MONOCLONAL ANTIBODY SPECIFIC TO A NOVEL MUCIN-LIKE GLYCOPROTEIN SURFACE ANTIGEN ON HUMAN CARCINOMA CELLS

Abstract: A monoclonal antibody specific to a unique antigen determinant on the surface of human breast carcinoma cells but which does not bind to normal human breast tissue. The antigen is characterized as a high molecular weight mucin-like glycoprotein complex having a molecular weight exceeding 400,000 daltons. Further, the monoclonal antibody is capable of being used in the diagnostic imaging and therapeutic treatment of breast cancer in humans.

Description: MONOCLONAL ANTIBODY SPECIFIC TO A NOVEL MUCIN-LIKE GLYCOPROTEIN SURFACE ANTIGEN ON HUMAN CARCINOMA CELLS TECHNICAL FIELD This invention relates to monoclonal antibodies which bind to surface antigens of human carcinoma and more particularly, relates to a monoclonal antibody which demonstrates activity with a mucin-like glycoprotein complex of very high molecular weight on the surface of breast carcinomas and some carcinomas of other tissues, but which does not bind to normal human breast epithelial cells. BACKGROUND ART Monoclonal antibodies have been developed that recognize a high molecular weight mucin-like glycoprotein complex present on the surface of normal human breast epithelial cells. Peterson et al., Im- perial Cancer Research Fund, London, England, March 2-3 (1981); Taylor-Papadimitriou et al. , Int. J. Cancer, 28:17-21 (1981); Ceriani et al., Somatic Cell Genetics, 9:415-427 (1983). Other investigators have developed monoclonal antibodies using both the human milk fat globule as the immunizing agent [Taylor-Papadimitriou et al., Int. J. Cancer, 28:17-21 (1981); Ceriani et al., Somatic Cell Genetics, 9:415-427 (1983)] and dif¬ ferent breast tumor cells as the immunizing agents [Papsidero et al., Cancer Research, 43:1741-1747 (1983); Kufe et al., Hybridoma, 3:223-232 (1984); Frankel et al., J. Biol. Response Mod., 4:273-286 (1985); Colcher et al., Proc. Natl. Acad. Sci. USA, 78:3199-3203 (1981); Foster et al., Virchows Arch, 394:279-293 (1982); Ellis et al., Histopathology, 8:501-516 (1984); Ashall et al., Lancet, 2:1-11 (1982)] which were determined to recognize a mucin-like glycoprotein also. These monoclonal antibodies were found to recognize such mucin-like glycoproteins that vary in mass from approximately 250,000 daltons to over one million daltons, depending on the immunogen prepa- ration. Shimizu et al., Biochem J. , 233:725-730 (1986). In biochemical studies of the large molecular weight mucin-like glycoproteins of the human milk fat globule membrane, it was suggested that these glycoproteins are complexes consisting of at least three distinct components which may represent at least three distinct molecular entities. Shimizu et al., Biochem J., 233:725-730 (1986). Thus, the mucin-like glycoproteins of the human milk fat globule membrane have been shown to be large molecular complexes which, due to their size, can be expected to have a myriad of epitopes. Monoclonal antibodies have been developed which bind to mucin-like glycoproteins on the surface of normal breast epithelial cells and malignant breast cells such as the Mc5 monoclonal antibody described in Ceriani et al., Somatic Cell Genetics, 9:415-427 (1983). Additionally, a monoclonal antibody D-274, specific to guinea pig milk fat globule membrane, was used to determine the distribution of mucin-like glycoproteins of greater than 400,000 daltons in both benign fibrocystic disease and infiltrating duct car¬ cinoma of the human breast. Greenwalt et al., Am. J. Pathol., 118:351-359 (1985). Prior art monoclonal antibodies have been devel¬ oped that will bind to normal breast cells, to breast carcinomas, as well as, to some epithelial cells of other tissues. However, these monoclonal antibodies exhibit binding specificities for normal breast tissue as well as carcinoma of the human breast. It would be highly advantageous to provide a monoclonal antibody which will bind to a unique epitope that is expressed on the surface of breast carcinomas and some carcinomas of other tissues but is not expressed on normal human breast epithelial cells. DISCLOSURE OF THE INVENTION A monoclonal antibody which binds to a novel mucin- l ike glycoprotein antigen on human breast car- cinoma cells but which does not bind normal human breast epithelial cells. The monoclonal antibody recognizes adenocarcinoma cells of the breast, ovary, endometrium, lung, and pancreas and will not bind normal epithelial cells of the heart, gastrointestinal tract, pituitary, prostate, mesenchymal tissues, liver, parathyroid, breast, spleen, thyroid, testis, or