Monoclonal antibodies against antigens expressed on human hepatocellular carcinoma cells

Monoclonal antibodies with selectivity for human hepatoma cell lines were produced by immunizing BALB/c mice with human hepatoma cell lines, HA22T/VGH or Hep 3B, and fusing sensitized mouse spleen cells with mouse myeloma cells. Two monoclonal antibodies recognizing antigens present only on human hepatoma cell lines were investigated. The monoclonal antibody IB1 was found to react with 3 of 9 hepatoma cell lines. Monoclonal antibody 9B2 reacted with all nine hepatoma cell lines. None of the other 20 cell lines tested was bound by IB1 and 9B2. The immunoperoxidase staining of monoclonal antibodies on frozen sections of paired hepatoma and normal liver tissues from the same individuals were studied. Antibody IB1 reacted with 3 of 13 hepatoma tissues, but with none of the normal liver and other tissues, and antibody 9B2 was reactive with antigens appearing on the bile canalicular domain of hepatoma and normal liver tissues. The antibody 9B2 stained no normal tissues with the exception of proximal tubules of kidney. Radioimmunoprecipitation tests identified two antigens reacting with 9B2. The major antigen had an apparent molecular weight of 140,000 and a minor one of 130,000. Therefore, antibody IB1 seems to be specific for antigens present on a group of human hepatoma cells and may be useful for classification and diagnosis of human hepatomas. Antibody 9B2 is quite specific to human liver cells and may be used to provide clues for the characterization of tumor cell lines, identification of metastatic tumors with hepatocytic origin, and study of the structure and function of bile canaliculi.     

Polyreactivity of Human Monoclonal Antibodies: Human Anti-Rh Monoclonal Antibodies of IgM Isotype Are Frequently Polyreactive

The specific aim of this study was to characterize human anti-Rh monoclonal antibodies cross-reacting with self-antigens. We studied supernatants from man-mouse hybridomas and from lymphoblastoid cell lines. Man-mouse hybridomas were established by fusion of peripheral blood lymphocytes from healthy individuals recently immunized against Rh alloantigens, with mouse myeloma (or man-mouse heteromyeloma) cell lines. Lymphoblastoid cell lines were produced by Epstein-Barr virus induction of lymphocytes from identical sources. Of the 55 monoclonal alloantibodies studied, 11 also reacted with intracellular self-antigens as demonstrated by immunofluorescence assay on cryostat sections of human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 11 cross-reacting mAbs 10 were IgM). The cross-reactivities of these monoclonal antibodies were ascertained by absorption of alloreacting antibodies with red blood cells. Similar results were obtained on a panel of purified cellular antigens by ELISA. The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells. Therefore, polyreactive autoantibodies present in sera from healthy individuals may be the result of an immune response against foreign antigens.     

Human prostate specific and shared differentiation antigens defined by monoclonal antibodies.

Splenic lymphocytes of BALB/c mice immunized with membrane-enriched fractions of human benign prostatic hyperplasia tissues were fused with the NS-1 light chain-secreting murine myeloma cell line. This generated hybridoma cultures that secreted immunoglobulins reactive in solid-phase radioimmunoassays with membrane preparations of prostatic tissues but not with membrane preparations of apparently normal human liver, spleen, thymus, or erythrocytes. After further screening of immunoglobulin reactivities and cloning of cultures, eight monoclonal antibodies were chosen that demonstrated reactivity with human prostate tissues. These monoclonal antibodies could be placed into at least three major groups--epithelium-specific, polyepithelial, and stroma-specific--on the basis of differential binding to the surfaces of various component cells in the prostate and other epithelia. Two antibodies defined unique protein antigens specific for prostate epithelia that were not crossreactive with prostatic acid phosphatase or the recently described "prostatic antigens." These antibodies also detected antigens on malignant prostate tissues as well as other malignant tissues. Four antibodies defined three unique polyepithelial protein antigens (two of the antibodies were different isotypes defining the same protein). Each of the polyepithelial antigens was expressed on a different spectrum of normal epithelial tissues. Two displayed brain tissue crossreactivity, one was present on pancreas, and one was present on platelets. The two antibodies that detected prostatic stromal protein antigens showed different spectra of reactivities. One antibody reacted with apparently all prostatic stromal cells as well as endothelial cells in the prostate and other organs. The other antibody apparently reacted with all prostatic stromal cells as well as myoepithelial and muscle cells in other organs.     

