Monoclonal antibody against human gallbladder carcinoma-associated antigen

Monoclonal antibody HI-531 of immunoglobulin G2b subclass was produced against a human gallbladder carcinoma cell line. HI-531 was investigated for reactivity with a panel comprising ten types of different origin in fluorescence-activated cell sorter analysis. The antibody reacted with the gallbladder carcinoma cell line G-415 used for immunization and with four unrelated tumors. HI-531 was further shown, with the use of the avidin-biotin complex-immunoperoxidase technique and surgically resected tissues, to be strongly reactive with carcinoma of the gallbladder, pancreas, bile duct, and gastrointestinal tract. The antibody was reacted with several types of normal epithelial cells but often more weakly expressed than on corresponding tumors. One of six fetal lung tissues was weakly stained. All other fetal organ tissues tested showed negative staining reactions. These observations suggest that HI-531 may be of value in identifying the tumor-associated antigen expressed in gallbladder carcinoma. HI-531 immunoprecipitated the Mr 43,000 molecule from extracts of Na125I- or [35S]methionine-labeled tumor cells, but not from those of [3H]glucosamine-labeled tumor cells. In addition, cytofluorometric analysis showed that cells treated with trypsin or protease greatly decreased a reactivity to the antibody. The findings suggest that the antibody recognizes a Mr 43,000 protein molecule. Sequential immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies and analyses by nonequilibrium pH gradient and polyacrylamide gel electrophoreses showed that the Mr 43,000 molecule defined by HI-531 was not a Mr 43,000 heavy chain of HLA-A,B,C antigens detected by monoclonal antibody W6/32.     

Monoclonal antibody against a membrane antigen characterizing leukemic human B-lymphocytes

In the diagnosis of non-Hodgkin's lymphomas, the ready characterization of the neoplastic cell lineage by analysis of cell surface markers is of great importance. We present evidence for the existence of a human B-leukemia-associated antigen recognized by a complement-fixing monoclonal antibody (anti-Y 29/55). A hybridoma was produced by fusing mouse myeloma cells and splenocytes of a mouse immunized against lymphoid cells of a patient with B-cell chronic lymphocytic leukemia. Characterization of anti-Y 29/55-reactive normal and malignant leukocytes was demonstrated by cytolysis and indirect immunofluorescence. This revealed reactivity with an antigen on B-lymphoma cells (11 patients), on leukemic lymphocytes in B-cell chronic lymphocytic leukemia (13 patients), and on malignant cells in hairy-cell leukemia (two patients) but not on leukemic cells of T-cell acute lymphoblastic leukemia (one patient), on T-lymphoma cells (one patient), on cells of acute myeloblastic leukemia (four patients), or of chronic myeloid leukemia (four patients). No specific cytolysis occurred with B- and T-peripheral blood lymphocytes from (a) healthy donors (16 individuals), (b) patient with reactive lymphocytosis (one patient), (c) nonleukemic multiple myeloma (six patients), or (d) Hodgkin's disease (three patients). Surface immunoglobulin-positive, sheep RBC-negative lymphocytes isolated from human spleen (three individuals), tonsils (seven individuals), and lymph nodes (one individual), however, were recognized. It is concluded that leukemic B-cells carry a marker characteristic of nonrecirculating sessile B-lymphocytes.     

Monoclonal antibody against human lung carcinoma

Spleen cells of BALB/c mice immunized with human pulmonary adenocarcinoma cell LTEP-a2 were fused with murine myeloma cell SP 2/0, from which 4 hybridomas (2 A7, 2 E9, 4 F2 and 5 F11) were obtained. Indirect immunofluorescence test showed that these 4 monoclonal antibodies reacted with human lung cancer cells, but not with 2 BS or the lymphocytes and red blood cells in 4 different ABO groups of 10 persons. Using ABC immunoperoxidase stain technique, these 4 antibodies showed negative reaction with 9 tissue types from the normal subject and fetus but could react with 52-83% of the 29 human lung carcinomas and 64-92% of the 24 non-small cell lung cancers (non-SCLC). When 5 F11 was combined with 2 A7 or 2 E9, the percentage of positive stain was 100% in 24 non-SCLC. The results of indirect immunofluorescence stain showed that strong membrane stain by 5 F11 and membrane stain by 4 F2 were obtained, indicating that these antibodies could recognize antigens on cancer cell membrane. It is suggested that a mixture of 5 F11 and other antibodies be useful in the diagnosis and treatment of lung cancer. Molecular weight of the antigens recognized by the 4 antibodies was determined by SDS-PAGE and immunoblot technique to be 47 KD (2 A7), 67 KD (2E9), 40 KD (4F2) and 56 KD (5 F11).     

