Component of bromelain

Abstract

The invention relates to a component of bromelain which is largely responsible for the ability of bromelain to interrupt the MAP kinase cascade. The component contains ananain and comosain and is useful in the treatment or prevention of diseases and conditions mediated by T cell activation or by activation of the MAP kinase pathway.

Claims

1. A method for the treatment of an inflammatory disease in a patient suffering from said inflammatory disease, said method comprising enterally administering to said patient a pharmaceutical composition comprising an effective amount of ananain to thereby treat said inflammatory disease in said patient, wherein the composition does not comprise a protein having (a) an N-terminal amino acid sequence of SEQ ID NO: 1 and (b) an isoelectric point of 9.7.

2. The method of claim 1, wherein the ananain is substantially pure ananain.

3. The method of claim 1, wherein the enteral administration is oral administration.

4. The method of claim 1, wherein the effective amount of ananain inhibits or suppresses T cell activation in said patient.

5. The method of claim 1, wherein T cell activation is inhibited or suppressed in said patient.

6. The method of claim 5, wherein a mitogen-activated protein kinase pathway is inhibited in said patient.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

(1) The invention will now be further described with reference to the following examples and to the drawings in which:

(2) FIG. 1 is a diagrammatic representation of signal transduction events associated with T cell activation that lead to IL-2 production.

(3) FIG. 2 is an ultra violet elution profile of crude bromelain after cation exchange chromatography on SP Sepharose high performance media.

(4) FIG. 3 is a plot showing the proteolytic activity and the protein content of crude bromelain fractions after cation exchange chromatography on SP Sepharose high performance media.

(5) FIG. 4 is an SDS-PAGE of SP Sepharose high performance chromatography pooled fractions run on 4-20% T gradient gels with lanes 1 to 4 and 6 to 9 containing proteins CCT, CCV, CCX and CCZ and CCY, CCW, CCU and CCS respectively and lanes 5 and 10 containing molecular weight markers.

(6) FIG. 5 shows isoelectric focusing of pooled fractions run on pH 3-11 gradient gels with Lanes 1, 11 and 12 showing high IEF markers, Lanes 2 and 13 showing crude bromelain and Lanes 3 to 10 showing proteins CCT, CCV, CCX, CCZ, CCY, CCW, CCU and CCS respectively.

(7) FIG. 6 is a Western blot using anti-phosphotyrosine mAb which demonstrates that CCS reduces tyrosine phosphorylation of p42 kDa (ERK-2) protein. Th0 cells were treated with bromelain fractions (50 g/ml) for 30 min, washed and then stimulated with combined PMA (20 ng/ml) and ionophore (1 M) for 5 min. Unstimulated cells served as controls. Cells were then lysed and postnuclear supernatants were subjected to SDS-PAGE and Western blotting. In this figure, closed symbols indicate proteins phosphorylated by combined PMA plus ionophore. Open symbols indicate ERK-2 protein reduced by CCS treatment.

(8) FIG. 7 is a Western blot using anti-phosphotyrosine mAb which demonstrates that CCS increases tyrosine phosphorylation of protein substrates. Th0 cells were treated with CCS, crude bromelain (Brom), stem bromelain protease (SBP) or CCT fraction (50 g/ml) for 30 min, washed and then stimulated with combined PMA (20 ng/ml) and ionophore (1 M) for 5 min. Unstimulated cells served as controls (Cont). Cells were then lysed and postnuclear supernatants were subjected to SDS-PAGE and Western blotting. In this figure, closed symbols indicate proteins phosphorylated by CCS but not by other treatments. Open symbols indicate phosphoproteins protein reduced by CCS and crude bromelain treatment.

(9) FIG. 8 is a Western blot using anti-phosphotyrosine mAb which shows that the inhibitory effect of CCS on ERK-2 is dependent on its proteolytic activity and occurs in a dose-dependent manner. Th0 cells were treated with CCS (0 to 25 g/ml) or CCS incubated with the selected protease inhibitor, E-64, for 30 min. Cells were then washed and then stimulated with combined PMA (20 ng/ml) and ionophore (11 M) for 5 min. Cells were then lysed and postnuclear supernatants were subjected to SDS-PAGE and Western blotting. In this figure, closed symbols indicate proteins phosphorylated by CCS. Open symbols indicate ERK-2 phosphoprotein inhibited by active CCS but not by inactivated CCS.

(10) FIG. 9 is an immunoblot which confirms that the 42 kDa phosphoprotein inhibited by CCS is ERK-2. Th0 cells were treated with CCS (50 g/ml) for 30 min, or untreated, washed and then stimulated with combined PMA (20 ng/ml) and ionophore (1 M) for 5 min. Cell lysates were immunoblotted with anti-ERK-2 mAb.

