1. Field of the Invention
The present invention relates to a composition for identifying and suppressing the growth of tumor cells.
2. Description of the Related Art
One of the commonest causes of illness in the western world is the occurrence of malignant tumors. Early diagnosis of tumors and subsequent treatment thereof is still problematical. A number of markers for identifying tumor growth and the formation of metastases have been described in recent years (cf. Potter-Jordan, K. and Lippman,. M., Hematol. Oncol. Clin. North Am. 8, 73 to 100 (1994)). Some of these markers, for example erbE-2, cathepsin D, have proved to be very useful in a large number of studies. Very recently, variants of the protein CD44 have become important (Kaufmann et al. Lancet 345, 615 to 619 (1995), EP 0531300, DE-A 4134982). Finally, EP 351313, CA121: 29448, CA 115: 69417, Medline 95085293, CA 111: 209896 disclose antigen-antibody systems using proteins comprising N-glycosidically bonded saccharides. It is known on the basis of these publications that poly- or monoclonal antibodies which specifically recognize particular epitopes on the tumor marker proteins and react therewith, and which can be labeled by radioisotopes and/or be conjugated with substances with cytocidal or cytotoxic activity can be used for diagnosing and treating metastasizing tumors.
Antibodies respond to specific epitopes. As a rule, different tumors express different markers and, on the other hand, one marker always covers only one subset. It is therefore desirable to have a plurality of different markers for tumor control within the framework of multicomponent treatment.
It was accordingly an object of the present invention to develop a composition for identifying and for suppressing the growth of tumors which can, if possible, be employed equally well for different tumors.
This object is achieved by a composition for identifying and suppressing the growth of tumor cells comprising antibodies which react with proteins comprising N-glycosidically bonded saccharides, wherein the saccharides belong to the blood group-specific antigens of type B2, 3, 4 or type A2.
Suitable for the purposes of the invention is any appropriate variant of proteins of animal or human origin modified with the specific saccharides.
The term antibody means mono- or polyvalent antibodies and poly- and monoclonal antibodies, but also those representing fragments thereof and derivatives thereof, including the F(abxe2x80x2)2, Fabxe2x80x2 and Fab fragments, but also chimeric antibodies or hybrid antibodies having at least two antigen or epitope binding sites, or bispecific recombinant antibodies (for example quadromas, triomas), interspecies hybrid antibodies, anti-idiotype antibodies and, of these, those which have been chemically modified and are to be regarded as derivatives of these antibodies and which can be produced either by the known conventional methods for obtaining antibodies or by DNA recombination, via hybridoma techniques or antibody engineering or synthetically or semisynthetically in a manner known per se and have neutralizing or binding properties in relation to the sugar chains described and defined above. From the diverse literature, reference may be made only by way of example to studies by Kxc3x6hler, G. and Milstein, C., Nature 256, 495 to 497, 1975; Biocca, S. et al., Embo, J. 9, 101 to 108, 1990; Bird, R. E. et al., Science 242, 423 to 426, 1988; Boss, M. A. et al., Nucl. Acids Res. 12, 3791 to 3806, 1984; Boulianne, G. L. et al., Nature 312, 643 to 646, 1984; Bukovsky, J. and Kennett, R. H., Hybridoma 6, 219 to 228, 1987; Diano, M. et al., Anal. Biochem. 166, 223 to 229, 1987; Huston, J. S. et al., Proc. Natl. Acad. Sci. USA 85, 5879 to 5883, 1988; Jones, P. T. et al., Nature 321,, 522 to 525, 1986; Langone, J. J. and Vunakis, H. V. (ed.), Methods Enzymol, 121, Academic Press, London, 1987; Morrison S. et al., Proc. Natl. Acad. Sci. USA 81, 6851 to 6855, 1984; Oi, V. T. and Morrison, S. L., Bio Techniques 4, 214 to 221, 1986; Riechmann, L. et al., Nature 332, 323 to 327, 1988; Tramontano, A. et al., Proc. Natl. Acad. Sci. USA 83, 6736 to 6740, 1986; Wood, C. R. et al., Nature 314, 446 to 449, 1985.
Concerning the production of polyclonal antibodies against epitopes, a number of methods are available. It is possible for this purpose, for example, to immunize in a manner known per se various animals by injection with the glycoprotein according to the invention, or fragments thereof, and to isolate and purify the required polyclonal antibodies by known methods from the sera obtained thereby. It is likewise possible to use various adjuvants to increase the immune response to the protein administration, depending on the animal selected for the immunizationxe2x80x94for example Freund""s adjuvant, mineral gels such as, for example, aluminum hydroxide, surface-active substances such as, for example, polyanions, peptides, oil emulsions, hemocyanins, dinitrophenol or lysolecithin. Monoclonal antibodies are preferred according to the invention. The antibody M-N#1 is particularly preferred and can be obtained from the cell culture DSM ACC 2333 which is deposited at the DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig.