Monoclonal antibody against sialylated Lewis(a) antigen

We isolated hybridoma cells, which secreted monoclonal antibody (MAb) 121 SLE, an IgM showing the following reactivities: (1) by immunodiffusion, MAb 121 SLE and MAb NS 19-9 (a monoclonal antibody directed against a sialylated Lewis(a) antigen called CA 19-9) showed an identical precipitin line with mucin preparation containing this CA 19-9; (2) by immunoradiometric assay, MAb 121 SLE totally inhibited fixation of radiolabelled MAb NS 19-9; (3) by immunoperoxidase, MAb 121 SLE stained the normal gastrointestinal mucosa of Le-positive individuals exclusively, and this staining disappeared after neuraminidase treatment, as observed using MAb NS 19-9. However, the pattern of the staining obtained with MAb 121 SLE differed slightly from that given by MAb 19-9 on the different positive areas of the gastrointestinal mucosae. These differences principally concerned the number of positive epithelial cells and the intensity of their staining; (4) moreover, antibodies against idiotype determinant of NS 19-9 antibody did not react with the antibody 121 SLE. We concluded that MAb 121 SLE is different from the MAb NS 19-9. However, both these antibodies were associated with the same molecular sialylated Lewis(a) structure.     

Monoclonal antibody directed to human T-cell malignancy antigen

A murine monoclonal antibody (B2D) against a cultured pre-T acute lymphoblastic leukemia (ALL) cell line THP-6 has been produced. The antibody reacted with seven out of eight cultured T-ALL cell lines and with leukemic cells from three out of four T-ALL/lymphoma patients. The antibody did not react with normal T and B lymphocytes, monocytes, granulocytes, platelets, erythrocytes, bone marrow lymphoid-like precursor cells, thymocytes and other acute and chronic leukemic cells of non-T cell origin. Furthermore, B2D did not react with phytohemagglutinin-activated T cells nor with concanavalin A-activated T cells. The molecules immunoprecipitated with B2D had molecular weights of 50-55 kD. Thus, B2D seems to be highly specific for T-cell malignancies. These results show that B2D defines one of human leukemia antigens which are expressed on the cell surface of T-ALL cells. Monoclonal antibody B2D may be useful for the subclassification of T-ALL cells and has therapeutic potential for a certain type of T-ALL.     

Monoclonal antibody against human ovarian tumor-associated antigens

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.     

Monoclonal antibody recognizing human melanoma-carcinoma cross-reacting oncofetal antigen epitopically associated with carcinoembryonic antigen

By fusion of mouse NS1 myeloma cells with splenocytes from a BALB/c mouse immunized with human melanoma cells, an IgG1 monoclonal antibody, designated as 140.72, was produced. By the mixed hemadsorption antibody binding assay, 140.72 was shown to react with 17 of 20 melanoma cell lines and with 5 of 14 carcinoma cell lines. This antibody also reacted with 3 of 3 normal melanocyte cultures in much lower titers. It did not react with any of 35 other normal and malignant lines, including neuroblastoma, glioblastoma, sarcoma, teratoma, fibroblast, and lymphoid cell lines. Absorption with fresh melanoma and carcinoma homogenates confirmed the results of direct tests. Fetal reactivity of antibody 140.72 was determined by positive absorption with 10 of 11 tissue homogenates derived from different fetuses of 10-16 weeks' gestation. The reactivity of this antibody was completely removed by absorption with a highly purified preparation of carcinoembryonic antigen (CEA) derived from a colon carcinoma. The antigenic activity was detected in the culture medium of reactive cell lines. Immunoprecipitation analyses of melanoma and carcinoma cells indicated that the antigenic determinant recognized by antibody 140.72 is on a glycoprotein with an apparent molecular weight of 95,000-150,000 common to both serologically reactive cell types. Additionally, a 200,000-molecular-weight glycoprotein corresponding to the CEA molecule was detected only on the reactive carcinoma cells. These data confirmed previous findings obtained with polyclonal anti-CEA antisera for the existence of shared CEA-related antigenic determinants on human carcinomas and melanomas and provided additional molecular characterization of these glycoproteins. Further characterization of the molecules bearing the antigenic determinant recognized by antibody 140.72 should be performed with a view to exploring its potential in the immunodiagnosis and immunotherapy of patients with melanoma.     

