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The immunoanatomy of the human kidney and urinary tract has been analyzed by a panel of mouse anti-human monoclonal antibodies that define specific domains and structures. The differentiation antigens detected by these monoclonal antibodies represent a series of glycoproteins characteristic of different cell types. They differ from the blood group antigens and appear to be distinct from other antigens previously described within the kidney or urinary tract. The antigens recognized by these monoclonal antibodies represent an immunohistologic dissection of the human nephron. These antibodies have a broad range of potential applications in studying embryogenesis and pathogenesis of nonneoplastic and neoplastic diseases of the human kidney and urothelium.

Three human differentiation antigens (MU78, MT334, and MQ49) have been defined by mouse monoclonal antibodies developed from mice immunized with ovarian carcinoma cell lines. Their distribution was determined on 148 cultured cell lines of various histologic types and on frozen sections of 16 normal tissues. MU78 was found in fibrillar structures in soft connective tissue with a distribution resembling that of elastin fibers; however, elastin fibers in elastic cartilage and in the aorta were nonreactive. MU78 was detected in cultured carcinoma cells of various histologic types, where it had a nonfibrillar, cytoplasmic distribution, but was not detected in normal epithelial cells in frozen sections. Cultured fibroblasts, astrocytomas, melanomas, and lymphomas did not contain MU78. In cell lines, MU78 appears to be a protein of 2000-5000 daltons. The other two antigens, MT334 and MQ49, are both mucin-like molecules, and the determinants are probably carbohydrate in nature. Of the normal tissues examined, MT334 was detected only in goblet cells of the colon, though it was present in a variety of carcinomas in culture. It was detected as both a cytoplasmic and secreted component. MQ49 was detected in various secretory epithelial cells, in Hassall's corpuscles in the thymus, and in cultured carcinomas of various histologic types. It was found on the cell surface as well as in the cytoplasm and is present on a glycolipid as well as on a sulfated mucin. These results, and results of other recent studies, demonstrate the importance of mucin-like molecules as antigens in epithelial cells and secretions.

The antigenic phenotype of malignant cells from ascites of patients with epithelial ovarian cancers was examined and compared to that of their primary and metastatic sites. Cell-surface antigens on frozen sections of primary and metastatic tumors and frozen cell pellets from ascites were analyzed with a panel of murine monoclonal antibodies using the indirect immunoperoxidase method. In addition, ascites cells cultured with and without autologous cell-free ascitic fluid were evaluated by immunofluorescence. The pattern of antigen expression detected on fresh and cultured ascitic epithelial cells was shown to be identical to the expression in autologous solid tumor tissues. When placed in culture, malignant epithelial cells generally persisted for a minimum of one, but no more than five, passages. Addition of autologous ascitlc fluid to cultures of ascites cells did not alter the phenotype of the epithelial tumor cell population and did not enhance the growth of these cells. From one culture of ascites cells a permanent malignant epithelial ovarian cancer cell line (designated SK-OV-8) was established. The demonstration that epithelial tumor cells found in ascites of patients with epithelial ovarian cancer have the identical antigenic phenotype as their solid tumor counterpart, at least for the panel of antigens studied, may be useful in planning imaging and therapeutic trials with monoclonal antibodies. Copyright 1993, 1999 Academic Press

Seventeen monoclonal antibodies derived from fusions with spleen cells of mice immunized with established culture lines of renal cancers identified nine cell-surface antigenic systems. Six of the systems (gp160, S25, gp120r, gp120nr, gp115, and V1) represent antigens not previously described. The other three systems are related to HLA-A, -B, and -C heavy chain and A and B blood group antigens. The most restricted of the newly described antigens are gp160, S25, and gp120r. These determinants are found only on cells of renal origin, both normal and malignant, and represent differentiation antigens of human kidney. In addition to the difference in the molecular weight of two of these antigens, gp160, S25, and gp120r can be distinguished on the basis of differential expression on a panel of cultured renal cancers and normal kidney epithelium and fetal kidney cells. Glycoproteins bearing gp120r share a determinant with renal gp120nr (as indicated by sequential precipitations with monoclonal antibodies that detect gp120r and gp120nr), but gp120nr is found on a broader range of cell types, including fibroblasts and cell lines derived from lung, bladder, and colon cancers. The two other new systems, gp115 and V1, have characteristics of broadly occurring differentiation antigens but can be distinguished from each other and from gp120nr by differences in molecular weight, heat stability (V1 is a heat-stable determinant), and differential expression on cell types of diverse origin.

