Cancer-associated carbohydrates identified by monoclonal antibodies

"New" carbohydrate structures on the surface of or secreted by cancer cells, identified as epitopes by monoclonal antibodies, are reviewed. These structures may represent the accumulation of precursor chains because of decreased activity of synthesizing enzymes, the production of new oligosaccharides due to increased or aberrant glycosylation of carbohydrate chains, a change in density of carbohydrates on the cell surface, or exposure of chains usually covered by other structures. Alterations in glycolipid synthesis include aberrant fucosylation and/or sialyation of the lacto series, sialylation or fucosylation of the globo series, and sialyation of the ganglio series. Many of these carbohydrate epitopes have become useful for the diagnosis, prognosis, and monitoring of patients with cancer. Some of the important markers include CA 15.3, CA 19.9, CA 50, CA 125, CA 242, MCA, SLEX, etc. Incomplete glycosylation of O-linked mucin oligosaccharide is recognized as the important "cancer antigen" B72.3, which is sialyated Tn. The oligosaccharide components of alpha-fetoprotein, carcinoembryonic antigen, and epidermal growth factor receptor are also reviewed. In many instances the glycosylation seen in cancer cells or their products reflects patterns seen during normal development. Thus, cancer-associated oligosaccharides are oncodevelopmental in nature. The biologic significance of carbohydrates on cell surfaces is not known, but several possibilities include a role in cell to cell recognition, intracellular processing of glycoproteins, cell activation, and ability of cancer cells to metastasize.     

Human tumor-associated antigens identified by monoclonal antibodies

Conclusions Monoclonal antibodies have been obtained that are specific for cell surface antigens of human tumors. Some of these antigens are tissue type specific tumor antigens, most strongly expressed on tumors of the same histological type. The antigens identified by the monoclonal antibodies are primarily normal differentiation antigens that are expressed more strongly on neoplastic cells than on most normal adult cells. For several antigens, the higher degree of antigen expression on neoplastic cells appear to be adequate for diagnostic and therapeutic purposes. Future work in this area is likely to have far-reaching consequences for the clinical handling of human cancer patients.This work was supported by grants CA 14135, 19148, 19149, CA 25558, CA 27841 from the National Institutes of Health, and IM 241A from the American Cancer Society. The authors wish to acknowledge collaboration with Drs. M.-Y. Yeh, R. G. Woodbury, S. M. Larson, and K. Nishiyama     

Human monocyte-histiocyte differentiation antigens identified by monoclonal antibodies

Two distinct differentiation antigens of human myelomonocytic cells are defined using murine monoclonal antibodies. The antigens recognized by antibodies 20.2 and 20.3 are expressed by all cells of the monocyte lineage in both peripheral blood and bone marrow. Cell-sorting experiments demonstrated that histiocytes and immature bone marrow cells with detectable alpha-naphthyl butyrate esterase activity also express both antigens. Within cells of other lineages, the antigens had distinct patterns of expression. Immature myeloid cells were 20.2 negative, but 20.3 positive; whereas mature myeloid cells were 20.2 positive, but 20.3 negative. Nucleated erythroid cells and platelets expressed only the 20.3 antigen. These results indicate that myeloid and monocytic cells share common differentiation antigens with cells of the erythroid and megakaryocytic lineages. The 20.2 and 20.3 antibodies reacted with the leukemic cells from some patients with acute nonlymphocytic leukemia (FAB, M1-M5) and with some cell lines derived from patients with nonlymphocytic leukemia, but not with blast cells from patients with lymphoid leukemia or with lymphoid leukemic cell lines. These antibodies may prove useful in studying the differentiation of bone marrow stem cells, in defining the cellular origins and classification of leukemias, and in the identification of distinct prognostic subgroups of acute nonlymphocytic leukemia.     