Monoclonal antibody against free beta-subunit of human chorionic gonadotropin

Spleen cells from BALB/c mice immunized with free native human chorionic gonadotropin hormone beta-subunit (beta hCG) were fused with mouse myeloma cells (P3/X63-Ag8) and one hybridoma secreting monoclonal antibodies (MAbs), was obtained. This hybridoma specifically recognizes beta hCG and does not cross-react with other human glycoprotein hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG). The MAb was of the IgG(1) subclass and ascitic fluid from this hybridoma was purified by affinity chromatography on Protein A-Sepharose CL-4B column to isolate the IgG(1) active fraction. The affinity constant of this MAb was 1.5 x 10(10)M(-1).     

Monoclonal anti-idiotypic antibody mimicking a tumor-associated sialoglycoprotein antigen induces humoral immune response against human small-cell lung carcinoma

We have previously described the tumor-associated sialoglycoprotein antigen sGP90-135 defined by the murine LAM8 MAb. The antigen is characterized by strong membrane expression in a proportion of small-cell carcinomas of the lung, but little or no expression on normal tissues of epithelial or neural origin or on blood cells. With the aim of obtaining anti-idiotypic antibodies which might be useful as surrogates for sGP90-135 in vaccination studies, LOU rats were immunized with LAM8 MAb and their spleen cells fused with Y3 rat myeloma cells. The LY8-229 hybrid was selected by an anti-idiotype competition radioimmunoassay on antigen-positive target cells. LY8-229 was shown to be a rat IgG1 with high specificity for LAM8 combining site. Solubilized small-cell carcinoma extract, as well as antibody SEN16, which recognizes the same tumor-associated antigen sGP90-135, selectively inhibited 125I-LY8-229 binding to LAM8. Serum from BALB/c mice and DA rats immunized with anti-idiotypic antibody LY8-229 showed reactivity with antigen-positive target cell lines, but not with antigen-negative control cell lines. The induction of a specific immune response in 2 different species by the anti-idiotypic antibody LY8-229 suggests that LY8-229 bears the internal image of the antigen sGP90-135 and that it might be a candidate for immunotherapy trials in cancer patients.     

Monoclonal antibody therapy directed against human acute myeloid leukemia stem cells

Accumulating evidence indicates that many human cancers are organized as a cellular hierarchy initiated and maintained by self-renewing cancer stem cells. This cancer stem cell model has been most conclusively established for human acute myeloid leukemia (AML), although controversies still exist regarding the identity of human AML stem cells (leukemia stem cell (LSC)). A major implication of this model is that, in order to eradicate the cancer and cure the patient, the cancer stem cells must be eliminated. Monoclonal antibodies have emerged as effective targeted therapies for the treatment of a number of human malignancies and, given their target antigen specificity and generally minimal toxicity, are well positioned as cancer stem cell-targeting therapies. One strategy for the development of monoclonal antibodies targeting human AML stem cells involves first identifying cell surface antigens preferentially expressed on AML LSC compared with normal hematopoietic stem cells. In recent years, a number of such antigens have been identified, including CD123, CD44, CLL-1, CD96, CD47, CD32, and CD25. Moreover, monoclonal antibodies targeting CD44, CD123, and CD47 have demonstrated efficacy against AML LSC in xenotransplantation models. Hopefully, these antibodies will ultimately prove to be effective in the treatment of human AML.     