Eighteen mouse monoclonal antibodies were selected for reactivity with cell surface antigens of the immunizing human melanoma cell line SK-MEL-28. Six distinct antigenic systems were defined by direct serological assays and absorption tests with a panel of 41 cell lines derived from normal and malignant human tissues. Biochemical analysis indicated that two of the antigens are glycoproteins with molecular sizes of 95,000 and 150,000 daltons (gp95 and gp150). Two other antigenic systems (O5 and the R24 group) are associated with heat-stable molecules having the characteristics of glycolipids. The remaining two antigens (M19 and R8) are heat labile, but molecular characterization has not been possible. Each of the antigenic systems has a distinctive pattern of distribution on various cell types, varying from a broad representation to a more restricted occurrence. O5 appears to be a species antigen, being present on virtually every human cell type tested. gp95, gp150, M19, and R8 are found on a characteristic proportion of melanomas, astrocytomas, and epithelial cancers and on normal kidney cells. The antigen defined by the R24 antibody has the most restricted distribution of all. Reactivity is found with melanomas and astrocytomas, whereas epithelial cell types, fibroblasts, and cells of hematopoietic origin lack R24. Although occurrence of gp95, gp150, M19, and R8 distinguishes a small subset of melanomas not expressing these antigens, R24 is found on all melanoma cells.

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAcalpha2-3Galbeta1-3GalNAc-ol, Galbeta1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-3]GalNAc- ol and NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-6[NeuAcalpha2 -3Galbeta1-4GlcNAcbet a1-3Galbeta1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.

The antigen recognized by a mouse monoclonal antibody (mAb S27) raised against a human renal cancer cell line has been identified as the adenosine deaminase binding protein. mAb S27 immunoprecipitates binding protein purified from a soluble fraction of human kidney. It also recognizes the mature 120,000-dalton membrane form of binding protein from [35S]methionine-labeled human fibroblasts, HepG2 cells, and the renal cancer cell line against which the antibody was raised. A rabbit polyclonal antibody raised against purified kidney binding protein completely precipitates mAb S27-reactive material from labeled membrane extracts. mAb S27 does not precipitate the initially synthesized 110,000 molecular weight precursor of binding protein in fibroblasts and recognizes only a small portion of binding protein precursor in labeled HepG2 cells suggesting that the antigenic determinant recognized by mAb S27 may be a post-translational modification present on the mature form of binding protein or that mAb S27 recognizes molecules in a certain conformation. Glycopeptides derived from purified soluble kidney binding protein or exogenously added adenosine deaminase do not inhibit the immunoprecipitation of binding protein by mAb S27, indicating that the mature oligosaccharide chains of binding protein are not the determinant recognized by mAb S27 and that bound adenosine deaminase does not mask the antigenic sites on binding protein. The fact that monoclonal antibody S27, previously shown (Ueda, R., Ogata, S., Morissey, D. M., Finstad, C. L., Szkudlavek, J., Whitmore, W. F., Oettgen, H. F., Lloyd, K. O., and Old, L. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5122-5126) to detect a cell surface antigen on cultured renal cancer cells, is directed against the adenosine deaminase binding protein confirms and extends the earlier observation (Andy, R.J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925) that binding protein is located on the cell surface.

