invited speakers' abstracts from the second annual congress for hybridoma research

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  • HYBRIDOMAVolume 2. Number 1, 1983Mary Ann Liebert, Inc., Publishers

    Invited Speakers'Abstracts from the Second Annual Congress

    for Hybridoma ResearchFebruary 6-10, 1983



  • IMMUNODIAGNOSTIC STUDIES OF HUMAN ADENOCARCINOMAS WITH MONO-CLONAL ANTIBODIESRichard S. Metzgar, Melissa T. Gaillard, Michael Borowitz,Vicki N. Daasch, Francis L. Tuck, Ned W. Rodriguez and PhilipD. FernstenDepartments of Microbiology and Immunology, and Pathology,Duke University Medical Center, Durham, NC 27710

    Murine monoclonal antibodies have been elicited to humanpancreatic adenocarcinoma cells. Preliminary studies on thespecificity of five of these monoclonal s (DU-PAN-1,2,3,4 and5) indicated restricted normal cell distribution and strongreactivity with some adenocarcinomas from a variety of tissueand organ sites (Cancer Research 42j_ 601-608, 1982). Thetumor and normal tissue distribution of 3 of the antigenshave been studied in more detail. DU-PAN-1 antigen is detect-ed on certain epithelial cells of normal prostate, stomachand bladder but is not detected on pancreatic ductal cells.This antigen is on a high percentage of pancreatic carcinomasand some adenocarcinomas of colon, prostate, bladder, breast,and ovary. DU-PAN-2 antigen is detected on normal pancreaticductal cells but is not detected on bladder or prostaticepithelium. This antigen is present on 14 of 16 pancreaticadenocarcinomas and 6 of 9 gastric carcinomas as well as onsome carcinomas from ovary, breast, colon and lung. DU-PAN-3antigen is present on pancreatic ductal cells and prostaticepithelium but is not found on bladder epithelial cells. Theantigen is present on some adenocarcinomas from pancreas,lung, colon, breast, ovary and prostate. DU-PAN-1 and DU-PAN-3antibodies are of the IgG2 isotype and immunoprecipitate glyco-peptides of 30-35,000 and 70-75,000 daltons respectively.These antigens are currently being purified by affinitychromatography and subjected to molecular analysis. TheDU-PAN-2 monoclonal is an IgM and reacts with a neuraminidasesensitive, mucin like antigen which has been purified fromascitic fluids of patients with pancreatic adenocarcinoma.A competition radioimmune assay has been developed for theDU-PAN-2 antigen which can readily detect this antigen in thepleural and ascitic fluids and sera of some patients with adeno-carcinomas originating from a variety of organ and tissue sites.Preliminary studies on the detection and serial monitoring ofserum levels of DU-PAN-2 antigen indicate that this test maybe valuable as a diagnostic and/or clinical management aid.

    This work was supported in part by grants CA 08975 andCA 32672 from the National Cancer Institute and by grantIM-326 from the American Cancer Society.


  • SUBPOPULATIONS OF MYELOID PROGENITOR CELLS DEFINED BY MONOCLONALANTIBODIES Dario Ferrero*, Hal E. Broxmeyer1", Giovanni L.Pagliardi*, Salvatore Venuta*, Beverly Lange+, Silvana Pessano*and Giovanni Rovera* *The Wistar Institute of Anatomy andBiology, Philadelphia, PA 19104; ^Laboratories of DevelopmentalHematopoiesis, SloanKettering Institute for Cancer Research, NewYork, NY 10021; division of Oncology, Children's Hospital ofPhiladelphia, Philadelphia, PA 19104

    Two types of progenitor cells of the human granulocytic andmonocytic lineages (CFU-GM) can be distinguished using mousemonoclonal antibodies against human hemopoietic cells. Type 1CFU-GM contributes all of the peripheral blood CFU-GM as well asa small fraction of bone marrow CFU-GM and express on theirsurface antigens recognized by several anti-lymphomonocyticantibodies but not antigens expressed on morphologically identi-fiable cells of the myelomonocytic lineage. Type 2 CFU-GM arepresent only in the marrow and react with a variety of monoclo-nal antibodies. In vitro culture of type 1 CFU-GM in the pre-sence of CSF generates colony-forming cells that have thesurface phenotype of type 2 CFU-GM. This finding supports theinterpretation that type 1 and type 2 CFU-GM represent two dif-ferent stages of maturation of myelomonocytic stem cells.


  • ANTIGENS OF HUMAN GASTRO-INTESTINAL TUMORS. Zenon Steplewski,Meenhard Herlyn, Magdalena Blaszczyk, Koon Yan Pak, AlonzoRoss, John Powe and Hilary Koprowski, The Wistar Institute,Philadelphia, Pennsylvania 19104.

