DE4205938A1 - New immuno-toxin contg. CD30 monoclonal antibody - is coupled to type 1 ribosome inactivating protein toxin, for selective elimination of CD30 cells, esp. treatment of Hodgkins disease - Google Patents

Abstract

New immunotoxin (I) comprises monoclonal CD30 antibodies (Ab) covalently bonded to a ribosome-inactivating protein type-1 toxin (II). Pref. Ab is of the Ber-H2 type while (II) is bryodin; the toxin purified from Asparagus officinalis; momorcochin-5; dianthin; gelonin; trichosanthin or pref. saporin; PAP (from Phytolacca americana seeds) and/or momordin. Ab, esp. Ber-H2 from the hybridoma ECACC 92012823, and (II) are coupled using a heterobifunctional reagent, e.g. N-succininidyl-3-(2-pyridyldithio)propionate; S-acetyl-mercaptosuccinic anhydride; a carbodiimide; glutaraldehyde or esp. 2-iminothiolan (2-IT; sec Br. J cancer, 60 (1989) 315). Ab is isolated from hybridoma culture supernatant (or ascites) by affinity chromatography on protein A and then hydroxyapatite chromatography. It is derivatised with 2-IT in pH9 borate buffer then incubated for 20 hr at room temp. with 2-IT-derivatised toxin (reduced with dithiothreitol). The reaction mixt. was gel. filtered on 'Sephacryl S-200' (RTM) and the fractions contg. the low molecular wt. conjugate isolated (esp. it has mole ratio (II): Ab = 1-3). USE/ADVANTAGE - (I) can be used (1) for treating tumours and (2) for selective elimination, in vivo or ex vivo, of CD-30 positive cells. They are more active than similar immuno toxins prepd. from ricin A chain and give partic. good results in cases of Hodgkin's disease. (I) is administered by intramuscular or intravenous injection (as a soln. or suspension) to provide a dose of 0.01-5 (pref. 0.01-0.5) mg/kg, given one or more times per day for one to several weeks.

Description

The present invention relates to novel immunotoxins which through covalent binding of plant toxins to a monoclonal Antibodies of the CD30 type can be generated.

Some plant toxins inhibit the inactivation of ribosomes the protein synthesis of euraryotic cells and unfold this way cytotoxic activity. These molecules are called "Ribosome inactivating proteins" classified and abbreviated referred to as RIPs. There are two types of RIPs divorce. One type (referred to as type 2) consists of Heterodimers made from two different ones by disulfide bridge linked peptide chains are assembled; one of the Peptide chains bind to the surface of target cells while the other peptide chain that has cytotoxic activity. The other Type of toxins (referred to as RIP Type 1) consists of one single peptide chain. To eliminate cytotoxicity is one Conjugation of this peptide chain to a carrier is necessary has the ability to bind to cell surface membranes.

Such carriers can e.g. B. Antibodies for a certain cell antigen are specific.  

Type 1 RIPs are used in the invention described here Application. These proteins have been used in scientific and Patent literature described several times including methods their extraction, purification and determination of the biological Activity. The type 1 RIPs used include members of the Saporin family such as B. Saporin 6 (SO6) (F. Stirpe et al., Biochem. J. 216, 433 (1986)) and other proteins such as e.g. B. Bryodin (Stirpe et al., Biochem. J. 240, 659 (1986)), Bryodin-L (European German patent publication 03 90 040), Momorchochin (European Patent publication 03 90 040), Dianthine 30 and 32 (F. Stirpe et al., Biochem J. 195, 399 (1981) and A.I. Falasca, Biochem. J. 1 95 399 (1981)), toxins extracted from Asparagus officinalis (Bolognesi et al., Biochem. Biophys. Acta 1087, 293, 1990 (1983)) and European patent publication 03 90 040, PAP-S (Barbie ri et al., 203, 55 (1982)), Gelonin (Stirpe et al., J. Biol. Chem. 255, 6947 (1989)) and Trichosanthin (Maraganore et al., J. Biol. Chem. 262, 11628 (1987)).

Any RIP Type 1 toxin can be used to manufacture immunocon jugates can be used. In addition, it is possible Glycoprotein toxins in sugar-free or in a partial use deglycolized form for coupling.