Monoclonal antibody against an antigen selectively assembled into vesicular stomatitis virus virions from HeLa cells

A mouse hybridoma cell line IIB9, secreting IgG2b antibody specific for a HeLa cell antigen, was obtained by fusion of a mouse myeloma cell line with spleen cells from mice immunized with purified VSV tsO45 mutant (defective in assembly of G protein) which had been reproduced at a non-permissive temperature in HeLa cells. The monoclonal antibody IIB9 was strictly specific for HeLa cells in two tests: (1) reaction with VSV or Chandipura virus phenotypically mixed with host cell antigen, (2) complement-dependent cytotoxicity test (51Cr-release).     

Monoclonal antibody (WI-2) reactive against human transformed and leukemic cells

A monoclonal antibody (WI-2) was produced against HL-60 cells.This antibody was lgM Kappa and reacted with human cell linesderived from fibroblasts and from hemopoetic, lymphoid, andneoplastic tissues. It also reacted with lymphocytes transformedby mitogens. WI-2 lacked reactivity against normal human RBC's,mature granulocytes, and T and B lymphocytes. The target antigenfor WI-2 had a molecular weight of 43,000 daltons. Specimensfrom patients with leukemia were tested with WI-2. The numberof immature cells was compared with the number of WI-2 reactivecells in the peripheral blood and bone marrow and subjectedto linear regression analysis. There was a highly significant(p<0.001) correlation between the two parameters. The antibodymay be useful in monitoring the progress of the patients andin detecting early relapse in leukemia. ^*Current Address: Tokyo Medical School Hospital, 1st Departmentof Internal Medicine, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo160, Japan.     

Monoclonal antibody characterization of sarcoma-associated antigen p102.

A membrane protein of Mr 102,000 Da (p102) was detected in 51 out of 53 human sarcomas, using a murine IgG1 monoclonal antibody 23-26. Antigen expression in sarcoma histologic subtypes appeared dependent on the amount of fibrous tissue matrix present in the original specimen. High levels of p102 antigen were also found in all sarcoma, carcinoma cell lines and neonatal skin fibroblasts tested. Low levels of p102 were also expressed in membranes from some specimens of melanoma, lung and colorectal carcinoma, first trimester fetus, fat, lung and liver while skin specimens expressed slightly higher levels of antigen. Lectin affinity absorption indicated p102 was mannose containing glycoprotein, isoelectric point (pI) 4.7 and affinity constant of 2.3 x 10(9) M-1, with 5.9 x 10(5) binding sites per cultured human HT-1080 fibrosarcoma cell. The overexpression of p102 in sarcomas and other neoplastic tissues suggests that this protein may be associated with the neoplastic state.     

Monoclonal antibody against mouse CAR following genetic immunization

To broaden our repertoire of monoclonal antibodies against CAR (coxsackievirus and adenovirus receptor), we inoculated mice with an expression vector containing the cDNA encoding human CAR extracellular and transmembrane sequence, and boosted the response by inoculation with soluble human CAR protein produced in E. coli. Of the hybridomas obtained following this immunization protocol, one secreted IgG with exceptional reactivity against mouse CAR. Since CAR has been shown to form dimers, expression of human CAR in cells that express mouse CAR may have stimulated the host immune system to recognize endogenous CAR in heterodimers.     

Monoclonal antibody against human trypsin: production,characterization, and use for diagnosis

A monoclonal antibody (MAb) directed against human immunoaffinity purified trypsinogen has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro-enzyme-linked immunosorbent assay (UMELISA). The MAb was purified by affinity chromatography on protein A-sepharose, and MAb had a high affinity for trypsinogen with the affinity constant equal 2.06 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 but did not react with the same protein after reduction. The antibody seem to be directed against conformational epitope. The MAb obtained was characterized immunologically and used to develop UMELISA for detection Trypsin. This monoclonal assay enabled the detection of 2.8 ng/mL.     

