IE61237B1 - Purified thymidine kinase of fetal type - Google Patents

Abstract

The invention relates to purified thymidine kinase of foetal type (abbreviated to TK-F) as a new industrial product. It also relates to the process for preparing the purified TK-F by a process which comprises coupling the TK-F with thymidine and then cleaving the resulting TK-F-thymidine complex. The purified TK-F according to the invention can be used in the preparation of antiTK-F antibody to be involved especially as a reagent in the field of dosages and as a medication in therapeutics.

Description

SCOPE OF THE INVENTION The present invention relates, by way of a new industrial produce, to purified thymidine kinase of ( fetal type. It also relates to the method for the preparation of this product and, more particularly, to the method for its purification. This product is useful (i) in the field of quantitative analyses, especially immunological analyses involving antigen-antibody reactions, and (ii) in therapy for the preparation of antibodies used as drugs.

PRIOR ART Thymidine kinase (abbreviated to TI<) , which is the enzyme responsible for the phosphorylation of thymidine to thymidine 5’-monophosphate (abbreviated to d-TMP), is known to play an important part in the synthesis of DNA and consequently in cell reproduction.

Its activity is high in tissues which are in the process of proliferating or regenerating and in cells infected by viruses. It is also known that two isoenzymes of TK have been detected in eucaryotic cells: thymidine kinase of fetal type (referred to hereafter as fetal thymidine kinase or TK-F) and thymidine kinase of adult type (referred to hereafter as adult thymidine kinase or TK—A).

TK-F is present in embryonic tissues and has been detected in cancerous tissues in cases of hormonedependent cancers, especially cancers of the breast and prostate; reference may be made here to the article by J.L. Javre et al. in Bull. Cancer (Paris) 73 (No. 1), pages 8-16 (1986), entitled Mise en evidence de la thymidine kinase de type foetal dans les cancers du sein (Detection of thymidine kinase of fetal type in breast cancers), and the article by J.L. Javre et al. submitted for publication in Cancer Research and entitled Presence of fetal thymidine kinase in human breast cancers.

It is also known that the synthesis of TK-F in vivo is greatly increased by sex hormones in their own target organs, namely the prostate and the uterus; reference may be made here to the article by M. Bourtourault et al. in J. Steroid Biochem., 21 (No. 5), pages 613-620 (1984), entitled: Stimulation by 17pestradiol of thymidine kinase activity in the rat uterus, and the article by G. Gayet et al. in The Prostate _7» pages 261-270 (1985), entitled: Induction of thymidine kinase of fetal type by androgens in the rat prostate.

The TK-F is separated from the TK-A by electrophoresis or chromatography using an anion exchange resin, for example by electrophoresis on acrylamide gel or, preferably, by chromatography on DEAE-cellulose gel or DEAE-Sephadex gel marketed by the company called Pharmacia, the fetal isoenzyme having a zero or slow mobility and the adult isoenzyme having a fast mobility.

The TK-F isolated in this way, which has an enzymatic activity per mg of the order of 2 international units, is unsuitable for (i) performing more precise quantitative analyses, and especially (ii) preparing polyclonal and particularly monoclonal anti-TK-F antibodies useful in the field of diagnosis on the one hand and in the field of therapy on the other.

SUBJECT OF THE INVENTION According to a first feature of the invention, a purified TK-F is proposed, by way of a new industrial product, which has a distinctly greater enzymatic activity than the TK-F isolated hitherto by the methods of the prior art.

There is a particular need for purified TK-F in the field of diagnosis. In fact, the activity of TK-F varies according to the type of cancer. As the activity of TK-F is low in cells in the Go phase and high in cells in the S phase, there is every reason to believe that cancers showing a low TK-F activity are in a quiescent state while cancers showing a high TK-F activity are f in the process of proliferating. Quantitative analysis of TK-F in breast and prostate cancers is therefore of great interest for measuring the state of proliferation of the cancers. Furthermore, quantitative analysis of TK-F by means of anti-TK-F antibodies raised against purified TK-F constitutes a factor in assessing the development of the cancerous disease and consequently in assisting the orientation of therapy.

According to a second feature of the invention, a method for the preparation of the said purified TK-F is recommended, the said method comprising . coupling with thymidine the TK-F to be purified and then splitting the resulting complex (thymidine/TK-F) in ordsr to collect the purified TK-F.

