An experimental vaccine expressing wild-type p53 induces protective immunity against glioblastoma cells with high levels of endogenous p53.

Inoculation of mice with a recombinant vaccinia virus expressing the full-length mouse wild-type p53 protein (Vp53-wt) was shown to induce partial protection against peripheral challenge with a mouse glioblastoma cell line, termed GL261, expressing high levels of nuclear, endogenous wild-type p53. In vivo experiments with knockout (KO) mice and mice treated with depleting doses of antibodies specific to lymphocyte subsets revealed that vaccine efficacy depended on CD4+ and CD8+ T cells as well as on natural killer (NK) cells. Vp53-wt virus-vaccinated mice that failed to develop tumours upon challenge with a minimal tumourigenic dose of GL261 cells remained completely resistant to further challenge with increased doses of GL261 cells. The efficacy of the Vp53-wt vaccine was improved by adding recombinant mouse interleukin-12 (rIL-12) as an adjuvant at the time of tumour challenge. The induction of T cells to p53 in Vp53-wt virus-immune mice was also demonstrated at the tumour site by immunochemistry and was further confirmed by a delayed-type hypersensitivity response to the p53 protein, although in vitro experiments using splenocytes from vaccinated mice failed to demonstrate CD4+ or CD8+ T-cell activity to p53.

A method that allows easy characterization of tumor-infiltrating lymphocytes.

A method was developed to compare the lymphocytic infiltrates in regressing vs. progressing experimental mouse tumors using a model for human papillomavirus-16 (HPV-16) oncoprotein-linked cancer. Tumor cells mixed with matrigel, composed of natural matrix substances that provide a basement membrane structure for adherent cells, were inoculated into mice vaccinated with an efficacious vaccine to the E7 oncoprotein or a vaccine to a control antigen. The tumor cells remained within the solidified gel and recruited a cellular infiltrate that could readily be analyzed upon removal of the gelatinous mass containing progressing or regressing tumors. The results show that tumors recruit activated CD8(+) T cells regardless of their antigen specificity. In regressing tumors expressing an appropriate target antigen for the vaccine-induced CD8(+) T cells, a strong increase of the tumor antigen-specific T cell population was observed over time. Progressing tumors that lacked the target antigen for the activated CD8(+) T cell population did not show this selective enrichment.

Interaction between heat shock proteins and antimicrobial peptides.

Drosocin, pyrrhocoricin, and apidaecin, representing the short (18-20 amino acid residues) proline-rich antibacterial peptide family, originally isolated from insects, were shown to act on a target bacterial protein in a stereospecific manner. Native pyrrhocoricin and one of its analogues designed for this purpose protect mice from bacterial challenge and, therefore, may represent alternatives to existing antimicrobial drugs. Furthermore, this mode of action can be a basis for the design of a completely novel set of antibacterial compounds, peptidic or peptidomimetic, if the interacting bacterial biopolymers are known. Recently, apidaecin was shown to enter Escherichia coli and subsequently kill bacteria through sequential interactions with diverse target macromolecules. In this paper report, we used biotin- and fluorescein-labeled pyrrhocoricin, drosocin, and apidaecin analogues to identify biopolymers that bind to these peptides and are potentially involved in the above-mentioned multistep killing process. Through use of a biotin-labeled pyrrhocoricin analogue, we isolated two interacting proteins from E. coli. According to mass spectrometry, Western blot, and fluorescence polarization, the short, proline-rich peptides bound to DnaK, the 70-kDa bacterial heat shock protein, both in solution and on the solid-phase. GroEL, the 60-kDa chaperonin, also bound in solution. Control experiments with an unrelated labeled peptide showed that while binding to DnaK was specific for the antibacterial peptides, binding to GroEL was not specific for these insect sequences. The killing of bacteria and DnaK binding are related events, as an inactive pyrrhocoricin analogue made of all-D-amino acids failed to bind. The pharmaceutical potential of the insect antibacterial peptides is underscored by the fact that pyrrhocoricin did not bind to Hsp70, the human equivalent of DnaK. Competition assay with unlabeled pyrrhocoricin indicated differences in GroEL and DnaK binding and a probable two-site interaction with DnaK. In addition, all three antibacterial peptides strongly interacted with two bacterial lipopolysaccharide (LPS) preparations in solution, indicating that the initial step of the bacterial killing cascade proceeds through LPS-mediated cell entry.

Peptide mimicking sialyl-Lewis(a) with anti-inflammatory activity.

Peptides mimicking carbohydrate structure sialyl-Lewis a (SA-Le(a)) have been selected from a diverse dodecapeptide library using monoclonal antibody (MAb) NS19-9. Families of peptides with a consensus sequence consisting of three to nine amino acids and peptides that do not show a conserved core amino acid region were identified. Peptide DLWDWVVGKPAG was selected based on the consensus sequence DXXDXXVG shared with other peptides and strong binding in Western blot. Peptide competes with antibody binding to its native carbohydrate antigen, SA-Le(a), at 50% inhibitory concentration (IC(50)), 700 microM, implying that it represents a structural mimic of the carbohydrate epitope recognized by MAb. Statistically significant reduction of neutrophil recruitment into the intraperitoneal cavity was observed upon administration of this peptide in a murine acute inflammation model in vivo. Results suggest that the peptide mimic of SA-Le(a) carbohydrate might bind to E-selectin and block its interaction with another ligand, sialyl-Lewis X (SA-LeX), expressed on neutrophils.

