MXPA98002129A - Monoclonal antibody br110 and uses of the mi - Google Patents

Abstract

The present invention provides interiorizing ligands (ie, ligands of BR110) that specifically recognize and bind to the BR110 antigen. After binding to the antigen, the ligand and the antigen form a complex. As a complex, the antigen can be detected using well-known commercial methods and systems and developed

Description

MONOCLONAL ANTIBODY BR110 AND USES OF THE SAME BACKGROUND OF THE INVENTION Cancer includes a wide range of diseases that affect one in four people worldwide. Clearly, in terms of the number of people affected, cancer poses a serious medical problem. Because of the seriousness of the problem, and its impact on society, therapeutic agents and effective diagnostics directed against cancer are highly appreciated. Antibodies to antigens associated with tumors are recent technological achievements, which promise to be important therapeutic agents and diagnostics against certain cancers. Antibodies to antigens associated with human tumors are well known and well described. One such tumor-associated antigen is designated as the GA733-2 antigen. The GA733-2 antigen has a molecular weight of about 34 kO-40 kD, and is a member of the GA733 family of antigens (Linenbach et al., PNSA USA 86: 27-31 (1989)). The biological function of the GA773-2 antigen is unknown (International Publication No. WO 93/0829, published April 29, 1993).

REF: 26915 Using the GA733-2 DNA probe, the researchers were able to examine a genomic library, and isolate a homologous sequence designated GA733-1. From the GA733-1 gene, the researchers were able to express the antigen. However, none has determined whether the antigen was an antigen associated with tumor. The fact that the GA733-1 gene was isolated from a genomic library was additionally added to the uncertainty of whether the gene and the antigen encoded by it were usually found in normal cells, tumor cells, or both, and the frequency with which they were. The molecular weight and the antigen function of GA733-1 are unknown. The GA733-1 antigen contains multiple epitopes, and a segment of the DNA encoding GA733-1 is known (US Patent No. 5, 185, 254, supra). The GA733-2 antigen is recognized by a monoclonal antibody designated MoAb GA733 (also known as GA733-2 antibody) (Herlyn, et al., Hybridoma, 5: S3-SIO (1986)). The MoAb GA733 has been evaluated for the diagnosis and therapy of human gastrointestinal tumors. It exhibits tumoricidal characteristics. Despite the fact that GA733-1 and GA733-2 share 49% homology in their amino acid sequence (US Patent No. ,185,254, filed February 9, 1993), MoAb GA733 binds to the GA733-2 antigen but does not bind to the GA733-1 antigen. MoAb GA733 binds to normal epithelial cells.

In addition to GA733, several independently derived mAbs, such as C017-1A, M77, M79, 323 / A3, all recognize and bind to the GA733-2 antigen (International Publication No. WO 93/0829, supra). The monoclonal antibody C017-1A (also known as the 17-1A antibody) binds to the GA733-2 antigen, but either recognizes and binds to the GA733-1 antigen. At present, there are no known monoclonal antibodies directed against the GA733-1 antigen. The ligands of BR110 of the invention recognize and bind to the GA733-1 antigen, and are internalized by the cells after binding to the antigen. Additionally, the ligands of BR110 of the invention appear to exhibit tumoricidal activity. Interiorizing antibodies are rare. At present, there is a need in therapeutic applications for such "interiorizing" antibodies, ie, antibodies that are readily absorbed by the tumor cells to which they bind. Such antibodies, for example, can be coupled to biological and / or chemical agents that are only effective when transported within the cell.

BRIEF DESCRIPTION OF THE INVENTION The ligands of BR110 of the invention, for example the monoclonal antibodies of BR110, satisfy the therapeutic and diagnostic needs discussed above. The ligands of BR110 share common characteristics. For example, each BR110 ligand recognizes and binds specifically to the BR110 antigen, and is targeted to at least a portion of the epitope to which the monoclonal antibody BR110 is directed (ATCC No. 11698). In one embodiment of the invention, the ligand of BR110 is the monoclonal antibody BR110 (ATCC No. 11698). Additionally, another embodiment of the BR110 ligand of the invention is a human / murine recombinant antibody, the antigen binding region from which competitively inhibits the in-munospecific binding of monoclonal antibody BR110 produced by hybridoma HB 11698 to its target antigen. Additionally, the invention provides uses for the BR110 ligands of the invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a graphic representation of the plasmid pSE 70.0.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS As used in this application, the following words and phrases have the meanings specified. As used herein, "ligand" includes an intact antibody molecule, and any molecule having at least one antigen binding region or portion thereof (ie, the variable portion of an antibody molecule), by example, a Fv molecule, Fab molecule, Fab 'molecule, F (ab') 2 molecule, a bispecific antibody, a fusion protein, or any genetically engineered molecule that recognizes and binds specifically to the BR110 antigen. As used herein, "antigen binding region means a portion of the molecule that recognizes the target antigen." As used herein, "competitively inhibiting" means being able to recognize and bind to a determinant site to which it is attached. directed the monoclonal antibody BR110, using conventional reciprocal antibody competition assays (Belanger, L., Sylvestre, C. and Dufour, D., (1973), Enzyme linked immunoassay for alpha fetoprotein by competitive and sandwich procedures Clinica Chimica Acta 48, 15) As used herein, "target antigen" is the antigen GA733-1 or portions thereof As used herein, "partner" means to join two or more entities or segments usually separated. , through a covalent bond or a non-covalent bond As used herein, "functionally active" means that portion of the molecule that is capable of recognizing and binding to its target. "biologically or chemically active écula" means any biological or chemical molecule that can inhibit or interrupt cell growth, or act otherwise to the detriment of the cell. As used herein, an "immunoconjugate" means any molecule or ligand such as an antibody chemically or biologically bound to a cytotoxin, a radioactive agent, enzyme, toxin, an anti-tumor drug or a therapeutic agent. The antibody can be bound to the cytotoxin, radioactive agent, antitumor drug or therapeutic agent anywhere along the molecule, as long as it is able to bind to its target. Examples of immunocytes played include antibody-toxin chemical conjugates and antibody-toxin fusion proteins. As used herein, an "effective amount" is an amount of the antibody, immunoconjugate, or recombinant molecule that kills target cells, or inhibits proliferation thereof. As used herein, a "fusion protein" means any chimeric protein wherein an antigen binding region is connected to a biologically active molecule, for example a toxin, enzyme, or protein drug. In order that the invention described herein may be more fully understood, the following description is set forth.

A. THE LIGANDS OF THE INVENTION The present invention provides interiorizing ligands (ie, ligands of BR110) that specifically recognize and bind to the BR110 antigen.

The ligand of the invention can be in any form, as long as it has an antigen-binding region that competitively inhibits the immunospecific binding of the BR110 monoclonal antibody produced by the hybridoma HB 11698 to its target antigen. Thus, any recombinant proteins (e.g., fusion proteins wherein the antibody is combined with a second protein, such as a lymphokine or a tumor-inhibitory growth factor), which has the same binding specificity as the BR110 antibody falls. within the scope of this invention. In one embodiment of the invention, the ligand is a monoclonal antibody BR110. The hybridoma that produces the monoclonal antibody BR110 has been deposited under the requirements of the Budapest Treaty on August 10, 1994 with the American Type Culture Collection ("ATCC"), 12301 Parklaw Drive, Rockville, MD 20852, and has been identified as the Accession No. of ATCC: HB 11698. In other embodiments, the ligand is an Fv molecule (such as a single chain Fv molecule), a Fab molecule, a Fab 'molecule, an F (ab') 2 molecule, a fusion protein, a bispecific antibody, a heterobody or any recombinant molecule having the antigen binding region of the BR110 antibody. The ligand of the invention is directed to the epitope to which the monoclonal antibody BR110 is directed (ATCC No. 11698). In one embodiment of the invention, the ligand of the invention is directed to the epitope to which the monoclonal antibody BR110 is directed, and exhibits an affinity of at least about 2 x 108 liters / mole to its target or, in the case of antibodies multivalent, an avidity or affinity that is at least as high as that resulting from the binding of a bivalent affinity antibody 108 liters / mol to an antigen. Different affinities are possible, and are included by the invention. The ligand of the invention can be modified, that is, by modification of amino acids within the molecule, to produce derived molecules. Chemical modifications may also be possible. The derived molecules would retain the functional property of the polypeptide, ie, the molecule having such substitutions will still allow the binding of the polypeptide to the GA733-1 antigen or portions thereof.

These amino acid substitutions include, but are not necessarily limited to, amino acid substitutions known in the art as "conservative".

For example, it is a well-established principle of the chemistry of proteins that certain substitutions of amino acids, called "conservative amino acid substitutions", can often be made on a protein without altering either the conformation or the function of the protein. Such changes include replacing either isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids; aspartic acid (D) by glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) by threonine (T) and vice versa. Other substitutions may also be considered conservative, depending on the environment of the particular amino acid, and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, such as alanine and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be exchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable at sites where the significant aspect of the amino acid residue is its charge, and the different pK 's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments. B. METHODS FOR MAKING THE LIGANDS OF THE INVENTION The ligands of the invention include (1) monoclonal antibodies and fragments thereof, (2) recombinant molecules having at least one antigen binding region or a portion thereof, for example , an Fv molecule, Fab molecule, Fab 'molecule, F (ab') 2 molecule, a bispecific antibody, a fusion protein, or (3) any genetically engineered molecule, which recognize and bind specifically to antigen of BR110. Monoclonal Antibodies of the Invention The hybridomas that produce the monoclonal antibodies of the present invention are produced following general procedures described by Kohier and Milstein. ((1975) Continued culture of fused cells secreting antibody of defined specificity, Nature 256, 495-497) with some modifications (M. Yeh et al., "Cell Surface Antigens Of Human Melanoma Identified By Monoclonal Antibody. "Proc. Nati, Acad. Sci. USA, 16 (6): 297-31 (1979); and Yeh et al., "A Cell-Surface Antigen Which is Present In the Ganglioside Fraction And Shared By Human Melanomas ", Int. J. Cancer, 29: 269-75 (1982).) In this procedure, hybridomas are prepared by fusing cells that produce antibodies (typically spleen cells from mice previously immunized with an immunogen) to cells of a line immortal tumor cell using somatic cell hybridization methods The novel monoclonal antibodies described herein were generated by immunizing mice with H3922 human breast carcinoma cells previously treated with neuraminidase For immunization with H3922 human breast carcinoma cells, the animals were inoculated intraperitoneally at least once with 107 cells of the immunogen.The animals were then boosted two or more times with the immunogen.The spleens were extracted from the animals several days after the last inoculation, and a suspension of spleen for fusion, using known fusion techniques, with murine myeloma cells. Ibridomas resulting from the fusion process were allowed to grow. After this, the resulting supernatants were examined using immunoassay methods, to detect antibodies present in the supernatants capable of binding to the specific antigen.

