Phylogenic distribution of human renal antigens defined by monoclonal antibodies

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.     

Canine lymphoma-associated antigens defined by murine monoclonal antibodies

Summary Lymphoma in dogs resembles human non-Hodgkin's lymphoma in pathological presentation, immunophenotype, and response to therapy, thus representing a good model for comparative studies with human disease. Monoclonal antibodies (MAbs) were derived from mice immunized with a dog lymphoma cell line. Three MAbs were selected for further application in immunophenotyping and immunotherapy. The binding specificities, antigen characterization, and isotypes for these MAbs are described.Supported by NCI grant CA-10815     

Heterogeneity in expression of human leukocyte antigens and melanoma-associated antigens in advanced melanoma

The study of tumor immunology has led to many innovative therapeutic strategies for the treatment of melanoma. The strategies are primarily dependent on melanoma-associated antigen peptide vaccination or T-cell-based therapy. These immunotherapies are totally reliant on proper copresentation of human leukocyte antigen class I molecules in sufficient quantity and the presence and availability of melanoma-associated antigenic peptides. Altered expression of either HLA class I molecules or melanoma antigens is known to occur. These defects lead to altered manufacture and copresentation of HLA class I molecules with melanoma-associated antigens to T-cells. Defects in any one combination can lead to loss of recognition of melanoma cells and their subsequent destruction by cytotoxic T-lymphocytes. Thus, these immunotherapy strategies can be thwarted by defects or heterogeneity of expression of human leukocyte antigen class I or of melanoma-associated antigens.     

Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies

Monoclonal antibodies directed against human cancer cells were prepared by the murine hybridoma technique. These antibodies detect Lewis blood group antigens as determined by indirect solid-phase radioimmunoassay, hapten inhibition studies, and chromatogram binding assay. One monoclonal antibody is specific for the Lea terminal carbohydrate of Gal beta 1----3Glc NAc(4----1 alpha Fuc) beta 1----3LacCer. Five monoclonal antibodies react with the Leb terminal carbohydrate sequence of Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer, and four of these antibodies are highly specific for this glycolipid and do not react with other similar di- and monofucosylated glycolipids. One of the anti-Leb antibodies cross-reacts with blood group H glycolipid and has binding properties similar to those of the previously described antibody NS-10-17 [M. Brockhaus, J. L. Magnani, M. Blaszczyk, Z. Steplewski, H. Koprowski, K.-A. Karlsson, G. Larson, and V. Ginsburg (1981) J. Biol. Chem. 256, 13223-13225]. Two antibodies react with both the Lea and Leb antigens, though both bind preferentially to Leb.     

The human repertoire of antibody specificities against Thomsen-Friedenreich and Tn-carcinoma-associated antigens as defined by human monoclonal antibodies

Summary Human monoclonal antibodies specific for tumour-associated Thomsen-Friedenreich (TF) [Gal(1–3)GalNAc()-O-] and Tn [GalNAc()-O-] glycoproteins were prepared using peripheral blood lymphocytes from healthy blood donors. The B lymphocytes were either directly transformed with Epstein-Barr virus (EBV) or transformed after an in vitro stimulation period with synthetic glycoproteins. The EBV-transformed lymphocytes were subsequently fused with a mouse-human heteromyeloma to secure antibody production and stability. IgM antibodies exhibiting different patterns of specificity for synthetic TF and Tn antigens were obtained, including antibodies specific for the and forms of different Gal(1–3)GalNAc-O- and GalNAc-O- conjugates and antibodies agglutinating neuraminidase-treated erythrocytes. Several of the human monoclonal antibodies showed an increased binding to cultured carcinoma cells as compared to melanoma cells. This straightforward approach for the production of human monoclonal antibodies demonstrates the possibility of investigating the reactivity pattern of tumour-binding antibodies from peripheral blood lymphocytes. The binding patterns of these monoclonal antibodies show that healthy donors carry different fine specificities against synthetic TF/Tn antigens and that these antibodies react with different tumour cells.     

