IE903233A1 - Novel medical use - Google Patents

Abstract

The use of thioredoxin in the treatment of B lymphocytic leukemia and certain other malignant diseases. The enzyme can be used either alone or in combination with co-factors such as anti-immunoglobulins, interferons or interleukin 1,2,3, or 4.

Description

The present invention relates to a novel strategy for the treatment of B lymphocyte leukemias and certain other malignant diseases, including a method for potentiating the reactivity of lymphocytes responsive towards cancer cells expressing surface structures recognized by the patient's own cytotoxic cells. Examples of such cancers are malignant melanomas and colon cancer.

It is known from WO88/06891 that B-cell growth factors, and antibodies that mimic these, can be used for the induction of differentiation in certain malignant disorders. We describe here the use of an enzyme belonging to the thioredoxin family, such as MP6 cell line derived thioredoxin (MP6/Trx) for such induction of differentiation. The said enzyme will be used either alone or in combination with co-factors.

General outline of the invention and introduction Cancer cells are characterized by uncontrolled growth. For some time there has been a concept that growth can be suppressed by inducing these cells to differentiate into a non proliferative state. Clinical trials have also been done in different leukemias with differentiation-inducing agents such as vitamins and interferons. However, no such trials have been done with more specific growth and differentiation factors, or antibodies, which only react with defined receptor structures. The present invention proposes to use such specific factors for cancer treatment, either alone or in combination with supporting, agents.

The development of normal cells into cancer cells is a multi-step process. During malignant transformation some cell types, for example some B lymphocytes (reference 1), acquire the ability to express receptors for defined growth factors and respond to these by proliferation or maturation. The tumor cells are thus ’’frozen at a specific differentiation stage, characterized by a specific set of surface receptors. This block is, however, not irreversible. We here present a method for the use of an enzyme belonging to the thioredoxin family, and analogues to thioredoxin containing the same active site Cys-GlyPro-Cys, including monoclonal antibodies binding to the target structure, to be used alone or in common with cofactors, for the induction of terminally differentiated cells (end cells) which do not further divide. The said enzyme and co-factors are described. The strategy of clinical treatment is exemplified with B-cell chronic lymphocytic leukemias (B-CLL), which were induced to further differentiation (to a more mature stage) signified by impaired capacity to proliferate and the expression of a plasmacytoid morphology, as judged by surface markers, cytoplasmic immunoglobulin, and endoplasmatic reticulum.

For a resting B-cell, the initial activation signal, elicited by the antigen - immunoglobulin (Ig) interaction, must be followed by a series of finely tuned receptorligand signals and cell-cell interactions with other immunocompetent cells (1), to allow terminal plasma cell maturation. Several ligands for receptors involved in the transmission of growth and differentiation controlling signals in human B cells have been defined and the genes cloned. These include interleukin 1 (IL-1) to interleukin 6 (IL-6), low molecular weight B cell growth factor (LMWBCGF), SCD23, lymphotoxin (LT), tumor necrosis factor (TNF), interferon-% (IFN-tf) (1,2).