Mouse monoclonal antibodies to several cell surface antigens of human ovarian and endometrial carcinomas have been produced. The distribution of the antigens was determined by mixed hemagglutination assays on 153 normal and malignant cell cultures of various types and by immuno-peroxidase staining of frozen sections of 27 normal adult and 24 fetal tissues. Five distinct antigens were characterized. MD144 antigen was detected on only a single ovarian carcinoma cell line and has the biochemical properties of a lipid. MH55 antigen is weakly expressed on ovarian and uterine cancer cell lines but not on other cells and tissues tested. MF61 antigen was detected on an ovarian carcinoma and some renal carcinoma cell lines but not on other cell lines tested. It was also detected by immunoperoxidase staining in the noncellular follicles of the thyroid and in uterine glandular epithelial cells. This antigen also has the properties of a lipid. MF116 antigen was detected on a proportion of ovarian, uterine, renal, and bladder carcinoma and neuroblastoma cell lines and on normal kidney epithelial cell cultures but not on other cell lines tested. It was not detected in sections of any normal tissue tested using the immunoperoxidase method. MF116 was readily detected in the spent culture medium but not in detergent-solubilized extracts of metabolically radiolabeled cells. This shed antigen is a glycoprotein of Mr 105,000 and isoelectric point lower than pH 4.0. MH94 antigen was detected on a proportion of ovarian, uterine, colon, breast, lung, cervical, and pancreatic carcinoma cell lines. In tissue sections it was detected in many but not all epithelia, predominantly in secretory epithelial cells. Antibody MH94 did not immunoprecipitate a detectable antigen.

Deoxyribonucleic acid (DNA) base composition, as measured by guanine plus cytosine (G + C) content, was determined in 17 strains of Sporothrix schenckiiand Ceratocystisspecies. The average G + C content for S. schenckiiwas 54.7 ± 0.2 mol%. No strain of Ceratocystis, with the exception of C. minor, gave values for G + C content similar to those obtained for S. schenckii. All DNA samples from strains of S. schenckiicross-hybridized with high percentages of relative binding. DNA samples from C. stenocerasand C. piliferashowed low degrees of homology with S. schenckiiDNA. The DNA from C. minorhybridized with 75% binding with that from a strain of S. schenckii, as compared with the homologous reaction.

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2—-8NeuGc alpha 2—-3Gal beta I—-4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2—-8NeuGc alpha 2—-3Gal beta 1—-4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2—-8NeuGc alpha 2—-3Gal beta 1—-4GlcNAc beta 1—-3Gal beta 1—-4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2—-8NeuGc alpha 2—-3Gal beta 1—-4GlcNAc beta 1—-3Gal beta 1—-4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.

Two-dimensional polyacrylamide gel electrophoresis has been used to compare the Nonidet P-40 soluble, [3H]glucosamine-labeled glycoproteins of human kidney cancer cell lines and short-term cultures of normal kidney epithelia. Two pairs of autologous combinations of tumor and normal cells and also six other cancer lines and four normal cultures were examined. Because autologous cells could be compared, possible differences due to allogeneic variations could be eliminated. The electrophoretic patterns given by tumor and normal cells were extremely similar. Nevertheless, more than 10 differences could be recognized when randomly selected combinations of cancer and normal cells were examined in detail. Of these differences, however, only one was consistently found in all possible combinations of cancer and normal cells. Cancer cells had three prominent components of molecular weight 27,500 and pIs of 5.7, 5.3, and 4.9, respectively. Normal cells have a fainter and different constellation of spots in this region, with the more acid component (pI 4.9) being absent (or barely detectable). The pattern of fetal kidney cells resembled that given by normal adult kidney.

The proton and 13C magnetic resonance (CMR) spectra of 10 Sporothrix schenckiiand three Ceratocystis stenocerasrhamnomannans were studied. On the basis of their CMR spectra, the rhamnomannans from S. schenckiistrains could be classified into two major types, I and II, readily distinguishable from the polysaccharides formed at the same temperature (25 C) by C. stenoceras. Rhamnomannans from S. schenckiisynthesized at 37 C gave CMR spectra (type III) differing from those of polysaccharides of types I and II and from those of C. stenoceraspolysaccharides. The contention that C. stenocerasmight be the perfect form of S. schenckiiis not supported by the present data. A presumptive pathogenic mutant of C. stenocerassynthesized a type I rhamnomannan characteristic of S. schenckii, clearly differing from the rhamnomannans from C. stenocerassynthesized in the same conditions. Nuclear magnetic resonance spectroscopy revealed differences in the structures of the polysaccharides from these species not previously recognized by methylation analysis.