Differentiation antigens of mouse teratocarcinoma stem cells defined by monoclonal antibodies

Three differentiation antigens of mouse teratocarcinoma stem cells are defined using a panel of ten IgM-class monoclonal antibodies raised against teratocarcinoma F9 cells. TEC-01 and four other antibodies define an antigen that corresponds to SSEA-1. TEC-02 antibody defines an antigen that is expressed on teratocarcinoma stem cells, parietal yolk sac cells PYS-2, unfertilized eggs including the zona pellucida and blastocysts. It is absent from all mouse adult tissues tested. Three other antibodies exhibit binding properties similar to TEC-02. TEC-03 antibody defines an antigen that is expressed on teratocarcinoma stem cells, PYS-2 cells and mouse blastocysts. It is absent from all mouse adult tissues except for lungs.     

Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.     

Differential expression of cell surface antigens of canine keratinocytes defined by monoclonal antibodies

Nine monoclonal antibodies (MAb) directed against cell surface antigens of canine keratinocytes define distinct keratinocyte subpopulations owing to the differential expression of these antigens during the process of differentiation and depending on the tissue location of the cells. There was distinct antigenic heterogeneity between the different layers of stratified squamous epithelium and between stratified squamous epithelial of different tissue origin. Two MAb reacted only with antigens expressed by esophageal mucosa. Three MAb bound to antigens on keratinocytes of the suprabasilar and granular layers of stratified squamous epithelia, and they crossreacted with the transitional epithelial cells of the urinary tract. Two MAb reacted with antigens only expressed on differentiated cells, superficially located in the stratified squamous epithelium. The use of these MAb as markers for keratinocytes in studies on the characterization and differentiation of keratinocytes, as well as in tumor diagnosis and allograft transplantation, is discussed.     

T-cell surface antigens defined by monoclonal antibodies, involved in the induction of human interferon-gamma and interleukin 2

Monoclonal antibodies specific for human T-cell-differentiation antigens were used to investigate the mechanism of induction of interferon-gamma (IFN-gamma) and interleukin 2 (IL-2). High levels of IFN-gamma, accompanied by IL-2 production, were detected in the lymphocyte cultures stimulated by pan T monoclonal antibodies that were mitogenic. These antibodies recognize an antigen complex Tp 19-29 (a complex of T-cell proteins of 19-29 kDa). However, it was possible to induce IL-2 without concomitant production of IFN-gamma using some antibodies specific for other T-cell surface antigens, e.g., Tp 32-45, Tp 41, and Tp 100-120. These antibodies were not mitogenic. The production of lymphokines, therefore, appears to be regulated at the cell surface by receptors or interaction molecules involved in cell triggering. Binding of antibodies to T3 receptor was obligatory for both IFN-gamma induction and mitogenesis but was not required in the induction of IL-2 activity.     

Structural characterization of human melanoma-associated antigen p97 with monoclonal antibodies

Monoclonal antibodies were used to study p97, a human melanoma-associated antigen (MAA). Four hybridomas, designated 4.1, 96.5, 118.1, and 8.2, were obtained by fusing mouse myeloma cells with spleen cells from mice immunized with human melanoma cells. Antibodies 4.1 and 8.2 were IgG1; antibodies 96.5 and 118.1 were IgG2a. Sequential immunoprecipitation (IP) and sodium-dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that all 4 antibodies recognized the same 97 kilodalton (kD) protein. Binding studies with 125I-labeled antibody showed that antibodies 4.1 and 96.5 bound the same epitope, p97a. Antibodies 118.1 and 8.2 defined epitopes p97b and p97c, respectively. Six monoclonal antibodies (M17, L1, L10, R10, I12, and K5) specific for gp95, a kD melanoma cell surface glycoprotein were also tested. Sequential IP showed that these antibodies bound p97; p97 and gp95 are thus identical. Binding studies showed that antibody m17 bound epitope p971, and antibodies L1, L10, and R19 bound epitope p97c. Antibodies I12 and K5 defined 2 other epitopes, p97d and p97e, respectively. SDS-PAGE under nonreducing conditions indicated that p97 is monomeric, probably with intrachain disulfide bonds. Cell-surface labeling of sialic acid residues and neuraminidase digestion showed that p97 is a sialoglycoprotein. Digestion of p97 with papain or trypsin produced a stable 40 kD fragment, which expressed epitopes p971, p97b, and p97c, but not p97d or p97e.     