To grasp the concept of differentiation therapy it is important to understand how normal cells develop. In the bone marrow, different functionally specialized cell types develop as a result of differentiation (commitment) of the multipotent stem cells. This differentiation gives rise to precursors of various cell linages (B-cell linage, T-cell linage, myeloid linage). Subsequent phenotypic changes of such unipotent cells into end cells is called maturation or terminal differentiation. The activation of human B-cells from a resting stage, leading into further differentiation and maturation and the terminal stage proceeds through at least two steps. 1) The activation step, where the cells are exposed to activating factors. For the B-cell series these are: Antigens; anti-immunoglobulins (anti-idiotypes); interleukin 1, 2 and 3 and sub-components thereof, interleukin 4 (IL4) and antibodies to the IL4-receptor; reagents acting on the C3d-receptor (CDllc), such as polymerized complement 3d or antibodies to the C3d receptor (anti-gpl40); anti-gp35 (CD20). Phorbol esters, such as TPA or PMA are used experimentally in vitro as potent competence-inducing agents, but these can however only serve as models since they are toxic and incompatible with clinical use. The phorbol esters act on protein kinase C (PKC) and in their function mimic biologically active agents. Other experimental competence-inducing agents of importance are: solid phase protein-A; inactivated Staphylococcus Aureus Cowan I (SAC); Poke-weed Mitogen (PWM); non-transforming or inactivated EpsteinBarr Virus (EBV) (from the non-transforming strain P3HR1 or UV-inactivated virus) lipopolysaccharides (LPS). 2) The progression step. The activation step induces receptors for various progression signals such as : IL-2; B-cell growth factor II or TRF, now called IL5; low molecular weight BCGF (12K BCGF); Namalwa-derived 60K BCGF; antibodies to CD23 (a p45 protein expressed on the B-cell surface of IgM+, IgD+ cells, FcE receptor 2 (FcER2) antibodies to CD40, a p50 antigen present mainly on BIE 903233 cells and on urinary bladder carcinoma cells, but also on cervical and lung carcinoma cells, furthermore IL-6 (previously called B-cell differentiation factor (BCDF). The following list is a brief explanation of abbreviations used in the present specification.

BCDF: B-cell differentiation factor BCGF: B-cell growth factor B-CLL: B-cell chronic lymphocytic leukemia BSF: B-cell stimulating factor C3d: Sub-component of complement factor C3 CD23: A p45 protein expressed on cells of the B- lymphocyte linage, CD40: A p50 protein expressed on B-cells and on bladder carcinoma cells EBV: Epstein-Barr virus gp35: Glycoprotein 35K molecular weight, belonging to the CD20 group (cluster of differentiation group) gpl40: Glycoprotein 140K molecular weight, with C3d- receptor function IgD: Immunoglobulin class D IgM: Immunoglobulin class M IL-1, IL-2, IL-3, IL-4, IL-5: Interleukin 1, 2, 3, 4, c LPS: O Lipopolysaccharides Molt4: A T-lymphoma derived cell line p45: A 45K molecular weight membrane protein PMA: 4-phorbol 12-myristate 13-acetate PWM: Poke weed mitogen SAC: Staphylococcus aureus Cowan I Solid phase protein-A: Matrix (Sepharose for example) - bound protein-A TPA: Tumor promoting agent TAF: T-cell replacing factor T-T hybridoma: A somatic cell hybrid between two different T-cells.

TNF Tumor necrosis factor MP6/Trx: MP6 T-T hybridoma cell line produced enzyme of thioredoxin family.

Detailed description of the invention A 12 kDa B call stimulatory factor CBSF) secreted by a human CD4* T cell hybridoma CXPO), «as previously shown to facilitate growth of normal and malignant human B lymphocytes.

Ve have no* purified this lyvphokIne and Identified It as a member of the human thioredoxin family and named it MP6/Trx Thioredoxin is a well-characterized enzyme catalyzing thiol25 disulphide interchange reactions and net protein disulphide reductions via a Cys-Gly-Pro-Cys active site. We used normal peripheral blood or tonsillar B lymphocytes as target cells for monitoring biological activity . But monoclonal B cells, Blymphoblastoid cell lines, or B cells derived from B-type of chronic lymphocytic leukemia (B-CLL), were target cells par excellence, since they required MP6/Trx for cytokine induced proliferation and differentiation in vitro, when tested under suboptimal cell culture conditions.

Pre-act1vated cells did proliferate In response to the 2- recombinant or natural Ugandss Interleukin 2 C IL-23, Interleukin 4 CIL-O, low molecular weight BCGF CLMW-BCGF?, tumor necrosis factor-a CTNF-ού, or antl-CD4O, only If MP6/Trx was added. Antibodies to thioredoxin blocked the effect. These results assign an important role to extracellular thioredoxin in the regulatory events involved in receptor-ligand interactions and subsequent signal transduction in normal B-cell activation and in B-CLL leukemogenesis.

The present invention relates to a novel method for the treatment of such malignantly transformed cells in mammals and in man, which are sensitive to the co-factors mentioned below and to thioredoxin. The method is characterized by the administration of a therapeutically adequate amount of thioredoxin. If necessary, said enzyme is administered following a period of pre-treatment with a co-factor capable of inducing the malignantly transformed cells to become sensitive to said enzyme. Example of such co-factors, are given in Table 1 below. It is foreseen that the administration of the enzyme thioredoxin can be made simultaneously with the co-factor.