Monoclonal antibodies to human pancreatic carcinoma

Hybridoma cultures were produced by the fusion of SP2 mouse myeloma cells with spleen cells from mice immunized with human pancreatic carcinoma cells. After limiting dilutions, three monoclonal antibodies, YPan1, YPan2, and YPan3, which bound to immunizing cells but not to normal human skin fibroblasts, were further characterized. The three monoclonal antibodies were found to bind to all seven pancreatic carcinoma cell lines but not to other carcinoma cell lines tested except some colon carcinoma cell lines. When human tissue sections were examined using immunohistochemical techniques, the three monoclonal antibodies identified antigens in the pancreatic carcinomas and some normal pancreases, but only YPan1 showed strong positive staining. No cross-reactivity was seen in sections of other carcinomas tested except some colon carcinomas. The results suggest that these monoclonal antibodies may be usefully applied to the detection of pancreatic carcinomas.     

Presence of a 16/6 related human anti-DNA common idiotype (SA1) in the anticardiolipin antibodies of patients with primary antiphospholipid syndrome.

Patients with primary antiphospholipid syndrome (PAPS) have few or no autoantibodies, other than the antiphospholipid antibodies (aPL) that could be natural autoantibodies encoded by germline genes. Some of the autoantibodies marked by the human anti-DNA common idiotype 16/6 have been found to be encoded by unmutated germline genes. Hence, we tested the sera of 19 patients with PAPS for the presence of the 16/6 idiotype which has also been found to be expressed on antibodies that bind cardiolipin. For this we used an ELISA method with antiserum against the SA1 idiotype which recognizes the 16/6. Five of our patients had the idiotype in at least one serum. Among the patients there was one with a variant of PAPS with hemolytic anemia and an IgM antibody to phosphatidylcholine that is akin to the natural autoantibody of normal mice encoded by germline genes VH11 and VH12. Inhibition studies with ssDNA, dsDNA and cardiolipin revealed that all 3 antigens decreased the serum levels of the SA1 idiotype despite absence of detectable anti-DNA antibodies by other methods. Our findings suggest that within the B cell clones that produce aPL in patients with PAPS there are some that produce immunoglobulins bearing 16/6 related idiotypes. This could indicate that some of the aPL present in patients with PAPS derive from natural autoantibody producing cell clones.     

Generation of multiple monoclonal antibodies for diagnostic use from a single hybridoma fusion

BALB/c mice were exposed simultaneously to three nonrelated immunogens, myelin basic protein, uridyl-galactosyl transferase, and tissue obtained from a formalin-fixed, paraffin-embedded block containing a pilomatrixoma. Standard hybridoma techniques were used and antibody generation assayed using an unlabelled antibody biotin-avidin method with sections of human cerebellum, liver, and pilomatrixoma as the substrates. Using the above assay, clones were selected that secreted antibodies with selective specificities for each of the immunogens. By ELISA assay, the monoclonal antibodies reacting with cerebellum also reacted to the myelin basic protein preparation used as immunogen and the monoclonal antibody reacting to hepatocytes bound to the preparation of uridyl-galactosyl transferase used as immunogen. Our data suggests that the generation of monoclonal antibodies with a variety of diagnostic applications can be obtained from a single fusion following immunization with multiple nonrelated antigens, requiring considerably less laboratory cost and effort than would be required to obtain similar monoclonal antibodies in separate fusions.     

Production of monoclonal antibodies to the cuticle of advanced third-stage larva of Gnathostoma spinigerum

This study has demonstrated that sera from Balb/c mice infected with live advanced third-stage larvae (aL3), but not those immunized with crude larval extract, immunoprecipitated the 25-kDa protein from surface-iodinated extract of aL3. Hybridoma cell lines derived from spleen cells of an infected mouse secreted antibodies that reacted with several tissue of aL3 including the esophagus, intestine, muscle and cuticle by immunofluorescence assay. However, none of the cuticle-positive hybridoma cell lines produced antibodies that recognized surface-iodinated protein of aL3 by immunoprecipitation. Western blot analysis showed that monoclonal antibodies (MAbs) secreted by clones derived from one of the cuticle-positive hybridoma lines recognized proteins of molecular weights ranging from 55-96 kDa. The MAbs most likely reacted with the collagenous component of the cuticle.     

Retention of an idiotypic determinant in a human B-cell lymphoma undergoing immunoglobulin variable-region mutation.