(11) FIG. 10 is a Western blot using anti-phosphotyrosine mAb which shows that crosslinked anti-CD3 mAb induces tyrosine phosphorylation of multiple proteins. Th0 cells were stimulated with crosslinked anti-CD3 for 0 to 20 min. Cells were then lysed and postnuclear supernatants were subjected to SDS-PAGE and Western blotting. Closed symbols denote anti-CD3 mAb-induced tyrosine phosphorylated proteins.

(12) FIG. 11 is a Western blot using anti-phosphotyrosine mAb which demonstrates that CCS inhibits tyrosine phosphorylation in TCR-stimulated T cells. Th0 cells were treated with CCS (0 to 5 g/ml) for 30 min, washed and then crosslinked anti-CDR mAb for 5 min. Cells were then lysed and postnuclear supernatants were subjected to SDS-PAGE and Western blotting. In this figure, the symbols denote anti-CD3 mAb-induced tyrosine phosphorylation of ERK-2, which is reduced by CCS.

(13) FIG. 12 is a Western blot using anti-Raf-1 mAb which shows that CCS inhibits the mobility shift of Raf-1. Th0 cells were treated with CCS (0 to 50 g/ml) for 30 min, washed and then stimulated with either (A) combined PMA plus ionophore or (B) then crosslinked anti-CD3 mAb for 5 min. Cells were then lysed and postnuclear supernatants were subjected to SDS-PAGE and Western blotting.

(14) FIG. 13 is a pair of plots showing that CCS decreases IL-2 production and proliferation in purified CD4.sup.+ T cells. T cells were treated with CCS (50 g/ml), washed and then cultured in either media alone or with immobilised anti-CD3 mAb and soluble anti-CD28 mAb. (A) IL-2 production was determined by the CTL-L assay as described in Example 5. (B) Proliferation was determined by the incorporation of .sup.3H-thymidine. CD4.sup.+ T cells cultured in the absence of mAb (stimuli) did not produce any detectable IL-2 and do no proliferate.

(15) FIG. 14 is a pair of plots showing that CCS decreases IL-2 production by splenocytes but does not inhibit splenocyte proliferation. Splenocytes were treated with CCS (50 g/ml), washed and then cultured in either media alone or with immobilised anti-CD3 mAb. (A) IL-2 production was determined by the CTL-L assay as described in Example 5. (B) Proliferation was determined by the incorporation of .sup.3H-thymidine. Splenocytes cultured in the absence of mAb (stimuli) did not produce any detectable IL-2 and do no proliferate).

(16) FIG. 15 is a plot which shows that CCS inhibits tumour cell growth in vitro. Cancer cell lines were treated with CCS (50, 10, 2.5, 1 and 0.25 g/ml) or water as a control. After 96 h treatment, the effect of CCS on tumour cell growth was evaluated. Columns represent the 50% inhibitory concentrations (IC.sub.50 g/ml) of CCS (the amount of CCS required to inhibit 50% of tumour cell growth.

EXAMPLE 1

Purification of Bromelain Proteins

(17) a. Materials

(18) Reagents: Bromelain (E.C 3.4.22.4; proteolytic activity, 1,541 nmol/min/mg) was obtained from Solvay Inc. (Germany). Fast Flow S Sepharose, Pharmalyte 3-10 Ampholine 9-11, Ready Mix IEF (acrylamide, bisacrylamide) and IEF markers were obtained from Pharmacia Biotech. Precast 4-20% acrylamide gels and broad range molecular weight markers were obtained from Bio-Rad Laboratories. All other reagents were of analytical grade and obtained from either Sigma Chemical Co. or British Drug House.

(19) b. Proteinase Assay

(20) The proteolytic activity of bromelain was determined by use of an in-house microtitre plate based assay using the synthetic substrate Z-Arg-Arg-pNA. This assay was based on that described by Filippova et al. in Anal. Biochem., 143: 293-297 (1984). The substrate was Z-Arg-Arg-pNA as described by Napper et al. in Biochem. J., 301: 727-735, (1994).

(21) c. Protein Assay

(22) Protein was measured using a kit supplied by Bio-Rad that is a modified method of Lowry et al. (J. Biol. Chem. (1951) 193: 265-275). Samples were compared to bovine serum albumin standards (0 to 1.5 mg/ml) prepared in either 0.9% saline or 20 mM acetate buffer pH 5.0, as appropriate.