Monoclonal antibodies to a human prostate antigen

Three monoclonal antibodies reactive with a purified extractable Mr 34,000 prostate antigen (PA) have been prepared by fusing splenocytes of BALB/c mice preimmunized with purified PA with the NS1 mouse myeloma cell line. The three antibodies were all of the IgG-1 subclass. The antibodies defined two noncross-blocking unique determinants on PA; each present as one site per molecule. IF3 defined one antigenic site and 2G7 and 1C5 defined another antigenic determinant. All of the antibodies reacted with PA in a solid-phase radioimmunoassay and immunoprecipitated 125I-labeled PA. Absorption and sandwich radioimmunoassays showed PA in prostate tissues but not in tonsil, liver, or kidney. Immunoperoxidase staining of formalin-fixed paraffin-embedded sections of benign prostatic hyperplasia and prostatic carcinoma revealed strong prostate epithelial reactivity. None of the antibodies showed reactivity with prostate membrane preparations. A sandwich radioimmunoassay used 2G7 as a plate coat. 125I-labeled 1F3 was used to detect 5 ng PA per ml in sera of patients with prostate cancer. These results confirm previous observations regarding the specificity of PA and shed new evidence for its intracellular localization.     

Tumor-associated antigen defined by a monoclonal antibody against neuraminidase-treated human cancer cells

A monoclonal antibody, MH-A6, was produced by immunization with a human gastric cancer cell line, MKN 74, treated with neuraminidase. The antigen defined by the monoclonal antibody was detected on various tumor tissues and a limited number of normal tissues in immunoperoxidase assay, and the expression of MH-A6 antigen was not influenced by neuraminidase treatment except for some cases of tumor tissues. Interestingly, neuraminidase treatment enhanced binding of the antibody on some adenocarcinomas, but diminished binding of the antibody on squamous cell carcinomas. Both treatment of the immunizing tissues with trypsin and periodic acid diminished binding of the antibody. In isolation of MH-A6 antigen from MKN 74 cells by the monoclonal antibody coupled-affinity column, the epitope exists on molecules with molecular weights of 30,000 and 72,000, and with an acidic pH range in two-dimensional electrophoresis. CEA and CA 19-9 activities were not detected in purified MH-A6 antigen by solid-phase radioimmunoassay, and the reactivity of the MH-A6 antibody with CEA and CA 19-9 was not detected in enzyme-linked immunosorbent assay. Hemagglutination observed between erythrocytes (Lewisa, Lewisb, or NE-treated) and anti-Lewisa, anti-Lewisb sera, or anti-T-agglutinin (peanut lectin), respectively, was not inhibited by MH-A6 antigen. The results suggest that MH-A6 antigen is a tumor-associated antigen, probably glycoprotein, and different from CEA, CA 19-9, Lewisa, Lewisb, and Thomsen-Friedreich (T) antigen.     

Monoclonal antibody production to a B16 melanoma associated antigen

Eight different rat hybridoma cell lines have been produced which secrete MAbs identifying the melanoma-specific B700 antigen. This antigen has been previously shown to have significant sequence homology to the mammalian serum albumins, but antibodies specifically raised against B700 cross-react only with murine albumin. Each of these MAbs has been shown by Western immunoblotting to recognize an independent epitope on the B700 protein; it is concluded not only that the B700 protein is a highly immunogenic antigen, but also that the sequence homologies between the B700 antigen and murine albumin must occur extensively throughout the molecule.     

Monoclonal antibody to human pancreatic carcinoma cells

A monoclonal antibody (MoAb, SK-930) of the IgG2a subclass to human pancreatic carcinoma cells (MIA-PaCa 2) was obtained by hybridization of spleen cells from immunized Balb/c mice with murine myeloma cells. SK-930 was investigated for reacting in indirect immunofluorescence on FACS against a panel comprising 12 types of different origin. SK-930 reacted with seven out of 11 tumor cells and with one PBL. Immunoperoxidase techniques (ABC method) showed that SK-930 antigen was present on pancreatic adenocarcinoma cells, but could not be detected on normal pancreatic tissue. Immunoprecipitation experiments and SDS-PAGE analysis revealed that SK-930 recognized 134K dalton peptide on tumor cells. These results suggest that SK-930 reacts with a novel pancreatic cancer-associated antigen.