Vol 163, May 1986 pp 1085- 1099. . Further studies of BCGF II including an amino acid sequence have been published by Kinashi et al, Nature, Vol 324, 6 November 1986, pp 70-73. In one aspect this invention provides eosinophil differentiation factor (EDF) having both significant eosinophil differential activity (EDA) and. a-lso significant B cell growth promoting activity of B cell growth factor II (BCGF II). In another aspect the invention provides eosinophil differentiation factor ., ( EDF) of mouse or human origin, having significant eosinophil differentiation activity (EDA) in both mice and humans. The invention also includes a novel composition comprising _E$ having an eosinophil differentiation activity (EDA) of at least 5 x 10 3 units. In accordance with the present invention, it has been found that murine ∑Df has the following characteristics. (a) It is a trypsin sensitive glycoprotein; (b ) It is stable at both pH2 and pH12; (c) The glycoprotein is relatively thermally stable, retaining full activity after incubation at 56°C for 30 minutes or more, and retaining partial activity after incubation at 70°C for 10 minutes; (d) It is stable in 2% SDS; (e) It is sensitive to 59» 2 mercaptoethanol; (f) It has an isoelectric point (pi) in the range 4.5 — 5.5 as determined by agarose gel ionising electrophoresis (IEF); (g) It binds reversibly to lentil lectin, and for instance, may be eluted therefrom with 0.15M < ~ methyl mannoside; (h) The glycoprotein has a mean molecular weight of about 46K (range 32-66K) when determind by gel filtration, and of about 44K when determined by SDS, non-reducing polyacrylamide gel electrophoresis (PAGE) (i) The glycoprotein comprises a peptide component of M r about 30 , i.e. corresponding to a polypeptide of about 300 amino acids in length. (When cells which secrete _T- e.g. the T cell clone NIMP-THI, are grown in the presence of the glycosylation inhibitor tunicamycin a polypeptide of M r about 30K is produced in the place of the 46K glycoprotein.) (j) It exhibits both the eosinophil differentiation activity of EDF and the B cell-growth promoting activity of BCGF II. Characteristically, however, murine Ε_*P is indentical to the interleukin which is produced by the murine T-cell hybrid (NIMP-THl). This hybrid cell line appears to produce only £D# and thus serves as a standard, identifying source of ΕS_ψ. This cell line was deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, Wiltshire, U.K., on the 14th January, 1986, and is identified as deposit no. ECACC 86011401. The finding, in accordance with the present invention, that EDF and BCGFII are identical was based on the co-ordinate expression of the two activities by a panel of alloreactive T cell clones and cell line cells, and co-purification through a series of protein separation techniques. The experiments which were conducted are described in detail in the accompanying examples. Eosinophil Differentiation Activity (EDA) may be measured in an in. vitro biological assay in which activity is measured as a function of the level of eosinophil production which it causes. In this assay bone marrow cells from mice previously infected with the helminth Mesocestoides corti are incubated with test material and after 5 days eosinophils produced in the culture are determined by an assay for eosinophil peroxidase. This assay is described in detail by Strath et . al . (Malcolm Strath, David J. Warren _ Colin J. Sanderson, Journal of Immunological Methods, Vol. 83, pp 209-215 (1985)). The Units of EDA activity, as . used above and elsewhere in the present description, correspond to the reciprocal of the dilution giving 50% of maximum activity in this assay. These Units may be expressed in the form of a pure number e.g. 5 X 10 Units, or in terms of the protein concentration when this is known e.g. 5 X 10 Units/μg. The Εbf containing compositions of the present invention may be prepared by culturing cells which produce H_ . Suitable cells include T cell clones such as NIMP-Tl and NIMP-T2 (Sanderson et , . al.., J. Exp. Med., Vol 162, July 1985, pp60-74) and ΕD-f producing T cell hybrids especially the T cell hybrid NIMP-THl (Warren D. J. _ Sanderson C. J., Immunology 1985, 54., 615-623) which produces no other lymphokine besides __*-. Methods for culturing T cell clones and T cell hybrids to produce __t are described in the above mentioned Sanderson e_t. al.. and Warren & Sanderson publications. ~ B_*t- may also be prepared by culturing host cells which have been transformed with genes which code for 1D£ production. IDf is recovered from the producing cells e.g. in the cell supernatant, and