Monoclonal antibody recognizing anthracycline glycoside specific epitope and hybridoma secerning said antibody

Abstract: A monoclonal antibody against Doxorubicin and other anthracycline derivatives, being said monoclonal antibody able to selectively recognize epitopes located in correspondence or in proximity of the aromatic D ring of the anthracycline molecule, is described.

Description: The present invention relates to a monoclonal antibody which is able to recognize specific anthracycline glycoside epitope and the hybridoma secerning said antibody. Monoclonal antibodies against Doxorubicin have been already disclosed in EP-A-0316776 and in Int. J. Cancer 42:798-802, 1988. The production of monoclonal antibodies against drugs, such as Doxorubicin and generally other antitumour anthracyclines, which are able to interfere with the immune system, turned out to be difficult, requiring the introduction of innovative procedures in the immunization process of the animals used as splenocyte source. Particularly, it has been noticed that only resorting to a short immunization cycle and only in animals that were also subjected to another antigenic stimulus it is possible to induce the formation of splenocytes which can be fused to give monoclonal antibodies against Doxorubicin and Doxorubicin derivatives. In the above-mentioned paper published in Int. J. Cancer the main features of a monoclonal antibody, abbreviated as MAD2, exhibiting a different affinity degree against several anthracycline derivatives, the highest affinity being anyway noticed against Doxorubicinol, a Doxorubicin metabolite, and Doxorubicin itself, are described in detail. The availability of a monoclonal antibody recognizing different epitopes on the anthracycline molecule would allow the development of analytical methods for a whole class of substances and, moreover, would constitute a useful tool for the study of drug-receptor interactions, also including phenomena occurring as toxic or unwanted effects. Said problems have been solved by the monoclonal antibody of the present invention, secreted by the hybridoma deposited at European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, Great Britain, on January 10 th , 1990, under accession number 90011003. The antibody object of the invention, hereinafter also named with the abbreviation MAD11, which can be prepared with the same somatic hybridization already described for MAD2 antibody, is able to recognize different antigenic determinants, particularly the one located at the positions C₄-C₅ of the anthracyclinone residue, in contrast to the hitherto described antibody (MAD2) which recognizes the determinant constituted by positions C₉-C₁₁ of the Doxorubicin molecule, whose structural formula with relative numeration is hereinbelow reported: The ring D, which is 4-methoxy substituted, is a constant characteristic of almost all well-known anthracycline derivatives and this explains the wide recognition spectrum of the antibody of the invention. As already mentioned, such characteristic is advantageous for the above-mentioned aims. The cross reactivity of MAD 11 was evaluated by inhibition tests, incubating (1 hour at 37°C) a constant amount of antibody with different amounts of compounds structurally related to Doxorubicin. Then, the samples were incubated for 45 minutes at 37°C in Doxorubicin coated wells and the binding was evaluated by adding ¹²⁵I-radiolabelled mouse anti-Ig antibodies (Amersham Lab.). The curves, calculated as the mean value of at least 3 tests, resulted superimposable for Doxcrubicin, Daunorubicin, Doxorubicinol, 5-imino-Doxorubicin, 11-deoxy-Daunorubicin, 4'-epi-Doxorubicin and 4'-deoxy-Doxorubicin. Instead it was noticed a substantially different inhibition behaviour for two compounds lacking of the 4-methoxy groups, namely Daunosamin and 4-demethoxy-Daunorubicin. The antibody of the invention may be prepared according to the known procedure previously described for MAD 2 antibody. 50 µg of Doxorubicin-Bovine-Serum-Albumine (BSA) conjugate emulsified with complete Freund's adjuvant were injected in BALB/c mice, (previously conditioned by an unrelated antigen), at day 0 and 15 by subcutaneous route. On day 25, (i.e. three days before the fusion) a third administration of the conjugate at the same dose was carried out intra-peritoneally. Cell fusion was accomplished using immunized spleen cells and mouse myeloma P₃/NSO cells with 40% (v/v) polyethylene glycol Mr 1000 (Merck, Darmstadt, West Germany). Growing hybridomas were tested for immunoglobulin production using immunofluorescence (IF) assay as reported in Cancer Res. 43:1295-1300, 1983. The Ig containing supernatants were screened for anti-Doxorubicin antibody by a RIA technique on 96-well plates seeded with 50 µl/well of Doxorubicin (2.5x10⁻ ⁶M) or BSA as negative controls, diluted in carbonate buffer (pH 9.6). Sodium phosphate buffer (pH 7.2) (PBS) supplemented with Tween® 20 (0.05%) was used as diluting and washing buffer. The antibodies producing hybridomas which reacted with Doxorubicin but not with BSA alone were cloned by the method of limiting dilution and subsequently expanded in BALB/c mice primed with pristane (tetramethylpentadecane). The antibody isolated from hybridoma N. 9001003 was purified by affinity chromatography on a Protein-A-Sepharose® CL4B (Pharmacia) column as described by EY et al (Immunochem. 15, 429-436, 1978). The antibody, eluted at pH 5.5, after dialysis against PBS was subjected to radiolabelling by lactoperoxidase-catalysed iodination. 1 mCi CiNa¹²⁵I (Amersham International, Little Chalfont, UK), 24 µg of lactoperoxidase (Calbiochem, Behring, La Jolla, CA) and 8 µl of 0.03% H₂O₂ were added to 50 µg of monoclonal antibody. After 10 minutes at room temperature the reaction was stopped by desalting the mixture on a Bio Gel® Pg column (Bio Rad, Richmond, CA). The labelled antibody was titrated by serial dilution (10⁶ to 7800 cpm/well) on Doxorubicin coated wells, incubating for 3 hours at 4°C. An affinity value of the antibody of 1.7x10⁻⁸M⁻¹, was obtained by scatchard analysis. The antibody MAD 11 was shown to belong to the IgG2 class unlike the antibody MAD2 which belongs to the IgG1 class, as resulted from direct immunofluorescence assay on hybridoma cells.

Claims: Claim for the following Contracting States : ES, GR A process for the preparation of the monoclonal antibody against anthracycline glycosides belonging to Ig G2 class characterized by culturing the hybridoma deposited at European Collection of Animal Cell Cultures (ECACC) under N. 9001003 in a suitable medium. Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE Monoclonal antibody against anthracycline glycosides belonging to IgG2 class secreted by the hybridoma deposited at European Collection of Animal Cell Cultures (ECACC) under N. 9001003. Hybridoma deposited at European Collection of Animal Cell Cultures (ECACC) under N. 9001003.

Inventors: BALSARI ANDREA, COLNAGHI MARIA INES, GHIONE MARIO, BALSARI, ANDREA, COLNAGHI, MARIA INES, GHIONE, MARIO
Applicants: IST NAZ STUD CURA DEI TUMORI, ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI
Application year: 1991
Main classification: C12P21
IPC codes: C07K16, C12N15, C12R1, G01N33, C12N5, C12P21