ovary. The monoclonal antibody does not bind to adenomas generally. The antigen recognized by the monoclonal antibody is termed "BrEl" and is characterized as having a high molecular weight which may exceed 400,000 daltons. The specificity of the BrEl monoclonal antibody enables ad¬ vantageous diagnostic and possible therapeutic applica- tions in the treatment of breast carcinomas, especially in view of the recognized great incidence of breast cancer throughout the world. The BrEl monoclonal antibody was developed using normal delipidated human fat globule as the immunizing agent. BEST MODE FOR CARRYING OUT THE INVENTION Development of the monoclonal antibody embodying the invention utilized standard procedures generally described by Kohler and Milstein, Nature, 256:495-497 (1975). Immunization of the host animal was done with whole delipidated human milk fat globule (HMFG) prepared as described in Ceriani et al., Proc. Natl. Acad. Sci. USA, 74:582-586 (1977). The host animals were Balb/c mice and after a suitable period of incuba- tion, the murine spleen cells were harvested. The harvested spleen cells thereafter were fused with P3-NSl/I-Ag4-l mouse myeloma using well known polyethylene glycol techniques. The screening for identifying the hybridoma or hybrid cell line which produced the monoclonal antibody of the invention was done using both a solid phase radioimmuno-plate binding assay and an ELISA assay using the HMFG and components of HMFG in wells of a microtiter plate as described in Ceriani et al., Somatic Cell Genetics, 9:415-427 (1983). Thereafter, the wells positive for HMFG were screened on cell lines from human breast carcinoma tis¬ sue, MCF-7 [Soule et al., J. Natl. Cancer Inst., 51:1409-1413 (1973)] and other cell lines from cervical carcinoma, colon carcinoma and a lymphoma, Bris. 8. A monoclonal antibody was identified which stained only the human breast carcinoma cell line and the hybrid cell which produced that antibody was isolated. This monoclonal antibody is identified herein as anti-BrEl. The molecular weight or Mr of the antigen identified by the BrEl monoclonal antibody was determined by Western Blot test as described by To bin et al., PNAS, 76:4350- 4354 (1979) and solid-phase binding assay as described by Ceriani et al., Monoclonal Antibodies and Functional Cell Lines, Plenum Press, New York, 398-402 (1984). In the Western blot test, the HMFG was separated by a 7% polyacrylamide gel electrophoresis and then electroblotted to nitrocellulose paper. The nitrocel- lulose was cut into strips and the strips incubated. High molecular weight standards were simultaneously run on parallel sections of the gel. In the solid-phase binding assay, HMFG was elec- trophoresed on 7% polyacrylamide gel as described by Laemmli VK, Nature, 227:680 (1970). The gel lane was sliced into fractions, the slices eluted, and the eluate dried onto microtiter plates. Binding of monoclonal antibody was tested by a radioimmunobinding assay technique. When the HMFG was electrophoresed, in both the Western blot test and the solid-phase binding assay, the monoclonal antibody BrEl identified a material found only at the origin of the polyacrylamide gel. Thus, the mucin-like glycoprotein antigen identi¬ fied by the monoclonal antibody BrEl remained at the origin and did not penetrate the polyacrylamide gel. The HMFG then was electrophoresed on less than 7% polyacrylamide gel using the solid-phase binding assay previously described. The molecular weight of the mucin-like glycoprotein antigen of HMFG identified by the monoclonal antibody BrEl was then calculated using high molecular weight standards. The molecular weight of this antigen was estimated to exceed 400,000 daltons. Studies were conducted between the BrEl and Mc5 monoclonal antibodies to study the competition be¬ tween them for the glycoprotein antigen. The monoclonal antibody BrEl was found not to compete for binding with the same epitope to which the Mc5 antibody bound. In assays of body fluids which contained breast carcinoma cells or molecules using the BrEl monoclonal antibody, we determined that there occurred binding also to a glycoprotein molecular entity which exhibited a molecular weight of less than 400,000 daltons. It is postulated that the high molecular weight mucin-like glycoprotein antigen to which the BrEl monoclonal antibody bound had become denatured in the body fluid such as to fragmentize. It appeared that the BrEl monoclonal antibody recognized a common epitope on the high molecular weight antigen and the molecular entity or fragment which would explain the