(Hardy, R. R., (1986) Purification and characterization of monoclonal antibodies In: Weir, D. M., and Herzenberg, L.

A., (eds.) Handbook of Experimental Immunology, 4th edition, Vol. 1, page 13. Oxford: Blackwell Scientific Publications; Engvall, E. and Perlmann, P. (1971). Enzyme linked immunosorbent assay (ELISA): quantitative assay of immunoglobulin G. Immunochemistry 8_, 871). Recombinant Proteins of the Invention It would be routine to make recombinant proteins capable of binding to the same antigenic determinant as the BR110 antibody. Identifying recombinant proteins that recognize the same binding site simply involves establishing competition assays in which the antibodies of the present invention compete with another antibody for the target (Belanger, L. Sylvestre, C. and Dufour, D. (1973). ) Enzyme linked immunoassay for alpha fetoprotein by competitive and sandwich procedures Clinica Chimica Acta 48, 15). Competition assays are routine, and their protocols are well established (E. Harlow and D. Lane, eds., "Antibodies, a Laboratory manual", 1988, pages 567-577). Variants of class, isotype and other variants of the antibody of the invention having the antigen binding region of the BR110 antibody can be constructed using recombinant class and fusion switching techniques that are known in the art. (Winter, G. and Milstein, C. (1991), Man-made antibodies, Nature 349, 293-299, Pluckthun, A., (1991), Antibody engineering: advances from the use of Escherichia coli expression systems. Technology 9, 545-551, Moore, GP, (1989), Geneticaly engineered antibodies, Clinical Chemistry 35, 1849-1853). The present invention provides fusion proteins of BR110. The general techniques for the construction of expression vectors for fusion proteins are well established (Ernst Winnacker, "From Genes to Clones: Introduction to Gene Technology" Chapter 7, 1987, pages 239-317). For example, a general approach for the construction of expression vectors directing the synthesis of fusion proteins is as follows: The starting material may be a cDNA clone, wherein the gene of interest has been removed from a vector. Using methods known in the art, a restriction site is placed near the start codon. The next stage is a digestion step that cuts DNA fragments asymmetrically. The mixture of DNA fragments obtained is then cloned into a vector. Of course, a hydrolysis site must be present within the polylinker of the selected vector. Since a broad spectrum of vectors is available, it would not be difficult to find a suitable vector containing the desired hydrolysis site. Once a suitable clone is identified, the hydrolysis site can be used for the insertion of the gene of interest, which can be obtained from the original cDNA clone. The present invention provides BR110 chimeric proteins. Making a chimeric BR110 protein capable of binding to the same antigenic determinant as the BR110 antibody is routine (Morpson, SL, Johnson, MJ, Herzenberg, LA, and Oi, VT, (1984), Chimeric human antibody molecules: Mouse antigen- binding domains with human constant region domains Proc. Nati. Acad. Sci., USA 8JL, 6851-6855; Morrison, S. L., (1985). Transfectomas provide novel chimeric antibodies. Science 229, 1202-1207).

Genetic engineering can be used to create chimeric immunoglobulins (1) by combining the segment (VH) of the heavy variable gene of rodent with the gene segments of the human heavy chain constant region, to make the heavy chain gene construct, ( 2) connecting the segment (VL) of the rodent variable light gene with a human constant light exon (CL), to create the light chain gene construct, and (3) transfecting both the heavy chain gene constructs and the light in a myeloma cell line (Fell et al., in Proc. Nati, Acad. Sci. USA 86: 8507-8511 (1989); Morrison, SL, Johnson, M.

J., Herzenberg, L. A., and Oi, V. T., (1984). Chimeric human antibody molecules: Mouse antigen-binding domains with human constant region domains. Proc. Nati Acad. Sci. USA 81: 6851-6855. In principle, any variable domain of a rodent can be paired with any isotype of a human constant region, so that the optimal combination of antigenic specificity and effector functions (such as complement fixation and ADCC (cell-mediated cytotoxicity) can be selected. antibodies) (Morrison, SL, (1985), Transfection provide novel chimeric antibodies, Science 229, 1202-1207.) If necessary, precise tuning of the constructs can be done by introducing point mutations in the variable region gene segments that alter the affinity of the chimeric antibody for its ligand (Kunkel, T. A., 1985, Proc. Nati, Acad. Sci. USA 32: 488-492, Kunkel, T. A., et al., 1987, Methods Enzymol. 367-382. To reduce or eliminate any murine characteristic of the BR110 ligand, humanized versions thereof can be produced using molecular manipulations and transfectoma technology (Co, M.S., Deschamps, M., Whitley, R.J., and Queen, C. (1991).

Humanized antibodies for antiviral therapy. Proc. Nati Acad. Sci. USA 8_8: 2869-2873). Different human heavy chain isotypes can be selected for the humanized antibody, depending on the desired therapeutic effect. For example, when destruction of the target cell is desired, the constant region of human IgGl can be selected, since it is recognized by FcgRI, FcgRII, and FcgRIII, and average ADCC (Anderson, CL, and Looney, RJ (1986)). Human leukocyte IgG Fc receptors, Im unol. Today 7, 264-266). Chimeric antibodies have been produced that have different effector functions, binding different sequences to those that encode the antigen binding region. These different sequences include enzymes (Neure berg et al., Nature 312: 604 (1984)), constant regions of immunoglobulins of other species, and constant regions of another immunoglobulin chain (Sharon et al., Nature 309: 364 (1984)).; Tan et al., J. Im unol. 135: 3565-3567 (1985)). In other situations, such as imaging for diagnosis, antibody-receptor blocking, or drug delivery mediated by antibodies, isotypes that bind to Fc receptors poorly (or do not bind) and / or are relatively ineffective to complement-mediated lysis (such as IgG2, IgG4, or IgA) may be the isotypes of choice. Molecular manipulations and transfectoma technology are popular means to "humanize" rodent antibodies with interesting specificities. It is expected that these chimeric rodent-human antibodies are less antigenic and more useful in human therapy (Co, MS, Deschamps, M., Whitley, RJ, and Queen, C. (1991) Humanized antibodies for antiviral therapy. Nati, Acad. Sci. USA 88, 2869-2873). In general, an example of a method that can be used to produce chimeric antibodies involves the following steps: a) identifying and cloning the correct gene segment encoding the antigen-binding portion of the antibody molecule; This gene segment (known as the VDJ, variable, diversity and association regions for heavy chains or VJ, variable, and association regions for light chains, or simply as the V or variable region) can be in any form, that of cDNA or genomics; b) cloning the gene segments encoding the constant region or the desired part thereof; c) ligating the variable region with the constant region, so that the complete chimeric antibody is encoded in a transcribable and translatable form; d) ligating this construct into a vector containing a selectable marker and genetic control regions such as promoters, amplifiers and addition signals of poly (A); e) amplify this construct in bacteria; f) introducing this DNA into eukaryotic cells (transfection), more frequently mammalian lymphocytes; g) selecting cells expressing the selectable marker; h) screening to find cells that express the desired chimeric antibody; and i) testing the antibody, to determine the binding specificity and the appropriate effector functions. Other methods for producing chimeric antibodies are well known in the art. Identifying chimeric antibodies that recognize the binding site of the BR110 antibody is routine, for example by competition assays (E. Harlow and D. Lane, eds., "Antibodies, a Laboratory manual", 1988, pages 567-577). The BR110 monoclonal antibody of the invention can also be made by chemically conjugating the variable regions of the BR110 ligand and a human constant region by chemical means, for example conjugation with (2) disulfide-generating agents, such as 3- (2-pyridylthio) propionate (SPDP), (2) thioether bonds, (3) activated chlorambucil, (4) acid-labile and photohydrolyzable crosslinkers, and (5) avidin-biotin linkages, (Stevenson, FK, Bell, AJ, Cusak, R., Hamblin, TJ, Sade, CJ, Spellerberg, MB, and Stevenson, GT (1991).) Preliminary studies for an immunotherapeutic approach To the treatment of human myeloma using chimeric anti-CD38 antibody. Blood 77, 1071-1079; S. Wong "Chemistry of Protein Conjugation and Cross-Linking" (1993) CRC Press, Inc.). Other methods for producing the ligand of the invention are possible (Liu, AY, Robinson, RR, Murray, ED Jr., Ledbetter, JA, Hellstrom, I., and Hellstrom, KE (1987a).) Production of a mouse-human chimeric monoclonal antibody to CD20 with potent Fc-dependent biologic activity, J. Immunol., 139, 3521-3526).