(23) d. Preparation of Bromelain

(24) All the following steps were performed at ambient temperature (20 to 25 C.). A solution of bromelain (30 mg/ml) was prepared by dissolving 450 mg of powder in 15 ml of 20 mM acetate buffer (pH 5.0) containing 0.1 mM EDTA, sodium. The solution was dispensed into 101.5 ml microcentrifuge tubes and centrifuged at 13,000g for 10 minutes to remove insoluble material. The clear supernatants were pooled and used for chromatography.

(25) e. Fast Flow S-Sepharose High Performance Chromatography

(26) A Fast flow S-sepharose column was prepared by packing 25 ml of media into an XK 16/20 column (Pharmacia Biotech) and equilibrated with 20 mM acetate buffer (pH 5.0) containing 0.1 mM EDTA on an FPLC system at 3 ml/min. 5 ml of bromelain solution was injected onto the column. Unbound protein was collected and the column washed with 100 ml of acetate buffer. Protein bound to the column was eluted with a linear gradient of 0 to 0.8 M NaCl in acetate buffer over 300 ml. 5 ml fractions were collected throughout the gradient and FIG. 2 shows a typical U.V. chromatogram of crude bromelain obtained from this procedure.

(27) The fractions were then analysed for protein and proteolytic activity as described above and FIG. 3 shows the proteolytic activity against the synthetic peptide Z-Arg-Arg-pNA and the protein content of the individual fractions. The protein content profile closely mirrors that of the U.V., as expected, but the main proteolytic activity is confined to the two major peaks that correspond to that of bromelain protease (SBP). Small activities are observed in other areas of the chromatogram that may correspond to other proteases distinct from SBP, such as the later eluting CCS fraction, which contains ananain and comosain.

(28) The main peaks identified from the U.V. profile were pooled from three successive runs and named as indicated in Table 1. Pooled fractions were used for physico-chemical characterisation. Pooled fractions were concentrated by ultrafiltration and buffer exchanged using PD10 columns into isotonic saline (0.9% w/v NaCl). The protein content and Z-Arg-Arg-pNA activity were calculated prior to biological testing and are shown in Table 2.

(29) The pooled fractions were processed for analysis as described below.

(30) TABLE-US-00001 TABLE 1 Summary of Pooled Fractions from SP Sepharose BP Fractionated Bromelain (QC2322) Fractions Pooled Component Description (Inclusive) CCT Flow through (unbound components) Unbound column flow through CCV First peak off column 8-9 CCX Second sharp peak off column 13-14 CCZ Small peak on ascending edge of the 19-20 third main bromelain peak CCY First main bromelain peak 23-24 CCW Second main bromelain peak 27-29 CCU Small peak on descending edge of the 33-34 second main bromelain peak CCS Last double peak off column 39-44

(31) TABLE-US-00002 TABLE 2 Calculated Protein Content and Z-Arg-Arg-pNA Activity of Pooled Fractions used for Testing Biological Activity. Z-Arg-Arg-pNA Activity Protein Content Pooled Fractions (Moles/min/ml) (mg/ml) CCT 11.30 1.00 CCV 9.78 1.00 CCX 71.71 1.00 CCZ 688.81 1.00 CCY 1500.0 0.574 CCW 1500.0 0.543 CCU 1500.0 0.421 CCS 379.76 1.00

(32) f. Processing of Pooled Fractions

(33) The proteolytic activity and protein content of pooled fractions were determined and the concentrations adjusted to approximately either 1.4 mg/ml of protein or 105 nmoles/min/ml of proteinase activity using a Filtron stirred cell containing an ultrafiltration membrane of nominal molecular weight cut-off of 10 kDa. The fractions were then buffer exchanged using PD10 columns (Pharmacia Biotech) into isotonic saline (0.9% w/v NaCl), sterile filtered (0.2 m) and adjusted for protein content or proteolytic activity. Samples were then frozen at 80 C. and used in the in vitro studies described below.

(34) g. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

(35) Pooled FPLC samples were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on precast 4 to 20% T gradient gels. Samples were prepared for electrophoresis by acid precipitation in which 100 l was mixed with an equal volume of 20% w/v trichloroacetic acid (TCA). Precipitated protein was collected by centrifugation at 13,000g for 10 minutes and the supernatant discarded. The pellet was washed twice with 0.5 ml of diethyl ether and left to dry in air at ambient temperature. The pellets were then dissolved in 300 l of SDS-PAGE sample buffer (62.5 mM Tris-HCl pH 6.8 containing 10% v/v glycerol, 2% w/v sodium dodecyl sulphate and 40 mM dithiothreitol) and heated at 95 C. in a water bath.