Tumor cells from a patient with B-cell lymphoma were fused with a mouse myeloma cell line. A set of heterohybridomas was thus derived, each of which represented a separate clonal derivative from the tumor cell population. The immunoglobulins secreted by these cell lines reacted variably with a panel of anti-idiotypic antibodies, indicating that the tumor was heterogeneous; however, one antibody, 4D6, reacted strongly with the product of all the heterohybridomas. cDNA for the immunoglobulin heavy chain variable-region genes expressed in these heterohybridomas was cloned and sequenced. Comparison of these sequences indicated that the cells expressing them were clonally related but that they had undergone considerable mutation. Despite mutation, the cells in this tumor population continued to express a functional immunoglobulin molecule and to retain, over a span of 3 years, the idiotypic determinant defined by the 4D6 monoclonal antibody. Thus a selective force existed within the host to retain tumor cells bearing an immunoglobulin molecule with a particular idiotypic structure.     

Analysis of two new leukemia-associated antigens detected on human T- cell acute lymphoblastic leukemia using monoclonal antibodies

Two monoclonal antibodies (anti-3-3 and anti-3-40) were produced, which identify two new leukemia-associated antigens. Both antibodies reacted with most cell lines derived from patients with T lymphoblastic leukemia (T-ALL), but were not detected on suspensions of normal hematopoietic cells (including thymocytes) by cytotoxicity, absorption, or indirect immunofluorescence assays. Analysis of fresh leukemic cells indicated that anti-3-3 only reacted with T-ALL cells, while anti-3-40 also reacted with some non-T, non-B ALL cells and a few acute myelocytic leukemia (AML) cells. The 3-40 antigen was also found histopathologically in frozen sections of several normal tissues, including the epithelial cells and a few lymphoid cells of the thymus, and some malignant tissues. The 3-3 antigen was not found in any tissue studied. A "double absorption"assay provided additional serologic evidence that the two antibodies identify different antigenic determinants. Biochemical analysis indicated that the molecules immunoprecipitated by anti-3-3 and anti-3-40 have molecular weights of 35,000-40,000 daltons. This study demonstrated that the 3-3 and 3-40 antigens are markers for human T-ALL and can be used along with the normal T-lymphocyte antigen, 3A1, to discriminate T-ALL from cutaneous T-cell lymphoma (CTCL), adult T-cell leukemia (ATL), and T-cell chronic lymphocytic leukemia (T-CLL).     

Specificity analysis of human monoclonal antibodies reactive with cell surface and intracellular antigens.

Over 4350 human Ig-secreting hybrids have been generated through the fusion of human lymphocytes with NS-1 (mouse), LICR-2, SKO-007, GM4672, or UC729-6 (human) myeloma and lymphoblastoid cell lines. NS-1 proved to be the most satisfactory fusion partner, and 83% of the stable Ig-secreting clones were derived from NS-1 fusions. Three hundred five hybrids produced human monoclonal antibodies (hmAb) reactive with cell surface or intracellular antigens expressed by cultured human tumor cell lines, and 111 of these have undergone detailed serological specificity analysis. Several general points have emerged from our study of hmAb: A significant proportion of the human B-cell clones produce antibody reactive with cellular antigens. The majority of these antigens have an intracellular location and are broadly distributed. Intracellular and cell surface differentiation antigens and other antigens with restricted distribution have been defined by hmAb, including two cell surface antigens not detected on normal cells. The relationship of these findings to cancer is unclear, as hmAb reactive with antigens showing distinctive distribution have been generated from the lymphocytes of normal individuals as well as tumor-bearing patients.     

Monoclonal antibody to a human osteogenic sarcoma cell line

Monoclonal antibodies against a human osteogenic sarcoma cell line were prepared by production of a somatic cell hybrids between the spleen cells from U-393OS--immunized mice and the mouse myeloma cells SP2/0. From 7 producing and well-growing clones only one--B-0S12--produced antibodies, reactive preferentially with osteosarcoma cells as identified by binding second antibodies and 125I-labeled Protein A. This antibody was tested against a panel of normal and tumor cell targets to determine the pattern of the antigen detected. The monoclonal antibody reacted strongly against U-3930S cells and another human sarcoma in vitro and more weakly against human fibroblasts, peripheral lymphocytes, red blood cells and was negative against mouse fibroblasts. When tested against a panel of unrelated human tumor cell lines, B-0S12 antibody was positive with melanoma cells and negative with cells from bladder, cervix and mammary carcinoma. These cross reactions suggested, that the antibody is reactive with a protein, expressed on different tumor types. This protein is not expressed on the cell surface and is probably associated with cytoskeleton, as revealed by immunofluorescence experiments. Western-blot analysis of a cytoskeletal preparation of U-3930S cells suggests, that B-0S12 antibody recognizes a protein with Mr 55 kD. Further studies are needed to characterize the molecules, carrying the epitope, identified by this monoclonal antibody.     