The present invention also provides bispecific BR110 monoclonal antibodies. The synthesis of bispecific BR110 monoclonal antibodies capable of binding to the same antigenic determinant as the BR110 antibody is routine (Haber et al., 1990; Wells, W., Harwerth, IM, Zwickl, M., Hardman, N., Groner , B., Hynes, NE (1992), Construction, bacterial expression and characterization of a bifunctional single-chain antibody phosphatase fusion protein targeted to the human ERBB-2 receptor, Biotchnology 10: 1128-1132, A. Traunecker et al., (1991) EMBO Journal 10 (12): 3655-3659 Frames of the BR110 antibody of the invention BR110 antibody fragments include the Fv molecule, Fab molecule, Fab 'molecule, F (ab ') 2. ATCC murine ligand BR110 No. 11698 can be purified by affinity chromatography from murine ascites fluid F (ab') 2 fragments can be generated by digesting BR110 monoclonal antibody purified with pepsin according to a Lamoyi, "Preparation of F (ab ') 2 Fragments from Mouse IgG of Various Subclasses ", Meth. Enzymol. 121: 652-663 (1986). Alternatively, fragments of F (ab ') 2 can be produced through genetic engineering means, using routine methodologies (Mayforth, R. D., Quintana, J. (1190) Current Concepts: Designer and catalytic antibodies. New Eng. J. Med. 323: 173-178; Waldman, T. A. (1991). Monoclonal antibodies in diagnosis and therapy. Science 252: 1657-1662; Winter, G., Milstein, C. (1991).

Man-made Antibodies. Nature 349: 293-299; Morrison, S. L. (1992). In Vitro antibodies: strategies for production and applcation. Ann. Rev. Immunol. 10: 239-266; Haber et al., 1990; Weis, W., Harwerth, I.M., Zwickl, M., Hard an, N., Groner, B., Hynes, N.E., (1992). Construction, bacterial expression and characterization of a bifunctional single-chain antibody phosphatase fusion protein targeted to the human ERBB-2 receptor. Biotechnology 10: 1128-1132; A. Traunecker et al., (1991). EMBO Journal 10 (12): 3655-3659). The binding of the complete BR110 ligand can be distinguished from the binding of the F (ab ') 2 fragments using protein A conjugated to horseradish peroxidase (HRP), which binds to the whole antibody, but not to F fragments. (ab ') 2. Fab fragments of BR110 can be produced through proteolytic hydrolysis of the intact BR110 antibody using, for example, papain (Parha, P., 1986. Preparation and purification of active fragments from mouse monoclonal antibodies. In four volumes "Volume 1" Immunochemistry "(Editors DW Weir et al.) pages 14.1-14.23, Blackwell Scientific Publishers, Oxford). Parham, P. (1986). Preparation and purification of active fragments from mouse monoclonal antibodies. In: Weir, D. M., and Herzenberg, L. A., (ed.) Handbook of Experimental Immunology, 4th edition, Vol. 1, page 14. Oxford: Blackwell Scientific Publications). Alternatively, Fab fragments can be produced through genetic engineering means, using routine techniques (Huse et al., 1989. Generation of a large combinatorial library of the immunoglobulin receptor in phage lambda.) Science 246: 1275-1281 ).

PROTOCOL TO BUILD AND PURIFY THE LINE OF BR110 CONJUGATES AND FUSION PROTEINS The invention provides immunoconjugates of BR110 comprising a ligand of BR110 associated with at least a portion of a biologically or chemically active molecule (Batra et al., Proc. Nati, Acad. Sci. USA 86: 8545-8549 (1989); et al., J. Biol. Chem., 263: 9470-9475 (1988); and Batra et al., Proc. Nati Acad. Sci. USA 86: 8545-8549 (1989). The biologically or chemically active molecule, such as a cytotoxic agent, can inhibit or interrupt cell growth, or act otherwise to the detriment of the cell. In accordance with the practice of the invention, biologically or chemically active molecules include, but are not limited to, an enzyme, lymphokine, a toxin, a paramagnetic isotope, biotin, a fluorophore, a chromophore, a heavy metal, a radioisotope, or a chemotherapeutic agent. Suitable examples of toxins include, but are not limited to, Pseudomonas exotoxin A, ricin, bryodin, and diphtheria toxin. Additional examples include bleomycin, dactinomycin, daunorubicin, doxorubicin, mitoxantrone, mitomycin, cisplatin, and procarbazine. Genetic engineering techniques known in the art can be used as described herein to prepare recombinant immunotoxins produced by ligating a DNA sequence encoding an antigen-binding region of BR110 with a DNA sequence that encodes a molecule biologically or chemically active at the DNA level, and express in a transformed host cell the cytotoxic molecule as a recombinant protein. Recombinant immunotoxins are homogeneous molecules that retain the specificity of the antibody binding portion of BR110 with the cytotoxic potential of the toxin (Kondo et al., J. Biol. Chem., 263: 9470-9475 (1988 ); Siegall et al., Proc. Nati Acad. Sci. USA 85: 9738-9742 (1988). Pastan, I., and FitzGerald, D (1919. Recombinant toxins for cancer treatment, Science 254, 1173-1177, Pastan, I., Willingham, M. C, and Fitzgerald, DJ (1986), I. Munotoxins, Cell 47, 641-648). Recombinant immunoconjugates, particularly single chain immunoconjugates, have an advantage over the drug / antibody conjugates in that they are produced more rapidly than these conjugates, and generate a population of homogeneous molecules, i.e. unique polypeptides composed of the same residues of amino acids (Trail, PA, et al., Cure of xenografted human carcinomas by BR96-doxorubicin immunoconjugates, Science, 261: 212-215, 1993). When the biologically or chemically active molecule is a toxin or drug, the conjugate is more potent than its unconjugated counterpart.

D. USES OF THE LIGANDS OF THE INVENTION The present invention provides a method for detecting BR110 antigen in tissue sections of a subject. In accordance with the practice of the invention, the subject can be a human, equine, porcine, bovine, murine, canine, feline, or avian subject. Other warm-blooded animals are also included. The method comprises contacting tissue sections with the monoclonal antibody of the invention. The conditions under which the tissue sections are contacted with the monoclonal antibody of the invention are such that the binding occurs in such a way that a complex is formed. After complex formation, the antigen can be detected using well-known and developed commercial methods and systems (Gatter, K. C, Falini, B. and Mason, DY (1984).) The use of monoclonal antibodies in histopathological diagnosis. Advances in Histopathology, 12, 35; Boekmann, E., Bau-m, R. 0., Schuldes, H., Kramer, W., Hertel, A., Baew-Christow, T., Hanke, P., Jonas , D., and Hor, G. (1990) .Tumor imaging of bladder carcinomas and their metastases with? NIn-labelled monoclonal anti-CEA antibody BW 431/26. British Journal of Cancer 62 (Suppl.), 81).

In one embodiment of the invention, the tissue section is breast tissue, and the neoplastic tissue is a breast carcinoma. Alternatively, the tissue is colon tissue, and the neoplastic tissue is adenocarcinoma. Additionally, the tissue is lung tissue, and the neoplastic tissue is lung carcinoma. Also, in another embodiment, the tissue is ovarian tissue, and the neoplastic tissue is ovarian carcinoma. Generally, the tissue sections are of a tissue in which normal tissue is characterized by the absence or low antigen concentrations of BR110, and the neoplastic tissue is characterized by the presence of high antigen concentrations of BR110. In accordance with the practice of the invention, the monoclonal antibody bound to the tissue sections can be detected directly, using a labeled label bound to the antibody. Alternatively, the monoclonal antibody bound to the tissue sections can be detected by contacting the monoclonal antibody with a second antibody. The second antibody can be labeled with a detectable label. After contact, the monoclonal antibody bound to the tissue sections forms a complex with the second antibody. The complex is detected by detecting the second antibody bound in this way. The present invention further provides a method for determining a difference in the amount of BR110 antigen distribution in tissue sections of a neoplastic tissue to be tested, relative to the amount and distribution of the BR110 antigen in tissue sections of a normal tissue This method comprises contacting both the tissue to be tested and the normal tissue with the monoclonal antibody of the invention, so that the detection can be carried out as described above. After the detection is made, a determination of the difference in the amount and distribution of BR110 antigen can be made. This determination is a quantitative determination, that is, counting the number of antigens detected in this way, or a visual determination, that is, determining which sample is darker or lighter, or has a particular color, etc. The invention additionally provides a method for diagnosing a neoplastic condition in a subject. In one embodiment, this method comprises obtaining a tissue sample from the subject. The tissue sample is then contacted with the antibody of the invention, so that the presence of the BR110 antigen in such tissue sections will cause the formation of a complex between the antibody and the antigen. The presence of the complex is detected. If a higher concentration of the complex is detected in the sample than in normal tissue, then that concentration is indicative of a neoplastic condition, and additional tests may be assured. Alternatively, the detection of a tumor can be performed using a biological fluid sample, to detect human carcinoma in a subject. Serological diagnostic techniques involve the detection and quantification of tumor-associated antigens that have been segregated or "poured" into the serum or other biological fluid of patients thought to be suffering from carcinoma. Such antigens can be detected in body fluids using techniques known in the art, such as radioimmunoassays (RIA), or enzyme-linked immunosorbent assays (ELISA), where an antibody reactive with the "spilled" antigen is used to detect the presence of an antigen in a fluid sample (Uotila et al., "Two-Site Sandwich ELISA With Monoclonal Antibodies To Human AFP." J. Immunol. Methods, 42: 11 (1981); and Allu et al., supra, on pages 48-51).

