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United States Patent   Patent Number: 4,522,918
Schlom et al.  Date of Patent: Jun. 11, 1985
 PROCESS FOR PRODUCING
MONOCLONAL ANTIBODIES REACTIVE
WITH HUMAN BREAST CANCER
 Inventors: Jeffery Schlom, 404 Victoria Ct,
NW.; David Colcher, 2587 Rambling
Rd., both of Vienna, Va. 22180;
Marianna Nuti, 9010 Lindale Dr.,
Bethesda, Md. 20034; Patricia H.
Hand, 1113 E. Capital St., SE.,
Washington, D.C. 20003; Faye
Austin, 8204 Toll House Rd.,
Annandale, Va. 22003
 Appl. No.: 330,959
 Filed: Dec. 15, 1981
 Int. C1.3 C12P 21/00; C12N 5/00;
C12N 15/00; G01N 33/54; A61K 49/00;
A61K 39/00; C12R 1/91
 U.S. CI 435/68; 435/172.2;
435/240; 435/7; 435/948; 436/548; 436/813; 424/85; 424/1.1; 935/93; 935/104; 935/107;
 Field of Search 435/7, 188, 810, 240,
435/241, 172, 68, 948; 424/85, 88, 1.1, 8, 12;
23/230 B; 436/548, 813, 64
 References Cited
U.S. PATENT DOCUMENTS
4,172,124 10/1979 Koprowski et al 435/172
4,196,265 4/1980 Koprowski et al 435/240
4,229,426 10/1980 Haagerisen, Jr 23/230 B
4,232,001 11/1980 Jensen et al 23/230 B
4,349,528 9/1982 Koprowski et al 424/85
Stahli et al, "High Frequencies of Antigen Specific Hybridomas: Dependence on Immunization Parameters and Prediction by Spleen Cell... ", Journal of Immunological Methods, 32, (1980), pp. 297-304. Sloane et al, "Distribution of Epithelial Membrane Antigen in Normal and Neoplastic Tissues and its Value in Diagnostic Tumor . . .. ", Cancer, 47(4), (1981), pp. 1786-1795.
Schlom et al, "Generation of Human Monoclonal Antibodies Reactive with Human Mammary Carcinoma
Cells", Proceedings of the National Academy of Sciences, 77(11), pp. 6841-6845, (1980). Kohler et al, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity", Nature, 256, (8-1975), pp. 895-897.
Herzenberg et al, "Cell Hybrids of Myelomas with Antibody Forming Cells and T-Lymphomas with T Cells", Handbook of Experimental Immunology, by Weir, (1979), pp. 25.1-25.7.
Edynak et al, "Fluorescent Antibody Studies of Human Breast Cancer", Journal of the National Cancer Institute, 48, (1972), pp. 1137-1141.
Sternberger et al, "The Unlabled Antibody Enzyme Method Immunocytochemistry of Horomone Receptors at Target Cells," First Int. Symposium on Immunoenzymatic Techniques, (2), (1976), pp. 43-58. Colcher et al, "A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells", Proceedings of the National Academy of Sciences, 78(5), (5-1981), pp. 3199-3203.
Colcher et al, "Use of Monoclonal Antibodies to Define the Diversity of Mammary Tumor Viral Gene Products in Virons and Tumors," Cancer Research, vol. 41(4), pp. 1253-1277.
Primary Examiner—Thomas G. Wiseman
Assistant Examiner—John Edward Tarcza
Attorney, Agent, or Firm—John S. Roberts, Jr.
Monoclonal antibodies demonstrating a reactivity with human breast cancer are produced. The hybridoma cultures secreting immunoglobins are produced by hydridoma technology. Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissue are fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. Screening of immunoglobulin reactivities and double cloning of cultures yielded 11 monoclonal antibodies that demonstrated activities with the surface of human mammary tumor cells and not with the surface of apparently normal human tissues. These monoclonal antibodies aid in the diagnosis, prognosis and treatment of human breast cancer.
7 Claims, 3 Drawing Figures
PROCESS FOR PRODUCING MONOCLONAL ANTIBODIES REACTIVE WITH HUMAN BREAST CANCER
PRIOR ART STATEMENT
Hybridoma technology was developed by Kohler and Milstein [Kohler, G. and Milstein, C. (1975) Nature (London), 256, 494-497]. 10
The technique is also set out in detail in the following excerpt from a basic immunology textbook, Herzenberg and Milstein, Handbook of Exerimental Immunology, ed. Weir (Blackwell Scientific, London), 1979, pp. 25.1-25.7. 15
Assaying for antibody in solid-phase RIA is described by the inventor in Colcher, Horan Hand, Teramoto, Wunderlich, and Schlom, Cancer Research, 41, 1451-1459 (1981).
Background art by the inventors is in Colcher, Horan 20 Hand, Nuti, and Schlom, J. Proc. Natl. Acad. Sci., May 1981, Vol. 78, No. 5, 3199-3203.
