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R A Reisfeld

Publications and source records attributed to R A Reisfeld.

At least 127 records · Page 7Linked to original sources
PubMed1989

Functional properties and effect on growth suppression of human neuroblastoma tumors by isotype switch variants of monoclonal antiganglioside GD2 antibody 14.18.

A complete family of IgG isotype switch variant hybridomas was generated from the anti-GD2 monoclonal IgG3-producing hybridoma, 14.18, with the aid of the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a monoclonal antibodies (Mabs) produced by respective isotype switch variant hybridomas 14G1, 14G2b, or 14G2a, have binding activities for the biochemically defined GD2 antigen and GD2-expressing neuroblastoma target cell lines identical to that of IgG3 Mabs produced by the 14.18 parent cell line. This permitted us to examine the relative in vitro and in vivo cytotoxic capacities of each of the anti-GD2 antibodies for GD2-expressing neuroblastoma cells independent of antibody binding affinity or specificity. Mabs produced by 14.18, 14G2a, or 14G2b, but not 14G1, can direct efficient complement-dependent cytotoxicity against neuroblastoma tumor cells in the presence of human complement. Mabs produced by the parent 14.18 or by 14G2a are more efficient in directing antibody-dependent cell-mediated cytotoxicity than Mabs produced by 14G2b, and Mabs of 14G1 are inactive. However, despite these noted in vitro differences, antibodies produced by each member of this switch variant family suppress the growth of human neuroblastoma tumor cells in BALB/c athymic nu/nu mice. These studies suggest that a mechanism(s) other than Fc-directed complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity may account for the in vivo antitumor effects of these particular antibodies.

Animals↗
PubMed1989

Post-translational addition of chondroitin sulfate glycosaminoglycans. Role of N-linked oligosaccharide addition, trimming, and processing.

A melanoma proteoglycan model system has been used to examine the role of core protein asparagine-linked (N-linked) oligosaccharides in the transport and assembly of proteoglycan molecules. The use of agents which block discrete steps in the trimming and processing of core oligosaccharides (castanospermine, 1-deoxynojirimycin, N-methyldeoxynojirimycin, 1-deoxymannojirimycin, and swainsonine) demonstrates that removal of glucose residues from the N-linked oligosaccharides is required for the cell surface expression of a melanoma proteoglycan core protein and for the conversion of the core protein to a chondroitin sulfate proteoglycan. However, complete maturation of the oligosaccharides to a "complex" form is not required for these events. Treatment of M21 human melanoma cells with the glucosidase inhibitors castanospermine, 1-deoxynojirimycin, or N-methyldeoxynojirimycin results in a dose-dependent inhibition of glycosaminoglycan (GAG) addition to the melanoma antigen recognized by monoclonal antibody 9.2.27. In contrast, treatment with the mannosidase inhibitors 1-deoxymannojirimycin and swainsonine does not effect GAG addition. Identical results are obtained when the major histocompatibility complex class II antigen gamma chain proteoglycan is examined in inhibitor-treated melanoma and B-lymphoblastoid cells. These data, in conjunction with the known effects of the glucosidase and mannosidase inhibitors on the transport and secretion of other glycoproteins support the hypothesis that the addition, trimming, and processing of N-linked oligosaccharides is involved in the transport of certain proteoglycan core proteins to the site of GAG addition and to the cell surface.

Antigens, Neoplasm↗
PubMed1989

Lysis of neuroblastoma cells by the ADCC-reaction: granulocytes of patients with chronic granulomatous disease are more effective than those of healthy donors.

Granulocytes from healthy donors lyse human neuroblastoma cells in the ADCC-reaction using antibody MAb 14.18 directed to ganglioside GD2 present on the surface of most neuroblastoma cells. Addition of catalase, superoxide dismutase and azide do not impair this process. Granulocytes from patients with chronic granulomatous disease (CGD) kill neuroblastoma cells even better than those collected from healthy donors. These results indicate that reactive oxygen intermediates (ROI) are not involved in killing of neuroblastoma cells using MAb 14.18, and that granulocytes from patients with CGD may compensate for defects in generation of reactive oxygen intermediates by more effective oxygen-independent killing mechanisms. One patient with CGD was treated with interferon-gamma. During and after treatment, generation of ROI could not be detected and neuroblastoma cell killing was not significantly altered.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1989

Rate of internalization of an immunotoxin correlates with cytotoxic activity against human tumor cells.