(36) SDS-PAGE broad range molecular weight standards diluted 1:20 in SDS-PAGE sample buffer were treated similarly and run with the samples. Gels were run on a mini Protean II electrophoresis system according to Bio-Rad's protocol at 240 V and until the dye front reached the end of the gel (30 to 45 min).

(37) After electrophoresis, separated proteins were stained overnight with orbital mixing in a solution of 0.075% w/v colloidal brilliant blue G-250 containing 1.5% v/v phosphoric acid, 11.25% w/v ammonium sulphate and 25% v/v methanol. Gels were destained, to obtain a clear background, in a solution of 25% v/v methanol and 10% v/v acetic acid.

(38) Results

(39) The purity of fractions is shown by SDS-PAGE in FIG. 4. All of the pooled fractions except the column flow through (CCT) showed that the major protein present was of molecular weight between approximately 25-28 kDa. This corresponds to the molecular weight of cysteine proteinases isolated from bromelain by other authors (Rowan et al., Methods in Enzymology, (1994), 244: 555-568). The purity of fractions CCX, CCZ, CCY and CCW appears to be high. Minor components of lower molecular weight can be observed in some fractions, particularly CCT, CCV, CCX and CCS. Pooled fractions CCU and CCS contain a doublet between 25-28 kDa; the higher gel loadings of fractions CCX, CCZ, CCY, and CCW means that doublet bands may also be present in these fractions. A summary of the components and their calculated molecular weights in pooled fractions, as determined by SDS-PAGE, is shown in Table 3.

(40) Proteins in pooled fractions CCX, CCZ, CCY+CCW and CCU were transferred onto nitro-cellulose after SDS-PAGE by Western blotting and probed with rabbit antisera raised against purified stem bromelain protease (SBP) (results not shown). All protein bands in these pooled fractions were recognised by antibodies in the sera, indicating immunologically similar proteins, probably belonging to the cysteine proteinase family of enzymes.

(41) TABLE-US-00003 TABLE 3 Summary of the Molecular Weights of Proteins found in SP Sepharose BP Pooled Fractions as Determined by SDS-PAGE. Pooled Molecular Weight (kDa) of Molecular Weight (kDa) of Fractions Major Protein Band(s) Minor Protein Band(s) CCT 76.03 15.07 CCV 15.07, 25.85, 28.28, 76.03 CCX 25.08 15.07, 76.03 CCZ 27.45 13.37, 16.49, 76.03 CCY 27.45 6.5 CCW 27.45 CCU 27.45, 28.28 CCS 15.07, 25.85, 27.45

(42) h. Isoelectric Focusing

(43) Pooled fractions (0.5 to 1.0 mg/ml) were diluted 1:3 with deionised water and run on gradient gels of pH 3 to 11. Gels were cast using Ready Mix IEF to produce a 5.5% T, 3% C polyacrylamide gel containing 10% v/v glycerol, 5.0% Pharmalyte 3-10 and 2.5% Ampholine 9-11. Briefly, 10 l of sample and high pI markers were loaded onto the gel after prefocusing at 700 V. Sample entry was at 500 V for 10 min, focusing was at 2500 V for 1.5 hour and band sharpening at 3000 V for 10 min. After electrophoresis the proteins were fixed with a solution of 20% w/v TCA for 30 min, washed in destain for 30 min to remove TCA and stained with brilliant blue G-250 as described for SDS-PAGE (see above).

(44) Results

(45) FIG. 5 shows that all fractions except CCX contained basic proteins focusing beyond the 9.3 l marker. Localised charge interactions with the chromatographic media functional groups may explain why proteins of pI 3.8 and 3.85 in CCX, adsorbed onto a cation exchange resin at pH 5.0. CCZ was present as a single band of pI 9.7, whilst pooled fractions CCY, CCW, and CCU contained multiple bands of isoelectric points in the range pH 9.5-9.8. At least part of this heterogeneity can be explained by variation in the carbohydrate moiety of a common stem bromelain protein backbone. The values are in agreement with those reported in the literature of pI 9.45-9.55 for bromelain (Rowan et al., Methods in Enzymology, (1994), 244; 555-568). Pooled fractions CCS contains two basic proteins of pI greater than 10.25. Estimates by extrapolation give pIs of 10.4 and 10.45. These correspond to ananain and comosain, and are in agreement with other estimates (Rowan et al., as above) of pIs greater than 10. The pIs of proteins in each of the pooled fractions are summarised in Table 4.