The other component of the immunotoxins presented here is a monoclonal CD30 antibody and especially the monoclonal Antibody Ber-H2 with the "cluster of differentiation" no. CD30. This antibody was short and detailed in 1987 by R. Schwarting et al. (Leukocyte Typing III, Blood...) Be wrote. The antibody Ber-H2 is replaced by the same name Hybridoma secreted by the European Collection of Animal Cell Culture (ECACC), Public Health Laboratory Service, Porton Down, Salisbury, Wiltshire U.K. according to the provisions of Budapest Convention under provisional number Ber-H2 92012823 was deposited on January 28, 1992.  

The CD30 antigen recognized by the monoclonal antibody Ber-H2 is a glycoprotein with a molecular mass of 120 kD if this is determined by SDS electrophoresis. The special thing about that CD30 antigen is that it is only very much in the normal organism few T cell and B cell blasts and only in these low density is present (Stein et al., "International Journal of Councer 1982 ", Blood 66, 848 (1985) Leukocyte Typing III and IV), while it occurs in a number of lymphatic systems proliferative processes is expressed in high concentration. The first CD30-positive malignant lymphomas Line the Hodgkin lymphoma, the anaplastic large cell Lymphoma as well as the acute form of adult T cell leukemia. Because of the extremely rare occurrence of the CD30 molecule in normal organism (apart from a few activated B and T cell Blasts are negative for all body cells) and high Expression of this molecule on the tumor cells of the above An immunotoxin containing Ber-H2 appears for the lymphoma Elimination of CD30 positive tumor cells excellent suitable.

Immunotoxins required for elimination of Hodgkin lymphoma cells or can be used by CD30-positive tumor cells known in literature. Such immunotoxins are e.g. B. in WO91 / 07437 and WO91 / 07941 or in Engert et al., Cancer Res. 50, 2929 (1990). These immunotoxins contain as cytotoxic component of the A chain of ricin, which is a toxin of RIP type 2. The effect of antibodies bound Ricin A chains have been studied in many studies; the clinical So far, application has given unsatisfactory results. The reason for this is the unfavorable in vivo pharmacokines tics of these immunotoxins (J. Biol. Resp. Modifiers., 7, 559 (1988)).

It has now been shown that immunotoxins are caused by Conjugation of the monoclonal antibody Ber-H2 with toxins of the RIP type 1 were produced, the immunotoxins with toxin RIP  Type 2 are superior. This applies to both the removal of CD30 positive cells in vitro as well as in vivo. In the through Experiments conducted showed the conjugates with toxins from RIP Type 1 has a significantly higher cytotoxic capacity than ricin A Chain-containing immunotoxins (Siena et al., Transplantation 43, 421 (1987), pp 69, 345 (1987)).

The immunotoxin described here was produced using conventional methods as described in EP-A-16 911 and in "Monoclonal antibody-toxin conjugates: liming the magic bullet ", Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pages 168-190 (1982).

The conjugation of the toxin with the antibody is suitable heterobifunctional reagents such as B. N-succinimidyl-3- (2- pyridyldithio) propionate (SPDP), 2-tminothiolan (Traut’s Reagent), S-acetyl-mercaptosuccinic anhydride (SAMSA), Carbodiimide, glutaraldehyde and similar reagents. The users approximately of these reagents is in the relevant literature described. For the production of the Immunoto presented here xins coupling with the help of iminothiolane is preferred. This reagent allows both the toxin and the antibody to derivatize separately.

The Ber-H2 toxin conjugates are said to be parenteral (intramuscular or intravenously). Therefore, the immunotoxin must be in a corresponding pharmaceutical preparation is present, e.g. B. as a sterile solution or soluble suspension or freeze dried substance with appropriate solvents can be reconstituted. The dose is different Factors depending on e.g. B. from the spread and the type of Er disease, the condition of the patient and others Therapy measures. The dose is in most cases between 0.01 mg / kg and 5 mg / kg, with a dose between 0.01 mg / kg and 0.5 mg / kg is preferred.  

The therapeutic application can be one or more applications per day for a period of one to several weeks include.

The derivatized toxins and antibody molecules are listed under Production of a disulfide bridge conjugated. The one in the front In the present invention, Ber-H2 antibodies of the CD30 type were used and the toxins are described below accordingly conventional methods cleaned and characterized.