then may be purified and concentrated to give the H_φ containing compositions of the invention. Thus the invention includes processes for the production of the IDf containing compositions of the invention comprising culturing ϊDf producing cells, recovering Εύ produced by the cultured cells and, if required, purifying and concentrating the recovered _t_ . Any suitable method may be used for the purification and concentration of Εbψ, including methods which are well known in the field of protein chemistry. However preferred methods for the purification of Hb-f from culture supernatants comprise: a first step involving treatment with ammonium sulphate and centrifugation; a second step involving lentil lectin chromatography; a third step involving high performance gel filtration; a fourth step involving reversed phase high performance liquid chromatography, and a fifth stage involving non-reducing polyacrylamide gel electrophoresis (PAGE) It will be appreciated, however, that variants of this procedure may also be used. Using such preferred methods, purified Erf compositions according to the invention have been prepared. Preferably such compositions have eosinophil differentiation activities (EDAs) in excess of 10,000 U/μg and especially comprise substantially pure IL4 e.g. compositions having EDAs of about 10 U/μg. Thus the compositions of the invention comprise ∑Df compositions having EDAs preferably greater than 10 U/μg, and especially Ε_*~ compositions comprising substantially pure —_ * V e.g. having an EDA of about 10 U/μg. The reason why H_£ has the widely differing biological activities that it does is not clear. However, we believe that EDf may be involved in antibody switching; for instance, as _ switch factor which controls switching of antibody isotype production. This holds up the possibility that __ may be useful in monoclonal antibody production; for instance to prime B cells into a particular antibody producing state prior to fusion with myeloma cells or EBV transformation. Generally also it is envisaged the ED** will have therapeutic applications; for instance for regulating the immune response e.g. promoting eosinophilia. In this latter respect we have found that murine D*f is active on human cells in a lineage specific way inducing proliferation, differentiation and activation of eosinophils with no detectable effect on neutrophils. Also we have found that unglycosylated iDf (30K) retains biological activity. Thus _)ψ from one species may be used for treatment of another species and therapeutic compositions may comprise the unglyocosylated form of _ * - . Thus the present invention includes therapeutic compositions comprising H> and also includes therapeutic and other uses of H>f. We have now applied the techniques of recombinant DNA technology to J32ψ, to obtain DNA sequences which code for E-Vf, to identify these sequences and the corresponding amino acid sequence (s) of IL4, and thus to provide processes for the production of ID4 5 of defined composition for further study of the biological function of t f as well as material for use in therapeutic applications. Accordingly, the invention also includes an B_Vf polypeptide produced in host cells which have been transformed with DNA which codes for production of the £D^ polypeptide. The _4_*f polypeptides of the invention characteristically comprise an £^p amino acid sequence; i.e. the amino acid sequence of an authentic mammalian ID 1 or an authentic mammalian EDf amino acid sequence substituted or modified to provide a functionally equivalent product. Preferably the J *f amino acid sequence comprises at least 90% homology, especially at least 95% or greater homology, with an authentic mammalian __*f amino acid sequence. The E> polypeptides may also comprise biologically active fragments of complete E_^ amino acid sequences which possess ΣDf functional activity. In addition to the E_> amino acid sequence, the _ ^ψ polypeptides may comprise one or more additional amino acids. Thus, for example, the _O$ polypeptide may comprise a methionine ID polypeptide, i.e. a polypeptide having an N-terminal methionine amino acid residue in addition to the bf amino acid sequence. Preferably the methionine residue is adjacent the N-terminal amino acid of the E&f amino acid sequence, though may be separated therefrom by one or more amino acids provided the product possesses E>P functional activity. When the _t)ψ polypeptide comprises a methionine Εbf polypeptide, the host cells are preferably bacteria, e.g. E.coli, or yeast, e.g. Saccharomvces Also, the _x_ € polypeptide may comprise an Eϋ^ precursor polypeptide. For