IMMUNOTOXIN FROM ANTI-CD5 MONOCLONAL ANTIBODIES

Abstract: The invention describes an immunotoxin that can be obtained by covalent conjugation of ribosome inactivating protein Type 1, to an anti-CD5 monoclonal antibody.

Description: IMMUNOTOXIN FROM .KTI-CD5 MONOCLONAL ANTIBODIES The present invention relates to an immunotoxin obtained by covalent conjugation of a toxin of vegeta¬ ble origin to an anti-CD5 monoclonal antibody. Toxins of vegetable origin inhibit protein synthe- sis in eukaryotic cells, thereby exerting a cytotoxic effect. These substances comprise either a single poly- peptide chain with catalytic activity that inactivates ribosomes (ribosome inactivating protein Type 1, RIPl), or two chains, namely one with a catalytic function and one responsible for binding to the cell surface (RIP2). The present invention provides immunotoxins the costituents of which are selected from ribosome inactivacting proteins (RIPss) Type 1 and anti-CD5 monoclonal antibodies, which are capable of recognising the surface antigen CD5 of human T lymphocytes (Reiter, C, Cluster report: CD5 in: Leukocyte Typing IV, ed. Knapp W. et al. , Oxford University p.33, 1989). These are commercially available from Hybritech (code T101), Becton-Dickinson (Leu 1), Xoma (code H65, ATCC HB 9286) and Ortho (code OKTl) . The latter antibody is described in US Patent Nos. 4,515,894, 4,657,760 and 4,363,799 and is secreted by the hybridoma deposited in the American Type Culture Collection ATCC) under number CRL 8000. This has been redeposited by the Applicant at the Institut Pasteur under No. 1-1025 on 1.10.1991 in accordance with EPC Regulations 28 and 28a. The RIPl toxins used in the immunotoxins according to the present invention are of the saporin type, such as, for example, εaporin-6 (SO-6) (F. Stirpe et al. , Bioche . J. , 216, 433, 1983), briodin (F. Stirpe et al., Biochem. J. , 240, 659, 1986), briodin L (publication of European patent application 390,040), mo ordin (F. Stirpe et al., Biochem. J. , 186, 443, 1980), mo orcochin S (publication of European patent application 390,040), dianthins 30 and 32 (F. Stirpe et al., Biochem. J. , 195, 399, 1981 and A.I. Falasca et al. , Biochem. J., 207, 505, 1982), the toxins extracted from Asparagus officinalis (asparin 1 and 2 ; A. Bolognesi et al. , Biochem. Biophys. Acta, 1087, 293, 1990 and publication of European patent application 390,040), PAP-S (L. Barbieri et al. , Biochem. J. , 203, 55, 1982), gelonin (F. Stirpe et al. , J. Biol. Chem. , 255, 6947, 1980), tricoxanthin (Maraganore et al., J. Biol. Chem., 262, 11628, 1987). However, any single chain protein or glycoprotein vegetable toxin can be used in the immunoconjugates according to the present invention. In addition, glycoprotein toxins can also be used in the corresponding partially or completely deglycosylated forms. The CD5 antigen is a glycoprotein with a molecular weight of 67,000 D that is expressed by 90% of T lym¬ phocytes (Reinherz E.L. et al., J. Immunol. 123, 1312, 1989) and by 15% of B lymphocytes (Casali P. et al. , Immunol. Today !£, 364, 19). The latter belong to a particular subpopulation of B lymphocytes that produce multireactive antibodies that appear to be involved in the genesis of a number of autoimmune diseases. CD5. is also expressed in various acute B forms and chronic T and B forms of leukaemia and in a number of B lympho- mas. The CD5 antigen therefore represents an ideal tar¬ get for anti-T lymphocyte antibodies. Anti-T cell immu¬ notoxins in which the toxin portion is derived from ri¬ bosome inactivating proteins of vegetable origin have already been described, for example in US Patent No. 4,590,071. The use of these immunotoxins, in particular of the immunotoxin containing ricin A, to treat autoim¬ mune-based diseases was proposed in W088/05309 (7.28.88), 089/00583 (1.26.89), W089/06967 (8.10.89) and W089/06968 (8.10.89). The