The following are the manufacture and characterization some Immunoto representative of the present invention xine described in more detail:

1. Production of the anti CD30 monoclonal antibody

For the production of the monoclonal antibody Ber-H2, the hybrid cell line of the same name. The Ber-H2 Antibody has the isotype IgGI and recognizes one for it CD30 molecule specific epitope. The hybridoma cell line will in cell culture using standard culture conditions in RPMI supplemented with 10% fetal calf serum 1640 medium expands.

In order to obtain a larger amount of antibody, 1-3 × 10 ⁶ hybridoma cells can also be injected intraperitoneally into nu / nu mice (Swiss background) treated with pristan (2,6,10,14-tetramethyl-pentadecane, Sigma); under these conditions, hybridoma cells in the mouse produce an intraperitoneal tumor with ascites formation. The ascites contains the antibody in a very high concentration. The binding activity of the Ber-H2 antibody secreted in the cell culture supernatant or in the ascites is tested in two different ways. The binding of the antibodies to the CD30-positive cell line L540 is analyzed using the indirect fluorescence technique (see FIG. 1).

In the other method, the selective binding of the obtained Ber-H2 antibody in CD30 positive tumor cells in Frozen sections checked using immunohistology.

2. Purification of the anti-CD30 monoclonal antibody Ber-E2

The antibody is made from culture supernatant or ascites Help using affinity chromatography Protein A columns and hydroxyapatite columns isolated and cleaned.

Affinity Chromatography (Ey P.L. et al., Immunochemistry 15, 420 (1972)). The sterile culture fluid or Ascitic fluid is removed by centrifugation and filtration via a nitrocellulose filter with a pore size of 0.45 µm clarified. The filtrate is 1: 2 in buffer (1.5 M glycine, 3 M NaCl, pH 9.8) and diluted with a Sepharose-Pro given an A pillar (Pharmacia). The antibody (IgG1) will then by applying a 0.1 M sodium citrate pouf heel, pH 6.0 eluted. For other subclasses, the Elution at pH 3.5 or 3.0. The IgG1 containing fractions using an ELISA test (enzyme linked immunosorbent assay).

Hydroxyapatite chromatography (Bio-Rad) (Stalkel et al., J. Immonol. Methods 76, 157, (1985)). Pooled Ber H2 antibody fractions containing are against a 50 mM Phos dialyzed phat buffer pH 6.8 and then over given the hydroxyapatite chromatography column. The antibody per becomes one with a linear potassium phosphate gradient Buffer (50 to 500 mM, pH 6.8) eluted. The antibodies containing fractions are collected, dialyzed against physiological saline, then with the help of Ultrafiltration concentrated and over a filter with the Pore size of 0.2 µm filtered.  

The purified antibody thus obtained is now through Affinity chromatography under sterile conditions and using an endotoxin-removing gel column (Pierce) made endotoxin free.

The endotoxin admixture, which must be below 0.125 EU, is determined with the LAL test (Wittaker). All solutions used to clean the antibody were made with pyrogen-free and autoclaved sterile water scheduled. The glass material was sterilized at 190 ° C.

Plastic materials were made possible by pyrogens Treatment with sodium hydroxide solution or detergents accordingly exempted from the instructions of the manufacturers. The purity of the Antibody was removed using electrophoresis denaturing conditions (Laemmli U.K., Nature 227, 680 (1970)) checked; the degree of purity was shown in each case higher than 98%. On an aliquod of the purified antibody the binding capacity was determined using fluorocytometry (Figure 1) evaluated. The purified antibody showed one similarly good binding to the L540 cell line or CD30 positive tumor cells in frozen sections of Hodgkin's disease Tissue such as the antibody from cell culture or the Ascites fluid.

3. Cytofluorometric and immunohistological analysis of the monoclonal CD30 antibody

The binding activity of the monoclonal antibody Ber-H2 was determined using indirect immunofluorescence using a cytofluorimeter on CD30-positive cell lines, e.g. B. the Hodgkin cell line L540, the HUT 102 and K562 as well as with the alkaline-phosphatase-anti-alkaline-phosphatase reaction on acetone-fixed frozen sections of Hodgkin's disease tissue were analyzed. Using the same methodology, the purified antibody was analyzed before and after its conjugation with the toxin. For the purpose of this test, 1 × 1 10 ⁶ CD30 positive cells were incubated in 100 μl solution for 30 minutes. After washing three times in an iso-osmolar buffer containing 1% serum albumin, the samples were conjugated with 100 µl of a fluoroescin isothiocyanate (FITC) conjugated anti-mouse immunoglobulin. After three washes, the analysis was carried out in a fluorescence activated fluorimeter. For tissue section analysis, frozen sections of CD30-positive lymphomas were fixed in acetone-pro-analyzes at room temperature for approx. 10 to minutes and then incubated with approx. 300 µl Ber-H2 antibody preparation. After a short wash, a rabbit anti-mouse Ig antibody was applied as a bridge antibody. After washing again, the Apap complex consisting of alkaline phosphatase and antibodies against alkaline phosphatase was then applied for 30 minutes. After washing again, the bound alkaline phosphatase molecules were visualized using the new fox inversion (Stein et al., Lab. Invest. (1985)).