example, the ΕH-f polypeptide may comprise a pre-E&£ polypeptide, i.e. a polypeptide having an N-terminal ED4- pre-amino acid sequence in addition to the JD^ amino acid sequence. When the polypeptide is a pre-∑T_^ polypeptide, the host cells are preferably capable of cleaving the ρre-ED polypeptide to produce a corresponding mature Ε f amino acid sequence polypeptide, and especially secrete the mature polypeptide to the culture medium. For example, the host cells may comprise mammalian tissue culture cells. Alternatively, the __>f polypeptide may comprise an J-D* fusion polypeptide i.e. a polypeptide comprising a heterologous polypeptide in addition to the Ε φ amino acid sequence. The heterologous polypeptide may comprise all or part of a polypeptide o protein capable of production, advantageously at high level, in the host cells. Suitable such proteins include β-galactosidase, chloramphenicol acetyl transferase (CAT) and the product of the trpE gene. The fusion polypeptide preferably includes a site susceptible to selective chemical or enzymic cleavage between the heterologous polypeptide and the E^f 1 amino acid sequence. Preferred host cells for production of ∑_yf fusion polypeptides are bacteria (e.g. E.coli) and yeast (e.g. S.cerevisiae) cells. In a particular embodiment when the polypeptide is a fusion polypeptide and the host cells are yeast cells, the heterologous polypeptide may be a yeast signal sequence. . In this embodiment the yeast host cells may advantageously cleave the fusion polypeptide to produce the corresponding mature _T_<f polypeptide. Thus in a further aspect the invention provides an Σ ^ polypeptide comprising a methionine _ύ*P polypeptide, a pre~ID4F polypeptide or an fl^f fusion polypeptide. Advantageously, the E>£ polypeptide of this aspect of the . appreciated, however, that —_~f polypeptides, in particular polypeptides comprising an ED amino acid sequence or a fragment thereof which possesses TDf functional activity, may be prepared by peptide synthesis techniques. Such synthetic ΕS polypeptides are included within the scope of the present invention. The Eύf polypeptide typically comprises an __lψ amino acid sequence of mammalian origin. For instance, the polypeptide may comprise the amino acid sequence of a murine I&f. Preferably, however, the polyp-ptide comprises a human E amino acid sequence. In another aspect, the invention provides DNA which codes for production of an E&f polypeptide. The DNA of this aspect of the invention preferably comprises DNA coding for an Ebf polypeptide according to the above aspect of the invention. DNA which codes for production of an Εbφ polypeptide typically comprises a gene coding for the corresponding ∑bf amino acid sequence. Such a gene may be prepared from cells which produce EDf. Any suitable cells may be used including T cell clones, such as the murine T cell clones NIMP-T1 and NIMP-T2 (Sanderson et __. J. Exp. Med., Vol .162, July 1985, pages 60-74), and T cell hybrids. We have used the murine T cell hybrid NIMP-THl for preparation of genes coding for ECv . This T cell hybrid is particularly advantageous as it appears to produce no other interleukin besides E f. Furthermore, this cell line serves as a standard, identifying source of _T- . This cell line has been described recently by Warren and Sanderson (Warren D.J. & Sanderson C.J. , Immunology, 1985, 54., 615-623) but has not been made publicly available in any way at all. This cell line was deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, Wiltshire, United Kingdom, on the 14th January, 1986, and is identified as desposit no. ECACC 86011401. __ - any suitable method including those which are known in the recombinant DNA field. Thus genomic DNA may be cloned from the E-t -producing cell lines or preferably cDNA clones may be prepared from mRNA obtained from the cells. Clones which code for Ε_ * production may be selected by any suitable method. Thus, for example, oligonucleotide probes synthesised on the basis of partial amino acid sequence determined for purified __)f, for instance purified as described above may be used to select positive clones. Alternatively cDNA coding for -ΕL^ψ may be prepared by an mRNA hybridisation-translation selection procedure; for instance, as described by Fung et , al., (M.C. Fung et. al,, Nature, Vol. 307, 19th January 1984, pages 233-237) for the molecular cloning of cDNA for murine interleukin 3 (IL3) . Preferably, however, ∑t^P cDNA may be prepared using a hybridisation arrest selection procedure, in which clones are selected by their ability to inhibit translation of ED mRNA. Alternatively, cDNA