The term thioredoxin as used in the present specification is understood to include the thioredoxin enzyme family and analogues of thioredoxin containing the active site Cys-Gly-Pro-Cys, specifically the MP6 cell line-derived thioredoxin.

More precisely, the novel method of treatment by the present invention can be applied to stem-cell disorders, hematopoetic malignancies, for example leukemias, B-cell leukemias and B-cell chronic lymphocytic leukemias, and other tumors which express co-factor receptors and respond to thioredoxin. For example, bladder carcinomas expressing the CD40 antigen can potentially be treated in the described fashion. Thioredoxin as well as the co-factors listed in Table 1 are substances which are known as such. They are, however, not in every instance known to have therapeutic utility.

The choice of a suitable co-factor is no critical parameter of the invention. There are experimental methods available which will enable the skilled worker to establish whether a specific co-factor as listed in Table 1 acts in synergy with the thioredoxin. It is, however, preferred to use IL-2 as co-factor. Also IL-4 and TNF-c< may be mentioned as preferred co-factors.

The invention in another aspect relates to thioredoxin for use in the treatment of malignantly transformed cells in animals and in man, in particular for use in such malignantly transformed cells which are sensitive to thioredoxin. Also in this aspect, if necessary, thioredoxin is administered following a period of pretreatment with a co-factor as described, which is capable of inducing the malignantly transformed cells to develop sensitivity for thioredoxin. Another aspect of the invention relates to the use of thioredoxin in the preparation of a medicament for treatment of malignancies. Such a medicament may comprise a co-factor as described above. Even though thioredoxin as well as co-factors as exemplified in Table 1 are known in the art, pharmaceutical preparations containing thioredoxin or of a combination of thioredoxin and a co-factor according to Table 1, are novel and represent as such an additional aspect of the present invention.

It is foreseen that such malignancies which are sensitive to treatment with IL-2, such as malignant melanomas, will be suitable targets for treatment with thioredoxin, suitably in combination with a co-factor.

It is also foreseen that cellular immunity (T-cells and NK-cells) can be strengthened by treatment with thioredoxin, optionally in combination with a co-factor as described.

In clinical practice, thioredoxin, co-factors or combinations thereof are administered in a manner which is analogous with known ways of administering medicaments for the treatment of cancer. Thus, administration will preferably be made by infusion or by intramuscular deposition.

The amount in which thioredoxin and/or co-factors is administered will vary within a wide range and will depend on various circumstances such as the severity of the disease and the age and the state of the patient. As an example of a suitable dosage interval can be mentioned a dosage which will provide a serum or plasma level of thioredoxin which is from about 2 to about 100 times the naturally occurring thioredoxin serum or plasma level.

The following Table 1 gives a list which exemplifies cofactors which may be used. The designation E indicates that the co-factor mainly is experimental and has possible use for diagnostic purposes. The designation C indicates that the co-factor has clinical use.

Table 1. Co-factors E Phorbol esters such as TPA E Antigens C Anti-Immunoglobulins (anti-idiotypes) C Interleukin 1 and sub-components thereof C Interleukin 2 and sub-components thereof C Interleukin 3 and sub-components thereof C Interleukin 4 (BSF1) C Anti-IL4-receptor antibodies E Poke weed mitogen E Lipopolysaccharidec E Epstein Barr virus, non-transforming or inactivated C C3d receptor (CDllc) reactive agents C' and anti30 receptor (gp 140) antibodies C Anti-gp35 (CD20) E SAC, Inactivated Staphylococcus aureus Cowan I E Solid-phase protein A C Interferons (alfa, beta and gamma) C Vitamins (in particular vitamin A, D, and biologically active derivatives C Leukotriene B4 C TNF-CV The enzyme thioredoxin as used in the present invention is preferably of human origin. It is an enzyme catalyzing thiol-disulphide interchange reactions and net protein disulphide reductions via Cys-Gly-Pro-Cys active site.