A chain of ricin - RIP Type 2 - is, in fact, the toxin that has been most widely studied with a view to preparing immunotoxins for therapeutic use. However, the results of application obtained so far are not at all satisfactory, partly because of the unfavourable in vivo pharmacokinetics that are characteristic of immu- notoxins containing the A chain of ricin (J. Biol. Re¬ sponse Modifiers, ]_ > 559 «* 1988). It has now been found that immunotoxins obtained by conjugating a known RIPl, preferably momordin, dianthin or PAP-S, to anti-CD5 monoclonal antibodies can be used advantageously in the ^ιι vivo or ex vivo treatment of conditions in which it ' is desirable to suppress T lymphocyte function, such as autoimmune- based diseases (rheumatoid arthritis) and Graft Versus Host Disease (GVHD) following a heterologous bone marrow transplant, or for the e_x vivo elimination from the bone marrow of leukaemic cells in the case of autologous transplants. In particular, the use of anti-T lymphocyte immunoconjugates in the in vivo control of the corti- coid-resistant form of GVHD (Kernan N.A. et al. , JAMA 259, 3154, 1988; Byers V.S. et al. , Blood Jl . 1426, 1990) and in the treatment of chronic lymphoid leukae- ia B (B-CLL) (Filipovich A.H. et al. , Transplantation 50, 410) seems especially promising. The immunotoxi s according to the invention are prepared by conventional methods such as those descri¬ bed in EP-A-169,111 and in "Monoclonal Antibody - Toxin Conjugates: Aiming the Magic Bullet", Thorpe et al. Mo¬ noclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982). A most preferred RIPl is momordin. For example, heterobifunctional reagents such as N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) , 2-iminothiolane (Traut's reagent), S-acetylmercapto- succinic anhydride (SAMSA), carbodiimide, glutar ldehyde, etc. are used to conjugate the toxin to the antibody. These reagents are used in accordance with the generally known methods. In particular, the use of 2-iminothiolane, with which the toxin and the antibody are derivatised separately in suitable condi¬ tions, is preferred. One then proceeds to conjugate the derivatised to- xin and antibody, obtaining an immunotoxin characteri¬ sed by a disulphide bridge. The monoclonal antibodies and the RIPl are pu¬ rified and prepared by conventional methods described briefly below. For the envisaged therapeutic applications, the immunotoxins according to the invention will be formu- lated in pharmaceutical compositions suitable for pa¬ renteral (i. . and i.v.), topical and transdermal administration. Examples of such formulations include sterile liquid suspensions or solutions, freeze-dried powders for reconstitution with suitable solvents, cre¬ ams, gels, ointments, etc. The dosage will obviously depend on a number of factors such as the severity and type of disease, the general condition of the patient, and concomitant treatment, but in the majority of cases it will be between 0.01 mg/kg and 5 mg/kg, preferably between 0.01 mg/kg and 0.5 mg/kg. The therapeutic regime can be based on one or two doses per day for periods of up to several weeks. The results that can be obtained with the immuno- toxins according to the invention have the advantages of reduced non-specific toxicity compared with the known immunotoxins derived from ricin A, and more fa¬ vourable pharmacokinetics. We shall now describe in detail the preparation and characteristics of the immunotoxin comprising anti¬ body 0KT1 and momordin derivatised with 2-iminothio- lane. 1) Production of the anti-CD5 monoclonal antibody The cell line of hybridoma OKTl produces the mono- clonal antibody (MoAb) of class IgG,K, which recognises the lymphocytic antigen CD5. This line acquired from the American Type Culture Collection (ATCC-CRL-8000) and redeposited on 1.10.1991 at the Institut Pasteur (1-1025) was expanded in culture using the