According to yet another feature of the inventions the use of purified TK-F is recommended for the preparation of anti-TK-F antibodies, these antibodies being intended for use in diagnosis on the one hand and in therapy on the other. It has actually been founds surprisingly, that the administration of anti-TK-F antibodies to patients suffering from a hormone-dependent cancer (especially a breast or prostate cancer) has a beneficial therapeutic effect.

DETAILED DISCLOSURE OF THE INVENTION According co the invention, a purified TK-F not obtained hitherto is recommended, by way of a new industrial product, which is useful in the field of quantitative Θ analyses and the field of the preparation of anti-TK-F antibodies, the said purified TK-F having a specific , enzymatic activity per mg greater than or equal to 5000 units, preferably greater than or equal to 8000 units and particularly preferably greater than or equal to ,000 units.

The electrophoretic Rf (at a pH of about 8.6) in a non-denaturing medium (for example in the buffers A and B described below) is about 0.15 for a purified TK-F having a specific enzymatic activity per mg of the order of about 11,800 units.

In accordance with convention, the specific enzymatic activity is expressed in enzymatic units per mg of protein. A so-called international unit of TK-F corresponds to 1 nanomol of nucleotides synthesized per mg of protein per minute. In other words, the enzymatic activity can be expressed in nmol/mg of protein/min.

By way of comparison, it will be seen in Example 1 below that; - the activity in the original biological medium is 1.03 nmol/mg of protein/min: - the activity after chromatography on DEAESephadex is 2.08 nmol/mg of protein/min; and - che activity of the purified TK-F is 11,875 nmol/mg of protein/min.

The method for the preparation of purified TK-F which is recommended according to the invention, wherein the TK-F to be purified is coupled with thymidine and the resulting thymidine/TK-F complex is then split, comprises; a) coupling the TK-F with thymidine by bringing the TK-F to be purified into contact with thymidine which has been fixed beforehand to a first suitable support, b) eluting the TK-F. bound in this way, by means of a suitable eluent containing thymidine, and then c) splitting the thymidine/TK-F complex, eluted in this way, by means of a second support which is different from the first support and selectively adsorbs the thymidine from the said thymidine/TK-F complex.

The best mode for carrying out this method consists of affinity chromatography comprising a socalled competition mechanism. Thus, in stage a), the TK-F to be purified is passed over an appropriate resin which has been coupled with thymidine beforehand. The thymidine bound to the said resin fixes the TK-F, which is retained, while the protein-type impurities polluting or contaminating the TK-F are not fixed to the thymidine and are not retained.

In stage b)s the TI<-F fixed to the support via the thymidine coupled to the said support beforehand is eluted by means of an eluent containing free thymidine. Stage b) comprises the so-called competition mechanism, whereby the TK-F bound to the thymidine fixed to the support couples with the free thymidine in the eluent. The eluate collected in this way contains the resulting thymidine/TK-F complex.

In stage c)s the said thymidine/TK-F complex is destabilized by adsorbing the thymidine on a second support which is selective for thymidine.

In practice, the preferred support for stage c) is an epoxy-Sepharose gel marketed by the company called Pharmacia under the name of epoxy-Sepharose 6B. This gel is first coupled with thymidine by a known method, il especially the one described by P. Grobner et al. in J. Biol. Chem.s 259„ pages 8012-8014 (1984), or a similar method. The preferred support for stage c) for fixing the thymidine is a composition comprising charcoal, dextran and gelatin. The purified TK-F is then isolated, especially by centrifugation of the eluate which contains it together with the said second support used in stage c).

The starting TK-F is a protein substance of animal origin (obtained from fetal animal tissue, especially the fetal liver of a rodent such as the rat), of human origin (obtained from cancerous human tissue) or alternatively of synthetic origin (obtained especially by genetic engineering).

The TK-F purified in this way is useful according to the invention for the preparation of anti-TK-F antibodies, these antibodies being monoclonal in preference to polyclonal. They are obtained by a con10 ventional method known per se. For example, a mouse is inoculated with the said purified TK-F. the spleen is removed from the immunized animal, the lymphocytes are extracted from the spleen, the said lymphocytes are fused with murine myeloma cells in the exponential growth phase, at a rate of 5 to 20 and preferably 10 lymphocytes per myeloma cell, the resulting secretory hybridomas are selected so as to obtain monoclonal antibodies which react selectively with TK-F but not with TK-A, ascitic tumors are developed in mice with selected clones, the ascitic fluids containing the antibodies are recovered and the resulting monoclonal antibodies are purified.