Human thioredoxin is preferably of human lymphocyte origin although other origins can be used. However, also the use of animal including mammal thioredoxin, procaryotic thioredoxin obtained e.g. from E. Coli and thioredoxins produced by genetically engineered expression vectors is included in the scope of the invention.

Thioredoxin, also known as thiol-oxidoreductase, is a ubiquitous 12 kDa protein with a redox-active disulphide (3); it is usually reduced by NADPH and the flavoprotein thioredoxin reductase. Reduced thioredoxin is a hydrogen donor for ribonucleotide reductase, an essential enzyme making deoxyribonucleotides for DNA synthesis. Thioredoxin is also involved in regulatory events (3), such as the light-dependent activation of photc-synthetic enzymes in the chloroplast of plant cells (4), and activation of glucocorticoid receptors to a steroid binding state (5). 15 Thioredoxin regulates enzyme activity by thiol redox control which involves reduction of protein disulphides with a rate that is about 10° times faster than that of dithiotreitol (DTT) (3). Mammalian thioredoxins have been isolated and 20 characterized (3,6). The distribution has been studied by immunohistochemical methods and thioredoxin was shown to be related to protein secretion and partly membrane associated (6). Recently a human thioredoxin gene was cloned by Wollman et al. (7). The gene was found to be expressed in activated, but not in resting lymphocytes. Originally the thioredoxin was reported to be an IL-1 like factor, derived from an Epstein-Barr virus containing B-cell line (7). Tagaya and co-workers (8) showed that the IL-2-receptor/Tac-inducing factor, also called adult T cell leukemia (HTLV-1) derived factor (ADF), was homologous to or identical with thioredoxin from analysis of a c-DNA clone.

The present invention assigns new biological functions for 10 the thioredoxin family of enzymes and expands its role in lymphocyte activation.

Target cells in clinical situations: Target cells in clinical situations are all such malignantly transformed cells that can respond to thioredoxin, especially MP6/Trx, by differentiation, including all those malignant cells that can be induced to express binding sites for thioredoxin and respond to this.

Such induction can be exerted by the co-factors described in Table 1 or by other means.

Experimental evidence The MP6 le a CD4* T helper cell hybridoma, previously Isolated and cloned by us (9). The MP6 clone constitutively secretes a 12-14 kDa B cell stimulatory factor (BSF-MP6) Inducing proliferation and IgM/IgG secretion in normal (9, 10), as well as in malignant pre-activated B cells of B-type chronic lymphocytic leukemia (B-CLL) (11). The IL-2 receptor expression was also enhanced by BSF-MP6 (12). Kishlmoto and Honjo et al. have demonstrated that mRNA from MP6 cells did not hybridise with cDNA probes for IL-lo, IL-lft, IL-4, IL-5 nor IL-6 (12). Using various cellular assays, the MP6 supernatant was shown to lack activities of LMW-BCGF, ThF-o, and -fi, IFN-o, -/?, -Y, granulocyte-monocyte colony stimulating factors (GM-CSF). IL-1. IL-2, IL-4, IL-5 and IL-6 (9).

In the experiments represented in Figures IA and IB. we utilized monoclonal cells derived from a patient with B-CLL. This clone (183) represents Gq arrested B-cells inducible to differentiation or differentiation accompanied by proliferation depending on the co-stimulatory signals (13). when activated by 12-O-Tetradecanoyl-phorbol 13-acetate (TPA) or by Staphylococcus aureus Cowan I (SAC). 183 cells were pre-activated by SAC for 2 days, to mimic antigen-triggered signals, or by a sub-optimal dose of TPA (1.6x10 7M) for 1 h.

The cells were refractory to any of the recombinant or natural B-lymphotropic lymphokines rIL-1/?, rIL-2, rIL-4, rIL-6. rTNFo>, LMW-BCGF, rIFN-y, anti-CD40, or combinations thereof. SAC-activated cells did, however, respond to the lymphokines rIL-2, rTNFo, LMW-BCGF, when BSF-MP6, was added (Figure IA).