(46) TABLE-US-00004 TABLE 4 Summary of the estimated Isoelectric points of Proteins found in SP Sepharose HP Pooled Fractions. Pooled Fractions Isoelectric Points of Proteins CCT Not detected CCV Not Detected CCX 3.8, 3.85 CCZ 9.7 CCY 9.6, 9.7 CCW 9.57, 9.6, 9.7 CCU 9.57, 9.6, 9.75 CCS 10.4, 10.45

EXAMPLE 2

NH2-Terminal Amino Acid Analysis of Bromelain Components

(47) In a separate experiment, pooled fractions of bromelain were run by SDS PAGE and blotted as above onto PVDF membrane. The membrane was stained with 0.025% w/v coomassie blue R-250, dissolved in 40% v/v methanol for 10 min, followed by destaining in 50% v/v methanol. The membrane was dried in air at room temperature and NH.sub.2-terminal amino acid sequencing of the stained proteins was carried out. Briefly, the protein band was cut from the membrane and placed in the upper cartridge of the sequencer. NH.sub.2-terminal amino acid analysis of bromelain components was determined by Edman degradation using a gas phase sequencer (Applied Biosystems), equipped with an on-line phenylthiohydantion amino acid analyser. Table 5 shows the first NH.sub.2-terminal amino acids of CCZ (SEQ ID NO: 1), CCX (SEQ ID NO: 2), stem bromelain protease (SEQ ID NO: 3), ananain (SEQ ID NO: 4) and comosain (SEQ ID NO: 5).

(48) TABLE-US-00005 TABLE 5 NH.sub.2-Terminal Sequence Similarities of CCZ Protein and Those of Known Proteinases Isolated from Bromelain. Proteinase Position from N-Terminus CCZ V L P D S I D W R Q K G A V T E V K N R G (SEQ ID NO: 1) 15101520 CCX V P Q S I D W R D Y G A V N E V K N (SEQ ID NO: 2) 4914 Stem Bromelain A V P Q S I D W R D Y G A V T S V K N Q N (SEQ ID NO: 3) Protease 15101520 Ananain V P Q S I D W R D S G A V T S V K N Q G (SEQ ID NO: 4) 1491419 Comosain V P Q S I D W R N Y G A V T S V K N Q G (SEQ ID NO: 5) 1491419

(49) All proteins share sequence homologies. Ananain and comosain differ by 2 out of 20 amino acids when compared to stem bromelain protease. CCZ differs by 8 out of 21 amino acids when compared to stem bromelain protease. CCZ differs from ananain and comosain by 6 out of 20 amino acids. Comosain differs by 2 amino acids from ananain. Whilst it is clear that these proteins are structurally related, they are all distinct, showing divergence from each other. These proteinases also differ in their proteinase substrate specificity and their biological activity.

EXAMPLE 3

Fraction CCS Inhibits Tyrosine Phosphorylation of p42 kda Phosphoprotein

(50) a. Materials

(51) Antibodies: Anti-CD3 -chain mAb (145-2C11) and anti-CD28 mAb (PV-1) were purchased from Pharmingen (San Diego, Calif.) and goat anti-hamster IgG Ab was from Sigma (Dorset, UK). Mouse anti-phosphotyrosine mAb (4G10), mouse anti-MAPk R2 (ERK-2) mAb and mouse anti-Raf-1 mAb were from UBI (Lake Placid, N.Y.). Goat anti-mouse and goat anti-rabbit IgG Ab conjugated to horse radish peroxidase (HRP) were from BioRad (Hemel Hemstead, Hertfordshire, UK). Rabbit polyclonal phospho-specific MAPk IgG which recognise tyrosine phosphorylated p44 and p42 MAPks were from New England BioLabs (Hitchin, Hertfordshire, UK).

(52) Reagents: Phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 were purchased from Sigma. Bromelain (E.C 3.4.22.4; proteolytic activity, 1,541 nmol/min/mg) was obtained from Solvay Inc. (Germany). E-64 (L-trans epoxysuccinylleucylamido-(4-guanidino)butane, a selective cysteine protease inhibitor, was from Sigma.

(53) Cells: The T cell hybridoma GA15 was a generous gift from B. Fox (ImmuLogic Pharmaceutical Corporation, Boston, Mass.). GA15 was generated by fusing the thymoma BW5147 with the T.sub.h2 clone F4 specific for KLH in association with I-A.sup.b, and were maintained as previously described (Fox, 1993, Int. Immunol., 5: 323-330). GA15 exhibit a T.sub.h0 cell phenotype as they produce IL-2, IL-4 and IFN- following stimulation with crosslinked anti-CD3 mAb (Fox, 1993).

(54) b. Stimulation of T cells.