coding for Ε_f nay be selected by a direct expression selection procedure; for instance, in which cDNA clones are transfected into cos cells and the cell supernatants are tested for __*p activity. Furthermore, antibody to __ may be used for selection of positive clones. DNA which codes for the production of an E_* polypeptide, such as the polypeptides of the above aspect of the invention may be prepared from __>f cDNA by inclusion of or ligation with additional DNA sequences e.g. pre-sequence DNA or heterologous polypeptide DNA. DNA manipulations may be carried out by methods well known in the art. Murine Σ& cDNA may be used for screening human DNA libraries, both genomic and cDNA, to obtain corresponding genes coding for human _ -f. In another aspect, the invention provides a vector containing DNA which codes for production of an ∑Df polypeptide . The vector is characteristically adapted for use in a given host cell type by the prov s on o su ta e se ecta e mar ers, promoters an ouer contro regions as appropriate. In another aspect, the invention provides host cells transformed with a vector containing DNA which codes for production of an Tϋf polypeptide . The host cells may be any cells which may be transformed by a vector including DNA which codes for an E f polypeptide such that expression of the DNA occurs. Suitable host cells include bacteria (e.g. E.coli) , yeasts (e.g. S .cerevisiae ) and mammalian tissue culture cells. Preferably where the host- ' cells are bacteria or yeast cells the vector includes DNA coding for a methionine E_» polypeptide or an —_ * *P fusion polypeptide, and when the host cells are mammalian tissue culture cells, the vector preferably includes a gene coding for a pre-B_»f polypeptide. It has been found that the non-glycosylated form of l_f (Mr^»30K) retains functional __*f activity. Thus bacterial cells may be used advantageously for production of EDf polypeptides, such as methionine-E&f polypeptides, in a non-glycosylated form. Preferably, however, if glycosylated forms of _H*p (Mr * - 46K) are required, yeast cells or especially mammalian tissue culture cells are used as host cells. ∑b prepared by the process of the present invention may be purified e.g. as described above and used, after further formulation as required, in therapeutic and other applications. Ε_<f may be used for study of haemopoiesis and B cell differentiation. Thus J f may be useful in monoclonal antibody production; for instance, to prime B cells into a particular antibody producing state prior to fusion with myeloma cells or EBV transformation. Generally also _- may be used therapeutically; for instance, for regulating the immune response and to promote eosinophilia. The invention is further described by way of illustration only in the following Examples, which refer to the accompanying diagrams: Figure 1, which are graphs giving the results of co-ordinate analysis of (a) EDA and BCGF II and (b) EDA and BCGFI activities produced by a panel of 39 T cell clones. Figure 2, which are graphs showing the factionation of NIMP-T2 and NIMP-THl conditioned medium by gel filtration, and Figure 3 which are graphs showing the fractionation of NIMP-THl conditioned medium by reversed phase HPLC. Figure 4 is a DNA sequence, for part of which a corresponding amino acid sequence is also shown. Figure 5 shows two amino acid sequences. The upper complete one (M) is for mouse EDF polypeptide. The lower incomplete one (H) is for human EDF polypeptide. Figure 6 shows the relative number of eosinophils as measured by assay for eosinophil peroxidase (A 4g2 ) after 5 days in mouse bone marrow cultures, by different dilutions of mouse EDF (o) , human EDF (•), and a control preparation (a). Figure 7 shows the relative number of human eosinophils as measured by assay for eosinophil peroxidase (A 4g2 ) after 21 days in human bone marrow cultures by different dilutions of mouse EDF (o), human EDF (•) and a control preparation (fl) . 1 6 This Example describes the experiments we conducted to show that EDF and BCGFII are the same molecule: MATERIALS AND METHODS T cell cloning. Alloreactive T cell clones were isolated as described (Sanderson, C. J. _ Strath, M. (1985) Immunology 54., 275-279) . Briefly BALB/C.Nimr spleen cells were cultured in microplates at limiting dilution (100 cells/well) with 2x10 s irradiated CBA.Nimr spleen cells/well and a source of IL-2. After 2 weeks in culture the clones were restimulated with irradiated T cell depleted CBA spleen cells in the absence of exogenous IL-2, and supernatants collected after ' 48 hours. Lvmphokine assays . All assays were carried out in microplates. IL-3 was assayed on the dependent cell line 32D using the uptake of H- thymidine to determine cell growth (Ihle ___. al.. (1982) J. Immunol. 