It is apparent from the foregoing that the BR110 antibodies of the invention can be used in most assays involving antigen-antibody reactions. These assays include, but are not limited to, standard RIA techniques, both in solid or liquid phase, as well as ELISA assays, immunofluorescence techniques and other immunocytochemical assays (Sikora et al. (Eds.), Monoclonal Antibodies, pages 32-52 (Blackwell Scientific Publications, 1984) ADMINISTRATION OF LIGANDS FROM BR110 TO ANIMALS The ligands of BR110 can be administered to animals in an amount sufficient to decrease the rate of proliferation of target cells, or to kill them completely. those skilled in the art that the optimal program for administering BR110 ligands will vary based on the subject, the height and weight of the subject, the severity of the disease (G. Goodman et al., J. Clin. Oncol., 8, 1083 (1990) (Nedelman, FD, Shealy, DJ, Boulin, R., Brunt, E., Seasholtz, JI, Alien, E., McCartney, JE, Warren, FD, Oppermann, H., Pang, RHL, Berger, H .. J. and Weisman, HF (1993 Rapid infant imaging with a Technetium-99m-labeled anti-myosin recombinant single-chain Fv: evaluation in a canine model of acute myocardial infarction. Journal of Nuclear Medicine 3: 234. Courtenay-Luck, N. S., and Epenetos, A. A. (1990). Targeting of monoclonal antibodies to tumors. Curr.

Opinion Immunol., 2, 880-883). Finally, the use and administration program will be decided by the doctor in charge. The clinical protocols for determining the dosing interval and programming are standard. COMPOSITIONS THAT INCLUDE ANTIBODIES OF THE INVENTION The present invention provides compositions comprising a monoclonal antibody of the invention, to which a biologically or chemically active agent is attached. In one embodiment, the biologically or chemically active agent is a toxin. The toxin is selected from a group that includes, but is not limited to, ricin, diphtheria toxin, pseudomonas exotoxin A, abrin, supporin, and gelonin. Alternatively, the biologically or chemically active agent comprises an enzyme, a drug, or a DNA fragment (Nedelman, MA, Shealy, DJ, Boulin, R., Brunt, E., Seasholtz, JI, Alien, E., McCartney, JE, Warren, FD, Oppermann, H., Pang, RHL, Berger, HJ and Weisman, HF (1993) Rapid infant imaging with a Technetium-99m-labeled anti-myosin recombinant single-chain Fv: evaluation in a canine model of acute myocardial infarction, Journal of Nuclear Medicine 34: 234. The binding of the enzyme, drug, or DNA fragment to monoclonal antibodies is well known (Pollard-Knight, D., Hawkins, E., Yeung, D., Pashby , DP, Simpson, M., McDougall, A., Buckle, P. and Charles, SA (1990), Immunoassays and nucleic acid detection with a biosensor based on plasmon resonance, Annales de Biologic Clinique, 48, 642; MJ, and Little, WA, (1974) A new immunoassay based on fluorescent excitation by infernal reflection spectroscopy Proceedings of the N ational Academy of Sciences USA 71, 4533). ADVANTAGES OF THE INVENTION. The antibodies of the present invention, like other antibodies that recognize antigens associated with human tumors, are additional members of the arsenal of diagnostic and therapeutic agents for the war against diseases. The BR110 ligand is useful not only because it is a tumor-specific antibody, but also because it is an internalizing antibody. Antibodies of this type find use in therapeutic methods to kill cells selectively, using antibody-drug or antibody-toxin conjugates ("immunotoxins"), wherein an anti-tumor therapeutic agent is chemically or biologically bound to an antibody or growth factor, for its delivery to the tumor, where the antibody binds to the antigen or receptor associated with the tumor with which it is reactive, and "supplies" the antitumor agent within tumor cells (Embleton et al., "Antibody Targeting of Anti-Cancer Agents," in Monoclonal Antibodies for Cancer Detection and Therapy, pages 317-44 (Academic Press, 1985)) . In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only, and should not be construed as limiting the scope of this invention in any way. EXAMPLE 1 Preparation of the BR110 M-onoclonal Antibody The BR110 monoclonal antibody of the invention was produced using hybridoma fusion techniques previously described by M. Yeh et al., Proc. Nati Acad. Sci. USA, (1979), supra, and Yeh et al., Int. J. Cancer (1982), supra.

Briefly, BALB / c mice were immunized using H3922 human breast carcinoma cells previously treated with neuraminidase (10 'cells). The mice received multiple immunizations (x6). On the first occasion, the mice received an intraperitoneal injection using RIBI adjuvant. From the second to the sixth time, the mice were given an intraperitoneal injection without the use of RIBI adjuvant. The total number of cells injected on each occasion was approximately 107 cells. Four days after the last immunization, the spleen was removed, and bar cells were suspended in RPMI culture medium. The spleen cells were then fused with mouse myeloma cells P2x63-Ag8.653, in the presence of polyethylene glycol (PEG), and the cell suspension was cultured in microtiter wells, in HAT-selective medium, as described by Yeh et al., Supra [see also, Kohier and Milstein, Nature, 256: 495-97 (1975) and Eur. J. Immunol., 6: 511-19 (1976)]. The mixture was seeded to form low density cultures, which originated from individual fused cells or clones. Supernatants of these hybridoma cultures were then examined for direct binding activity on the H3922 cancer cell line, using an ELISA assay similar to that described by Douillard et al., "Enzyme-Linked Immunosorbent Assay For Monoclonal Screening Antibody Production Using Enzyme-Labeled Second Antibody ", Meth. Enzymol. , 92: 168-74 (1983).

According to this assay, the antigen (with which the antibody being examined is reactive) is immobilized on microtiter plates, and then incubated with hybridoma supernatants. If a supernatant contains the desired antibody, the antibody will bind to the immobilized antigen, and is detected by the addition of an antibody-enzyme conjugate anti-immunoglobulin and a substrate for the enzyme that results in a change that can be measured in optical density . In the present studies, the H3922 line of human breast cancer cells and fibroblasts was dispensed into a 96-well tissue culture plate (Costar Cambridge, MA) and incubated overnight in a humid incubator at 37 ° C. , with 5% of C02. The cells were then fixed with 100 μl of freshly prepared 2% paraformaldehyde, incubated for 15 minutes at room temperature, followed by blocking with the specimen diluent (Genetic Systems, Seattle, WA) for 1 hour at room temperature, this being the incubation condition for the complete assay. After washing with P / BS, hybridoma supernatants or purified antibody were then added and incubated for 1 hour. The plates were washed with PBS, and in a second stage Mab (IgG / M anti goat mouse, Tango Burlingname, CA) diluted in culture medium (IMDM, Gibco, NY, 10% FBS) was added and incubated for 1 hour. hour. After a washing step, damped substrate (Genetic Systems) was added, and the reaction was stopped with interruption reagent (Genetic Systems) after 15 minutes of incubation time. The absorbance was read at an OD of 450/630 nm (Dynatech Microelisa Autoreader). The supernatants of the hybridoma cultures were then added in 100 ml / well, the wells were incubated for 1 hour at room temperature, and the cells were washed three times with PBS. Then, horseradish peroxidase bound to goat anti-mouse IgG / M (Zymed, CA) diluted in 0.1% BSA and PBS was added at a concentration of 100 ml / well. The reaction mixture was incubated either for 1 hour at room temperature, or 30 minutes at 37 ° C, and the cells were then washed 3 times with PBS, then o-phenylenediamine (OPD) was added in 100 ml / well, and the plates were incubated in the dark at room temperature for 5-45 minutes. The antibody that bound to the cells was detected by a color change in the wells, which occurred in 10-20 minutes. The reaction was stopped by adding 100 ml / well of HSS0, and the absorbance was read in a Dynatech (Alexandria, VA) Microelisa Autoreader ™ at 490 nm.

This assay can be performed using live cultured adherent cells, or with live fixed cells in suspension, or fixed purified soluble antigen, and cell extracts such as immobilized antigen. Hybridomas that produced antibodies with binding reactivity to the H3922 line of cancer cells and not human fibroblasts were thus selected, cloned, and the specificity of the subclones confirmed. The culture of hybridomas was then expanded in vitro, to determine the production of antibodies. As described above, the BR110 antibody was purified from the supernatant of the hybridoma by affinity chromatography on immobilized protein A (Repligen, Cambridge, MA). The buffer of the final formulation was PBS, the antibody was sterilized by filtration, and stored refrigerated or frozen at -70 ° C. Hybridomas that were non-reactive with PBL-s were cloned, expanded in vitro, and further tested to determine the specificity of the antibody. Those hybridomas that produced antibodies reactive with human breast cancer were re-cloned, expanded, and injected in 3-month-old BALB / c mice primed with pristane, where they grew as ascites.

Following this procedure, the BR110 hybridoma cell line was obtained, it was cloned and injected into mice, so that it developed as an ascitic tumor.

As described above, the BR110 hybridoma has been deposited with the ATCC. The BR110 monoclonal antibody was purified from ascites by affinity chromatography on immobilized recombinant protein A (Repligen, Cambridge, MA). The clarified ascites was diluted with an equal volume of binding buffer (1 M potassium phosphate, pH 8) and applied to a protein A column previously equilibrated with binding buffer. The column was washed extensively with binding buffer, and then the antibody was eluted with 50 mM phosphoric acid, pH 3. The purified antibody fraction was neutralized with 1 M Tris, pH 9, and then dialyzed against buffered saline with phosphate. The purified ligand of BR110 was finally sterilized by filtration, and stored refrigerated or frozen.