(55) Cells (2107) suspended in RPMI 1640 were treated with CCS (1 to 50 g/ml) diluted in saline (0.9% (w/v)) for 30 min at 37 C. Mock treated cells were treated with an equal volume of saline (diluent). At high concentrations of CCS (50 or 100 g/ml) cell aggregation occurred, as noted previously in studies with crude bromelain. Following treatment, cell aggregates were gently dispersed by washing cells 3 times and then resuspending in fresh RPMI. Cells were stimulated via the cell surface with crosslinked mAb to the TCR (anti-CD3), or directly, using combined PMA (20 ng/ml) and ionophore (1 M) for times indicated in figure legends and the text.

(56) Stimulation via the TCR was conducted by first incubating T cells on ice for 30 min with anti-CD3 mAb (20 g/ml). Excess mAb was then removed by washing once at 4 C. and anti-CD3 mAb was crosslinked with goat anti-hamster IgG (20 g/ml) at 37 C. Stimulation was terminated by the addition of ice-cold lysis buffer (25 mM Tris, pH 7.4, 75 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 74 g/ml leupeptin, 740 M PMSF and 74 g/ml aprotinin) for 30 min with continual rotation at 4 C. Lysates were clarified (14,000g for 10 min) and an equal volume of 2SDS-PAGE sample buffer (50 mM Tris, pH 7, 700 mM 2-ME, 50% (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) was added to postnuclear supernatants. Proteins were solubilised at 100 C. for 5 min and samples containing 110.sup.6 cell equivalents were resolved by SDS-PAGE.

(57) c. Immunoblotting.

(58) Separated proteins were transferred to nitrocellulose membranes (Bio-Rad) which were then blocked with 5% (w/v) bovine serum albumin (Sigma, fraction V; BSA), 0.1% Nonidet P40 in Tris-buffered saline (170 mM NaCl and 50 mM Tris, pH 7.4; TBS). Immunoblots were incubated with the appropriate antibodies as indicated in figure legends. Primary antibodies were diluted in antibody dilution buffer comprised of 0.5% (w/v) BSA, 0.1% (v/v) Tween-20 in TBS at 4 C. for 2 h followed by detection with the appropriate secondary antibody conjugated to horseradish peroxidase diluted in antibody dilution buffer at 4 C. for 1 h. Following each incubation step, membranes were washed extensively with 0.1% Tween-20 in TBS. Immunoreactivity was determined using the ECL chemiluminescence detection system (Amersham Corp., Arlington Heights, Ill.).

(59) d. Inhibition of Proteolytic Activity of CCS.

(60) A specific cysteine protease inhibitor, E-64, was used to inactivate the proteolytic activity of CCS. CCS (25 g/ml) diluted in 3 M dithiothreitol, 100 M E-64, 60 mM sodium acetate (pH 5) was incubated for 10 minutes at 30 C. The inactivated CCS was then dialysed overnight in saline at 4 C. Earlier studies with crude bromelain have shown that these conditions are sufficient to induce 99.5% inactivation of proteolytic activity as assayed with the Z-Arg-Arg-pNA substrate (see above). T cells were treated with E-64 inactivated CCS (25 g/ml) and compared with untreated CCS and mock-treated T cells stimulated with PMA plus ionophore.

(61) Results

(62) a. Fraction CCS Inhibits Tyrosine Phosphorylation of p42 kda Phosphoprotein.

(63) We have previously shown that bromelain blocks tyrosine phosphorylation of ERK-2 following stimulation of T cells with combined PMA plus calcium ionophore (WO-A-96/00082). Phorbol ester and ionophore stimulation of T cells act synergistically to reproduce many features of TCR stimulation such as IL-2 secretion, IL-2 receptor expression, and T cell proliferation (Truneh et al., 1985, Nature, 313: 318-320; Rayter et al., 1992, EMBO, 11: 4549-4556). Phorbol esters can mimic antigen receptor triggering and bypass TCR-induced protein tyrosine kinases to activate ERK-2 by a direct agonist action on PKC and p21.sup.Ras. Calcium ionophore A23187 induces increased intracellular release of Ca.sup.2+ and therefore mimics the action of inositol 1,4,5-trisphosphate (IP.sub.3). Phorbol esters and ionophore however, stimulate PKC pathways that are not controlled by the TCR (Izquierdo et al., 1992, Mol. Cell. Biol., 12: 3305-3312) suggesting separate intracellular pathways within T cells that regulate T cell function. We therefore investigated which fraction of bromelain could block T cell signalling via the TCR-independent pathway by examining its effect on PMA and ionophore-induced tyrosine phosphorylation.