4. Extraction and purification of the toxins

As already mentioned above, the toxins were derived from vegetable matter Materials extracted and then after in the literature communicated procedures cleaned. E.g. Saporin 6 was made Saporin officinalis seeds (Stirpe et al., Biochem. J. 216, 617 (1983)) and according to Barbieri et al (J. Chroma togr. 408, 235 (1987)). The purity of the toxin was analyzed using SDS electrophoresis. they turned out to be higher than 99%. Momordin was made from the seeds of the Momordica charantia (L Barbieri et al., Biochem. J. 186, 443 (1980)) extracted and according to the method of Barbieri et al. be written method (J. Chromagr., 408, 235 (1987)) nends. The purity of this toxin was also determined with the SDS electrophoresis examined. The degree of purity proved higher than 99%. PAP-S was made from Phytolacca seeds  americana extracted (Barbieri et al., Biochem. J. 203, 55 (1982)). The purity is determined using SDS gel electrophoresis turned out to be higher than 99%.

The activity of the toxins was studied by means of a protein synthesis inhibition test using an in vitro translation system with rabbit reticulocyte lysates (F. Stirpe et al., Biochem. J. 240, 659 (1986)). The IC ₅₀ (ie the molar concentration which causes a 50% inhibition of protein synthesis in the system described) was determined by the inhibition of incorporation of ¹⁴ C-leucine. The IC ₅₀ for saporin, momordin and PAP-S was 3.7 × 10 ^-11 M (F. Stirpe et al., Biochem. J. 216, 617 (1983)) and 6 × 10 ^-11 M (L. Barbieri et al., Biochem. J. 186, 334 (1980) and 3.7 × 10 ^-11 M (L. Barbieri et al., Biochem. J. 203, 55 (1982)).

5. Conjugation of the Ber-H2 antibody to the toxins

The toxin conjugation was by chemical reaction between receive the Ber-H2 antibodies and the RIP type 1 toxins, where both reactants with 2-iminothiolane (2-IT) (Dinota et al., Brit. Journal Cancer 60, 315 (1989)) were derivatized.

Derivatization of the toxins

The toxin solutions (9 mg / ml in 50 mM sodium borate, pH 9) were added 125 I-labeled toxin (10 ⁶ cpm) and 2-IT (final concentration 1.0 mM). After incubation for 30 minutes at room temperature, the reaction was stopped by adding glycine at a final concentration of 200 mM. After a further 30 minutes at room temperature, the Ellman's reagent in dimethylformamide was added in a final concentration of 2.5 mM. After a further 30 minutes of incubation, the mixture was passed through a Sephadex G-25 column (Pharmacia). Elution was carried out with phosphate buffered saline (PBS). The derivatization of the toxins was determined on an aliquat on the basis of the optical density at 280 nm and at 412 nm before and after the reaction with dit hiothreitol (DTT) in a final concentration of 55 mM.

Derivatization of the Ber-H2 antibody

The antibody solution (5 to 25 mg / ml in 50 mM sodium borate pH 9) was a solution of 2-IT in a final concentration of 0.6 mM added. The derivatization and the SH groups were introduced in the same way as for the Toxins determined.

conjugation

The derivatized toxins were in an Amicon conc concentrated and reduced in a nitrogen atmosphere by adding 1/10 of the volume of 0.55 mM DTT. The redu Ornate toxin was removed from the reducing reagent Passage over a Sephadex G25 column freed and directly in an Amicon concentrator, in which the derivatized antibody. The reaction mixture was concentrated to 1/4 of the volume and at room temperature for Incubated for 20 hours and then over a high resolution send gel filtration column with Sephacryl S-200 (Pharmacia) given and eluted with PBS. Those in the eluted fractions Radioactivity obtained was determined with the optical density 280 nm compared. The first peak correlated with one High molecular weight conjugate, the second peak with a low molecular weight conjugate; then followed Trimers, dimers and monomers of the toxin. The free antibody per was with the last fraction of the low-molecular Conjugate co-elutes as from the partially overlapping Peak of radioactivity and optical density measurement at 280 nm emerges (Figure 2).  