Analysis of the humoral immune response of patients with melanoma has identified a small group of individuals with antibody to cell surface antigens that are restricted to autologous melanoma cells. These antigens, referred to as Class I or unique tumor antigens, are demonstrated by reactions between serum and cultured melanoma cells from the same patient and absorption tests with autologous and allogeneic normal and malignant cells to determine antibody specificity. Five Class 1 melanoma antigens have been defined to date, but insight into the nature of these antigens has been limited because antibodies identifying these antigens lacked detectable immunoprecipitating activity. We have now defined a Class 1 melanoma antigen (designated FD) that is immunoprecipitated by autologous antibody. FD antigen is identified by an IgG antibody present in the sera of patient FD, and peak titers of this antibody in tests with cultured autologous melanoma cells are in the range of 1:2048. By absorption tests, FD antigen could not be detected on any other cell type, including 33 allogeneic melanomas. Prolonged culture of FD melanoma cells resulted in decreased expression of FD antigen, but sublines could be obtained with stable antigen expression. FD antigen is trypsin and heat sensitive, neuraminidase resistant, and is shed in the culture medium. Immunoprecipitation of 125I-labeled cell membrane preparations revealed a 90,000 dalton component of pI 5.5. Serum immunoprecipitating activity could be absorbed by autologous melanoma cells but not by autologous B cells or allogeneic cell lines. A component of the same molecular mass could be precipitated from lysates of cells metabolically labeled with [3H]mannose. The membrane form of the FD antigen binds strongly to Con A-Sepharose and can be eluted with methyl-alpha-D-mannoside. The identification of a precipitating Class I antigenic system of melanoma facilitates efforts to generate monoclonal antibodies to this tumor antigen and to clone its coding sequence.

Antibodies present in the serum of melanoma patient FD detect an antigenic determinant (FD) restricted to the autologous melanoma cell line SK-MEL-131. This cell-surface determinant is carried on a glycoprotein of 90 kDa, designated gp90. Mice were immunized with a partially purified preparation of gp90 derived from SK-MEL-131 clone 1.5, and three murine hybridomas (KF23, KF26, and KF104) secreting monoclonal antibodies (mAbs) detecting this antigen have been generated. Sequential immunoprecipitation experiments demonstrate that the three mAbs and human FD serum react with the same gp90 species. The mAbs, in contrast to FD serum, react with a broad range of cultured cells in assays for cell-surface antigens and immunoprecipitate a gp90 component from radiolabeled extracts of these cells, including autologous FD B cells. We conclude that gp90 from SK-MEL-131 has two types of determinants: restricted (detected by FD serum) and common (detected by the mouse mAbs). gp90 molecules from cell lines other than SK-MEL-131 carry only the common determinant(s). Immunoperoxidase analysis of frozen tissue sections with mAb KF23 demonstrated a restricted gp90 expression in normal tissues (capillary endothelial cells, duct epithelium in sweat glands, breast, and pancreas). Melanomas and sarcomas showed strong gp90 expression, suggesting up-regulation of gp90 synthesis in certain human cancers.

Mouse monoclonal antibodies were obtained by immunization with cultured human bladder cancer or lysates of bladder papilloma. They identify 11 distinct antigenic systems as defined by serological analysis of cultured cells and studies of antigen distribution in normal and neoplastic tissues. The most restricted of these antigens, Om5, defines a subset of bladder tumors. Om5 is not detected in normal bladder urothelium or in any other normal or malignant tissue. T101 and JP165 are also subset markers for bladder cancer that are not detected in normal tissues. T16, T43, T87, and J143 (antigens represented on many cultured cells) are found in specific areas of the normal urinary tract and in a distinctive range of other normal and malignant cell types--e.g., T16 expression in pluristratified epithelium of skin, exocervix, and esophagus. T138 antigen is also a common feature of cultured cell lines, but its expression in sections of normal tissues is restricted to endothelial cells. In contrast, T110 is poorly represented on cultured cells but can be detected in culture supernatants. Localization of T110 in normal tissues showed that it is a component of the extracellular matrix. All determinants detected by this series of antibodies are heat labile and not related to A, B, H, I, Lewis blood group antigens. Six of the antibodies immunoprecipitated glycoproteins from radiolabeled cell lysates: AbT16 (Mrs 48,000 and 42,000), AbT87 (Mr 60,000), AbT43 (Mr 85,000), AbJ143 (Mr 140,000, 120,000, 60,000), AbT43 (Mr 85,000), AbJ143 (Mr 140,000, 120,000, and 30,000), and AbT110 (Mr 240,000).