Genetic adjuvants.

In 1992, the era of DNA vaccines began with the report of antibody production upon intradermal injection of mice with a plasmid vector expressing a foreign antigen (1). A rapid succession of subsequent manuscripts showed stimulation of immune responses, including cytolytic T cells, upon inoculation of expression-vectors specific for antigens derived from viruses, bacteria, protozoa and tumor-associated antigens (2-7). Plasmid DNA can be applied through various routes of injection including: intradermal, intramuscular, subcutaneous, intravenous, or directly on mucosal membranes (1,2,8,9). The most commonly used methods of inoculation involve the use of DNA-coated gold beads propelled into the skin by a gene gun or intramuscular inoculation of the vector in saline solution.

Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses.

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell-independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53.

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.

Cytokines and costimulatory molecules as genetic adjuvants.

DNA vectors expressing an antigen derived from a pathogen or a cancerous cell have been shown, after inoculation into experimental animals, to trigger de novo synthesis of foreign proteins, which induce an immune response. This immune response can be modulated by coinoculation of vectors encoding either cytokines or costimulatory molecules. A variety of cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-2, IL-4, IL-12 and IFN-gamma, as well as the costimulatory molecule B7.1, have been tested to date for their ability to amplify the immune response to genetic vaccines. Although the results obtained thus far clearly show that coadministration of vectors expressing immunomodulatory molecules, such as cytokines, may increase the efficacy of genetic vaccines, this approach is currently considered unsuitable for use in human patients due to the potential side effects of persistent cytokine expression.

Molecular recognition of the Lewis Y antigen by monoclonal antibodies.

The murine monoclonal antibody BR55-2 is directed against the tumor-associated antigen Lewis Y oligosaccharide. The Lewis Y core antigen is a difucosylated structure consisting of four hexose units. Analysis of binding profiles of lactoseries isomeric structures by BR55-2 suggest that the binding epitope includes the OH-4 and OH-3 groups of the beta-D-galactose unit, the 6-CH3 groups of the two fucose units and the N-acetyl group of the subterminal beta-D-N-acetylglucosamine (beta DGlcNAc). To elucidate the molecular recognition properties of BR55-2 for the Y antigen, BR55-2 was cloned, sequenced and its three-dimensional structure was examined by molecular modeling. The crystal structure of BR96, another anti-Lewis Y antibody, solved in complex with a nonoate methyl ester Lewis Y tetrasaccharide, and the lectin IV protein in complex with a Lewis b tetrasaccharide core were used as a guide to probe the molecular basis for BR55-2 antigen recognition and specificity. Our modeling study shows that BR55-2 shares similar recognition features for the difucosylated type 2 lactoseries Lewis Y structure observed in the BR96-sugar complex. We observe that a major source of specificity for the Lewis Y structure by anti-Y antibodies emanates from interaction with the beta-D-N-acetylglucosamine residue and the nature of the structures extended at the reducing site of the fucosylated lactosoamine.

Porcine submaxillary gland GDP-L-fucose: beta-D-galactoside alpha-2-L-fucosyltransferase is likely a counterpart of the human Secretor gene-encoded blood group transferase.

Partial amino acid sequence of GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase purified from porcine submaxillary glands was determined. Amino acid sequence analysis yielded 100, 93.3, and 84.2%, and 75, 46.6, and 84.2% sequence identity between 12-, 15-, and 19- amino acid tryptic peptides generated from porcine enzyme and amino acid residues 61-72, 111-125, and 308-326 and 89-100, 139-153, and 338-356 of the human Secretor and H type alpha-2-fucosyltransferases, respectively. Higher amino acid sequence homology of the porcine enzyme with the predicted sequence for the human Secretor locus as compared with H gene-encoded blood group beta-D-galactoside alpha-2-L-fucosyltransferase suggests that porcine alpha-2-fucosyltransferase highly corresponds to the human Secretor gene-encoded enzyme.

Elevated expression of H type GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase is associated with human colon adenocarcinoma progression.

GDP-L-fucose:beta-D-galactoside alpha-2-L-fucosyltransferase (EC 2.4.1.69) is a key enzyme in the biosynthesis of fucosylated type 1 and 2 lactoseries structures, such as Lewis b and the H type 2 and Lewis Y, respectively, that are accumulated in colon adenocarcinoma. Analysis of the mRNA transcript level for the human H gene-encoded beta-D-galactoside alpha-2-L-fucosyltransferase revealed 40- and 340-fold increases in the mRNA levels in all adenocarcinomas and tumor cell lines, respectively, compared to normal colon mucosa where a low level of mRNA transcript was detected. A variable increase in mRNA transcript levels was observed in 50% of adenomatous polyps. Nucleotide sequence analysis of the protein coding region of the cDNAs derived from normal colon, adenoma, and colon adenocarcinoma revealed 100% homology, suggesting that there are no tumor-associated allelic variations within the H beta-D-galactoside alpha-2-L-fucosyltransferase cDNA. These results suggest that beta-D-galactoside alpha-2-L-fucosyltransferase expression highly correlates with malignant progression of colon adenocarcinoma.