The signal pathway for SAC and TPA are different, in that TPA provides a non-phyaiological signal directly activating proteinklnase C. moving the cells into the cell cycle. Figure IB illustrates that TPA activated cells responded to BSF-MP6 •lone, end that the combinations of BSF-MP6 with several different B lymphokines did not further increase DNA-synthesis. The exceptions ere IL-4 end TNF-a which showed significant increase. This is in line with recent findings that TNF-o is an autocrine growth factor for human B cells (14), and we have previously shown in a series of experiments that IL-4 is strongly synergistic with BSF-MP6 for the Induction of DNA-synthesis and for IgM secretion in TPA-activated cells (11).

A highly specific radioimmunoassay for human thioredoxin (13) (Figure 2), reveals that the BSF-MP6 factor is homologous to thioredoxin, or an analogue of thioredoxin as described earlier.

Serum-free medium of 24h conditioned medium MP6 contained 34 ng/ml of thioredoxin. Biological activity was monitored using the 183 B-CLL clone or normal tonsillar B cells- and. was -©’'fined to the 12--kDa region in gel filtration experiments. Mammalian thioredoxins, after air oxidation, forms extra structural intra-molecular disulphide bonds leading to inactivation and aggregation (6). During the purification procedures and storage, preparations of BSF-MP6 were also easily oxidized, by atmospheric O*, with a resulting loss of activity. When this fact was realized we started to perform the B-CLL activation experiments with a sub-cptimal dose of 0.2 pM /^-mercaptoethanol present during the cultivation period. Higher concentrations of /7-mercaptosthanol (50-200 pM) promoted an Increase in DNA-synthssis in Itself, however. Growth stimulation of leukemic cell· by thiols end disulphides in vitro Is a well-known phenomenon (22). Figure 3 demonstrates the reconstitution of full biological activity in an 6 months old and Inactive BSF-MP6 preparation after Incubation with DTT. The observation that BSF-MP6 could be revived by reduction, la a typical feature of thioredoxins (6). The sample (eerum-free, sterile. 24 h conditioned medium of MP6 stored at *4eC) was reduced for 30 minutes et 37eC with 2 mM DTT prior to HPLC-gelfiltretion. Almost ell biological activity was recovered in the 12 kDa region.

The evidence from the radioimmunoassay and the reconstitution experiments that BSF-MP6 is homologous to thioredoxin or an analogue of thioredoxin as described earlier, prompted us to demonstrate whether thioredoxin, derived from another source could replace BSF-MP6 in the biological assay. Homogeneous human thioredoxin derived from placenta (13) was tested at a concentration of 0.5 x to 0.5 x 10-i*M, and showed biological activity down to 0.5 x 10_B M.

The test results are given in Table 2 below.

The B-CLL cells were pre-treated with SAC 1:100 000 and IL 2 10 U/ml. 3H-Thy was added for the last 18h of a 72h incubation period. As is seen in Table 2, the thioredoxin was highly active.

Table 2. Stimulation of B-CLL cells by human placenta thioredoxin Thioredoxd n (M) DNA-synthesis 3H-Thymidine incorporation (cpm) 0.5 x 10-7 6327 control medium 1240 Biochemical characterization of this T-helper cell derived thioredoxin was performed by immunosorbent affinity chromatography, with Sepharose-coupled sheep anti-thioredoxin antibodies, combined with HPLC-gelfiltratlon. The procedure yielded highly purified thioredoxin as seen in Figure 4. The starting material was 24 h MP6 serum-free medium. The insert SDS-PAGE gel picture confirms the purity and molecular weight of the affinity purified material.

For the understanding of B cell differentiation, the clonal malignancy of B-CLL has proven to be a very useful model (11).

The low proliferative capacity of B-CLL fn vivo might, in pert, be the result of a deficiency in growth factor* produced by autologous non-B cells. BSF-MP6/thioredoxin 1* according to the evidence presented here one of the missing links. The evidence that BSF-MP6 with it* thioredoxin activity, facilitates a proper response to the T-cell derived IL-2. IL-4, LMW-BCGF, end TNF-«, provides for the first time a possible explanation for the growth arrest of B-CLL cells. The notorious dys-regulation of T helper lymphocytes in B-CLL patients (15) might result in a loss of thioredoxin production, necessary for the activation of the B-CLL cells, as shown by our results. Alternatively, the B-CLL cells themselves might be deficient for autocrine thioredoxin or require an Initial dose of externally supplied thioredoxin to initiate its autocrine production.