Xenopus NK cells identified by novel monoclonal antibodies

Early-thymectomized (Tx) Xenopus frogs, which are permanently deficient in T cells, are used as a model sytem for the characterization of novel monoclonal antibodies (mAb) which identify candidate NK cells at the amphibian level of evolution. Hybridomas, generated from mice immunized with splenocytes from Tx Xenopus following B cell and thrombocyte depletion, were screened by flow cytometry. Three mAb (1F8, 4D4 and 1G5) were identified that stained increased proportions of splenocytes from Tx compared with control frogs. These mAb identified lymphoid populations from Xenopus spleen, liver and gut which, after 48 h culture in growth factor-rich medium, exhibited spontanous killing of MHC-deficient allotumor targets. mAb-defined splenocytes also rapidly induced apoptosis of such tumor targets. Dual color analysis confirmed that NK cells are neither T nor B cells. Cytospins of splenocytes isolated with anti-NK mAb revealed large lymphoid cells with distinct pseudopodia. Immunohistology indicated each anti-NK mAb routinely labeled cells within the gut epithelium but NK cells were difficult to visualize in spleen sections. Western blotting of spleen, liver and intestinal lysates subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that 1G5 reacted strongly with protein bands of approximately 70 - 85 kDa, whereas mAb 1F8 and 4D4 stained less intensely, but identified similar protein bands.     

Phenotype of human alpha-interferon producing leucocytes identified by monoclonal antibodies.

Monoclonal antibodies with specificities for subsets of human leucocytes have been used for the characterization of alpha-interferon (alpha-IFN) producing cells. The production of alpha-IFN was demonstrated to be a function of Ia+ leucocytes. OKT3+ T lymphocytes, BA-1+ B lymphocytes and Leu 7+ natural killer (NK) cells did not contribute to the production of alpha-IFN. OKM1+ monocytes were essential for the production of alpha-IFN in response to bacterial products or leukaemia cells, but were not required for the synthesis of virus- or poly I:C-induced alpha-IFN. The results indicate that alpha-IFN producing cells represent a heterogenous population of cells of the myeloid lineage.     

Mononuclear phagocytes of normal and rheumatoid synovial membrane identified by monoclonal antibodies.

The presence of cells bearing epitopes of the mononuclear phagocyte series was studied immunohistochemically in synovium removed from joints involved by trauma (T), osteoarthritis (OA) and rheumatoid arthritis (RA). Mononuclear phagocytes were the most consistent feature of the inflamed rheumatoid synovium. They shared at least five epitopes expressed by mononuclear phagocytes in other tissues. In OA/T samples, cells bearing markers of the less mature monocyte were present at the surface of the synovial membrane, namely the intimal layer, while those bearing macrophage epitopes were apparent throughout the intimal layer and subintimally. This suggested that maturation of the monocyte population takes place after the monocytes have entered the synovial tissues, settled at the surface, then moved downward into the subintimal layer. The synovial monocytes accounted for all the HLA-D region positive cells in the lining layer.     

Features of synovial membrane identified with monoclonal antibodies.

As part of a study of the characteristics of the synovial membrane which make it susceptible to inflammatory reactions, we tested a number of monoclonal antibodies (MoAb) which revealed novel features of the synovium using tissue from rheumatoid, osteoarthritic and traumatized (mechanically deranged) joints. In a previous study we detected macrophages (Mph) lining the synovial membrane by means of Mph specific and HLA-DR specific MoAb. These may account for the type A synoviocytes. Type B synoviocytes are thought to be fibroblastic and we used an anti-Thy 1 MoAb to identify these cells. Many fibroblasts were seen in a subintimal position but only few in the lining layer, not in sufficient numbers to account for the type-B category of synoviocyte. Staining with a new MoAb, 67, was found to precisely delineate the lining layer. This MoAb was previously seen to react with dendritic reticulum cells (DRC) of germinal centres, cells involved in B lymphocyte activation. However, other MoAb which react with germinal centre DRC did not label the synovial lining layer. Several MoAb revealed features of the vasculature not previously recognized. In rheumatoid samples, MoAb10 labelled small capillaries near the synovial surface and larger vessels deeper in the intima were labelled with a second MoAb, SM phi. This dichotomy of staining was not so apparent in synovium from control osteoarthritis and trauma (OA/T) samples. In addition, the Thy 1 epitope, identified previously on a variety of human cells, was strongly expressed on all vascular endothelium. Finally a new vessel or duct like structure was identified in OA/T samples, located subintimally. These ducts contained keratin, detected with MoAb LE61 and may be the normal counterpart for the rare malignancy, biphasic synovial sarcoma.     