Multiplicity of newly established monoclonal antibodies against hepatocellular carcinomas

To identify tumour-associated cell surface antigens of human hepatocellular carcinoma (HCC), we established three monoclonal antibodies against a well established human HCC line, hu-H2. One of these, designated as KY-1, reacted with several HCC lines and a majority of peripheral blood lymphocytes. A second monoclonal antibody, KY-2, reacted only with HCC cell lines but not with others. With this KY-2 antibody, which was highly specific for HCC, HCC tissue was positively stained. A third monoclonal antibody, KY-3, reacted with HCC lines and many other malignant cell lines, but not with non-malignant cells. These results indicate that at least three different tumour-associated molecules are expressed on human HCC.     

Study of antibodies against human melanoma produced by somatic cell hybrids.

We fused spleen lymphocytes obtained from mice immunized against a human melanoma cell line and melanoma-mouse hybrid cells with the P3 X 63 Ag8 mouse myeloma in order to produce hybrids secreting antibodies against a human melanoma. Antibodies secreted by individual hybrids were tested for their reaction with a panel of human melanoma, colorectal carcinoma, and normal cells in an indirect radioimmunoassay, and they displayed different specificities and crossreactivities. Some reacted only with melanomas, whereas others crossreacted with normal human or human colorectal carcinoma cells. By analysis of competitive binding of mixtures of monoclonal antibodies, it was possible to delineate different epitopes on melanomas. Hybrids growing in nude mice and producing antimelanoma antibody suppressed growth of melanoma tumors.     

Spectrin autoantibodies in normal human serum and in polyclonal blood grouping sera

Naturally occurring spectrin autoantibody was detected in normal human sera and in polyclonal blood grouping sera by the immunoblotting technique. The autoantibody seems to be IgG, stable, of high titre and low affinity. It was detected in all sera tested. Preparations of monoclonal antibodies directed against some blood group antigens and anti-A1 lectin reagent were devoid of spectrin autoantibody as expected. This autoantibody may be instrumental in clearing red cell membrane components from the circulation during haemolysis. Care must be exercised in studies designed to determine the association of some blood group antigens with the red cell membrane skeleton, when using polyclonal sera which contain spectrin autoantibodies in addition to the antibodies against the blood group antigen in question.     

Monoclonal antibodies specific for Hantaan virus.

Six hybridoma cell line producing monoclonal antibodies to Hantaan virus were established by fusion of NS-1 mouse myeloma cells with spleen cells of mice immunized with Hantaan virus strain 76-118. The specificity of these monoclonal antibodies was established by immunoblotting analysis and immunofluorescence. Five of the clones reacted with antigens on the cell surface and in the cytoplasm, and one clone reacted with a determinant expressed only in the cytoplasm of the infected cells. Two of the clones produced antibodies that reacted with a Mr 50,000 polypeptide in virus-infected cellular extracts and purified virus preparations. The monoclonal antibodies were used to examine the antigenic relationship among Hantaan virus strains and between Hantaan virus and Prospect Hill virus and the virus of nephropathia epidemica. Three antibodies were capable of distinguishing between the Lee strain and the 760-118 strain of Hantaan virus and three additional antibodies reacted with determinants shared by both virus strains. None of the six reacted with Prospect Hill virus or the virus of nephropathia epidemica.     

Production of Stable Human-Mouse Hybridomas Secreting Monoclonal Antibodies against Rh D and c Antigens

Peripheral blood lymphocytes from donors immunized against Rh antigens were fused with mouse myelomas and heteromyelomas in order to obtain human-mouse hybridomas secreting antibodies specific for these antigens. Three cell lines secreting anti-D IgG and two secreting anti-c IgM were stabilized and produced immunoglobulins for several months. These human monoclonal antibodies were evaluated as reagents for Rh phenotyping. Their complementary activity towards weak D and partial D antigens is examined.     

Production and characterization of human monoclonal antibodies against core protein p25 and transmembrane glycoprotein gp41 of HIV-1

With a view to obtaining human monoclonal antibodies against HIV-1 antigens we used the Epstein-Barr virus immortalization technique to induce lymphoblastoid cell lines from peripheral blood lymphocytes of 10 people who were seropositive for HIV-1 and had no clinical symptoms. A number of polyclonal lines were obtained which synthesized antibodies against most of the major proteins and glycoproteins of HIV-1. Three stable clones were characterized for class, secretion characteristics and specificity. Two of these clones produce antibodies which react with gp41, and the third reacts with p25. One of the anti-gp41 antibodies was found to have neutralizing activity.     