Our present finding of identity between thioredoxin and a B cell stimulatory factor, strongly suggests a pivotal immunological role for this enzyme. It facilitates proper signal transduction and the well-known function of the enzyme to catalyze thiol-disulphide interchange reactions may (3) presumably allow dynamic three-dimensional .correct receptor docking events to take place, although to gain knowledge about the exact mechanism further studies are required.

A useful fn vitro model system for studies of B cell growth and differentiation controlling signals have been the human B-lymphotropic herpesvirus Epstein-Barr virus (EBV), since it induces proliferation and differentiation (16). by turning on genes obligatory for B cell growth (17). The B-CLL cells have, however, been refractory to attempts of EBV-transformation and one possible explanation to this resistance could be found in the fact that B-CLL cells, in addition to their low expression of EBV-receptors (CD21), might be defective in their thioredoxin gene expression as indicated in this report and by our preliminary immunofluorescence analysis.

Cellular thioredoxin, was recently suggested to be a principal hydrogen donor for herpes virus simplex-type 1 encoded ribonucleotide reductase (18). Thus, a lack of thioredoxin in the B-CLL cells could effectively block any herpesvirus multiplication in those cells.

Strategy of therapy 1) Thioredoxin by itself, especially MP6/Trx, should be 5 administered when malignant cells already express any of the binding sites for thioredoxin. 2) Thioredoxin plus compounds in Table 1 should be administered in combination when the malignant cells do not express any of the binding sites for thioredoxin. This includes any of the specific compounds listed.

Figure Legends Figure IA and IB NPO Induces signals for DNA synthesis Cells derived from the B-CLL clone 103. kept frozen in liquid nitrogen, were revived and Induced to DNA synthesis (Figure IA and IB) and immunoglobulin secretion (data not shown) with 10 either SAC (Figure IA) or TPA (Figure IB) as activating signals. To Induce proliferation and differentiation with SAC, cells were incubated with fixed bacteria for 2 days and then exposed to 100 U/ml of recombinant interleukins or natural B cell cytokines with or without 254 BSF-MP6 (v/v). Cells were cultured in flat-bottomed 96-well plates as 0.2 ml cultures (4 x 10° cells/well) or 2-ml cultures (4 x t0*/wel1)(Costar, 2Q Cambridge, MA) in RPMI 1640 medium (Flow Laboratories, Ayshire, GB), supplemented with 104 newborn calf serum (Gibco Glasgow, G.B.), 2mM L-glutamine, SO pg/ml gentamycin, 100 IU/ml of penicillin and 100 pg/ml of streptomycin. The cells were cultured for 6 days at 37°C in 54 COa-in air atmosphere. DNA synthesis was measured, essaying the incorporation of 1 pCi (s37kBq) per well of tritiated thymidine ((*H ]dThd; spec.act. 6.7 Ci/mmol; Dupont Scandinavia, Stockholm, Sweden), during the last 20-24 h of the cultivation period.

Heat-inactivated, formal in-fixed SAC-particles were used at a final concentration of 0.14; TPA (Sigma Chemical Co., St.

Louis, MO) m«s used, et 1.6 x 10 cM-concentration; BSF--MP6 was obtained from serum-free 24 h cultures of the MP6 T cell hybridoma grown in Iscoves medium supplemented with 400 pg/ml of BSA (Boerhinger-Mannheim, Mannheim, W.Germany), 12.5 pg/ml of human transferrin (Kabi, Stockholm, Sweden), 50 pM /t-mercaptoethanol, 2 mM L-glutamin, penicillin/streptomycln, concentrated on an Amicon device with an YM2 filter. rIL-2 wee purchased from Amgen (Amersham, Amersham, G.B). rXL-l/V (Genzyme, Boston. MA} had a specific activity of 10* U/mg and was used at 10 U/ml. rIL-4 was purchased from Genzyme (Boston. MA) and used at a final concentration of lOOU/ml. rIL6 was a gift from Dr. Klshlmotc, Osaka, Japan, and was used et 100 U/ml. Recombinant TNFa with a specific activity of 6 x 107 U/mg was used at 100 ng/ml and was a gift from Dr. G.R.