(64) Stimulation of T cells with combined ionophore and PMA induced tyrosine phosphorylation of several proteins including those of circa 100 kda, 85 kda, 42 kda and 38 kda. CCS (50 g/ml) pre-treatment reduced tyrosine phosphorylation of the p42 kda protein, and did not significantly inhibit phosphorylation of any other substrate (FIG. 6). In two experiments, CCS, but no other fraction, also increased tyrosine phosphorylation of circa 36 kda, 38 kda, 85 kda, 94 kda and 102 kda proteins (FIG. 7 and FIG. 8).

(65) The ability of CCS to block tyrosine phosphorylation of the 42 kda phosphoprotein was dose-dependent (FIG. 8) and dependent on its proteolytic activity, since E-64 completely abrogated the inhibitory effect of CCS on p42 kda phosphorylation (FIG. 8). E-64 treatment of T cells did not affect PMA and ionophore-induced T cell signalling.

(66) CCS Inhibits ERK-2 Tyrosine Phosphorylation.

(67) We suspected that the 42 kda phosphoprotein inhibited by CCS was the MAPk ERK-2, so we conducted immunoblot analysis with specific anti-ERK-2 mAb and anti-phospho MAPk antibodies, which specifically detects ERK-1 and ERK-2 only when catalytically activated by phosphorylation at Tyr204. Immunoblotting of CCS-treated cells that had been stimulated with PMA plus ionophore, confirmed that the p42 kda phosphoprotein was indeed ERK-2 (FIG. 9).

(68) CCS Reduces TCR-Induced Tyrosine Phosphorylation of ERK.

(69) We next investigated the effect of CCS on TCR-mediated signal transduction by assessing substrate tyrosine phosphorylation of GA15 stimulated with crosslinked anti-CDR mAb. Immunoblots of GA15 lysates, using specific anti-phosphotyrosine mAb, revealed increased tyrosine phosphorylation of multiple proteins including those of circa 120 kda, 100 kda, 85 kda, 76 kda, 70 kda, 42 kda and 40 kda, consistent with phosphoproteins observed in other T cell lines following TCR-ligation (June et al., 1990, J. Immunol., 144, 1591-1599 and Proc Natl. Acad. Sci. USA, 87, 7722-7726, reviewed by Cantrell, 1996, Annu. Rev. Immunol., 14, 259-274) (FIG. 10). Tyrosine phosphorylated proteins were readily detected between 2 and 5 min following stimulation and remained phosphorylated for at least 10 min (FIG. 10). GA15 cells stimulated with anti-CD3 mAb alone or cross-linking Ab, did not induce tyrosine phosphorylation of any cellular substrate (data not shown). Again, CCS pretreatment of GA15 for 30 min caused a reduction in TCR-induced protein tyrosine phosphorylation of ERK-2 in a dose-dependent manner (FIG. 11). CCS did not markedly affect tyrosine phosphorylation of other TCR-induced phosphoproteins, suggesting that CCS has a selective mode of action.

EXAMPLE 4

CCS Retards the Mobility Shift of Raf-1

(70) Raf-1 is an immediate upstream activator of MEK-1 which activates ERK-2. Raf-1 activation requires phosphorylation on specific serine and threonine residues (Avruch et al., 1994, TIBS, 19: 279-283). To investigate whether CCS affects any other substrates upstream from ERK-2 in the MAP kinase cascade, we investigated the effect of CCS on Raf-1. T cells were treated with CCS (0 to 50 g/ml) and then stimulated with either anti-CD3 mAb or combined PMA plus ionophore as described earlier. Results show that CCS blocks the mobility shift of Raf-1, indicating that it blocks its protein phosphorylation and thus activation. This data confirms that CCS has an effect on the MAP kinase cascade (FIG. 12) and that the effect of CCS may not be directly on ERK-2, but on upstream substrates in the MAPk cascade.

EXAMPLE 5

Effect of CCS on IL-2 Production and T Cell Proliferation

(71) a. Materials

(72) Cells: Splenocytes were isolated from female BALB/c mice (6-8 weeks old), as previously described in WO-A-96/00082. Highly purified CD4.sup.+ T cells were isolated from splenocytes using magnetic activated cell sorting (MACS).

(73) b. Interleukin 2 Production.