129., 1377-1383) . B cell growth factor I (BCGFI) was assayed by incubating the test material with 5x10 enriched B cells, with 5μg/ml affinity purified goat anti-mouse iπmunoglobulin, for 72 hours (Howard et . al,. (1982) , J. Exp. Med., 155, 914-923) . Cell growth was assayed by adding H-thymidine for the last 4 hours. BCGFII was assayed by incubating 1.5 x 10* BCLi cells/well with the test material for 48 hours (Dalton e_t. __. , J. Immunol. 132, 2451-2456). Growth was determined as in the BCGFI assay. EDA was assayed by incubating bone marrow cells from mesocestoides corti infected mice (10 /well) with test material. After 5 days eosinophils produced in the cultures were determined by assay for eosinophil peroxidase (Strath __. al.. (1985) J. Immunol. Methods, Vol. 83, pp 209-215). Fractionation. Gel filtration was carried out on a 300ml bed column of AcA54-Seρharose (LEB, Croydon, Surrey) in phosphate buffered saline (0.15M a , p . con a n ng μg m po ye y ene g yco --_ou«u; _._.__ Pharmaceuticals Ltd., Poole, Dorset). The column was calibrated with bovine serum albumin, M r 66.000; ovalbu in, M r 12,400 (all from Sigma) . Separation by binding to lentil lectin was carried out using LcH-Sepharose (Pharmacia, Uppsala, Sweden) and eluting with 0.1M *-methyl- mannnoside. Reverse phase, HPLC was carried out on a μ-Bondapak Cn column (Water Associates, Milford, MA.). The column was eluted with acetonitrile containing 0.1% trif luoroace tic acid, from 25% to 40% acetonitrile, and then from 40% to 60% acetonitrile. Conditioned medium from 39 alloreactive T cell clones were tested for their ability to stimulate eosinophil differentiation and compared with activity in the BCGFI (BSF-1) and BCGFII assays. The results obtained are shown in Figure 1, in which EDA is expressed as absorption units obtained by assay for eosinophil peroxidase. The estimated best fit is shown in each case. Regression analysis indicated that there was no significant correlation between EDA and BCGFI production (r = 0.24, slope 12.3 + 8.1, t-value for slope = 1.5 (P>0.05)), whereas the relationship between the production of EDA and BCGFII was highly significant (r = 0.77, slope 6 + 0.8 t-value for slope = 7.3 (P<0.001)). This positive correlation could indicate either that the two lymphokines we ' re under co-ordinate control or that a single molecule was involved. As shown in Table 1 below the T cell hybrid (NIMP-THl) which was known not to produce IL-2, IL-3 , CSF or γlFN (Warren _ Sanderson (1985) Immunology 54, 615-623) was found to have BCGFII activity. Two other T cell hybrids (NIM-THll, NIM-TH12) which had been selected for BCGFII activity were found to produce high levels of EDF. In addition, two sublines of the EL4 cell line, both producing high levels of IL-2 , but differing markedly in BCGFII production were found to show a similar difference in their capacity to produce EDF. Also the T cell clone (NIMP- T2) maintained as a high producer of EDF (Sanderson ___. ___. (1985) , J. Exp. Med. 162. 60-74) was a high producer of BCGFII. Activity of conditioned medium from different cell lines in EDA and BCGFII assays . Cell Line EDA (Units) BCGFII (cpm x 10 " ' ) NIMP-THl (T hybrid) 550 25 NIM-THll ( ' ) 40 9.6 NIM-TH12 ( ' ) >1000 34 NIM-TH13 ( )* <10 1.5 NIMP-T2 (T cell clone) >1000 38 EL4-17 (lymphoma) 50 16 EL4-23 (lymphoma) >1000 31 Control (medium only) 0 0.5 EDA is expressed as the reciprocal of the dilution givng 50% of maximum activity - values greater than 10 represent significant activity. BCGFII is expressed as the uptake of H-thy idine at a dilution of 1:10 - values above 1.5 represent significant activity. The sublines of EL4 were kindly supplied by Dr. H.R. MacDonald. Conditioned medium from EL4 cell and the T hybrids were obtained after stimulation with phorbol myristate acetate or Concanavalin A. Conditioned medium from NIMP-T2 was obtained after stimulation with allogeneic spleen cells (Sanderson e__ al, J. Exp. Med., vol. 162, July 1985, pages 60 - 74) . * The hybrid NIM-TH13 originally showed both EDA and BCGFII activities, however, on recloning both activities were lost simultaneously. It is included as a negative control. These results provided circumstantial evidence that EDF and BCGFII might be properties of a single molecule, and so fractionation experiments were undertaken. Firstly, gel filtration of NIMP-THl conditioned medium (THl-CM) and NIMP-T2 conditioned medium (T2-CM) on AcA54 showed that in each case the EDA and