Adolf. Ehrnst Boehringer Institute, (Vienna, Austria). LMV-BCGF was purchased from Cellular Products (Buffalo, NY) was used at a concentration of 104 v/v. Monoclonal anti-CD40 (G28-5 Mab) used at a concentration of 1 pg/ml was a gift from Dr. E. Clark (Seattle, VA). rIFN-y was obtained from Genentech (San Franslsco, CA). It had a specific activity of 3 x 107 U/mg and was used at 500 U/rel.

IE 903233 22 Figure 2 Radioimmunoassay for thioradoxin shows identity between 1ST-XF© and thioredoxin.

Solid line indicates pure human placenta thioredoxin.

Broken line indicates BSF-XPO.

The radioimmunoassay was performed as described previously (13). briefly: 0.1 ml (0.2 pmol) of I-labeled human plac enta thioredoxin was incubated with 0.1 ml of standard i0 human thioredoxin or unknown sample (ΜΡ6 supernatant concentrated 50-fold by ammonium sulphate precipitation), serialy diluted, and 0.1 ml (5 pg) of the IgG fraction of e rabbit antiserum against human thioredoxin, at 37*C with shaking for 4 hours. At the end of the incubation period 0.1 ml of a 1:5 diluted sheep anti-rabbit IgG antiserum was added and incubation was continued for 16 h at 4 C. The bound radioactivity (B) was separated from the free (F) by centrifugation for 30 min at 10 000 x g, followed by careful removal of the supernatant. The radioactivlXy was measured in both the pellet and the supernatant fractions using a LKB gamma counter (Bromma, Sweden). The ratio B/F was calculated and plotted against various standard thioredoxin concentration. The reactions in the absence of competing human thioredoxin and the rabbit anti-human thioredoxin antibody 25 were used as negative controls. The radioiodination of thioredoxin was performed according to the chloramln-T method (13). All dilutions and Incubations were carried out in phosphate buffered saline (PBS) containing 1 mg/ml of BSA. figure J.

Reconstitution of BSF-KPO activity by reduction with DTT The biological activity of BSF-MP6 could be recovered by reduction as shown above. The thioredoxin-expresslng T-hybridoma clone MP6 was cultured for 24 hours in Iscoves medium containing 400 pg/ml of BSA. 12.5 pg/ml of human transferrin, 50 PM /T-mercaptoethanol, 100 pg/ml of streptavidin, and 100 U/ml of penicillin and 2 mM of L-glut amin, but the biological activity of the supernatant was Q lost after two weeks of storage et +4 C. The chromatogram shows the protein profile measured at f ul 1 scale: A«0.5). of a 200 pi MP6 supernatant, pre-treated with 2 mM DTT, then separated on a Superose-12 FPLC gel filtration column (Pharmacia, Uppsala, Sweden), equilibrated with sterile phosphate buffered saline, pH 7.2. The flow rate was 0.4 ml/min. Fractions of 2 ml were collected and monitored for biological activity on B-CLL cells or on normal tonsillar B cells by measuring [*H-]thymidine incorporation, as indicated by the vertical bars in the chromatogram. The molecular weight markers indicated were bovine serum albumin (66K), and ribonuclease A (13.7K).

IE 903233 24 Figure 4.

! Chromatograa on BSF-KPO purified on anti-thioredoxin affinity column plus HPLC-gelf 11 tratlon.

HPLC-chromat©graphy was. performed on material that wee bound and eluted at pH 3.0 from a sheep anti-thioredoxin Sepharose-protein λ column (1.5 x 6 cm), coupled as previously described (21). Fractions of 1 ml were collected.

Equilibration buffer was PBS de-alrated in He*. The first peak at the 12 kDa contains the biological activity. The second peak is salt.