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., Köhlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.

Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases.

The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with a basic group on the enzyme which removes the developing proton during the glycosyl transfer reaction. Such groups are proposed to represent logical targets for irreversible covalent inactivation of this class of enzyme.

Purification of H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase from human serum.

The human serum enzyme, beta-galactoside alpha 1----2 fucosyltransferase, presumably blood group H gene-encoded, was purified to homogeneity from serum of AB and mixed secretor phenotype individuals. The purification procedure involved chromatography on phenyl-Sepharose, S-Sepharose, GDP-hexanolamine-Sepharose, and high pressure liquid chromatography gel filtration. The enzyme was purified 10 x 10(6)-fold, with a final specific activity of 23.6 units/mg for the phenyl-beta-O-galactoside acceptor. The apparent Mr of the H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase was determined as 200,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing and reducing conditions, respectively. The Mr of native enzyme was found by gel filtration chromatography to be 148,000. The subunit structure as well as the sensitivity of the enzymatic activity to beta-mercaptoethanol suggest that the native enzyme exists in polymeric form of covalently bound subunits. Lectin binding properties of the purified molecule indicate that the enzyme is glycosylated. Another human serum beta-galactoside alpha 1----2 fucosyltransferase, presumably Se gene-encoded, was separated from the H enzyme by adsorption on S-Sepharose cation exchange matrix. A comparison of the kinetic parameters of the initial rate data of both alpha 1----2 fucosyltransferases revealed differences between Km values for various oligosaccharide acceptors. Higher Km values for the phenyl-beta-O-galactoside acceptor and a lower Km for the lacto-N-tetraose-beta-O-PA8 type 1 acceptor for the enzyme that adsorbed to S-Sepharose compared with nonadsorbed enzyme were observed. The two enzymes also were differentiated by binding properties to S-Sepharose and electrophoretic mobilities on native gel electrophoresis. We, therefore, postulate that the enzyme which does not adsorb to S-Sepharose and adsorbed enzyme are structurally different molecules and they represent the H and Se gene-encoded beta-galactoside alpha 1----2 fucosyltransferases, respectively.

Oligosaccharide Y specific monoclonal antibody and its isotype switch variants.

Isotype switch variants of the IgG3 secreting hybridoma BR55-2 were developed with gamma 1, gamma 2b and gamma 2a heavy chains. The variants have the same affinity for antigen and expected affinities for the Fc receptor. The Y antigen defined by MAb BR55-2 has a restricted distribution to breast, colorectal, pancreatic, gastric and small cell lung carcinomas. The MAb BR55-2 also bound to prostatic and laryngeal carcinomas. The BR55-2 defined epitope is expressed by gastrointestinal cancer cell lines as a glycolipid, while the breast carcinoma cells express the same epitope on glycoproteins. These differences may influence tumor cell susceptibility to lysis by MAb BR55-2 and its isotype switch variants.

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay.

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.

Y and blood group B type 2 glycolipid antigens accumulate in a human gastric carcinoma cell line as detected by monoclonal antibody. Isolation and characterization by mass spectrometry and NMR spectroscopy.

A monoclonal antibody (mAb), BR55-2, was generated from mice immunized with MCF-7 human breast carcinoma cells. This mAb specifically detected glycolipids with the Y determinant Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)-beta 1----3Gal beta 1----4Glc beta 1----1 Cer and the Y-related B-active difucosylated determinant Gal alpha 1----3Gal(2----1 alpha Fuc) beta 1----4GlcNAc(3----1 alpha Fuc) beta 1----3Gal beta 1----4Glc beta 1----1 Cer, but was not reactive with related monofucosylated glycolipids of type 2 chain (X-antigen, blood group H), type 1 chain (Lea antigen, blood group H and B) or with difucosylated type 2 and type 1 chain structures (A blood group antigen or blood group B and Leb, respectively). A series of glycolipids with Y and blood group B type 2 determinants were detected in human gastric adenocarcinoma cell line KATO III with mAb BR55-2 and with a previously characterized anti-blood group B mAb PA83-52 (Hansson, G. C., Karlsson, K.-A., Larson, G., McKibbin, J. M., Blaszczyk, M., Herlyn, M., Steplewski, Z., and Koprowski, H. (1983) J. Biol. Chem. 258, 4091-4097). The isolated antigens were structurally characterized by mass spectrometry of permethylated and permethylated-reduced derivatives and by proton NMR spectroscopy. In a chromatogram binding assay, mAb BR55-2 and mAb PA83-52 detected minor components with slower mobility than the Y-6 and blood group B-7-type 2 structures. The detection of a B type 2 determinant is the first chemical evidence for the presence of an autologous difucosyl blood group B type 2 antigen in human adenocarcinoma cells.