Development of lymphocyte subpopulations identified by monoclonal antibodies in human fetuses

We have used a panel of monoclonal antibodies to examine the development of lymphoid and myeloid sub-populations of cells in thymus, bone marrow, and liver of 16 fetuses from 12 to 16 weeks of gestational age. Pre-B and IgM⁺ B cells were present at a ratio of approximately 2:1 in all of the fetal bone marrow and liver samples; cells of both phenotypes were HLA-DR⁺ but did not express the mature B-cell antigen, HB-2. Cells expressing the myelomonocytic antigen, MMA or Leu-M1, were more frequent in bone marrow (40%) than in fetal liver (10%), and cells expressing the HNK-1 or Leu-7 antigen were rare (<1%) in all of the fetal tissues examined. Each of the T-cell antigens, T1, 4, 5, 6, and 8, was expressed by a majority of thymocytes irrespective of the age of the fetal donor. In contrast, cells with the T1, 4, 5, and 8 antigens were not seen in bone marrow and liver before the 13th week of gestation, and T6⁺ cells were never seen in these hemopoietic tissues. These results suggest that fetal liver and bone marrow precursors in humans do not express these T-cell antigens prior to thymic entry and the onset of thymocyte differentiation.     

Immunocytochemical distribution of a breast carcinoma associated glycoprotein identified by monoclonal antibodies.

A glycoprotein, BCA-225 (Mr 225,000-250,000), has been identified in cells and spent medium of clone 11 T47D breast carcinoma cells by three murine monoclonal antibodies, CU18, CU26, and CU46. The antigen was localized in paraffin sections of 167/178 (94%) Bouin's-fixed human breast carcinoma tissues and few other carcinomas (1/8 lung [squamous], 4/4 uterine cervix) in an intracellular pattern, whereas an apical or glycocalyx distribution was seen in several normal tissues, benign lesions, and malignant tumors. Although the immunocytochemical staining patterns observed with these antibodies have many similarities to those described with other previously reported monoclonal antibodies, notable differences include the lack of reactivity of CU18, CU26, and CU46 with lactating mammary gland and with gastrointestinal malignancies. BCA-225 binds to wheat germ lectin, not to concanavalin A, but monoclonal antibody binding does not appear to involve the carbohydrate component of the molecule. The frequency of the immunocytochemical detection of BCA-225 in breast carcinomas and its restricted distribution in other human tissues suggest considerable clinical potential for this antigen and its corresponding monoclonal antibodies.     

Characterization of monoclonal antibodies to chicken gizzard talin.

Eleven monoclonal antibodies against chicken gizzard talin, a major focal adhesion protein, have been produced. In order to determine the degree of homology between talin molecules from different sources, these antibodies were used to immunolocalize talin in five different vertebrate species. Three representative talin antibodies are described in this report.     

T-lymphocyte subpopulations identified by monoclonal antibodies after human bone-marrow transplantation

Peripheral T-lymphocyte reconstitution after bone-marrow transplantation, 12 allogeneic and 13 autologous, was studied by indirect immunofluorescence assay using mouse monoclonal antibodies. Abnormal counts were detected in the two major sub-populations of T-cells i.e. helper and T cytotoxic-suppressor lymphocytes, defined by monoclonal antibodies (alpha Leu 3a and B 9.2), and in DR antigen-positive T-cells. The pattern of T-lymphocytes replenishment was identical for both types of transplant, and was not affected by Graft Versus Host disease (GVHD).     

Chicken thymocyte-specific antigens identified by monoclonal antibodies: characterization and distribution in normal tissues and in tumoral tissues from Marek's disease chicken

Monoclonal antibodies (MAbs) were obtained against purified thymocyte membrane extracts. Five MAbs TA3, TB1, TB6 (IgG1), TC4, and TA1 (IgG2a), were tested by immunofluorescence and by immunoperoxidase tests against normal cells from different organs, Marek's disease (MD) cell lines, and MD tumoral cells from chickens. Three of them, TA3, TB1, and TB6, reacted exclusively with lymphoid cells in both cortical and medullary areas of the thymus and with less than 8% bursa cells. They identified a protein of apparently 40 kD. The other two revealed antigenic determinants on most medullar thymocytes and some cortical thymocytes, and on some splenic and peripheral blood lymphocytes. They were positive with MD cell lines and cells deriving from MD tumors. TC4 and TA1 detected molecular masses of about 110 kD and 16 kD, respectively. No MAbs reacted with erythrocytes, bone marrow, liver, brain, and skin cells. Not all of the tested cells were stained after contact with an anti-chicken immunoglobulin serum. In this paper, we determine a specific antigen restricted to T cells from thymus and different markers belonging to the mature T cells. The latter are also present on MD cell lines and MD tumoral cells.     

