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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
  • C12Y108/01008—Protein-disulfide reductase (1.8.1.8), i.e. thioredoxin

Definitions

• thioredoxin in the treatment of malignantly transformed cells in animals and man.
• 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.
• cancers are malignant melanomas and colon cancer.
• B-cell growth factors and antibodies that mimic these, can be used for the induction of differentiation in certain malignant disorders.
• an enzyme belonging to the thioredoxin family such as MP6 cell line derived thioredoxin (MP6/Trx) for such induction of differentiation.
• MP6/Trx MP6 cell line derived thioredoxin
• 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.
• B lymphocytes reference 1
• the tumor cells are thus "frozen” at a speci ⁇ fic differentiation stage, characterized by a specific set of surface receptors.
• This block is, however, not irreversible.
• the said enzyme and co-factors are described.
• B-CLL B-cell chronic lymphocytic leukemias
• the initial activation signal elicited by the antigen - immunoglobulin (Ig) interaction
• the initial activation signal must be followed by a series of finely tuned receptor- ligand 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 (LMW- BCGF) , sCD23, lymphotoxin (LT) , tumor necrosis factor (TNF), interferon-£ (IFN-tf) (1,2).
• the activation step where the cells are exposed to activating factors.
• activating factors include 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.
• PPC protein kinase C
• 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 Epstein- Barr Virus (EBV) (from the non-transforming strain P3HR1 or UV-inactivated virus) lipopolysaccharides (LPS) .
• the progression 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 B- 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).
• BCDF B-cell differentiation factor
• BCDF B-cell differentiation factor
• BCGF B-cell growth factor
• B-CLL B-cell chronic lymphocytic leukemia
• BSF B-cell stimulating factor
• 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 14OK 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, 5
• Molt4 A T-lymphoma derived cell line p45 A 45K molecular weight membrane protein
• TPA Tumor promoting agent
• TRF T-cell replacing factor
• T-T hybridoma A somatic cell hybrid between two different T-cells.
• MP6/Trx MP6 T-T hybridoma cell line produced enzyme of thioredoxin family.
• BSF B cell stimulatory factor
• MP Thioredoxin is a well-characterized enzyme catalyzing thiol disulphide interchange reactions and net protein disulphide reductions via a Cys-Gly-Pro-Cys active site.
• MP Thioredoxin is a well-characterized enzyme catalyzing thiol disulphide interchange reactions and net protein disulphide reductions via a Cys-Gly-Pro-Cys active site.
• monoclonal B cells, B- lymphoblastoid cell lines, or B cells derived from B-type o chronic lymphocytic leukemia (B-C L) were target cells par excellence, since they required MP6/Trx for cytokine induce proliferation and differentiation in vitro, when tested unde suboptimal cell culture conditions.
• Pre-activated cells did proliferate in response to the recombinant or natural ligands: interleukin 2 (IL-2), interleukin 4 (IL 4), low molecular weight BCGF (LMW- BCGF), tumor necrosis factor- ⁇ , (T F- ⁇ ), or anti-CD40, only if MP6/Trx was added.
• IL-2 interleukin 2
• IL 4 interleukin 4
• LMW- BCGF low molecular weight BCGF
• T F- ⁇ tumor necrosis factor- ⁇
• anti-CD40 anti-CD40
• 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.
• 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.
• 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.
• 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.
• 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-od 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.
• thioredoxin is administered following a period of pre- treatment 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.
• a medicament may comprise a co-factor as described above.
• thioredoxin as well as co-factors as exemplified in Table 1 are known in the art, pharma ⁇ ceutical 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.
• 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.
• T-cells and NK-cells can be strengthened by treatment with thioredoxin, optionally in combination with a co-factor as described.
• 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.
• 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.
• 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 co- factors 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.
• BSF1 C Interleukin 4 (BSF1) C Anti-IL4-receptor antibodies
• CDllc C C3d receptor (CDllc) reactive agents C and anti- receptor (gp 140) antibodies
• Vitamins in particular vitamin A, D, and biologically active derivatives
• 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 N-ADPH 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 photo-synthetic enzymes in the chloroplast of plant cells (4), and activation of glucocorticoid receptors to a steroid binding state (5).
• regulatory events (3) such as the light-dependent activation of photo-synthetic enzymes in the chloroplast of plant cells (4), and activation of glucocorticoid receptors to a steroid binding state (5).