standard methods (culture medium: RPMI 1640 containing 10% foe¬ tal bovine serum). To achieve a massive production cf antibody, 1-3 10 hybridoma cells were injected i.p. into nu/nu mice (Swiss background) that had previously been treated with priεtane (2,6,10,14-tetramethyl-pentadecane, Sigma); in these conditions the hybridoma induced the formation of an ascitic tumour secreting antibodies into the peritoneal cavity. The binding of the antibodies secreted in the cul¬ ture medium and in the ascitic fluid to positive CD5 cells (human ononuclear cells from peripheral blood from healthy PBMC-donors) was evaluated by a technique using indirect immunofluorescence, as described below (Fig. la, lb, lc). The percentage of positive CD5 cells was between 72 and 75%. 2) Purification of the anti-CD5 monoclonal antibody The MoAb was purified from the ascitic fluid by affinity chromatography on protein A, followed by ad¬ sorption chromatography on hydroxyapatite. Affinity chromatography (Ey P.L. et al., Immuno- chemistry L5_, 420, 1972): the ascitic fluid, removed sterilely, was clarified by centrifugation and filtered through nitrocellulose filters with a 0.45 μ mesh. The filtrate, diluted 1:2 in the binding buffer (1.5M gly- cine, 3M NaCl, pH 9.8), was loaded into a column packed with Sepharose-protein A (Pharmacia). The MoAb (IgG. ) was eluted with 0.1M sodium citrate buffer, pH 6.0, with the other subclasses being eluted at pH 3.5 and 3.0. The fractions containing IgG. were identified by an ELISA test (enzyme linked immunosorbent assay) using a rabbit antibody recognizing the specific isotype i (Zymed) . Chromatography on hydroxyapatite (Bio Rad) (Stanlkel H. et al. , J. Immunol. Methods 16_, 157, 1985). The pool of fractions containing the anti-CD5 MoAb was dialysed against 50 mM phosphate buffer, pH 6.8, and loaded into a column containing hydroxyapa¬ tite. The MoAb was eluted with a linear gradient of 50- 500 mM potassium phosphate, pH 6.8. The fractions con¬ taining the antibody were collected, dialysed against a 0.9% sodium chloride solution, concentrated by ultra- filtration and filtered through a 0.2 μm mesh filter. The purified MoAb was finally rendered pyrogen- free by affinity chromatography using a column packed with Detoxigel Endotoxin Removing Gel (Pierce), fol- lowing the supplier's instructions, in sterile condi¬ tions. The level of endotoxins - less than 0.125 EU - was determined by the LAL test (Wittaker). All the solu¬ tions used in purifying the MoAb were prepared with apyrogenic water sterilised in an autoclave. The glas¬ sware was sterilised in the oven at 190°C and the re¬ sins were rendered pyrogen-free with NaOH or detergents according to the supplier's instructions. The purity of the Mo.Ab, as determined by elec- trophoresis in denaturing conditions (SDS-PAGE) (Laemmli U.K., Nature 227, 680, 1970), was greater than 98%. A sample aliquot of the MoAb was taken to deter¬ mine the capacity for binding to the CD5 + cell in cyto- fluorimetric conditions (Fig. Id). The binding capacity of the MoAb was 72-78%, which is therefore comparable to that of the antibody in the culture medium. 3) Cytofluorimetric analysis of the anti-CD5 monoclonal antibody - indirect immunofluorescence (Siena S. et al., Transplantation 4_6, 747, 1988) The percentage of binding of the monoclonal anti- body produced by the OKTl cells was analysed in indi¬ rect immunofluorescence with flow cytofl orimetry on CD5 + cell lines (Jurkat) and mononuclear cells isolated from peripheral blood from healthy donors (PBMC) sepa¬ rated by density gradient (Ficoll-Pharmacia) . The method described below was also used to deter¬ mine the binding of the antibody to the cell after conjugation to the toxin. 