phenomenon. Thus, although the BrEl monoclonal antibody binds specifical¬ ly to the high molecular weight antigen exceeding 400,000 daltons, it can bind to a common epitope of the antigen found on such a molecular entity. This is pos¬ sible since the high molecular weight antigen appears to be a complex of molecular entities. The BrEl monoclonal antigen isotype was determined using a mouse immunoglobulin kit to be IgG2a. The BrEl monoclonal antibody was tested extensive¬ ly for binding to histological sections in order to further characterize its tissue specificity. Standard immunoperoxidase assay procedures were used for binding to histological sections of normal and cancerous human tissue. The tissues were prepared in the form of multi-tumor tissue blocks as described by Battifora H., Lab Invest., 55:244-248 (1986). Each tissue block was prepared from a collection of strips of fixed tissues by wrapping the tissues in peritoneal membrane or in- testine and embedding in paraffin. The blocks were then sliced in preparation for the binding studies. In this manner, a large number of different tissues were assessed with a single staining. The breast block used to characterize the BrEl monoclonal antibody contained 21 different specimens from normal breast, 22 adenomas, and 33 breast car¬ cinomas. The BrEl monoclonal antibody bound to the breast carcinomas but did not bind to any of the normal breast sections that were tested, nor did it bind to the adenomas. This binding occurred in spite of the fact that the source of the immunogen, HMFG, was derived from normal breast epithelial cell membrane. This observation may indicate that a transformed phenotype, or an early precursor, that is expressed in the breast carcinoma already existed in the immunizing HMFG. The BrEl monoclonal antibody was also tested on a large panel of different normal tissues and tumor tis- sues other than breast carcinoma. In contrast to the characterization of BrEl using the breast block, the BrEl monoclonal antibody bound to normal lung tissue and a majority of lung carcinomas. Also, it bound equally to ovarian and breast carcinomas, but it did not bind to normal ovary or breast. The BrEl monoclonal antibody did not stain the following tumors: nasopharyngeal, melanoma, neuroblastoma, prostate, parathyroid, peritoneum, spleen, kidney, thyroid, or embryonal. Further, the major non-breast tumors it did stain were adenocar- cinomas of the ovary, endometrium, lung, and pancreas. A sample of the hybrid cell capable of producing BrEl monoclonal antibodies is on deposit with the Amer¬ ican Type Culture Collection, Rockville, Maryland 20852, as of June 9, 1988, and is assigned A.T.C.C. No. HB 9738. The BrEl monoclonal antibody is unique because of its exceptional specificity for a mucin-like glycoprotein complex of very high molecular weight present on the surface of breast carcinomas and which expresses no specificity for normal breast epithelial cells. Consequently, the BrEl antibody can be espe¬ cially useful in the diagnosis of breast cancer in hu- mans. For example, the monoclonal antibody can be tagged with a detectable label, such as a dye, fluores¬ cent molecules, or a radioactive tracer for tumor imag¬ ing. A suitable tracer would be Indium 111, Technetium 99 or Iodine 131. Further, the BrEl monoclonal antibody may be used for therapeutic applications where conjugated to a toxin, for instance, which can lyse tumor cells to which the BrEl monoclonal antibody binds and yet not impact on normal breast epithelial tissue cells. The same procedure would be feasible using a suitable radioactive label such as Indium 111, Tech¬ netium 99 or Iodine 131. Immunoassay in which microspheres are utilized in conjunction with antigens or antibodies coated thereon and suitably tagged or labeled, can be employed for in vitro diagnostic applications with the BrEl monoclonal antibody. The labels or tags may be varied as dis¬ cussed herein and known in the art. For in vitro ap¬ plications, the BrEl antibody may be provided in an as¬ say kit accompanied by other ingredients for completing the assay of a biological sample according to assay in¬ structions in insert literature, for instance. The same may be feasible for in vivo applications, both for diagnostic and therapeutic uses. The assay may be used with flow cytometric procedures to study cell differen- tiation and cell-type specificity. It may also be used as a prognostic tool in the histopathology of tumors. These examples of in vitro and in vivo applications should not be deemed to exclude other applications of the use of the BrEl monoclonal antibody.