BCGFII activity had a similar elution pattern with a peak equivalent to M r 46.000. The results obtained are shown in Figure 2. IL-3 also present in T2-CM eluted with a peak of M χ 29,000 as shown previously (Sanderson e_t. ___. (1985) J. Ex . Med. 162, 60-74). Fractions were assayed for EDA (Δ) , BCGFII (α) and IL3 (0). Fractions from T2-CM were tested at a final dilution of 1:100 for EDA and 1:10 for BCGFII and IL3 , Fractions from THl-CM were tested at 1:1000 for EDA and 1;10 for BCGFII. Fractionation of THl-Ol by lentil lectin affinity chromatography, flat bed isoelectric focussing, preparative gel electrophoresis and elution from phenyl-Sepharose and DEAE-Sepharose showed that in each case EDF and BCGFII had identical elution profiles (data not shown). Material first separated by gel filtration and then affinity ch: tography on lentil lectin was subjected to reverse phase HPLC. The ts obtained are given in the lower Figure 2 showing that the two activities .ded closely. The THI->- " i was first fractionated by gel filtration on AcA54 and fractions high in EDA pooled and passed through a lentil lectin column. All the EDA bound to the column and was eluted with < ~ - methyl mannoside. This material was then factionated by HPLC. The protein profile ( ) and the acetonitrile concentration in the eluting buffer ( ) is shown in the lower panel of Figure 3. The upper panel shows the results of assay for EDF (o) tested at a dilution of 1;5000, and assay for BCGF II (•) tested at a dilution of 1:1000 Example 2 Purification of *- * £ The procedure employed to purify _-f produced by the T cell hybrid NIMP-THl was as follows: NIMP-THl cells were grown to approximately 5 x 10 cells per ml in tissue culture medium containing 5% heat inactivated foetal calf serum. 4β - phorbol -12β-myristate 13°= - acetate (PMA) was added to a final concentration of 10 ng/ml. After 48 hours the cell culture medium was harvested and concentrated 10 fold. __t* was recovered from the concentrated medium by firstly adding ammonium sulphate at concentration 272 g/1. The resultant medium was clarified by centrifugation (5000g for 30 mins) and the medium discarded. Further ammonium sulphate was added at concentration 245 g/1 and the precipitate recovered after centrifugation as above. The precipitate was then dissolved in water adjusted to pH8 and passed through a lentil lectin column (LcH-sepharose, Pharmacia). ∑_\f was bound by the column and was eluted by 0.1M <= methyl mannoside in 0.02M ammonium bicarbonate. Fractions containing EDf were pooled and freeze dried. This freeze-dried material was then dissolved in 0.15M NaCl and separated by high performance gel filtration on a G3000 Sw LEB Ultrapac column. The active fraction was pooled and separated by reverse phase HPLC substantially as described in Example 1, eluting with a gradient of 25% - 60% acetonitrile containing 0.1% trifluoroacetic acid. The active fractions were pooled and freeze dried. This material was then separated by non-reducing PAGE using a 10% gel and standard procedures. The gel was then sliced and each slice tested for activity. EDf was recovered in a band of M r of approximately 46E. The I&€ end product had an EDA in excess of 10 U/μg, and preferably comprised substantially pure ED (e.g. having an EDA of about 10. U/μg). EXAMPLE 3 mRNA Preparation NIMP-THl cells were grown to approximately 5 x 10 cells per ml in tissue culture medium containing 5% heat inactivated foetal calf serum. 4β-phorbol-12β-myristate 13«-acetate (PMA) was added to a final concentration of lOng/ml. The cells were collected by centrifugation, resuspended in 0.1 volume of serum free medium and mRNA was prepared from the cells by the guanidine thiocyanate-CsCl method (R. McCandliss e_t , al. Meth. Enzy . 79., 51-59 (1981); J.M. Chirgwin e_t a_l, Biochemistry 18., 5294-5299 (1979)). The mRNA was then further enriched by oligo (dT)-cellulose chromatography (P.W. Gray et a_l , . Nature, 295, 503-508 (1982)) and by centrifugation on a sucrose gradient. Translation Enriched mRNA, prepared as above, was injected into X.laevis oocytes and biologically active IDf was detected in the oocyte medium* Hilf activity was detected by determination of eosinophil differentiation activity (EDA) being measured as a function of the eosinophil production which it caused. In this assay bone marrow cells obtained from mice previously infected with the helminth mesocestoides corti were incubated with the oocyte medium and after 5 days the eosinophils produced in the culture were determined by an assay for eosinophil peroxidase. This assay is described in detail by Strath e_t