6 , 1 x 10 CD5 cells were incubated in test tubes for 30 min in ice with 100 μl of antibody. After being washed 3 times in physiological saline containing 1% of bovine serum albumin (BSA) , the sam¬ ples were incubated for 30 min in ice with 100 μl of goat serum containing mice antiimmunoglobulins conjuga¬ ted to fluorescein isothiocyanate (anti-Ig FITC Becton Dickinson) . After being washed 3 times the cells were analysed in the cytofluorimeter (Becton Dickinson). 4) Purification of the vegetable toxin momordin The momordin was purified from Momordica charantia seeds by the methods described earlier (Barbieri L. et al. , Biochem. J., 186, 443, 1980; Barbieri L. et al. , J. Chromatogr. 408, 235, 1987). The purity of the momordin after chromatography on CM- Sepharose (Pharmacia) was estimated as >99% in SDS- PAGE. The activity of the toxin was determined as inhi¬ bition of protein synthesis in an in. vitro translation system based on lysate of rabbit reticulocytes (Stirpe et al. , 3iochem. J. 240, 659, 1986). The IC 50 value, i.e. the concentration capable of inhibiting 50% of protein synthesis, obtained in the above system by measuring the incorporation of 14C-leu- cine into the proteins was 10 " M. 5) Conjugation of the anti-CD5 MoAb to momordin The conjugate was obtained from the chemical reac¬ tion between the anti-CD5 MoAb and momordin, both deri- vatised with 2-iminothiolane (2-IT) (Dinota A. et al. , Brit. J. Cancer , 315, 1989). Derivatisation of the momordin: To a solution of 9 mg/ml momordin in 50 mM sodium borate, pH 9, 10 cpm of momordin labelled with 125I and 2-IT (prepared in the same buffer) at a final concentration of 1.5 mM were added. After 60 minutes' incubation the reaction "was blocked by the addition of glycine at a final concen¬ tration of 200 mM. After a further- 30 minutes' incuba¬ tion at room temperature the Ellman reagent in di- methylformamide (final concentration 2.5 mM) was added and the sample was incubated for 30 minutes at room temperature before being separated on a column of Sephadex G-25 (Pharmacia) eluted with PBS. The deriva¬ tisation ratio of the toxin was calculated on an aliquot of eluate from the column taking as a basis the optical density values of = 280 and A = 412 respectively before and after reduction with 55 mM dithiotreitol (DTT) (final concentration). Derivatisation of the IgG: To a solution of 5-25 mg/ml MoAb (weight ratio IgG-momordin 1:3) in 50 mM so¬ dium borate, pH 9, a solution of 2-IT at a final con- centration of 0.6 mM was added; the derivatisation was then carried out and the introduced SH-groups calcula¬ ted as described for the toxin. Conjugation: The sample of momordin eluted from the column was 'concentrated in an Amicon concentrator in a stream of nitrogen and reduced by adding 1/10 of the volume of 0.55 M DTT. The reduced toxin was separa¬ ted from the free reducing agent with the aid of a gel filtration on Sephadex G-25 and collected directly in an Amicon concentrator containing the derivatised MoAb. The reaction mixture was concentrated 4 times and incu¬ bated at room temperature for 20 hours. At the end of this period it was loaded into Sephacryl S-200 (Pharmacia) high resolution gel filtration column, and eluted with PBS. The radioactivity in the eluted fractions was de¬ termined and compared with the molecular weights of the various peaks read at λ= 280 nm. The first peak corre¬ sponded to a high molecular weight conjugate, the se- cond to a low molecular weight conjugate, followed by the trimers, di ers and monomers of the toxin. The free Igs co-eluted with the final fraction of the low mole¬ cular weight conjugate as shown by the fact that the radioactivity peak did not coincide with (being smaller than) that of the absorption at 280 nm (Fig.2). The low molecular weight fractions were combined and the momor¬ din:IgG ratio was calculated from the absorption at 280 nm and the cpm values of the peaks of low molecular weight conjugate and of momordin alone. The purity of the conjugate was evaluated using indirect immunofluorescence as described earlier (Section 3). As shown in Fig. le, the conjugation left intact the ability of the MoAb to bind to CD5 + cells (73-78%) of labelled cells. 