In order to perform analyses, either for detecting the presence or absence of TK-F in a tissue or for quantitatively evaluating the level of TK-F in the said tissue, the anti-TK-F antibody is fixed to an appropriate support (glass beads, polystyrene beads, container walls, etc.) to give the material: (1) suppor t/anti-TK-F ·, this material is then brought into contact with a medium which may contain TK-F, the presence of the said TK-F giving the complex: (2) support/anti-TK-F/TK-F.

This complex constitutes a signal which is amplified by a method known per se for visualization and/or detection with a conjugate labeled with anti-TK-F* [for example the EIA method (especially the EIA-sandwich or EIA-competition method), where the complex 2 is coupled with an appropriate enzymatic conjugates the RIA methods which comprises coupling the said complex 2 with a conjugate labeled by means of a radioactive isotopes or the fluorescence method, which comprises coupling the said complex 2 with a conjugate labeled with fluorescein]. The amplification can also be carried out by agglutination or alternatively by an ERICA method involving cells.

According to the inventions analysis kits are recommended which comprise the reagents required to carry out antigen-antibody reactions, especially the following reactions; (a) support/anti-TK-F 4- TK-F _y suppor t/anti-TK-F/TIC-F (b) support/anti-TK-F/TK-F + anti-TK-F* -> support/anti-TK-F/TK-F/anti-TK-F* According to the invention, a therapeutic composition is recommended which contains a therapeutically effective amount of anti-TK-F in association with a physiologically acceptable excipient.

The invent3.on relates in particular to the use of anti-TK-F antibodies for the preparation of an anticancer drug for therapeutic use in the treatment of breast and prostate cancers.

Further advantages and characteristics of the invention will be understood more clearly from the following description of embodiments, this information as a whole not implying a limitation but being given by way of illustration.

EXAMPLE 1 Isolation and purification of TK-F from fetal rat liver 1) Homogenization Fetal rat liver tissues are homogenized in a Polytron in the following buffer solution: 10 mM TrisHCl, 10 mM MgCl2s 5 jiM NaF and 0.4 mM ^-mercaptoethanol at pH 7.5 (buffer A). 2) Preparation of the cytosol The resulting homogenate is centrifuged successively at 800 x g and 12,000 x g for 10 min in a cooled centrifuge. The supernatant obtained at 12,000 x g is then centrifuged at 105.000 x g for 45 min at +5°C in a type L8-55 Beckman ultracentrifuge. The supernatant obtained at 105,000 x g (cytosol) constitutes the source of TK-F. A quantitative analysis of the specific enzymatic activity of the TK-F is performed on an aliquot of the cytosol; this activity is 1.03 nmol/mg/ min . 3) Isolation of the TK-F The TK-F is isolated by chromatography on DEAESephadex A50 gel (marketed by the company called Pharmacia). The Sephadex gel is swollen in buffer A. After removal of the fine particles by sedimentation, the gel is deposited in a 7 cm x 1 cm mini-column. 800 jil of gel are introduced into the column. After the column of gel has been prepared, an aliquot of the cytosol is deposited on the gel. The TK-F is eluted by successively passing through 4 volumes (4 x 500 jil) of buffer A. The successive elutions are carried out by centrifugation of the column at 200 x g in a cooled centrifuge. Centrifugation accelerates the elution process and prevents the TK-F from becoming denatured.