EXAMPLE 2 Characterization of the BR110 monoclonal antibody Determination of the isotype To determine the immunoglobulin class produced by the hybridoma of BR110, the following ELISA technique was used: plates were coated with 96 wells of Dynatech Immulon with antibodies of the anti-IgG subclass. goat mouse (Southern Biotech, Birmingham, AL) at a concentration of 1 mg / ml in PBS, and incubated overnight at 4 ° C. Based on this procedure, it was determined that the BR110 monoclonal antibody is of the gamma isotype 2a. Immunohistology The peroxidase-antiperoxidase (PAP) technique of LA Sternberg was used, as described in Immunochemistry, pages 104-69 (John Wiley &Sons, New York, 1979) and as modified by HJ Garrigues et al., " Detection of A Human Melanoma-Associated Antigen, page 97, in Histological Sections of Primary Human Melanomas, "Int. J. Cancer, 29: 511-15 (1982), for immunohistological studies. The target tissues for these tests were obtained by surgery, and they were frozen within 4 hours of extraction, using isopentane previously cooled in liquid nitrogen.

The tissues were then stored in liquid nitrogen or at -70 ° C until they were used. Frozen sections were prepared, air dried, treated with acetone, and dried again [see Garrigues et al., Supra]. The sections that were to be used for the histological evaluation were stained with hematoxylin. To decrease the non-specific background, the sections were previously incubated with normal human serum diluted 1/5 in PBS (HJ Garrigues et al., "Detection Of A Human Melanoma-Associated Antigen, page 97, In Histological Sections Of Primary Human Melanomas" , Int. J. Cancer, 29: 511-15 (1982). Mouse antibodies were diluted, Rabbit anti-mouse IgG, and mouse PAP in a solution of 10% normal human serum and 3% rabbit serum. The rabbit anti-mouse IgG (Sternberger-Meyer I munochemicals, Inc., Jarettsville, MD) was used at a 1/50 dilution. The PAP PAP complexes (Sternberger-Meyer Immunochemicals, Inc.) containing 2 mg / ml of purified PAP specifically were used in a 1/80 dilution. The staining procedure consisted in treating sections in series with either the specific antibody, ie BR110, or a control antibody for 2.5 hours, incubate the sections for 30 minutes at room temperature with rabbit anti-mouse IgG diluted 1/50, and then exposing the sections to mouse PAP complexes diluted 1/80 for 30 minutes at room temperature. After each antibody treatment, the sections were washed twice in PBS. The immunohistochemical reaction was developed by adding recently prepared 3, 3'-diaminobenzidine tetrahydrochloride 0.5% (Sigma Chemical Co., St. Louis, MO) and 0.01% H202 in 0.05 M Tris buffer, pH 7.6, for 8 minutes (Hellstrom et al., J. Immunol., 127: 157-60 (1981)). Subsequent exposure to 1% 0s04 solution in distilled water for 20 minutes intensified staining. The sections were rinsed with water, dehydrated in alcohol, rinsed in xylene, and mounted on slides. The parallel sections were stained with hematoxylin. The cuts were each evaluated under code, and the coded samples were reviewed by an independent investigator. The typical cuts were photographed using a differential interference contrast optical device (Zeiss-Nomanski). The degree of antibody staining was evaluated as 0 (non-reactivity), + (a few weakly positive cells), ++ (at least one third of positive cells), +++ (most positive cells, ++ ++ (approximately all strongly positive cells) Because the differences between + and 0 staining were less well defined than between staining + and ++, a staining classified as ++ or higher was considered "positive". Neoplastic and stromal cells were observed in tumor samples.The recorded staining is that of the tumor cells, because the stromal cells did not stain, or stained much weaker than the tumor cells Table 1 below demonstrates immunohistological staining of various specimens of tumor and normal tissue using the monoclonal antibody of BR110.As clearly shown in the table, the BR110 antibody reacts with a wide range of human carcinoma specimens. Binding: The recognition of cell surface antigens by BR 110 Mab was identified in a variety of immunofluorescence carcinoma cell lines, using a Coulter Epics C FACS. Aliquots of individual cells were placed in aliquots in culture medium (Gibco IMDM, NY, 10% FBS) in a concentration of 5 x 105 to 1 x 10 b cells. BR110 Mab conjugated with FITC was added to the pellets of resuspended cells, at a concentration of 10 μg / ml diluted in culture medium, followed by a 1 hour incubation on ice. After washing the cells with culture medium, the samples were analyzed to determine the average fluorescence intensity. The binding ratios were calculated by dividing the linear fluorescence equivalent (LFE) by the LFE of the negative control. The results are shown in Table 4. Determination of Antigen binding sites per cell Cell binding assays were performed with BR110 Mab conjugated to FITC, using the H3619 and H2987 cell lines, followed by FACS analysis. The average fluorescence intensity was determined by staining with double antibody dilutions of 50 to 0.38 mg / ml). The fluorescein beads with known size and intensity produced a standard curve, which was included in the computation of fluorescence equivalents through antibody binding. The number of binding sites was determined to be for both cell lines of 8 x 104 sites / cell. E-JEMPLO 3 Internalization of BR110 To determine if BR110 could be internalized by antigen-positive carcinoma cells with indirect inhibition, an assay was applied, using an antibody conjugated to the A chain of ricin. According to this assay, the inhibition of 3H-thymidine incorporation in cellular DNA is a measure of the cytotoxic effect of the given conjugate, and reflects the internalization of the ricin A chain by the carcinoma cells. Carcinoma cells were seeded in a 96-well microtiter plate at 5 x 10J cells / well, followed by overnight incubation at 37 ° C, and 5% C02. The supernatant of the hybridoma (pure and a dilution of 10 potencies) was added to the culture, and incubated for 1 hour on ice. After washing with culture medium, a goat anti-mouse IgG antibody and ricin A chain (Inland Laboratories, Austin, Tx) was added at a concentration of 5 mg / ml, for an incubation time of 42 hours, at 37 ° C, after which 50 μl of 3 H-thymidine was added in 1 mCi / well, and the plate was incubated for another 6 hours at 37 ° C. The test plates were then frozen at -70 ° C for several hours. hours, thawed, and the cells harvested on glass fiber filters (Wallac, Finland) using a cell collector (Wallac, LKB). After adding liquid for scintillation counting, the filter residues were counted with a liquid scintillation counter (Wallac, LKB). The results are expressed as the percentage of inhibition of the incorporation of 3H-thymidine in the experimental group versus a line of untreated control carcinoma cells, H3922 shows 90% and H3396 a 60% growth inhibition after treatment with Mab BR110, the antibody exhibits evidence of internalization. As another approach to study the internalization of BR110, the carcinoma cells were analyzed by confocal microscopy (Leica confocal laser scanning microscope). Carcinoma cells (H3619, H3396, H3922) grown in IMDM / 10% FBS were allowed to adhere on glass slides (NUNC chamber coverslips) for 4-8 hours. The cells were placed at 4 ° C for 15 minutes. Conjugate FITC was added to the BR110 antibody, or FITC conjugated to goat anti-mouse IgG and BR110 antibody (Tago, Burlingname, CA) as a control, at a concentration of μg / ml for 1 hour at 4 ° C. The unbound antibody was removed by extensive washing with cold culture medium. To detect surface staining, the cells were washed with cold PBS, and fixed with 2% paraformaldehyde for 15 minutes at room temperature, followed by a treatment with anti-decolorization reagent of Dithioerythritol (Sigma). To investigate the internalization of BR110 , culture medium was added at 37 ° C, and the cells were incubated for individual time units (time 0-3 hours). Incubation was terminated with cold PBS, and post fixation. The confocal microscope images showed that when the cells were first exposed to BR110 at 4 ° C, the Mab bound exclusively to the cell surface. When the temperature was shifted to 37 ° C and the cells were kept at this temperature for 15 minutes, cytoplasmic staining was observed. The binding of BR110 at that time was irregularly distributed in cytoplasmic compartments, and was largely absent from the cell surface. Neither the cell surface nor the cytoplasm were stained with the control Mab. The data demonstrate that BR110 internalized into carcinoma cells. We have tested Mab GA733 for internalization in the H3396 and SW948 carcinoma cell lines. The antibody bound exclusively to the cell surface at 4 ° C with an extremely weak internalization, or no internalization when tested at 37 ° C. Antibody-dependent and complement-dependent BR110 cytotoxicity ADCC and CDC tests were carried out, labeled target cells (H3396, H3922) with 51 Cr, and exposing them for 4 hours to human lymphocytes or human serum as a complement source and BR110. The release of 51 Cr from the target cells was measured as evidence of tumor cell lysis (cytotoxicity). It was found that BR110 is negative for ADCC and CDC, which can be attributed to its IgG2a isotype. Competition by ELISA Taking into account the linkage of GA733 genes, a competition test was conducted to establish the differences in specificity between Mabs BR110 and GA733.

The FACS analysis has shown that both proteins (GA733-1 and GA733-2) are present in the H3396 cell line. The binding studies on cells fixed with paraformaldehyde showed that these antibodies do not cross-block, and therefore bind to different epitopes.