6) Determination of the in vitro biological activity of the anti-CD5 MoAb momordin immunoconjugate (Tab.l) a) Inhibition of protein synthesis in a "cell- free" system In the first instance, the biological activity was determined as inhibition of the incorporation of C- leucine into the proteins in a reticulocyte lysate sy¬ stem as described in Section 4. In various batches of conjugate tested the mean IC50 value was 10 " M. b) Inhibition of protein and DNA synthesis in PBMC (Stirpe F. et al., Br. J. Cancer _5_8, 558, 1988; Siena A. et al., Blood 7_2, 756, 1988) 1 x 10 PBMC isolated from the blood of healthy donors by density gradient were incubated for 4 hours at 37 °C in the presence of increasing quantities of to- o xin, antibody or the corresponding immunotoxin (10~ - 10 "12 M) . After being washed twice in physiological saline the cells were seeded in 96 well plates (50,000 cells/well in RPMI 1640 with the addition of 10% of fo¬ etal bovine serum and 10 μg/ml PHA (phytohaemagglu- tinin) (Wellcome) and incubated at 37 β C for 48 hours. After incubation for a further 15 hours in the presence of 1 μCi/well of H-thymidine (to determine inhibition 3 of DNA synthesis) or H-leucine (to determine inhibition of protein synthesis), the cells were sacrificed using a cell harvester (Titertek-Flow) . The radioactivity incorporated by the cells was measured with the aid of a radioactivity counter (3- counter, LKB) and the IC50 of the various substances was determined by linear regression analysis. The IC50 values of the conjugate for protein and DNA synthesis were similar: 10 " -10 " M, that of the —8 toxin alone was 10 M less, and the anti-CD5 MoAb exhibited no toxicity. c) Inhibition of DNA synthesis in whole blood (Siena S. et al. , Blood 72 ^ , 756, 1988) Aliquots of 1 ml heparinised human blood were di¬ stributed in test tubes and incubated for 1 hour at —8 3377' β C in the presence of increasing quantities (10~ -10 * 12 M) of toxin, antibody or conjugate. Each sample was then separated by density gradient and the mononuclear cells obtained used in a test of DNA synthesis inhibi¬ tion as described above. The inhibition value obtained in non-fractionated blood was similar to that obtained in PBMC: IC50 = 10 "11 M. 7) Determination of the biological activity of the conjugate in vivo (Tab. 2) To analyse the biological activity of the conju¬ gate in vivo we used an experimental model of positive CD5 human leukaemia (Fig. 3a, 3b) stabilised in athymic mice: Jurkat cell line. Before the conjugate was used iri vivo, its acti¬ vity was determined in_ vitro on Jurkat by means of the test of protein synthesis inhibition, described below, which gave a mean IC50 value of 10 " M (Tab.l). The cells, seeded in plates with 96 wells (10,000 cells/well) in RPMI 1640 with the addition of 10% of foetal bovine serum, were incubated for 72 hours at 37°C in the presence of increasing quantities of toxin, antibody and conjugate. After being incubated for a further 15 hours in the presence of 1 μCi/well of H leucine, the cells were sacrificed and the radioacti- vity incorporated is measured with the aid of a radio¬ activity counter (β-σounter, LKB) . For the in vivo experiments female nu/nu mice (Swiss background) were used; these already had reduced reactivity in the T compartment and were further immu- nosuppressed by means of 1) splenectomy, 2) irradiation (450 rads) 72 hours before being inoculated and 3) tre¬ atment, 24 hours before being inoculated, with a titra¬ ted solution of anti-asialoganglioside serum GMl to abolish the Natural Killer (NK) activity, which is gre- ater in athymic mice than in other urine strains. Mice treated in this way are known as nu/nu SIA mice (Arian R. et al., Cancer Res. £8, 