A quantitative analysis of the enzymatic activity of the TK-F is performed on an aliquot of the eluate, and the resulting eluate is lyophilized. At this stage, the enzymatic activity is 2.08 nmol/mg/min. 4) Purification of the TK-F Purification is effected by affinity chromatography on epoxy-Sepharose 6B gel (marketed by the company called Pharmacia) coupled with thymidine. The thymidine is coupled with the Sepharose gel in an alkaline medium at pH 12.0 and at 25°CS for 24 h9 by a II technique derived from that of P. Grober et al. mentioned above. The degree of coupling of the thymidine is 9 jimol per ml of gel. The gel is washed with the following buffer: 10 mM Tris, 10 mM MgC^s 5 jiM NaF, 0.4 mM j2>-mercaptoethanol and 1 mM ATP at pH 7.5 (buffer B). 3 ml of gel are deposited in a column of height .6 cm and diameter 0.6 cm. The lyophilizate containing the TK-F is taken up with distilled water and deposited on the affinity gel. The sample is allowed to penetrate at a rate of 1.2 ml per hour, The affinity gel is washed with buffer B in order to remove the proteins not fixed to the gel. They are eluted at a rate of 1.2 ml per hour and then 4 ml per hour. The total amount of proteins not retained by the gel is eluted by a volume of 24 ml of buffer B.

The TK-F is eluted by passing 4.5 ml of the following buffer over the affinity gel: 10 mM Tris9 10 mM MgCl9a 5 jiM WaFs 0.4 mM p-mercaptoethanolP 1 mM ATP and 10 mM non-radioactive thymidine at pH 7.5.

Elution is carried out at a rate of 0.5 ml per hour.

The resulting eluate contains the thymidine/TK-F complex.

) Removal of the non-radioactive thymidine The non-radioactive thymidine is removed by passing the eluate obtained above over a plug of charcoal/ dextran/gelatin (1% by weight of charcoal, 0.1% by weight of dextran T70 and 0.2% by weight of gelatin in 10 mM tl Tris-HCl buffer at pH 7.5). The mixture is shaken on a Vortex for 10 min at 0°C and then centrifuged at 800 x g for 10 min. The supernatant contains the purified TK-F. It is analyzed on an aliquot of the supernatant. The enzymatic activity of the TK-F purified in this way is 11.875 nmol/mg/min.

The total activity yield is 7.4%. The purification factor is of the order of 11,530 and the electrophoretic Rf of the purified TK-F (determined in a non-denaturing medium) is 0.15 at pH 8.6.

EXAMPLE 2 Technique for determining the activity of the TK-F jil of 200 mM Tris-HCl buffer (pH 7.8), containing 76 mM MgC^, 160 mM phosphoglycerate, 40 mM ATP and 40 to 50 ^iM [ 2-^C ]-thvmidine (specific activity: 54-58 mCi/mmol5 i.e. about 2 x 10® to 2.15 x 10® Bq/ mmol), are added to 20 ul of the extract containing the TK-F. The reaction medium is incubated at 37°C for 25 min and the reaction is then stopped by the addition of 10 |il of 2.1 M HCIO^ and centrifugation at 2.400 x g for 10 min. 10 jil of the supernatant from centrifugation are then used for separation of the synthesized nucleotides by high-voltage electrophoresis. Separation of the d-TMP, d-TDP and d-TTP (thymidine monophosphate, diphosphate and triphosphate) is effected on Whatman 3MM paper. in citrate buffer at pH 4.1, under 47 volts per cm of paper length, for 30 min and a t 3°C. After drying of the eleetropherogram and then location of the standard n ucleotides under UVC the paper is cut into strips of 2.5 cm in width by 0.5 cm in height and these are transferred to radioactivity counting vials containing 10 ml of liquid scintillator (Ready-solv EP, Beckman ).

EXAMPLE 3 Checking the purity of the TK-F by electrophoresis on polyacrylamide gel Electrophoresis is carried out in a polyacrylamide gel in a linear gradient from 10 to 15% in the presence of sodium dodecyl-sulfate (SDS) on a vertical plate. Migration of the proteins is effected in the following buffer: 12 g%° of Tris, 28.8 g%oof glycine and 0.1 g% of SDS at pH 8.7. under 70 volts for 12 h.

The proteins originating from the cytosol and from the DEAE-Sephadex eluate are stained with a solution of Coomassie Blue R 250 at a concentration of 0.5 g in 250 ml of 90% methanol. The proteins obtained after affinity chromatography are stained by the silver staining method according to the technique of P. Tunon and K.E. Johansson, J. Biol. Chem. and Biophys. Methods, 9, pages 171-179 (1984).

EXAMPLES 4 and 5 By following the procedure indicated in Example 1, but replacing the fetal rat liver tissues with cancerous human breast and prostate tissues, a purified TK-F is obtained which is analogous to that obtained from fetal rat liver and has substantially the same properties (enzymatic activity, Rf) as the said purified TK-F from fetal rat liver.