• Mammalian thioredoxins have been isolated and 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) .
• 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.
• HTLV-1-1 adult T cell leukemia
• SUBSTITUTESHEET The present invention assigns new biological functions for the thioredoxin family of enzymes and expands its role in lymphocyte activation.
• 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.
• the MP6 is a CD4 "1" T helper cell hybridoma, previously isolated and cloned by us (9).
• the MP6 clone constitutively secretes as 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).
• BSF-MP6 B cell stimulatory factor
• the IL-2 receptor expression was also enhanced by BSF-MP6 (12). Kishimoto and Honjo et al. have demonstrated that mR A from MP6 cells did not hybridize with cDNA probes for IL-l ⁇ , IL-1B, IL-4, IL-5 nor IL-6 (12).
• the MP6 supernatant was shown to lack activities of LMW-BCGF, TNF- ⁇ , and - ⁇ , IFN- ⁇ , - ⁇ , - ?*, granulocyte-monocyte colony stimulating factors (GM-CSF), IL-1, IL-2, IL-4, IL-5 and IL-6 (9).
• GM-CSF granulocyte-monocyte colony stimulating factors
• _7 signals, or by a sub-optimal dose of TPA (1.6x10 M) for 1 h.
• the cells were refractory to any of the re ⁇ ombinant or natural B-lymphotropic lymphokines rIL-l ⁇ , rIL-2, rIL-4, rIL-6, rTNF ⁇ , LMW-BCGF, rlFN- ⁇ , anti-CD40, or combinations thereof.
• SAC-activated cells did, however, respond to the lymphokines rIL-2, rTNF ⁇ , LMW-BCGF, when BSF-MP6, was added (Figure 1A) .
• TPA provides a non- physiological signal directly activating proteinkinase C, moving the cells into the cell cycle.
• Figure IB illustrates that TPA activated cells responded to BSF-MP6 alone, and that the combinations of BSF-MP6 with several
• 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 confined to the 12 kDa region in gelfiltration experiments.
• Mammalian thioredoxins after air oxidation, forms extra structural intra-molecular disulphide bonds leading to inactivation and aggregation (6) .
• preparations of BSF- MP6 were also easily oxidized, by atmospheric 0-, with a resulting loss of activity.
• 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.
• BSF- MP6/thioredoxin is according to the evidence presented here one of the missing links.
• 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.
• 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.
• B-iymphotropic herpesvirus Epstein-Barr virus EBV
• 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
• SUBSTITUTESHEET EBV-receptors 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).
• thioredoxin in the B-CLL cells could effectively block an herpesvirus multiplication in those cells.
• 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.
• MP6 was obtained from serum-free 24 h cultures of the MP6 T cell hybridoma grown in Iscoves medium supplemented with 400 ⁇ g/ml of BSA (Boerhinger-Mannheim, Mannheim, W.Germany), 12.5 ⁇ g/ml of human transferrin (Kabi, Sweden), 50 ⁇ M ⁇ -mercaptoethanol, 2mM L-
• rIL-2 was purchased from Amgen (Amersham, Amersham, G.B. ) .
• rIL-l ⁇ (Genzyme, Boston, MA) had a specific activity of 10 8 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. Kishimoto, Osaka, Japan, and was used at 100 U/ml.
• Recombinant TNF ⁇ with a specific activity of 6 x 10 "7 U/mg was used at 100 ng/ml and was a gift from Dr. G.R.
• LMW-BCGF was purchased from Cellular Products (Buffalo, NY) was used at a concentration of 10% v/v.
• rIFN-j- was obtained from Genentech (San Fransisco, CA) . It had a specific activity of 3 x 10 7 U/mg and was used at 500 U/ml.
• Radioimmunoassay for thioredoxin shows identity between BSF-MP6 and thioredoxin.
• Solid line indicates pure human placenta thioredoxin.
• Broken line indicates BSF-MP6.
• the radioimmunoassay was performed as described previously (13), briefly: 0.1 ml (0.2 pmol) of " ⁇ -labeled human placenta thioredoxin was incubated with 0.1 ml of standard human thioredoxin or unkonwn sample (MP6 supernatant concentrated 50-fold by ammonium sulphate precipitation) , serialy diluted, and 0.1 ml (5 ⁇ g) of the IgG fraction of a rabbit antiserum against human thioredoxin, at 37° C with shaking for 4 hours.
• BSF-MP6 activity by reduction with DTT.
• the biological activity of BSF-MP6 could be recovered by reduction as shown above.
• the thioredoxin-expressing T- hybridoma clone MP6 was cultured for 24 hours in Iscoves medium containing 400 ⁇ g/ml of BSA, 12.5 ⁇ g/ml of human transferrin, 50 ⁇ M ⁇ -mercaptoethanol, 100 ⁇ g/ml of streptavidin, and 100 U/ml of penicillin and 2nM of L- glutamin, but the biological activity of the supernatant was lost after two weeks of storage at +4°C.
• 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 to bottom: 92.5 kDa, 67 kDa, 45 kDa, 30.1 kDa, 20.1 kDa, 14.7 kDa.
• IL-5 C. Azuma et al.. Nucleic Acid Res. 14. 9149 (1986);
• IL-6 T. Hirano et al.. Nature 234, 73 (1986);
• LMW-BCGF S. Sharma, S. Mehta, J. Morgan, and A.
• IFN-V D.V. Goeddel et al., Nature 287, 411, (1980); Lymphotoxin (TNF- ⁇ ): P.W. Gray, Nature 312, 712,
• TNF T. Shirai, H. Yamaguchi, H. Ito, C. W. Todd, and

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