The surface antigens of cultured human malignant astrocytomas were analyzed by using mouse monoclonal antibodies. BALB/c mice were immunized repeatedly with either SK-MG-1 [a glial fibrillary acidic protein (GFA)-negative astrocytoma line] or SK-AO2 (a GFA-positive astrocytoma line). After fusion with NS/1 mouse myeloma cells, 12 antibody-producing clones were selected for detailed study. Serological analysis permitted the identification of nine distinct antigenic systems. Four monoclonal antibodies (Ab AJ225, Ab AO10, Ab AJ8, and Ab AO122) identified cell surface antigens preferentially expressed on tumors of neuroectodermal origin, and these antibodies subdivided the astrocytoma panel into distinguishable subsets. The determinant detected by Ab AO10 and Ab AJ8 showed mutually exclusive expression on the astrocytoma lines. The AO10 and AJ8 phenotypes appeared to reflect the differentiation state of the cultured cells; 4/7 AO10-positive astrocytomas expressed GFA, an intracellular astrocyte differentiation antigen, whereas all AJ8-positive astrocytoma (9/9) were GFA-negative. Five antibodies (Ab AJ10, Ab AJ9, Ab AJ17, Ab AJ425, and Ab AJ2) recognized determinants widely distributed on normal and malignant cells. Four antibodies defined in this study precipitated proteins from reduced preparations of radioisotope-labeled SK-MG-1 and SK-AO2 cells: Ab AJ225 (Mr 145,000); Ab AO122 (Mr265,000); Ab AJ10 (Mrs 195,000 and 165,000); and Ab AJ2 (Mrs 170,000, 140,000, 140,000, and 28,000).

AU antigen is defined by reactions of sera from patient AU with cell-surface antigens of cultured autologous melanoma cells (SK-MEL-28). Past studies established that no available cell type other than AU melanoma expressed AU antigen. By use of antibody inhibition tests for antigen detection, limited papain digestion of AU melanoma cells was found to result in the solubilization of AU antigen along with beta2-microglobulin (beta 2m) and HLA allogeneic and xenogeneic specificities. Comparable papain treatment of other melanoma and non-melanoma cell lines solubilized beta 2m and HLA, but did not result in the release of antigen with AU reactivity. Maximum yield of AU antigen from AU melanoma cells was obtained after very short (5-15 min) digestion times in contrast to the more prolonged proteolysis required for maximum HLA and beta 2m release. AU antigen was not immunoprecipitated by rabbit antiserum against beta 2m or HLA under conditions leading to partial or complete removal of beta 2m and HLA. At least a proportion of the molecules with AU determinants appear to be glycoproteins, as indicated by specific affinity for Lens culinaris hemagglutinin (LcH). After affinity chromatography on LcH-agarose, the specific activity of AU antigen was increased 50-fold. As determined by gel filtration chromatography, AU antigen has a molecular weight in the range of 20,000-50,000.

Two mouse monoclonal antibodies (mAb), AJ2 and J143, define two related human cell surface protein complexes, very common antigen 1 (VCA-1) and very common antigen 2 (VCA-2). In the present report, these complexes have been defined with respect to: (i) subunit arrangement; (ii) monoclonal antibody binding sites; (iii) carbohydrate content; (iv) homology to other cell surface protein complexes; and (v) possible functional roles. Analysis of the antigens from a human melanoma cell line, MeWo, reveals that VCA-1 is a noncovalently linked heterodimer of 170- and 140 (designated 1401)-kDa polypeptides. mAb AJ2 reacts with an epitope on the 1401-kDa polypeptide. VCA-2 is composed of the same 1401-kDa polypeptide as VCA-1 and another 170-kDa species; this 170-kDa species consists of a second distinct 140-kDa (designated 140(2)) and a 30-kDa polypeptide which are disulfide-bonded. Indirect evidence indicates that mAb J143 reacts with an epitope on this 170-kDa complex. Peptide mapping has shown that the complexes are members of a family of cell surface proteins that include antigens present on activated T cells (designated very late activation antigens). Since VCA-2 is found predominantly on the basal membrane of adherent cells and its expression increases 12-fold when HUT-102 lymphoblastoid cells are grown as an adherent cell culture, we suggest that VCA-2 plays a role in cellular adherence.