Protein polymorphism of human IL-18 identified by monoclonal antibodies

Six mAbs were raised against human "functionally inactive" recombinant IL-18, ELISA for determination of "functionally inactive" forms of IL-18 were established using two of these mAbs (#21 and #132), and inactive species of IL-18 protein were examined with human blood plasma and macrophages (Mp). In 6-day GM-CSF-treated monocytes, namely Mp, the mAb #21 recognized the IL-18 proform (24 kDa) and a 48 kDa dimer by immunoblotting. In contrast, only the 24 kDa species was detected as a relatively faint band with a commercial mAb against "active" IL-18. No IL-18 species was detected in premature monocytes. Thus, the dimeric IL-18 was produced in Mp and detectable with the mAb we established. In blood plasma of normal subjects and patients, the #21-recognizable IL-18 was also detected by ELISA, the levels of which were not consistent with those obtained with the commercially available kit for determination of "functionally active" IL-18. We designated the former as type 2 and the latter as type 1. Strikingly, IL-18 type 1 was detected in all volunteers while type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 were high (10-100 ng/ml) compared to those of type 1 (0.02-0.55 ng/ml) in their blood plasma. In patients with atopic dermatitis, the mean value of type 1 was high (200 ng/ml) compared to those of normal subjects (0.122 ng/ml) and patients with lung cancer (0.113 ng/ml). Production of high type 1 may be associated with an immunomodulatory state in atopic dermatitis. The levels and frequencies of IL-18 type 2 were not significantly changed among these populations. Hence, large amounts of type 2 species are produced in monocyte-Mp differentiation, and their levels and frequencies are unchanged in blood plasma irrespective of the levels of type 1.     

Distinct epitopes on Ik gene products identified by monoclonal antibodies

Analysis of reactivity of monoclonal anti-Ia^k alloantibodies, obtained by fusion between NS 1 myeloma and spleen cells from alloimmune A.TH mice, permitted the identification of 9 distinct determinants of the I^k gene products. Competitive binding experiments indicated that 2 private epitopes (defined by H 8–109.13 and H 8–138.4 antibodies) of the I-A^k product could be separated, thereby apparently splitting the Ia.2 specificity. A public determinant of the I-A^k molecule (identified by H 8–15.9 antibody) was found expressed not only on the 1-A products of the H-2^b, H-2^d, H-2^ja, H-2^P and H-2^q murine haplotypes, but also on human HLA-DR antigens. Four determinants of the I-E/C^k antigen (defined by H 7–8.26, H 10–81.10, H 10–93.2 and H 8–86.2 antibodies) had a strain distribution analogous to the Ia.7 specificity. However, competitive binding experiments, and the cross-reactivity pattern with la-like antigens from other species (e.g. human HLA-DR antigens) indicated that these antibodies detected distinct determinants on the I-E/C^k molecule, thereby subdividing the broad Ia.7 specificity. Two other determinants (defined by H9–14.8 and H9–15.4 antibodies) had a strain distribution that did not permit a precise assignment to a given Ia antigen, even though preliminary data suggested that they could detect separate determinants on the I-E/C^k product. All these monoclonal antibodies identified membrane antigens with the expected Ia tissue distribution pattern, and most of them could precipitate a molecule containing two chains of 28 kD and 35 kD, from mouse spleen cell lysates.     

Characterization of an apparently conserved epitope in E- and P-selectin identified by dual-specific monoclonal antibodies.

E- and P-selectin recognize a wide and overlapping range of oligosaccharide ligands including sialyl-Lewis X (sLeX) through their highly homologous C-type lectin domains. We report that an epitope apparently conserved between E- and P-selectin is functionally involved in ligand recognition although distantly located from the conventional carbohydrate binding site. We found that a previously established anti-E-selectin monoclonal antibody (mAb), 1.2B6, is cross-reactive with P-selectin, and that the 1.2B6 epitope is in the C-type lectin domain and identical to or overlapping with an epitope recognized by other independently established anti-E- and P-selectin dual-specific mAb. The epitope has been mapped by others to a region distant from the previously identified carbohydrate binding site of E-selectin in its three-dimensional structure. Nevertheless, it is of note that all dual-specific mAb, including 1.2B6, inhibited E- or P-selectin-mediated cell adhesion and also binding to sLeX. Engagement of the apparently conserved epitope by the dual-specific mAb may lead to inhibition of the ligand binding ability of E- and P-selectin by a previously uncharacterized mechanism(s) rather than by direct inhibition of sLeX binding to the hitherto identified ligand binding site.     