The relationship between the cellular internalization of an anti-ganglioside GD2 monoclonal antibody (14.G2a) and the toxic effect of its ricin A-chain immunotoxin (14.G2a-RA) was examined on GD2-bearing M21 human melanoma and T293 small cell lung carcinoma cell lines. The capacity for ligand uptake was determined by examining the parameters that contribute to this constant, including the number of cell-surface binding sites and the internalization rate constant (ke). The maximum uptake of 14.G2a is 11-fold greater for M21 than for T293 cells, due to a 2.7-fold difference in binding sites and a 4-fold difference in the rate of antibody internalization. The capacity for ligand uptake correlates with the cytotoxic activity of the 14.G2a-RA immunotoxin against these two cell lines. Furthermore, we were able to demonstrate that the consequence of internalization of 14.G2a-RA is the intracellular release of undegraded ricin A-chain from the antibody. These studies indicate that the rate of internalization is a quantitative parameter that plays a key role in predicting the cytotoxic potency of this immunotoxin.

Antibodies, Monoclonal↗
PubMed1988

Pharmacokinetics and mechanism of action of a doxorubicin-monoclonal antibody 9.2.27 conjugate directed to a human melanoma proteoglycan.

Doxorubicin (DXR) conjugated to a monoclonal antibody (MAb), 9.2.27, which recognizes a human melanoma-associated proteoglycan, effectively suppresses the growth of human melanoma xenografts and prolongs the life span of tumor-bearing athymic nude (nu/nu) mice. We have investigated further the mechanism(s) of this in vivo antitumor activity. Our results indicate that following iv injection, the DXR-MAb 9.2.27 conjugate is cleared from the circulation with typical biphasic kinetics, similar to the clearance process of the unconjugated MAb 9.2.27. In contrast, less than 10% of injected dose per milliliter of blood is found in the circulation at any given time after ip injection. Toxicity studies further indicate that DXR-MAb 9.2.27 conjugate is less toxic in vivo than the freely administered DXR, which is known to cause considerable cardiotoxic effects. Direct autoradiography demonstrates that the DXR-MAb 9.2.27 conjugate binds specifically to the tissue sections derived from a human melanoma xenograft of a nude mouse. A critical evaluation is given of the relevance of these findings and their impact on the design of future strategies for the immunochemotherapy of malignant melanoma.

Animals↗
PubMed1988

A human brain glycoprotein related to the mouse cell adhesion molecule L1.

We have employed monoclonal antibody 5G3, an antibody used to label human tumor cells of neural origin (Mujoo, K., Spiro, R.C., and Reisfeld, R. A. (1986) J. Biol. Chem. 261, 10299-10305), to isolate and characterize a large glycoprotein from normal adult human brain. This protein was compared to mouse L1 (Rathjen, F. G., and Schachner, M. (1984) EMBO J. 3, 1-10), a neural cell surface glycoprotein implicated predominantly in neurite-neurite interactions. On the basis of the following results the 5G3 antigen is considered to be the human homologue of mouse L1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both proteins share similar molecular masses of their carbohydrate-depleted or undepleted components. In tryptic fingerprint analyses of the iodinated L1 and 5G3 components, 65% of the resolved peptides comigrated. Comparison of NH2-terminal amino acid sequences revealed a high degree of homology between human 5G3 and mouse L1, with 11 of 15 residues being identical. Furthermore, polyclonal antibodies to human 5G3 antigen were found to be cross-reactive with mouse L1 antigen and vice versa. All components of 5G3 and L1 antigens show considerable charge heterogeneity with partial overlapping of regions in isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These findings provide a basis for studying the role of the human L1 homologue in human diseases.

Amino Acid Sequence↗
PubMed1988

Biochemical characterization of a sulfated phosphoglycoprotein antigen expressed on human small cell lung carcinoma.

A murine monoclonal antibody (mAb A23-16) was produced that recognizes a glycoprotein antigen preferentially expressed on the surface of human small cell lung carcinoma cells. This antibody is of IgG 1 isotype, has an association constant of 5 x 10(7) M-1, and reacts preferentially with human small cell lung carcinoma cell lines and fresh frozen sections in enzyme-linked immunosorbent assays and immunoperoxidase assays, respectively. The antigen recognized by A23-16 is a sulfated glycoprotein with phosphorylated threonine residues. The mature 90-kDa molecule has intrachain disulfide bonds and appears to be derived from a 76-kDa precursor, that is neither sulfated nor phosphorylated, but contains N-linked oligosaccharides. Conversion of the 76-kDa precursor to the mature form is accompanied by processing of these oligosaccharides from the high mannose to the complex type, although the increase in molecular mass from 76 to 90 kDa cannot be accounted for by this modification alone. MAb A23-16 reacts with its target antigen independent of the N-linked oligosaccharides, but requires intact intrachain disulfide bond(s) for reactivity. These studies on the molecular characterization of a monoclonal antibody-defined glycoprotein, preferentially expressed by small cell lung cancer, provide a basis for further structural and functional studies that may eventually lead to a delineation of its biological relevance for neoplastic transformation.