1312, 1988). Briefly, the animals were inoculated εubcutaneou- 7 sly with 1 x 10 tumour cells each, then after 24 hours they were divided into 6 experimental groups which were given the following treatment: 1) physiological saline 2) MoAb 10 μg/mouse 3) toxin 5 μg/mouse 4) toxin 10 μg/mouse 5) conjugate 5 μg/mouse 6) conjugate 10 μg/mouse Starting on the day after the tumour cells were inoculated, the animals were treated 9 times (twice a week) and any antileukaemic effect was evaluated in terms of inhibition of the growth of the tumour mass and the change in the numbers of animals reaching the latency threshold (time taken for the tumour nodule to develop) and surival threshold (time taken for the tu¬ mour to bring about death) . The data show that there is a reduction in the numbers of animals reaching both these thresholds (Tab.2) in the groups treated with the conjugate compa¬ red with the numbers in the control group and in the groups treated with either the MoAb or the toxin alone. TABLE 1 Experimental Parameter IC 50 value system evaluated Cell free inhibition of 10 "10 M synthesis protein PBMC inhibition of 10 -10 -10 -11 M protein synthesis PBMC inhibition of DNA 10 -11 M synthesiε Whole blood inhibition of DNA 10 "11 M synthesis Jurkat inhibition of protein 10 "11 M synthesiε * Peripheral blood mononuclear cells TABLE 2 Treatment physiological εaline MoAb CD5 10 μg toxin 5 μg toxin 10 μg immunotoxin 5 μg immunotoxin 10 μg Biological activity of the anti-CD5 MoAb-momordin conjugate in_ vivo 8) Stability of the conjugate The stability of the conjugate when stored frozen at -80°C was evaluated with time as inhibition of DNA synthesis. No loss of activity was evident over a pe¬ riod of 12 months analysis.

Claims: CLAIMS 1. Immunotoxin obtained by covalent conjugation of a riboεome inactivacting protein Type 1, to an anti-CD5 monoclonal antibody. 2. Immunotoxin according to Claim 1, characterised in that the anti-CD5 monoclonal antibody is secreted by the hybridoma deposited at the Institut Pasteur under No. 1-1025 on 1.10.1991. 3. Immunotoxin according to Claims 1 and 2, characteri¬ sed in that the conjugation of the toxin to the anti¬ body is achieved using heterobifunctional reagentε such aε N-εucci-nimidyl-3-(2-pyridyldithio)-propionate, 2- iminothiolane, S-acetylmercaptosuccinic anhydride, car- bodiimides, glutaraldehyde. 4. Immunotoxin according to one of Claims 1-3, charac¬ terised in that the conjugation of the toxin to the an¬ tibody is achieved with 2-iminothiolane (Traut's rea¬ gent) . 5. Immunotoxin according to any one of claims 1-4 wherein the toxin is momordin, dianthin or PAP-S. 6. Immunotoxin .according to any one of claims 1-4 wherein the toxin is momordin. 7. Procesε for the preparation of the immunotoxin ac- cording to one of Clai ε 1-3, characteriεed in that the toxin and the antibody are first derivatised with a heterobifunctional reagent and then conjugated. 8. Process for the preparation of the immunotoxin ac- cording to Claim 4, characterised in that the toxin and the antibody are first derivatised with 2-iminothiolane and then conjugated. 9. Pharmaceutical compositions containing the immunoto¬ xin according to Claims 1-6, as active principle in the form of sterile liquid εuspenεions and/or solutions, freeze-dried powders, gels, creams, ointments, supposi- torieε. 10. Use of the immunotoxin according to Claims 1-6, to prepare a drug for the treatment of autoimmune disea¬ ses, Graft Versus Host Disease and forms of leukaemia. 11. Use of the immunotoxin according to Claims 1-6, to eliminate leukaemic cells or T lymphocytes from the bone marrow ex vivo.

Inventors: GROMO GIANNI, MODENA DANIELA, PORRO GIULIANA, STIRPE FIORENZO, GROMO, GIANNI, MODENA, DANIELA, PORRO, GIULIANA, STIRPE, FIORENZO
Applicants: ITALFARMACO SPA, ITALFARMACO S.P.A.
Application year: 1992
Main classification: A61K47
IPC codes: A61K47