Claims: CLAIMS 1. A cell line developed by hybridoma technique which produces a monoclonal antibody to an antigen of human breast carcinoma cells but which does not bind to normal human breast tissue cells. 2. The cell line according to claim 1 in which said antigen is expressed on a mucin-like glycoprotein molecular complex of human breast carcinoma cells. 3. The cell line according to claim 2 in which said antigen is characterized as the BrEl antigen hav¬ ing a molecular weight exceeding 400,000 daltons and produces mouse IgG2a isotype antibody to BrEl antigen. 4. The cell line according to claim 3 in which said antigen also expresses an epitope on a molecular entity thereof which has a molecular weight less than 400,000 daltons to which said monoclonal antibody can bind. 5. The cell line according to claim 2 in which said antigen is characterized as the BrEl antigen hav- ing a molecular weight exceeding 400,000 daltons and which expresses an epitope on a molecular entity there¬ of which has a molecular weight less than 400,000 daltons to which the monoclonal antibody also binds. 6. The cell line according to claim 1 wherein said monoclonal antibody producing cells are derived from mice immunized with normal delipidated human milk fat globule. 7. The cell line according to claim 1 wherein said monoclonal antibody producing cells are mouse spleen cells immunized with delipidated human milk fat globule. 8. The cell line according to claim 7 in which said mouse spleen cells were immunized with normal whole human milk fat globule membrane. 9. The cell line according to claim 7 in which said mouse spleen cells were immunized with normal hu¬ man milk fat globule components. 10. The cell line according to claim 2 in which said antigen is essentially undetectable by the monoclonal antibody on adenomas. 11. The cell line according to claim 2 in which said monoclonal antibody recognizes normal epithelial cells of the lung and kidney. 12. The cell line produced by hybridoma technique having the essential characteristics of the sample on deposit with American Type Culture Collection, Rock- ville, Maryland and assigned A.T.C.C. No. HB 9738. 13. A monoclonal antibody which binds specifical¬ ly to an antigenic determinant on a high molecular weight mucin-like glycoprotein complex molecule of hu¬ man breast tumor cells characterized as follows: A. the antigen is identified as BrEl having a molecular weight exceeding 400,000 daltons as determined by carrying out electrophoresis on the antigen and comparing its movement with that of marker proteins of known molecular weight; B. it is expressed on the surface or cytoplasm of the tumor cells; C. it is essentially undetectable on normal hu¬ man breast tissue. D. is of IgG2a isotype. 14. The monoclonal antibody according to claim 13 in which said antigen also expresses an epitope on a molecular entity thereof which has a molecular weight less than 400,000 daltons to which said monoclonal antibody also binds. 15. The monoclonal antibody according to claim 13 produced by the hybridoma having the essential charac¬ teristics of the samples on deposit with the American Type Culture Collection, Rockville, Maryland, A.T.C.C. No. HB 9738. 16. The monoclonal antibody according to claim 13 in which said BrEl antigen is essentially undetectable on adenomas generally. 17. The monoclonal antibody according to claim 13 in which said BrEl antigen is detectable on the surface of epithelial cells of adenocarcinoma cells of the ovary, endometrium, lung, pancreas and breast. 18. A method of detecting the BrEl antigen of BrEl antigen containing human carcinoma cells or molecules in a biological sample, said method compris¬ ing contacting said biological sample with a monoclonal antibody which specifically binds to BrEl antigen coupled to a label for a time and under conditions suf¬ ficient for the formation of immunological complexes between said monoclonal antibody and antigen and detecting said immunological complexes on a substrate reaction detectible product. 19. The method of claim 18 in which said monoclonal antibody and antigen immunological complex detected comprises a molecular entity of the antigen which expresses a common epitope to which the BrEl monoclonal antibody binds. 20. The method of claim 18 in which said label is coated on microspheres. 21. The method of claim 18 in which said label is selected from the group comprising a radioactive ele¬ ment, a fluorescent dye and an enzyme. 22. A murine monoclonal antibody of the mouse IgG2a isotype which specifically binds to the BrEl antigen conjugated to a chemotherapeutic, photoac- tivated toxin or radioactive agent. 23. A method for detecting the presence or lack thereof of BrEl antigen on BrEl antigen-containing hu¬ man breast carcinoma cells or molecules in a biological sample comprising, contacting said biological sample with a labeled monoclonal antibody which specifically binds to the BrEl antigen for a time and under condi¬ tions sufficient for an immunological reaction, if any, between the monoclonal antibody and said antigen and detecting immunological complexes, if any. 24. The method of claim 23 in which said label is selected from the group comprising a dye, a fluorescent dye, a radioactive element and an enzyme. 25. A method of detecting a human breast car- cinoma tumor in a patient suspected of having said tumor, said method comprising, infusing a monoclonal antibody which specifically binds to BrEl antigen, derivatized with a radioactive element, into said patient for a time and under conditions sufficient for the formation of immunological complexes between said monoclonal antibody and said tumor, thereby labeling said tumor with the derivatized monoclonal antibody for detecting said tumor. 26. The method of claim 25 wherein said radioac¬ tive agent is Indium 111, Technetium 99 or Iodine 131. 27. A method of killing tumor cells of a human breast carcinoma tumor in a patient suspected of having said tumor, said method comprising, infusing a monoclonal antibody-toxic agent conjugate which specif¬ ically binds to BrEl antigen into said patient for a time and under conditions sufficient for the formation of immunological complexes between said antibody-toxic agent conjugate and said tumor and causing tumor cell death. 28. The method of claim 27 wherein said toxic agent is a radioactive agent.

Inventors: CERIANI ROBERTO L, PETERSON JERRY A, CERIANI, ROBERTO, L., PETERSON, JERRY, A.
Applicants: MUIR JOHN CANCER & AGING INST, JOHN MUIR CANCER & AGING INSTITUTE
Application year: 1989
Main classification: G01N33
IPC codes: A61K38, A61K39, A61K47, A61K51, C07K16, C12N5, C12N15, C12P21, C12R1, G01N33