Determination of Avidity or Affinity Scatchard analyzes were performed to determine the avidity of BR110 Mab radioactively labeled with its binding to non-fixed adherent cells. The data indicated that BR110 has an approximate assocon constant (Ka) of 5 x 10"b moles / liter, determined on cell lines H3619 and H3396 Characterization of BR110 antigen Antigen of BR110 was isolated from line H2987 of cells of antigen-positive carcinoma. The cells were solubilized in 1% Triton X-100, and the antigen-antibody complexes were purified by chromatography on protein A-Sepharose and SDS-PAGE under non-reducing conditions, and recovered by electroelution or electroblotting. The protein of Mr = 66,000 was blocked aminoterminally.A single amino terminal sequence of 20 residues was obtained for the protein of Mr = 55,000, showing a complete identity with the GA733-1 antigen assocd with tumor, residues 88-107. Cyanogen bromide hydrolysis of the BR110 antigen of Mr = 66,000 generated a major fragment, which was purified by SDS-PAGE, recovered and per sequence was identical to the sequence of G5A733-1, starting at residue 63, preceded by a methionine residue. These data confirm that the BR110 antigen is amino-terminally blocked, and fragmentation was required to obtain sequence information. In addition, the data suggest that the glycoprotein of Mr == 55,000 is a natural hydrolysis product of the protein of Mr = 66,000 which is still capable of binding to Mab BR110. The GA733-1 antigen was purified from the human colorectal carcinoma cell line SW948, and its amino acid partial sequence was determined (Linnenbach, A. J. et al., PNAS 86, 27-31, 1989). Two recombinants were isolated from a total human genomic library, and the complete DNA sequence of the Ga733-1 and GA733-2 genes was determined. The GA733-1 antigen, which is recognized by Mab BR110 is closely related to the GA733-2 antigen (ie, the comparison of the partial amino acid sequence [45 a.] shows good homology) which is recognized by Mabs GA733 and C017-1A. Consequently, it appears that BR110 Mab has specificity for a related antigen, but only that it is encoded by a gene that shares homology with exon 8 of the thyroglobulin repeat unit type 1, invariant chain associated with HLA-DR, and the subunit of the IL-2 growth factor receptor. TABLE 1 Immunohistology of mouse Mabs (positive number / number tested) Mab BR110 13-66-6 13-66-7 Carcinomas Lung 25/28 25/28 25/28 Colon 18/28 17/25 17/25 Seno 22/23 19/22 20/22 Ovary 17/18 17/18 17/8 Melanoma 0/9 0/9 0/9 Normal Fabrics Bazo 0/12 0/7 0/7 Liver 0/11 0/11 0/3 Kidney 16/16 (1) 10/10 (1) 12/12 (i; Heart 0/11 0/11 0/11 Large Intestine 0/9 0/11 0/6 Stomach 0/13 0/14 0/14 Esophagus 7/7 (2) 5/5 (2) 5/5 (2) Pancreas 10/10 (3) 9/9 (3) 6/6 (3) Lymphatic Nodule 0/5 0/5 0/5 TABLE 1 (Continued) Mab BR110 13-66-6 13-66-7 Normal Adrenal Tissues 0/7 0/7 0/7 Ovary 0/7 0/7 0/5 Seno 0/10 0/5 0/5 Eye 0/1 0/1 0/1 Bladder N / T N / T N / T Thyroid 0/9 0/5 0/5 Skin 9/9 (4) 6/6 (4) 7/7 (4) Brain 0/10 0/7 0/7 Lung 19/24 (5) 11/12: (5) 14/14 (5) Amygdala 2/2 (2) 2/2 (2) 2/2 (2) Prostate 0/1 0/1 0/1 Uterus 0/5 0/5 0/5 Small intestine 0/2 0/2 0/2 (1) only distal tubule; (2) weak epithelial staining; (3) weak epithelial staining, without islet cells; (4) stained epithelium; (5) weak and diffuse staining.

TABLE 2 Immunohistology (positive number / negative number) Carcinomas Mab BR110 GA733 Lung 7/7 7/7 Colon 4/7 7/7 Sine 12/12 12/12 Ovary 3/4 4/4 Gastric 3/8 8/8 Wound Normal Large Intestine 0/7 7/7 Stomach 0/3 2/3 (weak) Esophagus 6/6 0/6 Pancreas 2/2 2/2 TABLE 3 Immunohistology of Mouse Monoclonal Antibodies Expression of homogeneous and heterogeneous antigens in tumor are staining Positive number / Number tested Homogeneous number Heterogeneous number Carcinoma Mab BR110 13-66-6 13-66-7 Lung 25/28 25/28 25/28 Homo 21 24 22 Hetero 4 1 3 Ovary 17/18 17/18 17/18 12 12 11 5 5 6 Colon 18/28 17/25 17/25 12 9 8 6 8 9 Seno 22/23 19/22 20/22 20 17 17 2 2 3 TABLE 4 RECOGNITION OF THE SURFACE ANTIGEN BY Mab BR110 (DIRECT STAINING METHOD) FACS FITC Union Relationship * -Mabs Cell line Chi L6 BR110 13-66-6 13-66-7 ca. of breast H3396 12 9 13 H3922 18 14 20 H3464 66 1 1 1 MCF-7 * 1.6 15 10.5 15.5 H3630 * 1.7 15 12 18 H3760 * 5 19 14 21 adeno ca. of colon H3347 67 H3719 34 H3619 * 5 H3737 * 2 20 15 21 ca. of lung H2981 13.5 1 1 1 ca. of ovary Table 4 (Continued) FACS FITC Union Relationship * -Mabs Cell Line Chi L6 BR110 13--66-6 13--66-7 H3723B 3 15 6 9 H3907 * 16 37 30 46 H3909 * 12 1 1 1 CASKI * 6 17 13 20 ca. of breast + ovary COS TABLE 5 RECOGNITION OF SURFACE ANTIGEN BY Mab BR110 and Mab 733 (INDIRECT STAINING METHOD) Cell lines BR110 GA733 Binding ratios ca. of lung H2987 35 24 ca. of breast H3922 45 26 ca. of breast H3396 20 52 ca. colorectal SW94Í 1 86 EXAMPLE 4 Internalization of BR110 sFv-PE40 within carcinoma cells Materials and Methods Reagents and Cell Culture - purified BR96sFv-PE40 immunotoxin, used as a control, has been previously described (Friedman et al., 1993). The membrane binding ELISAs were performed using either polyclonal rabbit BR110 anti-idiotypic serum (Bristol Myers Squibb) or EXA2-1H8, a mouse anti-PE monoclonal antibody (Sigma Chemical Co. (St. Louis, MO) The specimen diluent and the conjugate diluent were provided by Genetic Systems (Seattle, WA) The goat anti-mouse Ig (H + L) specific HRP conjugate was purchased from Southern Biotechnology (Birmingham, AL) The porous HQMR resin was purchased from Perceptive Biosystems (Framingham, MA) Restriction and DNA modification enzymes were from New England Biolabs, and [3H] -leucine was purchased from New England (Boston, MA). Other chemical compounds and reagents, including protease inhibitors and glutathione, were purchased from Sigma Chemical Co. (St. Louis, MO). Colon adenocarcinoma H3619, lung carcinoma H3754 and carcinomas of ovary H3907 were cultured in Dulbecco's medium modified by Iscoves (IDMEM) with 10% fetal calf serum. The MCF-7 breast carcinoma cell line was obtained from the ATCC, and cultured as described above. The BR110 monoclonal antibody (mAb) was developed by immunization of mice with the H3922 line of human breast carcinoma cells, according to established procedures. Determination of the Amino Terminal Sequence - approximately 10 mmoles of muBRUO IgG, purified from a continuous culture of hybridoma cells, were separated by 15% SDS-PAGE, and the heavy and light chains were isolated for NH2- sequence analysis terminal, using a Protein Sequence Determinator from Applied Biosystems 477 and a 120 PTH Analyzer from Applied Biosystem (Hewick et al., 1981; Matsudaira, P., 1987). Twenty-five cycles of Edman degradation were performed. However, only the amino terminal sequence of the light chain was obtained, since it was thought that the heavy chain was N-terminally blocked. RNA Isolation, cDNA Synthesis, and Amplification - RNA was prepared from 5 x 10 7 muBRUO hybridoma cells, as previously described (P. Chomezynski and N. Sacchi, 1987). The cDNA was obtained using total RNA and the thermostable rt polymerase (rTH-RT-RNA-PCRMR kit, Perkin Elmer Cetus) according to the manufacturer's instructions.

For the amplification of the heavy chain Fv gene we use the pair of primers, MVH P9 (MVH = Mouse Variable Weighing Region; 5'-ACTACACGGTACCCGGGATCCATGGc / ATTGGAA / cCTGCTATTCCTG-3 ') (Jones and Bendig 1991) and the constant primer 3 'VH (5'-N6GAATTCAT / CC-TCCACACACAGGA / GA / GCCAGTGGATAGAC-3') (Larrick et al., 1991). The 5 'primer for the variable light chain gene was designed according to the N-terminal amino acid sequence derived from the Edman degradation and alignment of the database to other related light chain mouse genes. The 5 'primer was (5'-CGATCCGAATTCGACATTGTGATGACCCAGTCTCA-3'), while the 3 'constant primer VL was mck-3 (5'-N4GAATTCCAAGAAGCACACGACTGAGGCA-3') (Gilland et al., 1991). The recognition sequences for Kpn 1 and EcoR I are underlined. Cloning Amplified cDNA - The products of heavy and light chain Fv amplification encoding DNA fragments were identified by agarose gel electrophoresis, and migrated with an apparent molecular size of 475 and 375 base pairs respectively. At their 5 'and 3' ends are the restriction endonuclease recognition sites Kpnl and EcoRI to clone the PCR products into pBluescript SK (+) (Stratagene, La Jolla, CA). The sequence of the nucleotides was determined for multiple clones of BR110 heavy and light chain gene segments, using double-stranded plasmid DNA as a template, and the MR kit of Sequenasa 2.0 reagents (United States Biochemical, Cleveland, OH) with primers of sequence determination T7 and T3 (Pharmacia, Piscataway, NJ). Construction of Expression Plasmid BR110 sFv x PE40 - The expression plasmid used for the production of BR110sFvxPE40 in E. coli uses the bacteriophage T7 RNA polymerase promoter (Studier and Moffat, 1986). The BR110 V regions were amplified by PCR, to modify their terminal restriction sites using a 5 'VH PCR primer (5' -GCA ATG CATATGCAG ATC CAG TTG GTG CAG TCT GGA C-3 ') which encoded the site of Nde I restriction, underlined, which includes an ATG translation start code (thick letter) and the first eight codons of the VH gene. The 3 'VL PCR primer (5' -CCA TGG ATGCAT CCG TTT CAG CTC CAG CGT GGT CCC AGC-3 '), which encoded the Nsi I restriction site (underlined) and the last nine codons of the Vx gene.