It is observed, in particular for all the products of Examples 11, 4 and 5, that, in contrast to the activity of TK-A, the activity of TK-F is not inhibited by d-CTP and that the electrophoretic mobility of TK-F in polyacrylamide gel at pH 8.6 is zero or very low, while TK-A shows a fast mobility. These two isoenzymes also behave differently on a column of DEAESephadex or DEAE-cellulose. TK-F is not retained on this type of resin and it is eluted by solutions of low ionic strength. TK-A is retained on these resins and can only be eluted by solutions of high ionic strength. The electrophoretic and chromatographic behavior therefore indicates that TK-F has an alkaline character and TK-A an acid character. According to current knowledge, the pH of TK-F would be 9.0-9.7 and that of TK-A would be 5.0-5.7.

According to the invention, the anti-TK-F antibodies raised against the purified TK-F according to the invention, obtained from any source, especially a purified TK-F of animal or human origin, are recommended by way of new industrial products.

As indicated above, the anti-TK-F antibodies, especially monoclonal anti-TK-F antibodies, raised against purified TK-F are particularly useful in the field of quantitative analyses. Preferably, the purified TK-F used for the preparation of the said monoclonal antibodies is of animal origin (especially from fetal rodent liver tissue) or human origin (especially from cancerous human tissue).

Claims (14)

WHAT IS CLAIMED IS:

1. A purified thymidine kinase of fetal type (TK-F) having an enzymatic activity per mg greater than or equal to 5000 units, preferably greater than or equal to 8000 units and particularly preferably greater than or equal to 10,000 units»

2. The purified TK-F according to claim 1, whose enzymatic activity per mg is of the order of about 11,800 units .

3. The purified TK-F according to claim 2, whose electrophoretic Rf in a non-denaturing medium is of the order of 0.15 at a pH of about 8.6.

4. A method for the preparation of purified TK-F according to any one of claims 1 to 3, wherein the TK-F to be purified is coupled with thymidine and the resulting thymidine/TK-F complex is then split, which comprises: a) coupling the TK-F with thymidine by bringing the TK-F to be purified into contact with thymidine which has been fixed beforehand to a first suitable support. b) eluting the TK-F, bound in this way, by means of a suitable eluent containing thymidine, and then c) splitting the thymidine/TK-F complex, eluted in this way, by means of a second support which is different from the first support and selectively adsorbs the thymidine from the said thymidine/TK-F complex.

5. The method according to claim 4, wherein the first support is epoxy-Sepharose.

6. The method according to claim 4 S wherein the proteintype impurities not retained on the first support coupled with thymidine are eluted with the following buffer: 10 mM Tris, 10 mM MgCl 2 . 5 ^iM NaF, 0.4 mM β-mercaptoethanol and 10 mM ATP at pH

7.5. 14 7. The method according to claim 4, wherein the TK-F fixed to the thymidine of the support coupled with the said thymidine is eluted with the following buffer: 10 mM Tris, 10 mM MgCl ? . 5 ^jM NaF. 0.4 mM ^-mercaptoethanol, 1 mM ATP and 10 mM thymidine at pH 7.5.

8. The method according to claim A, wherein che second support is a composition comprising 1% by weight of charcoal, 0.1% by weight of dextran and 0.2% by weight of gelatin in 10 mM Tris-HCl buffer at pH 7.5.

9. Use of the purified TK-F according to any one of claims 1 co 3, wherein the said TK-F is used for che preparation of anti-TK-F antibodies, the said antibodies preferably being monoclonal.

10. Use of monoclonal anti-TK-F antibodies, according to claim 9, which relates to che preparation of an anticancer drug for use in therapy in the treatment of prostate and breast cancers.

11. An anti-TK-F antibody which Is raised against the purified TK-F according to claim 1.

12. The anti-TK-F antibody according to claim 11, useful in che field of quantitative analyses, which is monoclonal and which has been raised against the purified TK-F according to claim 1, obtained from an animal or human source.

13. A method for the preparation of purified TK-F , as defined in claim 1 and substantially as hereinbefore described by way of example

14. A purified TK-F according to any of claims 1 to 3 whenever prepared by a method as claimed in any of claims 4 to 8 or 13.