Adult↗
PubMed1988

Doxorubicin conjugated with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenografts in nude mice.

Doxorubicin (DXR) was covalently conjugated to a monoclonal antibody (mAb), 9.2.27 (IgG2a), which recognizes a chondroitin sulfate proteoglycan expressed preferentially on the surface of human melanoma cells. Immunoconjugates with a molar ratio of DXR to mAb ranging from 2:1 to 10:1 were obtained by coupling the drug via an acid-sensitive linker, cis-aconitic anhydride. The immunoreactivity of mAb 9.2.27 was well retained after conjugation. DXR-mAb 9.2.27 conjugates were found to be 2 orders of magnitude more potent in killing tumor cells in vitro (IC50 = 0.1 microM) than free drug targeted to drug receptor(s). Most significantly, DXR-mAb 9.2.27 immunoconjugates specifically suppressed the growth of established tumors in vivo and prolonged the life-span of tumor-bearing nude mice. This suppression of melanoma growth achieved by the immunoconjugate was both tumor and antibody specific. A biodistribution study indicated that DXR-mAb 9.2.27 conjugates delivered at least 4 times more DXR (3.7% total injected dose per g of tumor) as compared to free DXR alone (0.8% total injected dose per g of tumor) in tumor-bearing nude mice 48 hr postinjection. The tumor-suppressive effects of DXR-mAb 9.2.27 conjugates are even more remarkable since free DXR did not suppress tumor growth in vivo and also because this drug per se is known to be quite ineffective for the treatment of human melanoma.

Adenocarcinoma↗
PubMed1987

Disialoganglioside GD2 on human neuroblastoma cells: target antigen for monoclonal antibody-mediated cytolysis and suppression of tumor growth.

A murine monoclonal antibody 14.18 specifically recognizes disialoganglioside GD2, the major ganglioside expressed on the surface of human neuroblastoma cells. This monoclonal antibody (Mab) is of immunoglobulin G3 isotype, has an affinity constant (KA) of 3.5 X 10(8) M-1, and reacts preferentially with tumor cells and fresh frozen tumor tissues of neuroectodermal origin in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Mab 14.18 effectively lyses a number of human neuroblastoma cell lines by two distinct mechanisms, i.e., antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. There is a good correlation between the average number of antibody-binding sites per neuroblastoma cell and the amount of cell lysis observed in complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. In addition, Mab 14.18 suppresses establishment as well as growth of progressively growing, established human neuroblastoma tumors in nude mice when injected 24 h and 9 days, respectively, after the initial s.c. inoculation of tumor cells. These data suggest that Mab 14.18 can mediate tumor cell killing in vivo and in vitro and may thereby prove useful for immunotherapy of human neuroblastoma.

Antibodies, Monoclonal↗
PubMed1987

An Arg-Gly-Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2.

The disialogangliosides GD2 and GD3 play a major role in the ability of human melanoma cells to attach to Arg-Gly-Asp-containing substrates such as fibronectin and vitronectin, since pretreatment of these cells with monoclonal antibodies to the oligosaccharide of GD2 and GD3 can inhibit their attachment and spreading on such adhesive proteins. This report demonstrates that human melanoma cells (M21) synthesize and express a glycoprotein receptor that shares antigenic epitopes with the vitronectin receptor on human fibroblasts and is capable of specifically recognizing the Gly-Arg-Gly-Asp-Ser-Pro sequence. In the presence of calcium, GD2, the major ganglioside of M21 cells, colocalized with this receptor on the surface of human melanoma cells and their focal adhesion plaques as demonstrated by double-label transmission immunoelectron microscopy and indirect immunofluorescence. Biochemical evidence is presented indicating that the vitronectin receptor on M21 human melanoma cells contains associated calcium and GD2. This ganglioside copurified with the glycoprotein receptor for vitronectin on affinity columns containing either an Arg-Gly-Asp-containing peptide, concanavalin A, or lentil lectin. This major Arg-Gly-Asp-directed receptor on M21 cells could be metabolically labeled with 45Ca2+. Chelation of this ion with EDTA caused the dissociation of GD2 from the receptor and rendered the remaining glycoprotein incapable of binding to an Arg-Gly-Asp-containing peptide. Reconstitution experiments demonstrated a requirement for calcium, and not magnesium, for receptor binding to Arg-Gly-Asp and indicated that addition of ganglioside can enhance this interaction.