We also insert a flexible linker, (Gly4Ser) 3 between the two domains of V (Huston et al., 1988; Chaudhary et al., 1989) by synthesizing the 3 'VH PCR primer (5' -CCG GCC GGA TCC GCC TCC GCC TGA TGA GGA GAC GGT GAC CGT GGT CCC T-3 ') which was designed to have the restriction site Bam H I (underlined), the first half of the linker (thick letter) and the last eight codons of the VH gene. The 5 'VL PCR primer (5' -CCG GCC GGA TCC GGC GGT GGC GGT TCT GGC GGT GGC GGT TCT GAC ATT GTG ATG ACC CAG TCT CAC-3 '), encodes the Bam HI restriction site (underlined ), the second half of the linker (thick letter) and the first eight codons of the VL gene. The restriction site of Bam H I was used to ligate the two PCR products together, and then re-amplified via PCR, using the 5 'VH and 3' VL primers (Sambrook et al., 1989). The BR110 sFv was then replaced by the BR96 sFv (McAndrew, 1995), in pBW7.0, a vector based on pMS8 (+), by digestion with Ndel + Nsi I followed by ligation. The resulting expression plasmid directs the fusion protein pBR110sFv-PE40 to the cytoplasm, and was confirmed by DNA sequence analysis. Expression and purification of BR110 sFv-PE40 - The single-chain immunotoxin fusion protein BR110sFv-PE40 was expressed in E. coli as described above. Cells of E. coli BL21 (IDE3) (Studier &Moffat, 1986) were transformed with the expression plasmid pBR110sFv-PE40, cultured in "Terrific-Broth" medium (Gibco-BRL, Gaithersburg, MD) containing 100 μg / ml of ampicillin at 37 ° C, and were induced with 1 mTGT IPTG in the logarithmic phase, at an OD650 of 1.0. The cells were harvested ny minutes later. For the analytical examination, samples of 1 ml were harvested by centrifugation, and subjected to osmotic shock in cold H20 for 10 minutes. The cells were again centrifuged, and the cell pellets were resuspended in 10 mM Tris-HCl, pH 7.4. Aliquots prepared from approximately 108 spheroplasts were subjected to SDS-PAGE, and sta (Laemmli, 1970) or subjected to analysis by immunoblotting, using the polyclonal rabbit anti-BRUO idiotypic serum. A pellet of bulk bacterial cells prepared from a stirred 6-liter flask was processed as described above, and the inclusion bodies were isolated as previously described (Friedman et al., 1993). The extensively washed inclusion bodies were then denatured in guanid7 M. Cloning, Expression and Antigen Purification - The antigen of BR110 (GA733-1) was isolated from H2987 cells, and the amino terminal sequence of 20 residues was obta. The residues were complementary to residues 88-107 of the GA733-1 antigen associated with tumors. GA733-1 is an isolated genetic sequence based on its homology to GA733-2, which encodes the gastrointestinal-associated antigen recognized by Mabs GA73.3 and C017-1A. GA733-1 is 50% homologous to the GA733-2 antigen. Northern analysis was performed on three carcinoma cell l, H3907 ovarian carcinoma, H3619 colon adenocarcinoma and H3754 lung carcinoma, which had demonstrated the BR110 GA733-1 antigen via FACS analysis. RNA was isolated from 20-30 million cells of each, using the method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987 (Anal. Biochem. 162: 156-159)). An oligonucleotide probe was made based on the known oligonucleotide sequence of GA733-1. Northern analysis was conducted as described in Mantianis. The cDNA of the antigen was isolated using sequence-specific primers, total RNA and reverse transcriptase polymerase (RT-RNA PCR, Perkin Elmer Cetus) according to the manufacturer's instructions, except for the addition of 10% DMSO, used because the GA733-1 antigen is more than 50% GC rich. The specific RT primers used were 110-FP (5 '-GCC AAG CTT GTC CGG TCC GCG TCC GCG TTC CTC CGC CCC ACC-3 '), 110-RP (5' -CGG TCT AGA CTC GAG GCC GGG TAC CTA CAA GCT CGG TTC-3 '), 110-RP1 (5 '-GCG GGG ATC CGT GAG GCG CTT CAT GGA GAA CTT CGG-3'). The restriction site for each primer is underlined, and they are Hind III, Xho I, and Bam Hl respectively. Subsequent to reverse transcription with either Random Hexanucleotides provided with the kit, or the 3'-110-RPl primer, 32 cycles of PCR were conducted, with appropriate sized products recovered. The 1.15 kB gene fragments were digested with Hind III and Bam Hl before being ligated into pCD40ThrRgI (in a CDM7 Background) while the 0.86 kB gene fragments were ligated as HindIII-XhoI fragments in pCDM8. Plasmid DNAs containing the appropriate sized inserts were identified and subjected to a DNA sequence analysis. The DNA fragment encoding the extracellular domain was subcloned upstream of the human Fc region, and transiently expressed in COS cells. The resulting fusion protein was purified by affinity chromatography on protein A. Cell Membrane ELISA - H3619 colon adenocarcinoma cell membranes were prepared (Spira et al., "The Identification of Monoclonal Class Switch Variants by Subselection and ELISA Assay" J. Immunol., Meth., 74: 307-15 (1984) Uotila et al., "Two-Site Sandwich ELISA With Monoclonal Antibodies To Human AFP", J. Immunol. Methods, 42: 11 (1981), Sikora et al. (eds.), Monoclonal Antibodies, pages 32-52 (Blackwell Scientific Publications, (1984)).

Membranes were deposited on the surface of 96 well plates with Immulon IIMR (Dynatech Labs., Chantilly, VA) in 10 μg / ml protein in sodium carbonate / 0.5 M sodium bicarbonate, pH 9.6, and incubated all the overnight at 4 ° C. Plates were washed three times with PBS, blocked with specimen diluent at 1:10 (Genetic Systems, Seattle, WA) for 1 hour at RT The plates were washed three times with PBS, and then incubated with primary antibody overnight at 4 ° C in conjugate diluent (Genetic Systems, Seattle, WA). The plates were then washed three times with PBS, followed by the addition of secondary antibody, either goat anti-mouse IgG HRP or polyclonal rabbit BR110 anti-idiotypic antibodies, in 2.5 μg / ml in conjugate diluent. The plates were incubated for 1 hour at T.A., and then washed three times with PBS. The plates were washed three times with PBS, then incubated either 1 hour at T.A. with specific HRP of goat anti-rabbit IgG Fc in 1: 1000 in conjugate diluent, or washed two additional times, after which the reaction was developed with tetramethyl benzidine (TMB) for 15 minutes at RT, and stopped with H2SO4 1.3 M. After incubation, the plates with polyclonal serum were also washed five times in PBS, and treated as described above. Absorbance was quantified at a dual wavelength 450/630 nm, using a Bio-Tek microplate reader (Winooski, VT). Cytotoxicity analysis. The cells were incubated with BR110 sFv-PE40 for 24 hours at 37 ° C. Wells were then washed twice with phosphate buffered saline (PBS), 200 μl / well of calcein-AM 1.5 μM were added.