We have previously reported the identification and characterization of two related human cell surface protein complexes, very common antigens 1 and 2 (VCA-1, VCA-2) (Kantor, R. R. S., Mattes, M. J., Lloyd, K. O., Old, L. J., and Albino, A. P. (1987) J. Biol. Chem. 262, 15158-15165). We now report the transfection of DNA sequences encoding the 170-kilodalton heterodimer of VCA-2 from human SK-RC-41 renal cancer cells to B78H1 mouse melanoma cells. B78H1 cells were cotransfected with high molecular weight renal cancer DNA and a plasmid vector containing the neomycin resistance gene. Antibiotic-resistant transfectants were screened for the expression of the 170-kDa heterodimer with mouse monoclonal antibody (mAb) J143. Analysis of mAb J143-positive (J143+) transfectants showed that they expressed a 170-kDa heterodimer with an identical molecular weight, isoelectric point, two-dimensional peptide map, and spatial orientation of surface-exposed epitopes to the homologous 170-kDa species seen in human donor cells. The 170-kDa heterodimer in SK-RC-41 cells is associated with a 140-kDa (designated 140(1] polypeptide to form the VCA-2 complex. The 170-kDa complex and the 140(1)-kDa polypeptides are encoded by genes located on different human chromosomes. J143+ transfectants display a molecule of 140 kDa associated with the 170-kDa complex which is biochemically similar, but non-identical, to the human 140(1)-kDa polypeptide on VCA-2. This evidence supports our interpretation that the transfected human 170-kDa heterodimer associates with a murine counterpart of the human 140(1)-kDa polypeptide in J143+ transfectants.

Human monoclonal antibodies (mAbs) were derived from lymph node lymphocytes and peripheral blood lymphocytes (PBL) from patients with melanoma. Four methods for generating human mAbs were compared: fusion with human [LICR-LON-HMy-2 (LICR-2)] or mouse (NS-1) cells; transformation by Epstein-Barr virus (EBV); and EBV transformation followed by NS-1 fusion. NS-1 fusion with lymph node lymphocytes resulted in a higher number of growing hybrids than LICR-2 fusion. Virtually no hybrids were obtained from NS-1 or LICR-2 fusions with PBL. EBV transformed lymphocytes from lymph node and peripheral blood with equal efficiency, and the yield of proliferating cultures for antibody screening was more than 10- to 30-fold greater than that obtained by fusion techniques. However, once antibody-producing cultures had been identified, stability and clonability of EBV-transformed cells were poorer than that of NS-1 hybrid cells. To combine the strengths of both methods, cultures of EBV-transformed cells were fused with NS-1; and hybrid clones were isolated that showed vigorous growth, clonability, and stable antibody secretion. Detailed specificity analysis of the mAbs produced by six of these clones indicated detection of a class 1 (unique) melanoma antigen, a class 3 melanoma antigen, and four ganglioside antigens (GD3, GM3, and two other, as yet uncharacterized, heterophile antigens).

Mouse monoclonal antibody AbR24 has a high degree of specificity for human melanoma cells when tested on viable cultured cells using the protein A mixed hemagglutinin serological assay. The antigen detected by this antibody has been isolated from melanoma cells and shown to be GD3 ganglioside by compositional and partial structural analysis and by comparison with authentic GD3 in thin layer chromatography (TLC). AbR24 reacts with authentic GD3, but not with any other ganglioside tested. Using TLC and reactivity with AbR24, a wide range of cells and tissues was examined for the presence of GD3. A new serological assay, termed glycolipid-mediated immune adherence, was devised for assaying the reactivity of AbR24 with gangliosides. Melanomas (cultured cells or tumor tissue) were shown to have GD3 and GM3 as major gangliosides. Other cells and tissues examined also contained GD3, but usually only in low amounts. Melanomas (and MOLT-4, a T cell line) were characterized by a simplified ganglioside profile with GD3 and GM3 as major components. The apparent discrepancy between the ubiquitous presence of GD3 and the serological specificity of AbR24 for melanoma cells can be explained in terms of localization and concentration of GD3 in different cells.