Insert of SDS-polyacrylamide gel» An 8-254 gradient SDS-polyacrylamide minigel (Pharmacia Phast gel system) was used for analysis of the purity. The samples are from left to right: MP6 serum free supernatant, before the affinity column; Affinity purified BSF-MP6/Thioredoxin; Human placenta thioredoxin; Molecular weight markers (Pharmacia) from top Xo bottom: 9?,5 kDa, 67 kDa,—45 kDa, 30.1 kDa, 20.1 kDa, 14.7 kDa.

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Claims (24)

1. What we claim is:

1. A pharmaceutical composition for the treatment of malignantly transformed cells in animals and man, which comprises thioredoxin or an analogue thereof containing the active site Cys-Gly-Pro-Cys, especially MP6/Trx, together with a pharmaceutically acceptable carrier or diluent.

2. A composition according to claim 1 wherein the origin of the thioredoxin is animal, human or other mammalian, procaryotic or recombinant.

3. A composition according to claims 1 or 2 wherein the thioredoxin is of human lymphocyte or other human origin.

4. A composition according to any one of the preceding claims, wherein the thioredoxin is MP6/Trx.

5. A composition according to any one of the preceding claims, including a co-factor capable of inducing binding sites for thioredoxin on the malignantly transformed cells.

6. A composition according to claim 4, wherein the said co-factor is selected from: (a) anti-immunoglobulins (anti-idiotypes) (b) interleukin 1 and sub-components thereof (c) interleukin 2 and sub-components thereof (d) interleukin 3 and sub-components thereof (e) interleukin 4 (BSF1) (f) anti-IL4-receptor antibodies - 29 (g) C3d receptor (CDllc) reactive C' agents and antireceptor (gp 140) antibodies (h) anti-gp35 (CD20) (i) interferons (alfa, beta and gamma) (j) vitamins (k) leukotriene B4 (l) TNF-α.

7. A composition according to claim 5 wherein the co-factor is interleukin -2.

8. A composition according to claim 1 substantially as hereinbefore described in any one of the Examples.

9. Thioredoxin or an analogue thereof containing the active site Cys-Gly-Pro-Cys for use in the treatment of the human or animal body having malignantly transformed cells .

10. The thioredoxin according to claim 9 which is as defined in any one of claims 2 to 4.

11. The thioredoxin according to claim 9 or 10 for use in conjunction with a co-factor capable of inducing the malignantly transformed cells to express binding sites for thioredoxin.

12. The thioredoxin according to claim 11 for use in conjunction with a co-factor as listed under (a)-(1) in claim 6. - 30

13. The thioredoxin according to any one of claims 9 to 12 for use in the treatment of stem cell disorders.

14. The thioredoxin according to any one of claims 9 to 12 for use in treatment of hematopoetic malignancies.

15. The thioredoxin according to any one of claims 9 to 12 for use in treatment of B-cell leukemias.

16. The thioredoxin according to any one of claims 9 to 12 for use in the treatment of B-cell chronic lymphocytic leukemia.

17. The thioredoxin according to any one of claims 9 to 12 for use in the treatment of tumors which express binding sites/receptors for, and respond to, the thioredoxin.

18. The thioredoxin according to claim 9 substantially as hereinbefore described in any one of the Examples.

19. Thioredoxin or an analogue thereof containing the active site Cys-Gly-Pro-Cys, particularly the MP6/Trx variant, for use in a method of treatment of the human or animal body in therapy, optionally in conjunction with a co-factor capable of inducing malignantly transformed cells to express sites for the said thioredoxin.

20. The use of thioredoxin or an analogue thereof containing the active site Cys-Gly-Pro-Cys, particularly the MP6/Trx variant, in the preparation of a medicament for the treatment of malignantly transformed cells in animals and man.

21. The use according to claim 20 wherein the thioredoxin is as defined in any one of claims 2 to 4.

22. The use according to claim 20 wherein the medicament includes a co-factor as defined in any one of claims 5 to 7.

23. The use according to claim 20 to 22 wherein the treatment is as defined in any one of claims 13 to 17.

24. The use according to claim 20 substantially as hereinbefore described in any one of the Examples.