Amino Acid Sequence↗
PubMed1986

Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells.

In order to develop a molecular probe to delineate chemical and biological characteristics of human neuroblastoma cells, a murine monoclonal antibody (Mab 5G3) was produced that is directed to a glycoprotein, preferentially expressed on the surface of such cells. This antibody is of IgG2a isotype, has an association constant of 8 X 10(9) M-1, and reacts preferentially with human neuroblastoma cell lines and fresh frozen tissue sections in enzyme-linked immunosorbent assay and immunoperoxidase assays, respectively. Minimal reactivity is observed with a variety of lymphoblastoid cell lines and normal fetal and adult tissues. Mab 5G3 specifically recognizes a neuroblastoma target glycoprotein antigen of 215 kDa that is derived from a 200-kDa precursor, as evident from pulse-chase biosynthetic studies. Treatment with tunicamycin revealed that both molecules contain N-asparagine-linked oligosaccharides; however, only the 215-kDa species is resistant to treatment with endo-beta-N-acetylglucosaminidase H and sensitive to neuraminidase, indicating that it contains trimmed and terminally sialylated oligosaccharides of the "complex" type. In contrast, the 200-kDa precursor is sensitive to endo-beta-N-acetylglucosaminidase H and resistant to neuraminidase treatment indicating that it contains high-mannose non-processed oligosaccharides. The 215-kDa molecule is sulfated, phosphorylated at serine residues, and expressed on the cell surface. A molecule of 200 kDa is detected by Mab 5G3 in spent culture medium of human neuroblastoma cells which is neither sulfated nor phosphorylated.

Animals↗
PubMed1986

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonium Chloride↗
PubMed1986

Ammonium chloride interferes with a distinct step in the biosynthesis and cell surface expression of human melanoma-type chondroitin sulfate proteoglycan.

Human melanoma cells synthesize a cell-associated chondroitin sulfate-rich proteoglycan, whose core protein is recognized by monoclonal antibody 9.2.27. We report that the core protein is present on the surface of melanoma cells in two forms, either free or modified by the addition of chondroitin sulfate chains, suggesting that the addition of glycosaminoglycan chains may not be a prerequisite for cell surface expression of the proteoglycan core protein. Free core protein found at the cell surface does not seem to represent an overflow of the proteoglycan synthetic pathway, since experiments using a beta-D-xyloside acceptor suggest that core protein is, in fact, limiting proteoglycan synthesis. NH4Cl inhibits the synthesis of melanoma-type proteoglycan, shifting the balance of surface core protein toward the free form. The inhibition of proteoglycan synthesis is apparently not due to a disruption of enzymes and precursors involved in glycosaminoglycan synthesis, since cells treated with NH4Cl retain their ability to initiate and elongate chondroitin 4-sulfate chains on a beta-D-xyloside acceptor. In contrast, the divalent ionophore monensin inhibited core protein maturation and synthesis of glycosaminoglycan chains. The effects of both NH4Cl and monensin were reversible; thus, experiments using the drugs sequentially indicated that monensin temporally precedes NH4Cl in interfering with proteoglycan biosynthesis. Since the NH4Cl and monensin share the property of inhibiting the acidification of intracellular vesicles within cells, the present findings raise the possibility that the accessibility of proteoglycan core protein to the Golgi site of glycosaminoglycan addition is regulated in melanoma cells by acidification of intracellular compartments.

Aggrecans↗
PubMed1986

Activation of human peripheral blood mononuclear cells by anti-T3: killing of tumor target cells coated with anti-target-anti-T3 conjugates.

Cytotoxic T-cell clones have been shown to be capable of killing unrelated target cells in the presence of heteroconjugates consisting of OKT3 (monoclonal antibody against cell surface antigen T3) and targeting antibody. In contrast, human peripheral blood T cells were not known to have this ability, although these cells have been shown to undergo proliferation in response to OKT3 and accessory signals. Here we report that OKT3-stimulated human peripheral blood mononuclear cells effectively killed human melanoma or Raji cells coated with anti-target-cell--OKT3 conjugates. The extent of target-cell killing by stimulated effector cells was much greater than that observed with unstimulated cells, in spite of down-regulation of the T3 structure on the OKT3-treated cells. Target-cell lysis occurred at physiological concentrations of effector cells and was inhibited by monomeric OKT3, indicating that killing was T-cell-mediated.

Antibodies, Monoclonal↗