The fractions that the low molecular conjugate contained were collected; the ratio of toxin to H2 antibody was determined by the absorption at 280 nm and the radioactivity of the peaks of the low-molecular laren conjugate and the free toxin related were. With the technique described, immunotoxins groomed; they are representative of the invention Immunotoxin Ber-H2-Saporin 6 (Ber-H2 / SO-6), Ber-H2 / PAP-S and Ber-H2 / Momordin. The molar ratio of toxin and Antibody moved - depending on the toxin and the derivatizing reagent - between 1 and 3. In detail this ratio is on average for the immunoto xin Ber-H2 / SO-6 2.25, for the immunotoxin Ber-H2 / PAP-S 1.55 and for the immunotoxin Ber-H2 / Momordin 1.75.

The binding activity as well as the biological activity of the Conjugates were determined with that described under 3 Technology. As illustrated in Figure 1, the Conjugation is the binding characteristic of the Ber-H2 antibody pers in no way.

6. Characterization of the biological activity of the immunocon jugate Ber-H2 / RIP type 1 toxins a) Inhibition of protein synthesis in a cell-free system

The biological activity is measured by the inhibition of the incorporation of ^14- leucine into proteins which are produced in a reticulocyte lysate system as described under 4. The IC ₅₀ values of the immunotoxins described here characterize the invention (Table 1).

b) Inhibition of protein synthesis in a cell-containing system

The method was described as described by Stirpe et al., Biochem. J. 240, 659 (carried out in 1986). 2 × 10 ⁴ COLE cells, L428 cells or L540 cells were in 200 μl RPMI 1640 medium in a 96-microtiter plate at 37 ° C. for 24 hours with either the antibody, the toxin or the corresponding immunotoxin in concentrations between 5 Incubated × 10 ^-15 (5 × 10 ^-13 for COLE cells) and 5 × 10 ^-8 M. Then H3 leucine (2 µCi / well) was added; after an incubation of 18 hours at 37 ° C., the cells were harvested using a cell harvester (Titertek flow). The incorporated activity was measured in a β-counter (LKB) and the IC _{50 of} the various molecules was calculated using a linear regression analysis. The results are shown in Table 1.

It should be noted that the IC ₅₀ free toxin is greater than 10 ^-8 M, while the Ber-H2 antibody exhibits no toxicity at all, even at the highest concentrations.

Table 1

Inhibition of protein synthesis by immunotoxins

Claims (10)

1. Immunotoxins, characterized by a content of monoclonal CD30 antibodies, covalently bound to RIP type 1 toxins. 2. Immunotoxins according to claim 1, characterized in that the monoclonal antibodies are of the Ber-H2 type. 3. Immunotoxins according to claims 1 or 2, characterized records that the RIP type 1 toxins are selected from the Group consisting of Saporin, Bryodin, from Asparagus offici nalis purified toxin, PAP, Momordin, Momorcochin-S, Dinathin, Gelonin and Trichosanthin. 4. Immunotoxin according to claim 3, characterized in that the RIP type 1 toxin is saporin, PAP and / or Momordin. 5. A method for producing the immunotoxins according to the An sayings 1 to 4, characterized in that the toxin via heterobifunctional reagents to the antibody binds. 6. The method according to claim 5, characterized in that one as heterobifunctional reagents N-succinimidyl-3- (2- pyridyldithio) propionate, 2-iminothiolane, S-acetyl-mercapto succinic anhydride, carbodiimide or glutaraldehyde used. 7. The method according to claim 6, characterized in that one used as heterobifunctional reagent 2-iminothiolane. 8. Pharmaceutical preparations for the treatment of tumors, characterized in that they act as immunotoxins according to claims 1 to 4 included.   9. Pharmaceutical preparations according to claim 8 for selective in vivo or ex vivo elimination of CD30 positive cells. 10. Pharmaceutical preparations according to claims 8 or 9, characterized in that it contains the active ingredient in solution or contain as a suspension in a therapeutically effective dosage ten.