(74) T cells diluted in RPMI were treated with CCS (50 g/ml) or saline at 37 C. for 30 min, washed in fresh RPMI and then resuspended in culture medium. T cells were stimulated to produce cytokine mRNA by immobilised anti-CD3 (4 g/ml) and soluble anti-CD28 (10 g/ml). Anti-CDR mAb diluted in PBS was immobilised to 24-well, flat bottom, microculture plates (Corning, Corning, N.Y.) by incubation for 16 hours at 4 C. Wells were then washed three times in PBS prior to addition of triplicate cultures of either splenocytes or purified CD4.sup.+ T cells (2.5-510.sup.6 cells per well) which were incubated at 37 C. in humidified 5% CO.sub.2 for 24 h. IL-2 levels in the culture supernatant were measured using the CTL-L bioassay (Gillis et al., 1978, J. Immunol., 120: 2027-2032).

(75) c. T Cell Proliferation.

(76) T cells were treated with CCS (50 g/ml) for 30 min, washed in RPMI then stimulated with immobilised anti-CD3 mAb alone or combined anti-CD3 mAb plus anti-CD28 mAb. Cells were then cultured in 96 well, flat-bottom plates (Nunc) at 10.sup.5 cells per well for 36 h. Cultures were pulsed with 0.5 Ci of [.sup.3H]TdR 12 h prior to harvesting onto glass fibre filters.

(77) Results

(78) a. CCS Inhibits IL-2 Production and Proliferation of CDC4.sup.+ Cells.

(79) Activation of p21.sup.Ras, Raf-1, MEK-1 and ERKs are essential for induction of IL-2 transcription in T cells (Izquierdo et al., 1993, J. Exp. Med., 178: 1199). IL-2 is the major autocrine T cell growth factor which induces proliferation of T cells. The defect in ERK activation demonstrated here could therefore be expected to inhibit IL-2 production and T cell proliferation. We therefore investigated whether CCS could effect a functional outcome of T cell signalling, namely IL-2 production and proliferation in murine splenocytes and highly purified CD4.sup.+ T cells. CCS (50 g/ml) treatment of purified CD4.sup.+ T cells reduced both IL-2 production and proliferation when the ERK pathway was stimulated with anti-CDR mAb (FIGS. 13a and 13b). CCS also blocked IL-2 production by splenocytes, however it did not affect splenocyte proliferation (FIGS. 14a and 14b), suggesting that an as yet unidentified component in CCS was acting on accessory cell populations in splenocyte cultures, such as B cells or macrophages. Bromelain can increase costimulatory signals to T cells via an action on B cells. Regardless of the putative effect of CCS on accessory cells, data clearly indicate that CCS blocks IL-2 production and proliferation of purified CD4.sup.+ T cells, suggesting that CCS blocks T cell activation. IL-2 production and proliferation were dependent on cell stimulation with anti-TCR antibodies as no cytokine was detected in cells cultured in tissue culture media alone (FIGS. 13 and 14).

EXAMPLE 6

Effect of CCS on Human Tumour Cell Growth in Vitro

(80) a. Materials

(81) Cells: Tumour cell lines were provided by L. Kelland (Institute of Cancer Research, Sutton, UK) and were as follows; ovarian (SKOV-3, CH-1, A2780), colon (HT29, BE, LOVO), breast (MCF-7, MDA231, MDA361), lung (A549, CORL23, MOR) and melanoma (G361, B008, SKMe124).

(82) b. Growth Inhibition of Human Tumour Cell Lines.

(83) Studies were conducted by L. Kelland (Institute of Cancer Research, Sutton, UK). Cell lines were trypsinised and single viable cells were seeded into 96-well microtitre plates at a density of 410.sup.3 cells/well in 160 l growth medium. After allowing for attachment overnight, CCS was then added to quadruplicate wells in 40 l of growth medium to give a range of final concentrations in wells of 50, 10, 2.5, 1 and 0.25 g/ml. Eight wells served as control, untreated wells. CCS was diluted immediately prior to addition to cells in sterile water. CCS exposure to cells was for 96 h whereupon the cell number in each well was determined by staining with 0.4% sulforhodamine B in 1% acetic acid as described previously (Kelland et al., 1993, Cancer Res., 53: 2581-2586). 50% inhibitory concentrations (IC.sub.50 values in g/ml) were then calculated from plots of concentration versus control (%) absorbance (read at 540.sub.nm).

(84) Results

(85) a. CCS Inhibits Human Tumour Growth In Vitro.

(86) p21.sup.Ras and Raf-1 are important oncogenes, which when mutated cause uncontrolled cell growth and proliferation, leading to cancer. Since we have shown that CCS can block the effects of the p21.sup.Ras/Raf-1/MEK1/ERK kinase signalling cascade, we investigated whether CCS could block tumour growth. CCS treatment of human tumour cells resulted in a reduction in the growth of several different ovarian, lung, colon, breast and melanoma tumour cell lines in vitro (FIG. 15). CCS did not affect all cell lines equally, suggesting that CCS has a selective action.