    Hybridoma derived murine monoclonal antibodies generatedagainst human gastrointestinal tumors are used to characterizetumor associated antigens (Herlyn, M. et al. PNAS 76,1438,1979;Koprowski et al. Somat. Cell Genetics 5,957,1979). Monoclonalantibody defined antigens of gastrointestinal tumors were foundto belong to glycolipid and glycoprotein groups. The glyco-proteins include CEA and CEA related molecules, and a bimolec-ular protein which consists of^chain with MW 28,000 dal tonsand chain with MW of 22,000 dal tons. Two newly establishedhybridomas (G7-6 and G7-9) secrete antibodies directed against90,000/27,000 daltons molecules restricted to gastrointestinaltumors.The majority, however, of the antigens expressed on thesurface of gastrointestinal tumors and immunogenic in mice areof glycolipid nature. These include blood group B glycolipidchains Type I and II and Lewis blood group antigens Lea andLe. Two such antibodies react with both Lea and LeD moleculesTwo monoclonal antibodies directed against Le'3 hapten immuno-precipitated similar protein molecules with MW of 220,000,150,000 and 36,000. Monoclonal antibody specific for bothLea and Le^ haptens immunoprecipitates 220,000 and 150,000glycolipid molecules from colon carcinoma cells. These mole-cules are present only in gastrointestinal tumors. Finally,one of the antigens, a monosialoganglioside (Magnani et al.Science 215,55,1981), was found to be shed by tumor cells(Steplewski et al. Cancer Res. 41,2723,1981) and present insera of cancer patients (Koprowski et al. Science 215,53,1981).This antibody was used to construct a srodiagnostic assaymethod. Another antibody, of IgG2a isotype, directed againstantigen not shed by tumor cells is used for immunotherapyexperiment and as carrier of radioactive isotopes for nuclearimaging. The selection of antigen-antibody pairs at presentis based not only on their specific distribution but alsoaccording to their future application - antigens shed vs non-shed by tumor cells.



    J. Schlom, D. Colcher, P. Horan Hand, D. Wunderlich,D. Stramignoni, M. Nuti, B. Atkinson, R. Bowen.National Cancer Institute, NIH, Bethesda, Md, 20205

    Murine monoclonal antibodies reactive with human mammarytumor associated antigens have been generated and character-ized. The immunogens used in these studies were membrane-enriched fractions of human metastatic mammary carcinomalesions. The thirteen monoclonal antibodies characterizedcould be divided into five major groups based on differentialreactivity to various mammary carcinoma lesions, binding tothe surface of carcinoma cell lines, and the molecular weightof the reactive antigen precipitated. Two monoclonals ( F5.5) were reactive with purified 180,000d carcinoembry-onic antigen. Monoclonals B6.2 and B72.3 were extensivelycharacterized as to their range of reactivities, and wereshown to immunoprecipitate a 90,000d protein, and a 220,000d-400,000d high molecular weight protein complex, respectively.B72.3 clearly showed the most restricted pattern of reactivityfor carcinoma vs. normal human cells. Using the monoclonalantibodies and the immunoperoxidase technique on sections ofhuman mammary carcinomas, a spectrum of antigenic phenotypeswas observed, as was antigenic diversity of mammary tumor cellpopulations within a given tumor mass. Antigenic phenotypicdiversity and modulation was also observed in establishedmammary tumor cell lines and cloned cell populations. B6.2IgG was purified, FCab')? and Fab' fragments were generatedand radiolabeled with *25l; all were successful in localizinghuman mammary tumors transplanted in athymic mice. TheF(ab')2 fragments gave the highest tumor to tissue ratios.Scanning experiments confirmed the ability of radiolabeledmonoclonal antibody B6.2 and its fragments to detect thepresence of transplanted human mammary tumor lesions withoutthe aid of background subtraction manipulations.

    The reactivity of monoclonal antibodies B72.3, B6.2 andBl.l with fixed tissue sections of human colon carcinomas andadenomas was also examined. B72.3 demonstrated the most se-lective reactivity for carcinomas; 82% (14 of 17) of carci-nomas were positive while none of 45 adenomas or normal colonepithelium examined showed reactivity with greater than a fewpercent of adenoma cells positive. Monoclonal B72.3 alsoreacted with cells in areas of "atypia" within adenomas.Based on the expression of antigens reactive with the threemonclonal antibodies employed, both colon adenocarcinomas andadenomas can now be placed in several distinct phenotypes.


  • EFFECTOR CELLS FOR MURINE IgG2a MONOCLONAL ANTIBODIES.Hilary Koprowski, Dorothe Herlyn and Zenon Steplewski, TheWistar Institute, Philadelphia, Pennsylvania 19104.

    Murine monoclonal IgG2a antibodies directed against tumorassociated antigens are specifically cytotoxic in antibodydependent cell mediated cytotoxicity assay (ADCC) in vitro(Herlyn, D. et al. Eur. J. Immunol. 9:657, 1979) and specific-ally inhibit growth of human tumors in athymic mice (Koprowskiet al. PNAS, 75:3405, 1978; Herlyn D. et al. Cancer Res. 40:717, 1980). Monoclonal antibodies against these tumors but ofother than IgG2a isotypes showed no inhibition of tumor growthin nude mice. Complement depletion of nude mice had no effecton tumor suppression by IgG2a antibody. Silica treatment ofnude mice on the other hand, abolished tumoricidal activity of

    Analysis of macrophage mediated IgG2a depend-in vitro and above described ex


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