The monoclonal antibodies preferred for the use according to the invention can be obtained by any appropriate technique available for producing antibodies by cultivation of cell lines. The known techniques of this type include, for example, the methods described by Kxc3x6hler, G. and Milstein, C., 1975, loc. cit., or Taggart and Samloff, Science 219, 1228 to 1230, 1983, with hybridoma cells or those with human B cell hybridomas (Kozbor et al., Immunology Today 4, 72 to 79, 1983). These methods also include the use of gene libraries from B lymphocytes (Ward et al., 1989; Nature 341, 544-546) and xe2x80x9ccombinatorialxe2x80x9d libraries on phage vectors (McCafferty et al., 1990, Nature 348, 552-554; Krang et al., 1991, PNAS88, 4363-4366).
The antibodies can be purified by known methods, for example by immunoabsorption or immunoaffinity chromatography, by HPLC (high performance liquid chromatography) or combinations thereof. Antibody fragments containing the idiotype of the molecule can likewise be produced by known methods. For example, F(abxe2x80x2)2 fragments can be obtained by pepsin digestion of the complete poly- or monoclonal antibody. Fabxe2x80x2 fragments can be obtained by, for example, reducing the disulfide bridges of the relevant F(abxe2x80x2)2 fragment, and Fab fragments can be produced, for example, by treating the antibody molecules with papain and a reducing agent.
Any known method for identifying and selecting antibodies, fragments or derivatives thereof can be used. For example in that these can be detected after appropriate labeling when they have bound to isolated or purified antigen, or by immunoprecipitation of the antigen which has been purified, for example, on polyacrylamide gels, or by antibodies against the blood group antigens competing with other blood group-specific antibodies for binding to sugar side chains.
However, the present invention also relates to the use of hybridoma cell lines for producing the antibodies or a preparation for the use according to the invention and to a method for producing a preparation for the use according to the invention.
Compositions with such antibodies are used according to the invention for oncoses in animals and humans comprising prevention or prophylaxis, control, diagnosis or treatment thereof.
The use according to the invention of the antibodies is based on the surprising observation that proteins with N-glycosidically bonded saccharides belonging to the blood group-specific antigens of type B2, 3, 4 or type A2 allow various types of tumors to be identified and treated. Experiments have shown that it is not possible to employ any desired glycoproteins for producing the antibodies. Thus, proteins with O-glycosidically bonded saccharides have proven unsuitable. The clinical use of the antibodies according to the invention is thus particularly advantageous especially for a large number of disorders, illnesses and conditions.
Depending on the nature and cause of the illness or disorder to be treated or the condition to be influenced in an animal or human body, it may be desirable to administer the antibody preparation systemically, locally or topically onto or into the relevant tissue or organ. A systemic mode of action is desirable, for example, when different organs or organ systems require treatment. By contrast, a local effect would be considered if only the local manifestations of a tumor are to be influenced.
The relevant antibodies can be administered by every enteral or parenteral administration route known to the skilled worker. Suitable for systemic administration is, for example, the intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, oral or intrathecal route. Possible examples of more local administration are subcutaneous, intracutaneous, intracardiac, intralobar, intramedullary, intrapulmonary or into or onto the tissue to be treated (connective, muscle, nerve, epithelial or bone tissue). Depending on the duration and strength of the immunosuppressant effect to be achieved, the antibody preparations can be administered one or more times, also intermittently, a day for several days, weeks or months and in various doses. An antibody preparation suitable for said administrations can be produced by using the injectable physiologically tolerated solutions in sterile form which are known to the skilled worker. The well known aqueous isotonic solutions, for example saline or an appropriate plasma protein solution without gamma-globulin, are available for producing a solution ready for use for parenteral injection or infusion. The preparation can, however, also be in the form of a lyophilizate or dry product which can be reconstituted with one of the well-known injectable solutions immediately before use under sterile conditions, for example as kit of parts. Final production of an antibody preparation to be used according to the invention for injection, infusion or perfusion takes place by mixing antibodies purified by known methods and complying with the definitions given above with one of said physiologically tolerated solutions which may, where appropriate, be supplemented with known vehicles or ancillary substances (for example serum albumins, dextrose, sodium bisulfite, EDTA).
The amount of antibodies to be administered depends on the nature and severity of the illness or disorder to be treated or of the condition to be influenced and on the type and weight of the relevant patient (animal or human). However, the basis is use of a dosage of 0.5-2, preferably 0.7-1.5, mg/kg of body weight of the relevant antibody per dose unit.