al. (M. Strath e_t al, J. Immunol. Meth., Vol. 83, pp 209-215 (1985)). Screening cDNA Library The enriched mRNA, prepared as above, was used to construct a cDNA library in bacterio-phage M13. The phage was propogated in E.coli and pools of 32 clones were tested by hybridisation-arrest for their ability to inhibit the translation of IL4 mRNA in X.laevis oocytes, the presence of IDf activity in the oocyte supernatant being detected as described above. - 27- All pools positive in the hybridisation-arrest assay were subdivided into pools of 8 and further screened for hybridisation—arrest. Finally, individual clones were tested and positive clones sequenced using the Sanger dideoxy chain termination method (Sanger e_t a ., Proc . Nat. Acad. Sci. U.S.A., 74, 5463-5467 (1977)). In order to isolate a full length clone carrying the full coding sequence for H>f, the sequence of positive clones was used to prepare hybridisation probes for. screening of a size factionated cDNA library. The full length cDNA was then sub-cloned into a suitable cDNA expression vector, and transfected into mammaliam cells. The transfected cells were then cultured and the culture supernatants tested for eosinophil differentiation activity (EDA), as described above. This procedure was used to verify that the cDNA clones coded for EDF. The above procedures yield an ΣDf cDNA clone of about 900 bp in length corresponding to a polypeptide of about 300 amino acids in length (Mr-*»30K). Murine IDf cDNA clones obtained as described above may be used to probe human DNA libraries (genomic or cDNA) to obtain DNA coding for human J-Df. The clones so obtained (both murine and human) may be used for construction of expression vectors for transformation of host cells to produce H> . Example 4 The cloning and sequencing of the human EDF gene was carried out as described for the cloning and sequencing of the rat interleukin-3 gene (Cohen, D. R., Hapel, A. J. and Young, I. G. Nucleic Acid Research, Vol 14, Number 9, pages 3641-3658), except that DNA was obtained from human placenta. The DNA sequence is shown in Figure 4. A 3rd BamHI fragment carrying the entire EDF gene was recloned into the expression vector pCEXV 3 (Miller, J., Malek, T. R., Leonard, W. J., Greene, C , Shevach, E. M. and Germain, R. N. J. Immunol. Vol 134, June 1985, pages 4212-4217). Two clones were isolated, one for each orientation of the gene. Both of these clones were introduced into monkey C0S7 cells by transportation (Potter E., Weir, L. and Leder P., Proc. natl. Acad. Sci. USA. Vol 81 , 1984, pages 7161- 7165), and supernatants from cells transfected with one of these was found to contain recombinant human EDF (hEDF). This material has been partially sequenced, and the sequence is shown in Figure 5 together for comparison with the complete sequence reported by T. Kinashi et al (Nature, Vol 324, 6 November 1986 pp 70- 73-) for the polypeptide of murine BCGF II. Example 5 Human Bone marrow cultures Bone marrow was obtained from normal subjects donating bone marrow for transplantation following a protocol approved by the Ethics Committee at the Hammersmith Hospital, London. Low density cells were recovered after centrifugation over Ficoll-Pague (Pharmacia, Uppsala, Sweden). Liquid cultures were carried out as described previously (Sanderson, Warren and Strath, 1985). Briefly 10 5 cells were cultured in 100 ul of RP I 1640 with 155. fetal calf serum in 96 well microplates (Nune, Roskilee, Denmark). After 21 days total cells were counted and differential counts performed after staining with Luxol Fast Blue and haematoxyliπ or by assay for eosinophil peroxidase (Strath, M. , Warren, D. J., and Sanderson, C. J. J. Immunol Methods Vol 83, 1985 pages 209-215). Results are shown in Figures 6 and 7 which are in effect graphs of EDA against EDF dilution. From Figure 6 it is apparent that EDF of mouse origin shows activity in bone marrow cells, not only of mice but also of human. From Figure 7 it is apparent that EDF of human origin shows activity in bone marrow cells, not only of humans but also of mice. Example 6 EDF was tested for its ability to stimulate peripheral blood eosinophils and neutrophils in an ADCC assay. Eosinophils showed very low levels of ADCC against target cells in the absence of EDF, however, the addition of EDF resulted in a highly increased capacity to kill target cells by eosinophils from all six individuals tested. Neutrophils tested in parallel, while showing a higher level of basal ADCC activity, were not activated by EDF. This experiment is reported in more detail in Lopez et al. J. Exp. Med, Vol 163, May 1986, pp 1085-1099.