(Molecular Probes, Inc., Eugene, OR), and the plates were incubated for 40 minutes at T.A. Calcein-AM is permeable to the membrane, and not fluorescent. When hydrolysis occurs by intracellular esterases, the intensely fluorescent calcein product is formed. At the end of the incubation, the fluorescence was measured using a Fluorescence Concentration Analyzer ™ (Baxter Heathcare Corp., Mundelein, IL) at the excitation / emission wavelengths of 485/530 nm. EXAMPLE 5 Construction of the BR110 sFv-PE40 Immunotoxin (pSE70.0) The original BR110 sFv-PE40 construct was subcloned into a vector containing the kanamycin resistance gene and a second lac repressor constructed in the expression vector. This was done by digesting pET29c (Novagen Inc.) with EcoRI and Xbal, and inserting a similarly digested BR110 sFv-PE40 between the two sites. The resulting construct was called pSE 70.0 (Figure 1), and was under the control of the T7 promoter. Expression, Refolding and Purification of BR110 sFv-PE40 The BR110 sFv-PE40 single chain immunotoxin fusion protein was expressed in E. coli as inclusion bodies. Cell paste of 1 liter of start culture, containing the expressed fusion protein BR110 sFv-PE40, was resuspended in 50 mM Tris-HCl, pH 8.0 + 1 mM EDTA. The sample was subjected to centrifugation at 8 K for 15 minutes, and the supernatant was decanted. The pellet was resuspended in 40 ml of H20, kept at 4 ° C for 10 minutes, and subjected to centrifugation at 8 K for 15 minutes. The pellet was resuspended in 50 mM Tris-HCl, pH 8.0 + 1 mM EDTA, and 0.5 mg / ml lysozyme was added to a final concentration of 0.25 mg / ml. The sample was kept on ice for 30 minutes. The detergent Tergitol was then added to the sample, at a final concentration of 4%. The sample was kept on ice for 60 minutes, and subjected to centrifugation at 18 K for 30 minutes. The sample was washed three times with 50 mM Tris-HCl + 1 mM EDTA, The pellet was sonicated in 3 ml of guanidine solution (7 M guanidine-HCl, 0.1 M Tris-HCl, pH 7.4, 5 mM EDTA) , and left at 4 ° C for 48 hours. The sample was then subjected to centrifugation at 30 K for 30 minutes, and the supernatant was collected. The sample was then refolded at 100 μg / ml, by slow addition of refolding buffer (0.1 M Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 mM GSSH, 1 μM GSH, 1 M urea). The refolded protein was maintained at 4 ° C for 48 hours, and dialyzed against 40 mM HCl of pH 8.0 until the conductivity was below 5 mS / cm. The sample was loaded onto an anionic exchange column of DEAE, and eluted with a linear gradient of 40 mM Tris-HCl, pH 8.0 + 1M NaCl. The fractions corresponding to a fusion protein of BR110 sFv-PE40 were collected. 67 kDa by SDS-PAGE, were diluted in 250 ml of 40 mM Tris-HCl, pH 8.0, and reapplied to an Mono-Q anion exchange column with the same NaCl gradient as for the DEAE column . The fractions corresponding to the monomer of BR110 sFv-PE40 were collected and quantified by the Coomasie-Plus assay (Pierce Chem. Co.). Construction and Purification of the BR110-BD1 Immunotoxin BR110 Mab was thiollated by addition of a triple molar excess of 2-iminothiolane in 0.2 M sodium phosphate buffer solution (pH 8.0), 1 mM EDTA for 1 hour at 37 ° C. A derivative of BD1 with a triple molar excess of SMPT (4-succinimidoyloxycarbonyl-α-methyl-α- (2-pyridylthio) -toluene) was prepared in 0.2 M sodium phosphate buffer (pH 8.0), 1 mM EDTA, at room temperature for 60 minutes. BR110-BD1 was applied to a size exclusion column (TSK-3000) and separated from the free toxin. The immunotoxin (180 kDa) and the free antibody (150 kDa) eluted together, and were further purified by affinity chromatography on Blue-Sepharose. BR110-BD1 was adsorbed on Blue-Sepharose (5 ml resin / 5 mg conjugate) in 0.1 M sodium phosphate, pH 7.0 (wash buffer) for 16 hours at 4 ° C. The mixture was packed in an Econocolumn of 5 ml (Bio-Rad, Richmond, CA), and fractions of 1 ml were eluted with a 2-step gradient of increasing concentrations of NaCl in wash buffer (400 mM NaCl, step 1; 800 mM NaCl, step 2). The immunotoxin conjugate was quantified at a D02 C (1.4 = 1 mg / ml) and analyzed by non-reducing SDS-PAGE. Cytotoxicity Analysis of BR110-Immunotoxin Tumor cells (10 5 cells / ml) in growth medium were added to plates for 96-well flat bottom tissue culture (0.1 ml / well), and incubated at 37 ° C for 16 days. hours. Dilutions of BR110 sFv-PE40 or BR110-BD1 were made in leucine-free RPMI (test medium), and 0.1 ml was added to each well for 20 hours at 37 ° C (for BR110 sFv-PE40) or for 20 and 44 hours (for BR110-BD1). Cells were pulsed with [3 H] -leucine (1 μCi / well) for an additional 4 hours at 37 ° C. Cells were used by freeze-thaw, and harvested using a Tomtec cell harvester (Orange, CT). The incorporation of [JH] -leucine into the cellular protein was determined using a liquid scintillation counter LKB Beta-Plate. An excess of BR110 IgG (50 μg / ml) was added to the 48 hour BR110-BD1 cytotoxicity study to demonstrate the specificity of BR110 in the assay. Except for the addition of BR110 IgG, the assay was done exactly as described above. TABLE 6 BR110 sFv-PE40 at 24 hours. Cytotoxicity and FACS analysis on selected cell lines BR110 cell line sFv-PE40 EC5n FACS of BR110 binding ratio H2987 0.7 ng / ml 18.28 MCF-7 2.85 ng / ml 13.17 H3396 9 ng / ml 17.1 H2981 > 1 μg / ml 1 TABLE 7 Activity of the Conjugate of BR110 IgG-SMPT-BDl on Different Lines of Carcinoma Cells. Incubation of 24 and 48 hours. 48 hr FACS line 24 hr 48 hr + BR110 Cellular (BR110) EC; .o (μg / ml) ECso (μg / ml) (50 μg / ml) H3619 33 0.07 0.06 2 H2987 20 0.1 0.001 4 MCF-7 15 5 0.05 > 5 H3396 12 nd 0.09 > 5 H2981 1 > 5 > 5 > 5 nd = not done INDICATIONS RELATED TO A DEPOSITED AGENCY (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description throughout the text on the page, line B. IDENTIFICATION OF THE DEPOSIT Additional deposits are identified on an additional sheet D Name of the institution deposited AMERICAN TYPE CULTURE COLLECTION Address of the depository institution (including code and country) 12301 Parklawn Drive Rockville, MD 10852 United States of America Date of deposit: August 10, 1994 Accession Number: HB 11698 C. ADDITIONAL INDICATIONS (leave blank if it is not applicable) This information continues on an additional sheet D D. DESIGNATED STATEMENTS FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated states) E. SEPARATE SUPPLY OF INDICATIONS (leave blank if not applicable) The indications listed below will be sent to the International Office plus late (specifying the general nature of the indications, for example "Deposit Access Number") For the use of the receiving Office only 3D This sheet was received with the international application Officially authorized: G. Hill (SIGNED) For use by the International Office only D This sheet was received by the International Bureau on the date: Authorized Official: It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (32)

1. A monoclonal antibody that binds to the GA733-1 antigen, the antibody is characterized in that it has an antigen-binding site that competitively inhibits the binding of the BR110 monoclonal antibody produced by a hybridoma designated ATCC No. HB11698 to the GA733-1 antigen. The antibody according to claim 1, characterized in that the antibody preferably binds to tumorigenic tissue of a cell lineage, but does not bind to normal tissue of the same lineage. 3. The antibody according to claim 1, characterized in that the antibody forms a complex with the GA733-1 antigen, and is internalized in a cell when the GA733-1 antigen is located on the cell surface. The antibody according to claim 1, characterized in that the antibody binds to the colon carcinoma tissue, but does not bind to normal intestinal tissue. 5. The antibody according to claim 1, characterized in that the antibody binds to breast carcinoma tissue, but does not bind to normal breast tissue. 6. The antibody according to claim 1, characterized in that the antibody does not bind to normal liver, breast or thyroid tissue. 7. A hybridoma producing the monoclonal antibody according to any of claims 1 to 6. 8. A hybridoma, characterized in that it is designated ATCC No. HB11698. 9. A conjugate characterized in that it comprises the antibody according to claim 1, linked to at least a portion of a cytotoxic agent, the portion includes at least one functionally active segment. 10. The conjugate according to claim 9, characterized in that the cytotoxic agent is an enzyme, lymphokine, oncostatin or toxin. 11. The monoclonal antibody according to claim 1, characterized in that it is labeled with a detectable marker. The monoclonal antibody according to claim 11, characterized in that the detectable label is an enzyme, a paramagnetic isotope, biotin, a fluorophore, a chromophore, a heavy metal, or a radioisotope. 13. An Fv molecule characterized in that it has the antigen binding site of the monoclonal antibody according to claim 1. 14. The Fv molecule according to claim 13, characterized in that it is an Fv molecule. 15. A Fab molecule characterized in that it has the antigen binding site of the monoclonal antibody according to claim 1. 16. A Fab 'molecule characterized in that it has the antigen binding site of the monoclonal antibody according to claim 1. 17. An F (ab ') 2 molecule characterized in that it has the antigen binding site of the monoclonal antibody according to claim 1. 18. A bispecific antibody characterized in that it has a binding specificity for two different antigens, one of the antigens are those with which the monoclonal antibody according to claim 1 is linked. 19. A recombinant human / murine antibody, characterized in that the antigen binding region from which competitively inhibits the immunospecific binding of the BR110 monoclonal antibody produced. by hybridoma HB 11698 to its target antigen. 20. A method for detecting the antigen recognized by BR110 in a tissue section of a subject, characterized in that it comprises contacting the tissue section with the monoclonal antibody according to claim 1 under conditions such that the antibody binds to the tissue section, and detect the antibody bound to the tissue section, and thereby detect the antigen recognized by BR110 in the tissue section. The method according to claim 20, characterized in that the tissue section is a tissue in which the normal tissue is characterized by the absence of the antigen recognized by BR110, and the neoplastic tissue is characterized by the presence of the antigen recognized by BR110. 2

2. The method according to claim 21, characterized in that the tissue is an adenocarcinoma, a gastrointestinal tissue, a breast tissue, a colon tissue or a lung tissue. The method according to claim 21, characterized in that the detection comprises contacting the monoclonal antibody with a second antibody that is labeled with a detectable label. 24. A method for determining a difference in the amount of distribution of the antigen recognized by BR110 in tissue sections of a neoplastic tissue to be tested relative to the amount and distribution of the antigen recognized by BR110 in tissue sections of a normal tissue . 25. Use of the monoclonal antibody according to any of claims 10-11, characterized in that it is given in a method for diagnosing a neoplastic condition in a subject. 26. A composition characterized in that it comprises a monoclonal antibody according to any of claims 1 to 6, to which a detectable agent binds. The composition according to claim 26, characterized in that the detectable agent comprises a toxin selected from a group consisting of ricin, diphtheria toxin, bryodin, pseudomonas, exotoxin A, abrin, supporin, and gelonin. 28. The composition according to claim 26, characterized in that the detectable agent comprises an enzyme, a drug, or a DNA fragment. 29. A nucleic acid molecule, characterized in that it encodes the antibody according to any of claims 1 to 6. 30. A plasmid characterized in that it comprises the nucleic acid molecule according to claim 29. 31. A host vector system , characterized in that it comprises a plasmid according to claim 30, in a suitable host cell. 32. A method for producing a protein, characterized in that it comprises culturing the host vector system according to claim 31, for producing the protein in the host, and recovering the protein produced in this manner.