Analysis of antibodies present in the serum of melanoma patient FD has shown that they detect a unique tumor epitope present only on the autologous melanoma cell line SK-MEL-131. Previous results had shown that the unique FD epitope is carried on a common glycoprotein of approximately 90 kD, widely expressed on melanoma and a few other cell types. We now show by sequential radioimmunoprecipitation and partial amino acid sequencing that this common molecule is a previously recognized melanoma antigen, originally identified by mouse mAbs, designated gp95 or p97 (and also known as melanotransferrin). Thus, FD is the first of the class I (unique) melanoma antigens that has been characterized and related to a known cell surface molecule.

The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.

Three established lines of melanoma cells were derived from anatomically distinct metastases occurring in a single patient (DX). The lines, DX-1, DX-2, and DX-3, showed marked phenotypic diversity, as indicated by characteristic differences in growth rate, morphology, pigmentation, and the expression of surface antigens and glycoproteins. DX-1 and DX-3 expressed HLA-DR products, whereas DX-2 lacked HLA-DR expression. DX-1, DX-2, and DX-3 could also be distinguished on the basis of the profile of radiolabeled glycoproteins. Additional quantitative differences in the surface antigenic phenotype of the three cell lines were revealed by serological tests with a battery of monoclonal and conventional antibodies defining melanoma differentiation antigens. In tests for autologous humoral immunity to melanoma cells, sera from patient DX were found to have IgG antibody that reacted with surface antigens of DX-2 cells; no autologous reactivity was seen with DX-1 or DX-3 target cells or with three more recently established melanoma cell lines from patient DX. Absorption analysis indicated that the antigen detected by DX sera on DX-2 cells is a class 1 melanoma antigen, having been detected only on DX-2 cells and in much lower but demonstrable amounts on DX-1 and DX-3 cells. No other cell type, including DX normal fibroblasts, DX B cells, or 45 allogeneic melanoma cell lines expressed the class 1 antigen of DX melanoma. The fact that only one of the melanoma cell lines derived from patient DX was suitable target for the detection of autologous class 1 reactivity has implications for the study of human tumor antigens and may explain why antibody to class 1 antigens has been found so infrequently in past studies of melanoma patients.

AH antigen, initially defined by an antibody present in a melanoma patient, is a cell surface antigen found on approximately 65% of melanoma cell lines. Absorption analysis indicates that AH is a differentiation antigen marking normal and malignant cells of neuroectodermal origin. The AH determinant has been found to be related to GD2 ganglioside.

Lymph node lymphocytes from patients with primary lung cancer were immortalized with Epstein-Barr virus, and culture supernatants were screened for cell-surface reactivity against allogeneic cancer cell lines. The percentage of wells containing detectable antibodies in initial screening ranged from 1 to 17%, but the vast majority of the cultures lost antibody activity on subsequent expansion. Two antibody-secreting clones, J309 and D579, derived from separate individuals and reactive with anaplastic lung cancer cell lines, were successfully expanded and fused with the NS-1 mouse myeloma cell line. The antibodies produced by these clones exhibited identical restricted serologic reactivity against cultured cell lines and detected a carbohydrate antigen present in the neutral glycolipid fraction of MCF-7 breast cancer cells. Serologic, immunochemical, and chemical analyses revealed that the antigen recognized by antibodies J309 and D579 is galactosylgloboside [Gal(beta 1----3)GalNAc(beta 1----3)Gal(alpha 1----4)Gal(beta 1----4)- GlcCer]. Conclusions regarding the significance of these findings with respect to the biology of lung cancer await further information concerning the distribution of galactosylgloboside in normal and malignant tissues and the frequency of antibodies to this structure in normal and tumor-bearing individuals.

Human malignant melanoma cell lines were found to bear Ia-like cell surface determinants demonstrable by hetero- or alloantisera and by direct identification of the characteristic bimolecular glycoprotein complex. Immunoprecipitation confirmed the presence of Ia determinants on the bimolecular complex. The quantity of Ia molecules determined by these methods and by absorption experiments was relatively constant for each cell line. Among different lines, however, the amount of Ia antigens ranged from a level equal to that expressed by B-cell lines to a small fraction of this amount. This variation in level of Ia contrasted with the more uniform amount of beta2-microglobulin detected on the cell surface. The Ia alloantigen specificity DRw2 was the most frequently encountered specificity. Ia determinants were also found on the surface of an epidermoid carcinoma line, but not on various other cell lines of normal or neoplastic origin.