Claims: C L A I S 1. Eosinophil differentiation factor (EDF) having both significant eosinophil differentiation (EDA) and also significant B cell growth promoting activity of B cell growth factor II (BCGF II). 2. EDF as claimed in claim 1 having the following characteristi cs : - (a) It is a trypsin sensitive glycoprotein; (b) It is stable at both pH2 and pH12; (c) The glycoprotein is relatively thermally stable, retaining full activity after incubation at 56°C for 30 minutes or more, and retaining partial activity after incubation at 70°C for 10 minutes; (d) It is stable in 2% SDS; (e) It is sensitive to 5 % 2-mercaptoethaπol ; (f) It has an isoelectric point (pi) in the range 4.5 - 5.5 as determined by agarose gel ionising electrophoresis (IEF); (g) It binds reversibly to lentil lectin, and for instance may be eluted therefrom with 0.15M < -methyl mannoside. (h) The glycoprotein has a mean molecular weight of about 46K (range 32-66K) when determined by gel filtration, and of about 44K when determined by SDS, non-reducing polyacrylamide gel electrophoresis (PAGE); (i) The glycoprotein comprises a peptide component of p about 30K, i.e. corresponding to a polypeptide of about 300 amino acids in length. (When cells which secrete EDF e.g. the T cell clone NIMP-THl, are grown in the presence of the glycosylation inhibitor tunicamycin a polypeptide of M about 30K is produced in the place of the 46K glycoprotein.) 3. EDF as claimed in claim 1 or claim 2, which is human EDF. 5 4. EDF as claimed in claim 3, including a polypeptide comprising a chain of about 133 amino acids including from about position 59 to about position 133 the sequence shown in Figure 5. 5. EDF as claimed in claim 3 or claim 4, having 0 significant EDA in mice as well as human. 6. EDF as claimed in claim 1 or claim 2, which is murine EDF 7. EDF as claimed in claim 6, including a polypeptide having substantially the amino acid sequence shown in 5 Figure 5. 8. EDF as claimed in claim 6 or claim 7, having significant EDA activity in humans as well as mice. 9. EDF as claimed in any one of claims 1 to 8 having also significant selective activation of eosinophil 0 function. 10. Eosinophil differential factor (EDF) of mouse or human origin, having significant eosinophil differentiation activity (EDA) in both mice and humans. 11. EDF as claimed in claim 10, having also 5 significant B cell growth promoting activity of B cell growth factor II (BCGF II) . 12. A composition comprising EDF as claimed in any one 3 of claims 1 to 11 and having an EDA of at least 5 x 10 units. ° 13. An EDF polypeptide prepared by recombinant DNA technology, having both significant EDA and also significant B cell growth promoting activity of BCGF II. 14. An EDF polypeptide " prepared by recombinant DNA 5 technology, having significant EDA in both mice and humans. 15. An EDF polypeptide as claimed in claim 13, which is a methionine EDR polypeptide, a pre-EDF polypeptide or an EDF fusion polypeptide. 16. An EDF polypeptide as claimed in any one of claims 13 to 15 comprising a chain of about 133 amino acids including from about position 59 to about position 133 the sequence shown in Figure 5. 17. An EDF polypeptide as claimed in any one of claims 13 to 15 having substantially the amino acid sequence shown in Figure 5. 18. DNA which codes for the production of an EDF polypeptide according to any one of claims 13 to 17. 19. DNA as claimed in claim 18 having substantially the sequence shown in Figure 4. 20. A vector containing DNA according to claim 18 or claim 19. 21. Host cells transformed with the vector of claim 20.

Inventors: SANDERSON COLIN JOHN, YOUNG IAN GORDON, SANDERSON, COLIN, JOHN, YOUNG, IAN, GORDON
Applicants: AMERSHAM INT PLC, AMERSHAM INTERNATIONAL PLC
Application year: 1987
Main classification: C12P21
IPC codes: C07K14