Monocytes and other infiltrating cells in human colorectal tumours identified by monoclonal antibodies

Monoclonal antibodies have been used to examine the patterns of infiltrating cells in colorectal tumours staged according to Dukes' classification. MAbs reacting with monocytes, but not tissue Mph, revealed a six-fold increase in monocytes in metastasizing C tumours compared to normal gut. The non-metastasizing B tumours could be divided into one group containing increased numbers of monocytes, and a second group comparable to control gut. T-cell numbers were increased in all tumour stages by an average 1.4-fold, which disguised the lack of consistent pattern in T-cell subset ratios in the tumour stromal tissue. However, in the tumour epithelium, there was a constant decrease in the Ts + c cell subset and a subsequent alteration in T-cell subset ratio in favour of Th + i cells. With the progression from Dukes' Stage B to C, there was an increase in the proportion of monocytes and T cells which were activated as detected by mAbs to the C3b receptor and IL-2 receptor, respectively. These observations suggest that an immune response is in progress in these colorectal tumours and that it is most active in the metastasizing Dukes' C tumours. Whether this response is elicited by the tumour or other elements, whether it is detrimental to tumour growth, or whether it is actively assisting tumour growth and possibly dissemination, are matters of conjecture.     

Stage-specific, strain-specific, and cross-reactive antigens of Leishmania species identified by monoclonal antibodies.

The fusion of NS-1 myeloma cells with spleen cells from mice chronically infected with Leishmania tropica resulted in nine clones of hybridomas producing monospecific antibodies to membrane antigens of L. tropica. One of the antibodies (L-5-85) bound specifically to the promastigote form of the parasite, and the remaining eight recognized antigens shared by the promastigote and amastigote of L. tropica. Four of the antibodies (L-5-16, L-5-34, L-5-44, and L-2-3F11) detected parasite antigens on the surface of L. tropica-infected macrophages. Common antigens shared by L. tropica, L. mexicana, and L. donovani were identified as well as one antigen apparent on most Leishmania spp. and present also in Crithidia fasciculata. Two monoclonal antibodies (L-5-27 and L-5-41) were found to bind only to strains of L. tropica from simple cutaneous leishmaniasis. A special property shared by these two antibodies was the inhibition of parasite growth in macrophages in vitro.     

Two distinct antigens on chicken thymocytes defined by monoclonal antibodies

Two distinct antigens expressed on chicken thymocytes were defined by monoclonal antibodies designated as Lc-1 and Lc-2. Lc-1 (IgGl) reacted with 80% of the thymocytes (mostly cortical and medullary thymocytes) and Lc-2 (IgM) reacted with 40% of the thymocytes (mainly cortical thymocytes). Lc-1 reacted with 1% of the spleen lymphocytes, but the antibody was nearly nonreactive with cells from peripheral blood leukocytes, bursa and bone marrow. Lc-2 reacted with only small percentages of spleen and bursa cells, and with very few cells from peripheral blood leukocytes and bone marrow. This antibody reacted with a portion of concanavalin A stimulated spleen lymphocytes. When Marek's disease-derived T-lymphoblastoid cell lines were tested for their reactivities with monoclonal antibodies, Lc-1 reacted with none of the cell lines tested, whereas Lc-2 reacted with four of the six cell lines tested. Antigens recognised by Lc-1 and Lc-2 were first found in chick embryonic thymus on day 13 of incubation, after which the number of cells positive for Lc-1 and Lc-2 rapidly increased, reaching young adult levels by days 15 and 14 of embryonic life, respectively. Lc-1 precipitated materials with apparent molecular weights of 60 and 120 kDa from radioiodinated thymocytes.     

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.     

A pomona serogroup-specific, agglutinating antigen in Leptospira, identified by monoclonal antibodies

Monoclonal antibodies were produced by hybridoma cell lines derived by fusion of mouse NS-1 myeloma cells with splenocytes from mice immunized with Leptospira interrogans serovar pomona. One hybridoma (A3) produced an IgG2a antibody which agglutinated all leptospires of the Pomona serogroup but not leptospires representative of serovars of any other serogroup. The antibody precipitated in immunodiffusions with either alkali- or phenol-extracted lipopolysaccharide and also with the TM antigen of Yanagawa et al., indicating a common determinant on both antigens. A3 antibody opsonized viable leptospires for phagocytosis by mouse macrophages in vitro.