Monoclonal antibodies to membrane components of human breast cancer cell line MDA-MB-231. Production and reactivity with various cells in culture

Hybridoma clones were established by fusing spleen cells from mice hyperimmunized with human breast cancer cells of MDA-MB-231 line with murine myeloma cells P3-X63-Ag8-653. Ten permanent hybridomas were stabilized. The monoclonal antibodies of three of them, i.e. HBCA-6, HBCA-4 and HBCA-12 were tested against 20 various established cell lines. The most restricted binding properties showed HBCA-12 antibody which reacted positively only with two types of target cells. The cross-reactivity of HBCA-12 with human breast cancer cell line MDA-MB-231 and human myeloma derived cells ARH-77 is discussed in view of the pertinent target structure, i.e. differentiation antigens, allospecific antigens, hormone receptors and shared tumor associated antigens. It was shown that the target structure for HBCA-12 is localized on the cell surface.     

Monoclonal antibodies against MHC class II antigens elicited with a human non-T, non-B acute lymphoblastic leukemia cell line

A series of seven monoclonal antibodies recognizing cell surface antigens of similar distribution on a panel of leukemia/lymphoma and lymphoblastoid human cell lines was prepared by hybridoma technique after immunization with non-T, non-B acute lymphoblastic leukemia cell line REH. Immune reactivity of these monoclonal antibodies with B-lymphoblastoid and lymphoma cell lines, as well as with non-T, non-B leukemia cell lines but not with T-leukemic and myeloid leukemic cell lines (with the exception of myeloid leukemia cell line KG 1) was demonstrated by indirect immunofluorescence. No reactivity was observed with the examined human non-hemopoietic tumor cell lines, except melanoma cell lines HMB-2 and SK 1477. These antibodies immunoprecipitated a similar cell surface bimolecular glycoprotein complex consisting of two glycosylated chains (gp30, 35), as demonstrated by immunoprecipitation of cell surface 125I-lactoperoxidase radioiodinated, periodate/tritiated borohydride radiolabeled and 35S-methionine metabolically radiolabeled proteins of REH cells. These properties, typical of MHC class II antigens correspond also to immunoperoxidase staining of lymph nodes with some selected antibodies.     

Immunochemical diagnosis of hepatic localizations in malignant lymphoid hematologic diseases. Study of 80 cases

The diagnostic value of immunohistochemistry using monoclonal antibodies was assessed in 100 liver biopsy specimens. The majority of these cases were hepatic localizations of lymphoid malignancies. Ten normal and reactive inflammatory liver biopsies were used as controls. Some monoclonal antibodies directed against leukocyte antigens revealed unexpected reactivities with normal liver structures: biliary tract (anti-CD10, anti-B MB2) and hepatocytes (anti-B LN1). In 12/17 cases of hepatic involvement by large cell malignancy, immunohistochemistry allowed the diagnosis of non Hodgkin's lymphoma (NHL); the remaining 5 cases were metastatic undifferentiated carcinoma. It was difficult to differentiate small cell liver NHL from reactive inflammatory infiltration. New anti-B (MB1, MB2, 4KB5, LN1 and LN2) and anti-T (MT1 and UCHL1) monoclonal antibodies suitable for use on paraffin sections were of value to phenotype NHL when only fixed material was available. But, information was too limited to distinguish malignant from reactive infiltrates. Immunohistochemistry on frozen sections was often necessary to diagnose inflammatory infiltrates and to phenotype NHL. Most NHL were of B cell origin (11/13 cases) and showed monotypic surface immunoglobulins as well as B cell-associated antigens (CD22+). The expression of the T CD5 antigen by B-cell NHL may have some diagnostic value. When monotypic surface immunoglobulins could not be demonstrated (due to background staining) the expression of this antigen by B lymphocytes was considered to be highly indicative of their neoplastic nature. Hairy cell leukemia exhibited a pathognomonic phenotype on frozen sections (CD11c+, CD22+, CD25+). T NHL were rare (2 cases) and difficult to diagnose due to the lack of clonal markers. The diagnosis of Hodgkin's disease in liver (15/20 cases) was facilitated by using paraffin sections of both monoclonal antibodies anti-CD15 (Leu M1) and anti-CD30 (Ber-H2) which detect fixation-resistant antigens expressed by Sternberg cells.