The specificities of two human monoclonal antibodies (2-39M and 32-27M), produced by hybridomas derived from the lymphocytes of melanoma patients (Yamaguchi, H., Furukawa, K., Fortunato, S. R., Livingston, P. O., Lloyd, K. O., Oettgen, H. F., and Old, L. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2416-2420) have been elucidated. Using a large panel of glycolipids, it has been shown that the two monoclonal antibodies (mAbs) identified a number of N-glycolylneuraminic acid (NeuGc)-containing gangliosides. mAb 2-39M reacted with (NeuGc)GM3, (NeuGc)sialylparagloboside, and (NeuGc)sialylhexaglycosylceramide; no reactivity was observed with gangliosides containing only N-acetylneuraminic acid (NeuAc) or with disialogangliosides. These reactive species have the NeuGc alpha 2—-3Gal- sequence in common. mAb 32-27M reacted strongly with (NeuGc)2 GD3 and (NeuGc)2disialylparagloboside, and moderately with (NeuAc-NeuGc-)GD3 and (NeuAc-NeuGc-)disialylparagloboside. The reactive species have sialic acid alpha 2—-8NeuGc alpha 2—-3Gal- sequences in common. These two antibodies were used to demonstrate the species-related presence of different NeuGc-containing gangliosides in various animal erythrocytes by thin layer chromatography immunostaining. No reactivity of either mAb was observed with gangliosides isolated from fresh human colon cancer, melanoma specimens, or some normal tissues, including brain. On the other hand, it was shown that mAb 32-27M reacted with gangliosides isolated from human melanoma and astrocytoma cells grown in fetal bovine serum but not from those grown in synthetic medium. Within the sensitivities of the methods used, these data, and related chemical analyses, do not support the presence of NeuGc-containing gangliosides in human tumors.

A clinical isolate of Trichophyton mentagrophytes which produces a diffusible red-brown pigment as well as typical microconidia en grappe and abundant spirals on Sabouraud's Dextrose Agar has been studied. It splits urea and perforates hair in vitro but does not produce pigment on cornmeal agar or require vitamins for growth. A low molecular weight, red pigment, partially purified from culture filtrates by gel-filtration, was characterized as 2-carboxy 2,3-dihydroindole-5,6-quinone, an intermediate in melanin biosynthesis. It is water soluble, lacks free amino groups, unstable in alkaline buffers, and has spectral absorption peaks at 320-340 nm and 450-490 nm.

Rhamnomannans from several isolates of Sporothrix schenckiiand Ceratocystisspecies were compared. No major differences emerged in the analysis of the carbohydrate composition of polysaccharides from either species. Methylation analysis for the identification of the glycosidic linkages present in the polysaccharides showed that they all were very similar with minor differences being observed among strains and between polysaccharides obtained at 25 and 37 C. Partially methylated derivatives were identified by gas-liquid chromatography and mass spectrometry. Their proportions suggest that polysaccharides from S. schenckiiand C. stenocerashave a general structure similar to that of C. ulmi: a (1 → 6)-linked α-d-mannopyranosyl main chain substituted in the 3-positions by α-l-rhamnopyranosyl and in many cases by α-l-rhamnopyranosyl (1→2)-l-rhamnopyranosyl side chains. Differences among polysaccharides resided in the proportions of the dirhamnosyl side chains and of 4-O-substituted and 2, 4-di-O-substituted mannose units. Increased proportions of dirhamnosyl side chains were observed in the polysaccharides from S. schenckiistrains and from a pathogenic variant of C. stenocerasgrown at 25 C. Proton magnetic resonance spectra (H-1 region) of the rhamnomannans showed that S. schenckiistrains could be placed in two different groups similar but not identical to any Ceratocystisspecies. These spectra were very close to those of the polysaccharides from the C. clavataand C. ambrosiagroups of Ceratocystisspecies.