Glucocorticoid receptors in subpopulations of human lymphocytes defined by monoclonal antibodies

Glucocorticoid receptors (GR) were investigated in subpopulations of lymphocytes identified by monoclonal antibodies. Purified T (OKT3+) and non-T lymphocyte subpopulations were isolated from human peripheral blood using Degalan bead columns coated with rabbit anti-human IgG. Purified subpopulations of OKT4+ and OKT8+ lymphocytes were obtained by coating the nonadherent population (T cells) from the first column with OKT4+ or OKT8+ and pouring it into a second Degalan column, coated with goat anti-mouse IgG. GR content and affinity were analyzed by a whole cell assay with [3H]dexamethasone as tracer. The numbers of GR in lymphocyte subpopulations (OKT3+ cells, non-T cells, OKT4+, and OKT8+ cells) were nearly equal. It is concluded that the differential effects of glucocorticoids on the circulatory kinetics of OKT4+ and OKT8+ cells probably are not related to differences in glucocorticoid receptors of these T-cell subpopulations.     

Epitopes of human intestinal alkaline phosphatases, defined by monoclonal antibodies

Antibody additivity assayTopology     

Epitope clusters of Qa-2 antigens defined by a panel of new monoclonal antibodies

A recently derived intra-MHC recombinant mouse strain, the C3H.KBR was found to produce a surprisingly high titer of anti-Qa antibodies when immunized with C3H.SW lymphocytes. By using this immunization combination, a panel of 10 mAb with specificity for determinants encoded by the Q region was produced. These reagents were analyzed for strain distribution by microcytotoxicity, immunofluorescence, and flow cytometry assays. Competitive inhibition analyses, performed by using fluorescein-labeled antibodies and normal spleen cells, defined at least three epitope clusters, or groups of spatially related determinants, detected by this panel. One epitope cluster was unique to this new series of antibodies in that it was not detected with seven previously described anti-Qa mAb. These antibodies also have been analyzed for reactivity with products of isolated Q-region genes by using transfected cell lines. The data indicate that the Q6d, Q7d, and Q10d genes encode determinants reactive with one or more mAb and that two of the three epitope clusters defined with normal cells map to the N and/or C2 domains of these molecules. The third epitope cluster is presumed to map to the C2 domain. These reagents should be useful in determining the number of Q-region genes expressed and in analyses of Q gene expression in subpopulations of normal cells, in transfected cell lines, and during differentiation and ontogeny.     

Two surface antigens expressed on proliferating mouse T lymphocytes defined by rat monoclonal antibodies

A hybrid cell line resulting from the fusion of a Con A-activated normal mouse spleen cell and a transformed mouse T cell (EL-4BU) has been used to prepare and select rat monoclonal antibodies reactive with molecules expressed on the surface of proliferating, as opposed to resting, mouse T cells. In this report, the characterization of two such antigens identified in this way is described. One antigen is a membrane component common to mitogen-activated T and B cells, some bone marrow cells, and various transformed cell lines but is not detectable on either normal thymocytes or the majority of spleen cells by radioimmunoassay or FACS analysis. It has a m.w. of approximately 200,000 daltons under nonreducing conditions and 100,000 daltons under reducing conditions. Antibodies to this antigen precipitate cell-bound transferrin but do not react directly with transferrin itself. It would thus appear that the antigen is the transferrin receptor molecule. The second antigen is not detectable on normal thymocytes, spleen cells, bone marrow cells, or mitogen-stimulated spleen cells but is expressed at high levels on some transformed T cell lines. It, too, appears to be a dimer, with a m.w. of 95,000 daltons under nonreducing conditions, decreasing to 50,000 daltons under reducing conditions. Although the function of the 95,000-dalton antigen is not yet known, its lack of expression on adult T cell populations both before and after activation suggests either a short-lived role at a very early stage of T cell development and/or an association with T cell transformation.     

Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies

A series of seven monoclonal antibodies directed at determinants on human peripheral blood monocytes were produced and characterized. The antibodies were separated into three groups based on cell distribution and percentages of monocytes bearing antigen. Hybridoma antibodies, termed OKM1, OKM9, and OKM10, recognized antigen(s) expressed on the majority of adherent monocytes, null cells, and granulocytes. The second group, comprising OKM5 and OKM8, reacted with most adherent monocytes and platelets. OKM3 and OKM6, comprising a third group of antibodies, reacted with a subpopulation of adherent monocytes and platelets. OKM antibodies were not expressed on lymphocytes, thymocytes, and lymphoblastoid cells, with the exception of OKM3 which reacted with three B-cell lines. SDS gels of immunoprecipitates formed with OKM antibodies yielded the following tentative molecular weight results: OKM1 and OKM9 antigens appeared to be 160,000 (nonreduced) and 170,000 (reduced); OKM10 precipitated two polypeptides of 170,000 and 115,000 (reduced); OKM5 and OKM8 precipitated a single polypeptide of 88,000 (reduced, nonreduced); OKM6 antigen appeared to be 116,000 (nonreduced) and 130,000 (reduced).     

Membrane antigens of human bronchial epithelial cells identified by monoclonal antibodies

Summary Subpopulations of normal bronchial epithelial cells were identified using a series of murine monoclonal antibodies. These antibodies were used to stain intact bronchial epithelial cells in culture by indirect immunofluorescence. LAM 2 reacted with 80%, LAM 6 with 75%, LAM 7 with 60%, and LAM 8 with 5% of these cells. Sections of human bronchial epithelium were also stained with these antibodies by immunoperoxidase methods. LAM 2 was found to bind with 80%, LAM 6 with 65%, LAM 7 with 50%, and LAM 8 with less than 1% of bronchial epithelial cells. LAM 2 stained both columnar epithelial cells and basal cells; LAM 6 stained mainly basal cells and only a small proportion of columnar cells; LAM 7 showed specificity for basal cells; LAM 8 distinctly stained single cells in the basal cell layer. These antibodies were previously shown to react with the surface membrane of human lung carcinomas, ranging from the broad reactivity of LAM 2 with small cell and non-small cell lung cancers to the highly restricted reactivity of LAM 8 with small cell carcinomas of the lung. Thus, membrane antigens have been identified in bronchial epithelial cells by monoclonal antibodies which exhibit a similar range of cellular reactivity in vitro as in vivo. Inasmuch as these antibodies recognize subsets of cells which could not be easily distinguished by morphologic characteristics, these reagents may be useful in classifying bronchial epithelial cells.     

Molecular properties of ECMA 2 and ECMA 3 antigens defined by monoclonal antibodies against embryonal carcinoma cells

ECMA 2 and ECMA 3 antigens defined by two monoclonal antibodies are preferentially expressed in early embryonic cells of the mouse. The antigens were isolated from F9 embryonal carcinoma cells by detergent solubilization followed by indirect immunoprecipitation. Both antigens were glycoproteins, which, upon extensive pronase digestion, released the high-molecular-weight glycan (embryoglycan). The immunoprecipitation reactions were inhibited by the glycan, indicating that the two antigens were carried by it. Furthermore, binding of anti-ECMA 2 antibody to the glycan was directly demonstrated by a modified Farr's assay. The antigenic determinant of ECMA 2 antigen was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases, abolished the antigenic activity. Serological experiments indicated that ECMA 2 antigen is different from other alpha-galactosyl antigens, namely blood group B and P1 antigens and an antigen defined by antibodies in the sera of patients with ovarian germ cell tumors.