Patents relating to other tumors produced by hybridoma technology are U.S. Pat. Nos. 4,172,124 and 4,196,265. 25
These eleven monoclonal antibodies aid in the diagnosis, prognosis, and treatment of human breast cancer. 3Q These antibodies are activated only by tumor cells from human mammary cells and not by apparently normal human tissues.
STATEMENT OF DEPOSIT
The following hybridoma cell lines were deposited in the ATCC with the following accession numbers: B6.2, ATCC #HB8106; B25.2, #HB8107; B72.3, #HB8108; F25.2, #HB8109; B38.1, #HB8110; and B50.4, #HB8111. 40
SUMMARY OF THE INVENTION
Mice were immunized with membrane-enriched fractions of human metastatic mammary carcinoma cells from either of two involved livers (designated Met 1 45 and Met 2). Spleens of immunized mice were fused with NS-1 myeloma cells to generate 4250 primary hybridoma cultures. Supernatant fluids from these cultures were screened in solid phase RIAs for the presence of immunoglobulin reactive with extracts of metastatic 50 mammary tumor cells from involved livers and not reactive with similar extracts of apparently normal liver. Whereas many cultures demonstrated immunoglobulin reactive with all test antigens, 370 cultures 55 contained immunoglobulin reactive only with the metastatic carcinoma cell extracts. Following passage and double cloning of these cultures by endpoint dilution, the monoclonal immunoglobulins from eleven hybridoma cell lines were chosen for further study. The g0 isotypes of all eleven antibodies were determined; ten were IgG of various subclasses and one was an IgM.
DETAILS OF THE INVENTION
FIG. 1 shows tissue sections and the detection of 65 binding of monoclonal antibodies to human mammary adenocarcinoma cells using the immunoperoxidase technique.
MATERIALS AND METHODS
Cell extracts were prepared from breast tumor metastases to the liver from two patients as well as from apparently normal liver. Tissues were minced finely and homogenized for 2 to 3 min. at 4° C. in 10 raM Tris-HCl, (pH 7.2), 0.2 mM CaCl2 (10 gm/100 ml). The homogenate was then subjected to nitrogen cavitation using a cell disruption bomb (Parr Instrument Co., Moline, IL) for 5 min. at 1000 lg/in2 and then clarified at lOOOxg for 5 min. Cell membrane enriched fractions were prepared from this extract by centrifugation of the lOOOXg supernatant in a discontinuous 20%/40% (w/w/, 10 ml of each) sucrose gradient in 10 mM Tris-HCl, (pH 7.2), containing 2 mM CaCL2 for 16 hr at 130,000 Xg. The material obtained from the 20-40% interface was diluted and pelleted at 130,000Xg for 60 min (SW27 rotor, Beckman). The pellet was resuspended in PBS buffer and sonicated at 4° C. for 1 min. at 15 sec. intervals (Branson Sonifier, setting 7). The sonicate was centrifuged at 10,000Xg for 10 min. and the supernatant was used for immunizations. The balance of the cell extract was repeatedly passed through columns of Protein A-Sepharose (Pharmacie) to remove cell associated Ig that would interfere with the solid phase radioimmunoassays. Protein concentrations were determined by the Lowry method (Lowry, O. H., et al, J. Biol. Chem., 193, 265-275, 1951).
Four-week old C57BL/6, BABL/c and C3HfC57BL mice were immunized by intraperitoneal inoculation with 100 jxg of membrane-enriched fractions of either of two breast tumor metastases to the liver (termed Met 1 and Met 2) mixed with an equal volume of complete Freund's adjuvant. Fourteen and 28 days later, mice were boosted with an intraperitoneal inoculation of 100 /xg of the immunogen mixed with incomplete Freund's adjuvant. After an additional 14 days, 10 u,g immunogen was administered intravenously. Spleens were removed for cell fusion 3 to 4 days later.
The preferred embodiment of this procedure requires HL clones. H and L are the heavy and light chains contributed by the antibody producing parental cell. In the past, HL clones were derived from MOPC-21 through the X63-Ag8 myeloma cell line (which secretes an IgGl myeloma protein). X63-Ag8 also secretes heavy and light chains of G and K. Thus, the hybrid produces a variety of tetrameric molecules with different H, L, G, and K combinations. Only some of these molecules bind to the antigen and even fewer are detectable by a complement dependent reaction.
Fortunately, it has not been difficult to obtain variants of the selected hybrids which have lost the ability to make G and K. By subcloning HLGK lines, HLK clones can be detected and by subcloning these, HL variant clones can be found. The frequency of variants differs from clone to clone but is often in the order of 1-3 percent for each step.
Screening for HL clones can be done in favorable cases simply by haemolytic or cytolytic activity assays, since HL and HLK have greater activity than HLGK. A more general method of screening for HL clones is chain analysis of secreted immunoglobulins (Ig) using